CN101085091A - Traditional Chinese medicine extraction for preventing and curing osteoporosis and its preparation method - Google Patents

Abstract

The invention relates to a Chinese traditional medicine extract for preventing and treating osteoporosis and its preparation method and application. The extract mainly comprises rhizoma atractylodis volatile oil, total alkaloid of Phellodendron amurense, and total saponins of radix achyranthis bidentatae and total sterone of radix achyranthis bidentatae; can be used for preventing and/or treating osteoporosis and relevant disorder, which has gradually become important pathogenesis influencing life-quality. The extract has easily-accessible raw materials, simple preparation, high extraction efficiency, strong pharmacological action, stable quality, high safety, low toxicity, and controllable quality. The invention comprises three Chinese traditional medicines in classical prescription, come to optima activity for resisting osteoporosis, reduces adverse side effect, and provides a new medicament source for preventing, diagnostic ting, detecting, protecting, treating and researching these diseases and their relevant diseases.

Description

A kind of Chinese medicine extract that is used for protect against osteoporosis and preparation method thereof

Technical field

The present invention relates to medicine, food, technical field of beverage, specifically relate to a kind of Chinese medicine extract and its production and use, more particularly relating to a kind of is Chinese medicine extract of feedstock production and its production and use with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae.

Background technology

(1) research overview of osteoporosis

Osteoporosis (Osteoporosis) is a kind of common, multiple Senile disease.Along with the aged tendency of population phenomenon is on the rise, osteoporosis is having a strong impact on old people's quality of life, especially postmenopausal women.It is reported that existing more than 8,000 ten thousand people of China suffer from this disease and need treatment, the U.S. suffers from this disease person according to statistics 2,500 ten thousand people is arranged approximately, and Japan then has 8,000,000 people approximately.In bedfast old people, have 20% to be that osteoporosis causes, because of causing fragility fractures, this disease finally causes dead the 12nd of having leapt to various cause of disease mortality rates, only in the sick for this reason annual medical expense that will pay more than 100 hundred million dollars at least of the U.S..There are some researches show, in following 60 years, the hip fracture number that is caused by osteoporosis in the world wide will reach 6,000,000, the huge spending of medical expense, to society and family all is a kind of white elephant, gives patient's human body and has also produced very big misery mentally.

The etiology and pathology of osteoporosis is learned and be studies show that, postmenopausal osteoporosis mainly is that (Estrogen, be called for short: E) level decline causes owing to estrogen.Estrogen can increase calcitonin (Calcitonin, be called for short: secretion CT), suppress parathyroid hormone (be called for short: activity PTH), melt thereby suppress bone calcium.In addition, estrogen can be adjusted to the activity and the function of osteocyte and osteoclast.Osteoblast (osteoblast during estrogen loss, be called for short: OB) formation minimizing and osteogenesis function go down, and osteoclast (osteoclast is called for short: formation OC) and raise increase, broken bone effect strengthens, owing to occur negative balance between osteoblast and the osteoclast bone amount is reduced.Therefore, early prevention osteoporosis and the miscellaneous fracture that causes thereof then seem even more important.Though the medicine of protect against osteoporosis is more, as estrogen, calcium preparation and bis phosphoric acid salt etc., certain side effect is arranged all, as estrogen the danger of bringing out carcinoma of endometrium and breast carcinoma is arranged, its clinical practice is restricted.Chinese medicine and natural product thereof have multiple mechanism of action, and few side effects uses various modern technologies to seek from Chinese medicine and compound recipe and has active lead compound of this respect or new drug, will be an important research direction.

(2) research overview of prescription Chinese medicine

1, general introduction

Ermiao san, three wonderful balls are the Chinese traditional patent formulation that Chinese Pharmacopoeia (2005 editions) records.Wherein, three wonderful balls are made up of Cortex Phellodendri, Rhizoma Atractylodis, Radix Achyranthis Bidentatae three flavor Chinese medicine (3: 2: 1) preparations, and Cortex Phellodendri, Rhizoma Atractylodis (1: 1) are formed ermiao san.Ermiao san side's name comes from " danxi's experiential therapy " of Zhu Zhenheng (1281～1358), and " medical science main story " volume of then showing 1515 Yu Tuans 5 adds Radix Cyathulae with we, is smalls, batter is ball, and is big as Semen Firmianae, name three wonderful balls, cure mainly damp and hot dirty, numbness of feet, or as the heat that fire irons, three wonderful balls, the Triple Magic Powder (soup) used till today all come from this, after adding Radix Achyranthis Bidentatae in the side, then descending power strengthens, and more is longer than vitamin B1 deficiency, the waist knee joint controlling damp invasion of lower energizer and cause and aches eczema and leukorrhagia, stranguria with turbid discharge.

Three wonderful ball tradition are used for heat clearing and damp drying, can be used for treating children's's chickenpox, limbs joint pain and damp-heat in lower-JIAO, pelvic inflammatory disease etc. clinically.

Studies show that the Triple Magic Powder extracting solution can improve the carbon clearance speed of mice, improve lymhocyte transformation rate and erythropoiesis, can improve immunity, the partial symptoms that prompting causes aging is effective.The Radix Achyranthis Bidentatae that contains in the side has the effect of invigorating the liver and kidney, bone and muscle strengthening, removing blood stasis collateral dredging, conducting blood to flow downwards, contain compositions such as saponin, ecdysterone, oleanolic acid, glucuronic acid, wherein contained sterols composition on chemical constitution with estrogenic structural similarity, it can combine with the estrogen receptor on the osteoblast, activate the regulator gene on the osteoblast, be adjusted to the activity of osteocyte and osteoclast, promote bone formation, suppress bone resorption.Thereby reach the effect that prevents bone loss.The phytoestrogen constituents is existing uses protect against osteoporosis clinically, and side effect is less.

2, the research overview of Cortex Phellodendri, Rhizoma Atractylodis, Radix Achyranthis Bidentatae

(1) Cortex Phellodendri

Cortex Phellodendri is the dry bark of rutaceae wampee Phellodendron chinense Schneid. or Cortex Phellodendri Phellodendron amurenseRupr., and the former practises title " Cortex Phellodendri ", and the latter practises title " Cortex Phellodendri "; " Cortex Phellodendri " main product in Sichuan, Guizhou, Hubei, Yunnan, also produce in Hunan, Gansu, Guangxi." Cortex Phellodendri " main product in Jilin, Liaoning, Hebei, also produce in Heilungkiang, the Inner Mongol.Cortex Phellodendri has another name called nest wood (Shennong's Herbal), LIPI (treatise on Febrile Diseases), yellow poly-(Collective Notes to the Canon of Materia medica), wampee (Sichuan), and head is stated from Shennong's Herbal.Bitter in the mouth, cold in nature.Return kidney, urinary bladder channel.Energy heat clearing and damp drying, pathogenic fire purging are removed and are steamed, and skin ulcer is treated in detoxifcation.Be used for damp-heat dysentery, yellow cellulitis, leukorrhagia, pyretic stranguria, beriberi, hectic fever due to YIN-deficiency consumptive fever, night sweat, seminal emission, sore swollen toxin, eczema pruritus.Salt Cortex Phellodendri (processed) nourishing YIN to lower pathogenic fire is used for hyperactivity of fire caused by deficiency of YIN, the night sweat hectic fever due to YIN-deficiency.

Chemical constituent

Mainly contain alkaloid, it is mainly formed berberine (berberine), palmatine (palmatine, palmatine), jateorhizine (jatrorrhizine), magnoflorine (magnoflorine) etc., other contains phellodendrine (phelldendrine), Dauricine (menisperine), N monomethyl hordenine (candicine) etc.Still contain in addition obakulactone (obaculactone), obacunone (obacunone), limonin (limonin), cupreol (β-sitosterol), campesterol (campesterol), 7-Dehydrostigmaslerol (7-dehydrostigmasterol), Dictamnolide (dictamnolide), obacunonic acid (obacunonic acid), lumicaeruleic acid (and lumicaeruleic acid, berrubine), chlorogenic acid compounds, the single glucoside of lignanoid, fatty oil, volatile oil, trace element etc.Contained chemical constituent is basic identical in Cortex Phellodendri root bark and the bark, but the content of little poly-alkali, root bark is higher than bark.Outside the Herb disleaf, fruit and xylem also all contain fruitlet alkali.Place of production difference, the fruitlet alkali content is variant.Root bark also contains Xiao Li alkali, jateorhizine, phellodendrine, candicine.Main component of essential oil is myrcene (myrcene) in the Cortex Phellodendri fruit, accounts for 60～70% of total oil mass.

Pharmacological action

1. antibacterial action Cortex Phellodendri water decoction or pure preserved material have in various degree inhibitory action to multiple pathogenic bacteria, as staphylococcus aureus, Staphylococcus albus, Staphylococcus citreus, Hemolytic streptococcus, Diplococcus pneumoniae, anthrax bacillus, vibrio cholera, diphtheria corynebacterium, bacillus subtilis, escherichia coli, bacillus pyocyaneus, Bacillus typhi, Salmonella paratyphi, meningococcus, Bacillus foecalis alkaligenes etc. and various dysentery bacterium in various degree inhibitory action are arranged all.In addition, Cortex Phellodendri also has stronger inhibition or killing action to tubercule bacillus, leptospira etc., but invalid to the experimental mycobacterium tuberculosis infection of Cavia porcellus.Research is closely arranged, and Cortex Phellodendri can also significantly suppress the growth of Streptococcus mutans.Planting alkaloid more than contained is the Cortex Phellodendri antibiotic effective ingredient, as nidulus alkali, palmatine, jateorhizine etc. stronger antibacterial activity is arranged all, and the antibacterial activity of palmatine and little fine strain of millet base are originally identical or lower slightly.Cortex Phellodendri all has stronger inhibitory action external to the various skin pathogenic fungus, and Candida albicans, trichomonas vaginitis are also had remarkable inhibiting activity.In addition, Cortex Phellodendri also has effects such as resisiting influenza virus.Cortex Phellodendri also has the obvious selectivity inhibitory action for hepatitis B surface antigen.

Berberine is the topmost antimicrobial component of Cortex Phellodendri, and it has broad-spectrum in-vitro antibacterial effect, and it is antibacterial when low concentration, sterilizes during high concentration.Anti-dysentery bacterium action intensity is close with sulfanilamide, but its effect is not subjected to influence of serum.Drug resistance strain produces more, and does not have complete intersection drug resistance.Other there are some researches show that the shuttle process is taken off in the oxidation of berberine energy strong inhibition yeast and antibacterial carbohydrate metabolism intermediate link acetone acid, and its antibacterial action can be by vitamin B6, institute's antagonisms such as vitamin PP, para-amino benzoic acid.Berberine also suppresses protein of bacteria, nucleic acid metabolism, and RNA of vibrio cholera and protein synthesis are suppressed.It is synthetic relevant with concentration that berberine suppresses bacterial nucleic acid.Berberine also has the antibacterium detoxifying function, studies show that, berberine can resist hyperfunction, the diarrhoea and dead of intestinal secretion due to vibrio cholera and the escherichia coli, and can resist the edema of intestinal villus due to the cholera toxin, significantly suppresses the caused local inflammation of subcutaneous injection cholera toxin.

Except that berberine, contained other the multiple alkaloid of Cortex Phellodendri also has remarkable antibacterial activity.The antibacterial action that the report palmatine is arranged is a little less than berberine or similar to it, and 0.05% palmatine is 1.07cm to the inhibition zone of golden Portugal bacterium, and is that the 1.26cmo jateorhizine also has certain resisting pathogenic microbes effect with the little poly-alkali of concentration.

2. hypotensive effect Cortex Phellodendri fluid extract or alcohol extract alkaline matter lumbar injection all have significant antihypertensive effect.Cortex Phellodendri 2g/kg gavages guilt ball excision hypertensive rat blood pressure is reduced.By Cortex Phellodendri compatibility Rhizoma Curculiginis, excessive sheep flower bud, Radix Morindae Officinalis, the Rhizoma Anemarrhenae, when the two celestial mixture duodenal administrations that are grouped into, anesthetized cat and chronic renal type hypertension also there is certain hypotensive effect, the square for this reason blood pressure lowering principal agent of Cortex Phellodendri.The contained multiple composition of Cortex Phellodendri all has in various degree antihypertensive activity as berberine, phellodendrine, palmatine etc.Its blood pressure lowering mechanism and block nerves joint, suppress the blood vessel maincenter and resist neurotransmitter such as sympathetic relevant.The antihypertensive effect of jateorhizine then may be relevant with anti-sympathetic nerve medium.In addition, magnoflorine also has hypotensive effect.Rhizoma Coptidis toxic materials clearing away decoction also has remarkable antihypertensive effect, and Cortex Phellodendri is one of its blood pressure lowering principal agent, has therefrom got the active substance with α-epinephrine and beta-adrenaline.

3. the Cortex Phellodendri that influences to digestive system has inhibitory action to tryptic activity, and this effect berberine contained with it does not have obvious relation.For rat experiment gastric ulcer due to hydrochloric acid one ethanol, Cortex Phellodendri 50% methanolic extract has remarkable protective effect, and berberine is also effective, but the total extract antiulcer activity is strong than berberine.For intestinal smooth muscle, Cortex Phellodendri can strengthen the rabbit intestine in vitro and shrink, and its contained berberine also increases shrinkage amplitude, the also excited intestinal smooth muscle of obacunone, and obakulactone is then counter to relax intestinal tube.In addition, Cortex Phellodendri water extract can promote hungry tame lapin bile and pancreatic secretion.After have the experiment show that Cortex Phellodendri truly has choleretic effect, can promote bile secretion, and promote bilirubinic discharge.

4. contain berberine in other effect Cortex Phellodendris; can strengthen the ability that experimental staphylococcus aureus septicemia canine leucocyte is engulfed golden Portugal bacterium; and watch for animals and avoid death, 2～5mg/kg is quiet to be annotated in rabbit, also can strengthen the phagocytic activity of its reticuloendothelial system.Berberine 50mg/kg gavages 1 time or continuous 7 daily can reduce normal mouse blood glucose, deliver medicine to medicine for 1 time after effect in 2～4 hours the strongest, and experimental hyperglycemia disease mice due to glucose and the epinephrine had blood sugar decreasing effect.For spontaneous diabetes KK mice and alloxan diabetes mice, the continuous first quarter moon of 50mg/kg berberine also has remarkable result, can improve the glucose tolerance of KK mice.

5. the contained alkaloid of toxicity Cortex Phellodendri is few in intestinal absorption, so toxicity is low, its contained multiple quaternary amine alkali gastrointestinal administration toxicity is also little, but drug administration by injection then can have big toxicity.The LD of Cortex Phellodendri mouse peritoneal injection ₅₀Be 2.7g/kg.

(2) Rhizoma Atractylodis

Dry rhizome for feverfew Atractylodes lancea (Thunb.) DC. Atractylodes lancea (Thunb.) DC. or Atractylis chinensis Atractylodes chinensis (DC.) Koidz.Acrid in the mouth, hardship, warm in nature.Return spleen, stomach, Liver Channel.Can be drying damp and strengthening spleen, dispel the wind, cold expelling makes eye bright.Be used to take off distention and fullness in the abdomen, have loose bowels, edema, a beriberi paralysis ancient piece of jade, round, flat and with a hole in its centre.Rheumatic arthralgia, anemofrigid cold, nyctalopia nyctalopia.

Rhizoma Atractylodis have another name called red art (TAO Hong-Jing), horse Ji (" saying that literary composition system passes "), blue or green art (open and fill " water south writing brush note "), magic arts (Compendium of Material Medica), thatch art (Jiangsu), magnificent Rhizoma Atractylodis (Ningxia).The Rhizoma Atractylodis beginning is stated from Shennong's Herbal, classifies as top grade.

Atractylodes lancea (Thunb.) DC. main product Jiangsu, Hubei, Henan.In addition, Zhejiang, Anhui, Jiangxi etc. are also economized and are produced.Provinces such as Atractylis chinensis main product Hebei, Shanxi, Shaanxi.In addition, also produce in Liaoning, Jilin, Henan, Shandong, the Inner Mongol, Gansu.

Chemical constituent

Rhizome contains volatile oil, and main component has atisine chloride atractydin (atractylodin), hinesol (hinesol), eucalyptol (eudesmol), elemi oleyl alcohol (elemol), atractylone (atractylon), P-cymene (cymol), bisabolol (bisabolol), hydroxyl atractylone (hydroxyatractylon), acetoxyl group atractylone (acetoxyatractylon), atisine chloride atractydin alcohol (atractylodinol), acetyl atisine chloride atractydin alcohol (acetylatractylodinol) in the oil.Atractylodes lancea (Thunb.) DC. also contains atractylodes lactone (butenolide), sesquiterpene glucosides and trace.Ou Cangshu (A.gummidera L.) contains atractyloside (atractyloside), and the Atractylodes lancea (Thunb.) DC. volatile oil content is 5～9%, and Atractylis chinensis content is 1～2.5%.

Pharmacological action

1. to blood glucose influence rabbit subcutaneous injection Rhizoma Atractylodis water decoction 8g (crude drug)/kg or pure extractum 8g (crude drug)/kg the time, tame rabbit blood glucose is risen.When rabbit is irritated stomach Rhizoma Atractylodis water decoction 8g (crude drug)/kg, blood glucose is risen.Atractyloside then has hypoglycemic activity to mice, rat, rabbit and dog, and its blood sugar lowering and the effect of reduction hepatic glycogen reduce oxygen consumption, but the blood lactic acid content is increased, may be because atractyloside has promoted glucolytic cause.

2. antiulcer action Rhizoma Atractylodis 50% methanolic extract has antiulcer action.When 200mg/kg gives the rat oral gavage administration, to pyloric ligation ulcers, histamine's type, aspirin type and stress fixed antiulcer action all be arranged the type gastric ulcer, ulcer index is reduced, gastric juice amount, gastric acidity and pepsin activity are all had obvious inhibitory action.Serum ulcer system is because of the gastric mucosa ischemia, circulatory disturbance and the energy shortage that causes, and the mucosa resistance weakens, and causes tissue necrosis and the ulcer that forms, and Rhizoma Atractylodis have positive effect to this.

3. the Rhizoma Atractylodis that influence to gastrointestinal motility have regulating action to gastrointestinal motility, advance experiment to find that the Rhizoma Atractylodis acetone extract can obviously promote gastrointestinal motility to whole animal with carbon powder, and its main component is eudesmol and hinesol.Activity all has than the obvious suppression effect to the rabbit duodenum for the alcohol extract of Rhizoma Atractylodis and aqueous solution, has the intestinal tube smooth muscle contraction that anti-acetylcholine is caused, the pipe smooth muscle after speeding is then had slight enhancing contraction.

4. other effects show that with potassium cyanide induced mice anoxia model Rhizoma Atractylodis acetone extract 750mg/kg gavages and can obviously improve the mice time-to-live, reduce mortality rate.Rhzoma Atractylodis Lanceae volatile oil, hinesol and eudesmol have inhibitory action external to esophageal cancer cell, and be wherein the strongest with the effect of hinesol.Rhzoma Atractylodis Lanceae volatile oil has sedation to the frog on a small quantity, makes spinal reflex hyperfunction simultaneously; Then be inhibitory action in a large number, finally make respiratory paralysis and death.

(3) Radix Achyranthis Bidentatae

Radix Achyranthis Bidentatae has another name called Radix Achyranthis Bidentatae, Radix Achyranthis Bidentatae, chicken glue bone, is the root of Amaranthaceae (Amarathaceae) plant Radix Achyranthis Bidentatae Achyranthes bidentataBL..Pharmacopoeia of People's Republic of China (version in 2005) records and is conventional Chinese medicine.Be distributed widely in ground such as Chinese Henan, Shanxi, Shandong, Jiangsu, Anhui.Property is put down, and the sweet in the mouth picric acid is gone into liver, kidney channel.Give birth to the congestion that looses the detumescence carbuncle; Ripely use invigorating the liver and kidney, bone and muscle strengthening.Has the effect that benefiting essence and marrow, tonifying YIN are invigorated blood circulation.Control waist kneecap pain, spasm of the limbs.

Chemical constituent

Radix Achyranthis Bidentatae contains multiple oligosaccharide, peptide polysaccharide and polysaccharide.Also contain multiple saponin component in the Radix Achyranthis Bidentatae, wherein based on the oleanolic acid type triterpene saponin.As radix achyranthis bidentatae saponin, Radix Achyranthis Bidentatae saponin.Also have ecdysterone and inokosterone, metamorphosis hormone sterone in addition.

Radix Achyranthis Bidentatae also contains flavonoid and alkaloids and Coumarins composition.As Quercetin-3-O-rutinoside, Quercetin-3-O-glucoside, kaempferol-3-O-glucoside.

In addition, still contain volatile oil, sitosterol, succinic acid, allantoin, chrysophanol, dibutyl phthalate, palmitic acid, daucosterol, ibuprofen and physcione and weight element etc.

Pharmacological action

1. the immunoregulation effect Inokopolyose (is called for short: ABPS) in the external secretion that can improve lymphocytic multiplication capacity of old Mus T and IL-2.Body is internal energy to significantly improve TNF and the generation of NO and the activity of NOS in senile rat T lymphocyte and the serum, has immunoregulation effect.Have the natural killer cell activity of enhancing and promote that (be called for short: Con A) inductive tumor necrosis factor produces concanavalin A external, strengthen inductive delayed hypersensitivity of dinitrofluorobenzene and antagonism cyclophosphamide to the active inhibitory action of NK, but the generation of inductive T cell proliferative response of Con A and IL-2 is not had obvious influence.ABPS ip 50mg/kg can obviously improve the content of total IgG and specific antibody hemolysin, and increases index and spleen index, can also resist specific antibody hemolysin that Ciclosporin A causes and the decline of IgG.ABPS is external to stimulate mouse boosting cell propagation, also can strengthen the inductive bone-marrow-derived lymphocyte propagation of LPS, has the humoral immunization enhancement function.

2. tumor inhibition effect Radix Achyranthis Bidentatae total saponins has the tumor cell inhibitory action, can significantly suppress mouse transplanted sarcoma 180 growths, improve low serum 1 g-G content and antibody forming cell's quantity and the splenic lymphocytes of tumor-bearing mice, can also improve the serum T NT-a generation that mice with tumor NK cytoactive and LPS induce, external do not have direct cytotoxicity to 5180 cells, but can strengthen among the M lethal effect to S180.Its antitumor action is relevant with its enhancing host immune function.

3. antiinflammatory, antibiotic different processed products with the analgesic activity Radix Achyranthis Bidentatae all have analgesic activity to a certain degree, and are wherein strong and lasting with the effect of wine Radix Achyranthis Bidentatae (processed).Radix Achyranthis Bidentatae has stronger resist inflammation on repercussive function.Because of its no thyroliberin sample effect, therefore antiinflammatory action is not by due to the adrenal cortex release 17-hydroxy-11-dehydrocorticosterone, but the human body immunity improving function, activate the phagocytosis of mouse macrophage system pair cell, and blood vessel dilating, improvement circulation promote inflammatory lesion absorption etc.

4. nootropics, anti-aging effects Radix Achyranthis Bidentatae decocting liquid gavage 7d for mice continuously, can obviously improve the dysmnesia due to the pentobarbital, with the diving tower method obviously prolonged the incubation period of jumping off first, errors number obviously reduces in the 5min, the 3rd day correct response rate of Y type arm method obviously improved, and can obviously prolong mice load swimming time.The Radix Achyranthis Bidentatae decocting liquid can significantly improve aging model mice blood SOD vigor and mice plasma due to the D-galactose, liver homogenate LPO level, and rising aging model mice blood hydrogen peroxide (is called for short: vigor CAT).

5. the effect for reducing blood fat Radix Achyranthis Bidentatae can make with high fat and bring out the Atherosclerosis Model crane quail that feedstuff causes, and serum TC, TG level significantly reduce, and reduces serum LPO level, has study of anti-atherogenic effect.

6. the function of resisting osteoporosis Radix Achyranthis Bidentatae is a kind of invigorating the kidney and strengthening the bones Chinese medicine, the kidney-nourishing tcm drug prescription water decoction that contains medicines such as the Cortex Eucommiae, Radix Achyranthis Bidentatae, Concha Ostreae, Radix Rehmanniae Preparata can alleviate rat uterus weight, increase bone trabecula density, area, cumulative volume and Compact bone area, reduce the medullary cavity area.

Radix Achyranthis Bidentatae also has uteri excitation effect and antifertility action

(3) extraction separation method of Chinese herbal medicine effective ingredients commonly used

1, solvent extraction method

(1) principle: solvent extraction method is according to the dissolution properties of various chemical constituents in solvent in the Chinese herbal medicine, select for use the active component dissolubility big, to not needing the little solvent of composition dissolubility of stripping, the method that effective ingredient is dissolved out in the medical material tissue.When solvent is added in the herbal raw material (needing suitably to pulverize), solvent is owing to diffusion, osmosis penetrate in the cell by cell wall gradually, dissolved solable matter, and cause concentration difference inside and outside the cell, so intracellular concentrated solution is constantly to external diffusion, solvent constantly enters in the medical material histiocyte again, so repeatedly come and go, when solution concentration reaches dynamic equilibrium inside and outside cell, this saturated solution is leached, continue repeatedly to add novel solvent, just can be bordering on complete stripping or the stripping of big portion to desirable ingredients.The dissolubility of medicinal herb components in solvent is directly relevant with solvent property.Solvent can be divided into hydrophilic organic solvent and lipotropy organic solvent, and dissolved material also has hydrophilic and lipophilic difference.Hydrophilic radical is many in the organic compound molecule structure, and its polarity is negligent of oil greatly; The hydrophilic radical that has is few, and its polarity is little and be negligent of water.The character of each kind solvent, equally also relevant with its molecular structure.Like this, the inventor just can remove to estimate their this type of character and the solvent of selecting for use by to the medicinal herb components structural analysis.Generally speaking,, bigger dissolubility will be arranged therein, i.e. the rule of so-called " similar mixing " as long as this character of the hydrophilic of medicinal herb components and lipotropy and solvent is suitable.This is to select appropriate solvent to extract one of foundation of required composition in Chinese herbal medicine.

(2) choice of Solvent: the key of utilization solvent extraction method is to select appropriate solvent.Solvent is selected suitably, just can be more successfully the composition of needs be extracted.Selective solvent will be noted following 3 points: 1. solvent is big to the effective ingredient dissolubility, and is little to the impurity dissolubility; 2. solvent can not play chemical change with the composition of Chinese medicine; 3. solvent want economical, be easy to get, safe in utilization etc.Common extraction solvent can be divided into following three classes:

1. water: water is a kind of strong polar solvent.Hydrophilic composition in the Chinese herbal medicine can both be gone out by water-soluble as the not too big polysaccharide of inorganic salt, saccharide, molecule, tannin, aminoacid, protein, acylate, alkaloid salt and glycoside etc.In order to increase the dissolubility of some composition, also often adopt sour water and aqueous alkali as extracting solvent.

Ethanol), methanol (but also claims: another name for), acetone etc., the most frequently used with ethanol 2. hydrophilic organic solvent: just general said and the miscible organic solvent of water (claim not only: as ethanol.Alcoholic acid solubility property is relatively good, and is stronger to the penetration capacity of Chinese herbal medicine cell.Outside hydrophilic composition isolating protein, phlegmatic temperament, pectin, starch and the part polysaccharide etc., big multipotency dissolves in ethanol.Be insoluble in the low-polarity component of water, the dissolubility in ethanol is also bigger.Can also adopt Different concentrations of alcohol to extract according to the character that is extracted material.More less than water consumption with ethanol extraction, extraction time is short, and it is also few to dissolve the water-solubility impurity that.Ethanol is organic solvent, though inflammable, toxicity is little, low price, and convenient sources has a locking equipment can reclaim repeatedly and use, and alcoholic acid extracting solution is difficult for moldy metamorphism.Owing to these reasons, be one of always the most frequently used method with the method for ethanol extraction.The character of methanol is similar with ethanol, boiling point lower (64 ℃), but toxic, should note during use.

3. lipophilic organic solvent: the organic solvent that just general said and water can not be miscible, as petroleum ether, benzene, chloroform, ether, ethyl acetate, dichloroethanes etc.These choice of Solvent performances are strong, can not or be not easy to propose hydrophilic impurities.But this kind solvent volatility is big, how inflammable (except the chloroform), generally poisonous, price is more expensive, equipment requirements is higher, and they penetrate plant tissue ability a little less than, often need to extract repeatedly for a long time and could extract fully.If contain more water in the medical material, just be difficult to leach its effective ingredient with this kind solvent, therefore, when extracting herbal raw material in a large number, directly using this kind solvent has certain limitation.

(3) extracting method: use the solvent extraction medicinal herb components, infusion process commonly used, percolation, decocting method, reflux extraction and continuous backflow extraction method etc.Simultaneously, factors such as the degree of grinding of raw material, extraction time, extraction temperature, appointed condition also can both influence extraction efficiency, must take in.

1. (be called for short: infusion process): the dipping genealogy of law is packed herbal powder or fragment in the proper container, adds The suitable solvent (as ethanol, rare alcohol or water), and the dipping medical material is with the stripping method of composition wherein to flood extraction method.This law is relatively simple, but leaching rate is relatively poor, and is solvent as water, and the easy moldy metamorphism of its extracting solution must note adding suitable preservatives.

2. (be called for short: percolation): percolation is that herbal powder is contained in the percolator to the percolation extraction method, constantly adds novel solvent, makes it penetrate medical material, flows out a kind of leaching method of leachate from top to bottom from the percolator bottom.When moving down when solvent infilters medicated powder, stripping composition proportion strengthens, its position is just replaced in the solution on upper strata or rare immersion, causes good concentration difference, and diffusion energy is carried out preferably, so leaching effect is better than infusion process.But should control flow velocity, in oozing transient, on powder, replenish novel solvent at any time, make till effective ingredient fully leaches in the medical material.Maybe extremely shallow or when oozing the volume that gushes liquid and being equivalent to heavy 10 times of crude drug when oozing the dropping liquid color, just can think and extract basically fully.The rare leachate that often will collect in mass production is as the usefulness of the solvent of another batch new raw material.

3. (be called for short: decocting method): decocting method is traditional leaching method that China uses the earliest to decoct extraction method.Used container is generally pottery, sand jar or copper, enamel ware, should not use iron pan, in order to avoid the medicinal liquid variable color.Preferably stir often during straight fire heating, in order to avoid that local medical material is heated is too high, burnt easily the paste.Big reaction pot, big copper pot, barrel are adopted in the pharmaceutical factory that steam-heating apparatus is arranged more, or feed Steam Heating in the pond of cement block.It is interconnection by pipeline also several can be decocted device, fries in shallow oil continuously and soaks.

4. heating and refluxing extraction method: use the organic solvent heating extraction, need to adopt the reflux device, in order to avoid the solvent evaporates loss.When operating in a small amount, can on round-bottomed flask, connect reflux condenser.The powder charge material is about 20%～60% of capacity in the bottle, and solvent soaked the about 1～2cm in medical material surface.Reflux in water-bath, the general maintenance, seethed with excitement 3～6 hours, puts cold filtration, and solubilizer in medicinal residues is made second and third time reflux and is made an appointment with half an hour respectively again, or to carrying till the most effective ingredient substantially.This method extraction efficiency adopts continuous extractions than the cold-maceration height more in the mass production.

5. continuous backflow extraction method: use volatile organic solvent and extract Chinese herbal medicine effective ingredients, no matter small test or large-scale production, all with continuous extraction for well, and need with quantity of solvent lessly, the extraction composition is also more complete.Laboratory fat-extraction device commonly used or title apparatus,Soxhlet's.Continuous extraction generally needs a few hours could extract fully.It is longer to extract the composition heated time, and the labile composition of case of thermal instability should not adopt this method.

2, separation and purification process

Resulting extracts of Chinese herbal medicine of said extracted method or extract remain mixture, need further remove impurity, separate and make with extra care.

(1) solvent segregation: generally be with above-mentioned total extract, select three for use, the solvent of four kind of opposed polarity, by low polarity to high polarity proceed step by step extraction separation.Aqueous extract or ethanol extract often are jelly, be difficult to be dispersed in the low polar solvent, so can not extract fully, can admix an amount of inert filler, as kieselguhr or fiber powder etc., low temperature or natural drying then, after the pulverizing, to select for use solvent to extract successively, make each constituent in the total extract again, obtain according to the difference of its dissolubility in the opposed polarity solvent separating.Utilize the Chinese herbal medicine chemical constituent, the dissolubility in the opposed polarity solvent carries out separation and purification, is the most frequently used method.

(2) solvent extraction:

1. extraction: the solvent extraction extraction is called for short extraction again, is to utilize the difference of each composition partition coefficient in two kinds of immiscible solvents in the mixture and reach isolating method.If each composition partition coefficient in solvent differs big more during extraction, then separation efficiency is high more; If the effective ingredient in aqueous extract is lipophilic material, the general lipotropy organic solvent of using more, extract as benzene, chloroform or ether, if effective ingredient is to be partial to hydrophilic material, indissoluble is separated in lipophilic solvent, just need use weak lipophilic solvent, for example ethyl acetate, butanols etc. instead.Can also in chloroform, ether, add an amount of ethanol or methanol to increase its hydrophilic.When extracting flavones ingredient, how to extract with ethyl acetate and water.Then multiselect n-butyl alcohol, isoamyl alcohol and water extract to extract the strong saponin of hydrophilic.But, the common organic solvents hydrophilic is big more, and the effect of doing extraction with water is just bad more, and is because more hydrophilic impurities is followed, very big to the further refining influence of effective ingredient.

2. counter current continuous extraction method: be a kind of successive solvent extraction.Its device can have one, several or more extracting tube.Fill the contact surface during with increase solvent extraction in the pipe with little porcelain circle or little rustless steel wire ring.If a kind of infusion of Chinese herbal medicine need extract with the benzene lighter than water, ethyl acetate etc., then need water extracting liquid is contained in the extracting tube, and benzene, ethyl acetate are stored in the high-level container.Extract whether complete, but sample thief is analysed with thin layer chromatography, ply of paper and chromogenic reaction or precipitation are checked.

3. counter-current distribution method: counter-current distribution method claims CCD method, counter-current distribution or countercurrent distribution again.Counter-current distribution method is consistent with solvent counter-current extraction principle, but the application of sample amount is certain, and continuous in the solvent of a constant volume, reaches the separation of mixture through repeatedly being shifted the extraction distribution.

4. drop counter-current distribution method: the drop counter-current distribution method claims the droplet countercurrent chromatography method again.Be improved solvent extraction on the counter-current distribution method basis in recent years.To the same substantially counter-current distribution method of the selection of solvent system, but requirement can be separated at short notice, and can generate effective drop.Because mobile phase forms drop, contacting effectively with immobile phase in thin distribution extracting tube, rubbing constantly forms new surface, promotes the distribution of solute in solvent, so its separating effect is often good than counter-current distribution method.

(3) macroporous adsorbent resin method: macroporous adsorbent resin is a class organic polymer adsorbent that grows up the sixties in 20th century, have the good adsorption performance, be applied to the development of the extraction separation and the new Chinese medicine of Chinese herbal medicine chemical constituent surplus in the of nearly ten over year gradually.

Macroporous adsorbent resin is for absorption and screen the parting material that principle combines.Its adsorptivity is because the result of Van der Waals force or generation hydrogen bond.The screening principle is because itself cellular structure determines.Because absorption and screening principle, organic compound separates through certain solvent elution on macroporous adsorbent resin according to the difference of absorption affinity and the size of molecular weight.This make organic compound especially the purification of water soluble compound simplified greatly.The skeleton of macroporous adsorbent resin is generated by styrene and divinylbenzene polycondensation, because the adding of modifier, the polarity of macroporous adsorbent resin changes, and according to the surface nature of resin, that adsorbent resin generally is divided into is nonpolar, Semi-polarity and polarity three classes.

Nonpolar adsorption resin be by the very little monomer-polymer of dipole moment make not with the adsorbent resin of any functional group.Typical example is the adsorbent resin of styrene-divinylbenzene system, as D101, XAD-1, DiaionHP-10 macroporous adsorbent resin.

The Semi-polarity adsorbent resin refers to contain the adsorbent resin of ester group, as an acrylate or a crosslinked analog copolymer such as methacrylate and double methyl methacrylate.It is on the basis of nonpolar macroporous adsorption resin, adds acrylic acid methyl ester. or acrylonitrile polycondensation and forms, as the AB-8 macroporous adsorbent resin of the domestic frequent use of China.

Polar Adsorbent Resin is meant that amide-containing, itrile group, phenolic hydroxyl group etc. are nitrogenous, the adsorbent resin of oxygen, sulfur polar functionalities base.In addition, sometimes the ion exchange resin of ligand groups such as nitrogenous, oxygen, sulfur is called strong Polar Adsorbent Resin, the boundary of strong Polar Adsorbent Resin and ion exchange resin is difficult to difference.Polar macroporous adsorption resin can be formed by methyl methacrylate, acrylamide or the polycondensation of sulfoxide class, as the Diaion HP 2MG of Mitsubishi chemical industry, the XAD-10 of U.S. Rohm-hass company, XAD-9 macroporous adsorbent resin.

Compare with other adsorbent with active carbon, macroporous adsorbent resin has a lot of advantages, and is higher as the adsorptive selectivity to certain material; Physical and chemical stability and mechanical strength are better; Description is more, can change resin physics or chemical constitution as required; Adsorbent resin is generally spherical particle, and fluid resistance is less or the like.Thereby be widely used in chemical industry, medicine and other fields, more and more about the applied research report of macroporous adsorbent resin in natural product extraction is separated in recent years.Macroporous adsorbent resin centering herbal chemistry composition such as alkaloid, flavone, saponin, coumarin and some other glycoside compositions all have certain adsorption.Absorbability to sugar is very poor, and is stronger to the absorbability of pigment.

(4) sedimentation method: be in extracts of Chinese herbal medicine, to add some reagent to make the generation precipitation, with the method that obtains effective ingredient or remove impurity.As lead salt precipitation: lead salt precipitation is one of classical way of separating some medicinal herb components.Because lead acetate and Lead monosubacetate in water and alcoholic solution, can generate the lead salt or the complex salt precipitation of indissoluble with multiple medicinal herb components, so can utilize this character that effective ingredient is separated with impurity.Then lead salt precipitation is suspended in the novel solvent, passes to hydrogen sulfide gas, make and decompose and transfer insoluble vulcanized lead to and precipitate.

(5) salting out method: salting out method is in the water extract of Chinese herbal medicine, adds inorganic salt to finite concentration, or the state that reaches capacity, and can make the dissolubility of some composition in water reduce precipitation and separate out, and separate with the big impurity of water solublity.Be commonly used for the inorganic salt of saltouing sodium chloride, sodium sulfate, magnesium sulfate, ammonium sulfate etc. are arranged.

(6) dialysis: dialysis is to utilize small-molecule substance can pass through semipermeable membrane in solution, and macromolecular substances can not reach isolating method by the character of semipermeable membrane.Otherwise also macromolecular impurity can be stayed in the semipermeable membrane, and micromolecular material is entered in the outer solution of film by semipermeable membrane, and separation and purification in addition.

(7) crystallization, recrystallization and Steppecd crystallization: identify the Chinese herbal medicine chemical constituent, study its chemical constitution, must at first medicinal herb components be prepared into the pure product of monomer.At normal temperatures, the character of material own is the chemical compound of liquid, can carry out separation and purification with fractionating process or chromatography respectively.In general, the Chinese herbal medicine chemical constituent is solid material at normal temperatures mostly, all has the general character of crystalline solid, can reach the purpose of separation and purification according to the difference of dissolubility with crystallization process.

3, conventional drying method

(1) vacuum drying: be based on such ultimate principle: water saturation vapour pressure and temperature are closely related, under vacuum state, the boiling point lowering of water, i.e. operation operation at low temperatures just under vacuum, can avoid the destruction of nutritional labeling such as vitamin etc. at high temperature, improve rate of drying simultaneously.Vacuum drying is widely used in industries such as food, pharmacy, chemical industry, and China also develops and introduced various vacuum dryers, and its version is varied.Form commonly used mainly contains box type vacuum exsiccator, bipyramid formula vacuum desiccator, belt vacuum desiccator etc.These traditional Minton dryers mainly adopt heating such as hot blast, steam or electricity, utilize conduction of heat, convection current or radiation theory that heat is passed to material inside from the outside.It is low that vacuum drying has a baking temperature, and anoxia relatively in the hothouse can be avoided fat oxidation, and series of advantages such as pigment brown stain are suitable for the drying of heat sensitivity food material, and equipment cost, dry expense are also relatively low in addition.

(2) spray drying: be that fluidization technique is used for the exsiccant a kind of method of liquid material.Because of being wink-dry, be specially adapted to heat sensitive material, so the products obtained therefrom quality is good, keep original color, smell and taste, and easily dissolving.The research that utilizes spray drying to prepare microcapsule is carried out, it is that heart material is suspended in the solution of dress material, through centrifugal atomizer it is sprayed in the thermal current, the product of gained is the microcapsule that dress material bag heart material forms, this microcapsule powder can be used in direct compression, also can prepare capsule, syrup or suspensoid.

(3) lyophilization: be that the dry liquid material is frozen into solid, under the low-temperature reduced-pressure condition, utilize the distillation performance of icing, make the low-temperature material dehydration and reach exsiccant a kind of method.Because material is dry under high vacuum and cryogenic conditions, so the drying of some extremely thermo-labile article is well suited for.Wang Dalin has reported a kind of spraying ventilation lyophilizing new technique, be to utilize cold air or nitrogen as medium, the scars of flowing through rapidly make water sublimate, the product microgranule that makes of spraying lyophilizing is little, fast drying, time are short, evenly, good fluidity, and the good instant capacity of tool.In recent years, plaster material and the exsiccant research of sticky material have been obtained bigger progress, fluidization technology, spraying technique, inert carrier technology then grow up on this research basis.Rotatingandflashstreamingdrier, thermojet pneumatic drier, inert carrier drying machine all are fit to the drying of heat sensitive material and plaster material.These new achievements in research are used for Chinese medicine preparation production, with improving the technical merit of Chinese medicine processing greatly, enhance productivity.

(4) far infrared heating drying method: be a new dry technology, its drying principles is to change electric energy into far infrared radiation, thereby by the molecule absorption of medical material, produce resonance, cause the vibration and the rotation of molecule and atom, cause the object heating,, finally reach exsiccant purpose through thermal diffusion, evaporation and chemical change.Far-infrared ray drying can be saved electric energy 20%～50%, and effect is better.

(5) micro-wave drying method: be the new technique that develops rapidly a sixties in 20th century, microwave drying is actually by eddy-current heating and medium heating, make moisture and fat in the thing that is dried absorb microwave energy to some extent, thereby and it is changed into heat reach exsiccant purpose.But microwave drying killing microorganisms and mycete, and has disinfective action.The microwave heating installation of China's production at present has 915mhz and two frequencies of 2450mhz.

4, supercritical CO ₂Extracting process

(1) extracting method about the animal and plant fatty oil component has: that water-boiling method, hydrophily partition method, molecular distillation and distillation under vacuum, metal add is legal, extractive crystallization process, branch coil freezing milling process and organic solvent method etc.

Adopt the decocting in water heating, long because of heat time heating time, temperature is high, easily make heat-labile volatile ingredient is changed, as the oxidation of some unsaturated fatty acids.Hydrophily partition method, the obvious advantage of this partition method are to have eliminated high cost and public hazards that organic solvent method causes, but the shortcoming of this technology is that the purity of the satisfied fatty acid that obtains and unsaturated fatty acid or rank are not as organic solvent partition method height.Molecular distillation and distillation under vacuum need to remove most of satisfied fatty acid with methods such as urea adducts for reducing thermal denaturation, must be removed in the product that obtains; Though can obtain purer fatty acid, need several steps just can reach target, more time-consuming, effort has increased production cost.Metal adds the lower K cryogenic treatment of legal separation needs, also will manage to remove metal ion at last, removes and not to the utmost also can cause metal ion pollution.It is higher that extractive crystallization process obtains fatty acid purity, carries out under lower temperature, can prevent oxidation of fatty acids, but the time is longer, not too is applicable to large-scale industrial production.Divide the freezing milling process shortcoming of dish to be, the refrigerating chamber of laying plate rail and hydraulic press needs thousands of square metres area and huge cooling system, and big facility needs many people's operations, the labor intensity height, and separating rate is slow, and efficient is low, can not continued operation.Organic solvent method is to adopt petroleum ether or ether organic solvent under heating state in the water-bath, carries out reflux cycle and extracts; Because the organic solvent such as petroleum ether or the ether etc. that are adopted are a kind of low-boiling point material, inflammable and explosive, simultaneously, the use of organic solvent pollutes the environment on the one hand, organic solvent might have residually in extract on the other hand, can influence extract in pharmaceutically further application.

(2) supercritical CO of relevant animal and plant fatty oil component ₂Extracting process: a kind of fluid is called as supercritical fluid when being in its critical temperature and pressure state.Because the density of supercritical fluid approaches liquid, has the extracting power suitable with liquid flux.Have the low viscosity suitable with gas again, diffusivity is bigger 100 times than liquid, thereby has higher mass transfer performances.When adopting supercritical extraction, utilize these character of supercritical fluid, making it to contact with material to be separated penetrates matrix, therefrom extracts target substance.Utilize the way of blood pressure lowering and/or intensification to reduce its density then, thereby reduce solute dissolubility therein, make extract and separated from solvent.Because different materials dissolubility under identical extraction conditions is different, thereby might be by this species diversity with them further separately.Supercritical fluid extraction is usually selected CO for use ₂Low and chemically inert material is an extractant Deng critical temperature, and it is specially adapted to separating of heat-sensitive materials and readily oxidizable substance, therefore is highly suitable for the extraction separation for animal oil.Therefore, the present invention takes this method, can prepare the fatty oil of highly purified extract.

Supercritical CO ₂Extracting process is to extract like this: the coarse powder of exsiccant extract is placed in the supercritical extraction equipment uses CO ₂Extract, promptly get the fatty oil of extract after the decompression, be light yellow transparent oily under the room temperature condition; The purity of total fatty acids is better than other extracting method more than 40 in the fatty oil of the extract that extracts; Total fatty acids generally comprises other components of oleic acid, linoleic acid, Palmic acid and surplus etc.

Supercritical CO ₂The method of the fatty oil of extraction extract comprises the steps:

(1) exsiccant extract is crushed to 10～30 orders after, place supercritical extraction reactor, feed the CO under the supercriticality continuously ₂Extract, preferred condition generally is: CO ₂Flow 10～55kg/hkg raw material, pressure 5～50Mpa, 15～75 ℃ of temperature, time 0.5～6h; Further preferred condition generally is: CO ₂Flow 45kg/hkg raw material, pressure 35Mpa, 50 ℃ of temperature, time 1h;

(2) will extract the CO of the fatty oil of extract ₂Be decompressed to 1～12MPa, can obtain the fatty oil of extract at ambient temperature, the purity of total fatty acids is more than 40% in the fatty oil of extract, and the physicochemical character of extract and fatty acid purity all are better than other extracting method.

The selection of extraction conditions realizes by following way: at first selected four factors, three levels, according to orthogonal design method, press L9 (3 ⁴) hand over design to show experiment arrangement (" medicostatistics " with reference to the Guo Zuchao chief editor, People's Medical Officer Press, front page in 1999 are arranged in the orthogonal experiment design).From Orthogonal experiment results, analyze the extraction process condition that obtains.

To supercritical CO ₂After the fatty oil of the extract of extraction carries out methyl esterification of fatty acid, carry out the gas chromatography and mass spectromentry analysis.

Supercritical CO ₂The preparation method of fatty oil of extraction extract does not with an organic solvent wait material, does not pollute the environment, and does not have problems such as being harmful to dissolvent residual yet, and easy and simple to handle, the extraction time is short, product purity is high, the CO of use ₂But cheap and repetitive cycling is used, and commercial production is with low cost.

By literature search etc., up to the present, still find no being the relevant report of the new compound of feedstock production extract with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae.

Summary of the invention

The technical problem that will solve required for the present invention is to disclose a kind of new Chinese medicine extract and preparation method and purposes, to overcome the above-mentioned defective that prior art exists.

That is to say, the invention is intended in clear and definite a kind of new be Chinese medicine extract of feedstock production and its production and use with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae; Promptly the present invention relates to a kind ofly adopt all medicines to close to carry or single medicinal material extracts Chinese medicine extract of the blended process preparation in refining back and its production and use.

Described osteoporosis disease product is meant the product that is used for osteoporosis disease and associated conditions, the product of diagnosis, detection, prevention, protection, treatment or research osteoporosis and associated conditions; Wherein, described osteoporosis disease product is to comprise in medicine, the Foods or drinks field product one or more, in preferred agents, reagent, food, health food, additive or the beverage one or more, in further preferred anti-osteoporosis agents, osteoporosis disease reagent, osteoporosis disease food or the osteoporosis disease beverage etc. one or more, most preferably anti-osteoporosis agents.

(1) technical conceive

The independent development original new drug is a present urgent task of China.China's Chinese medicine and pharmacy has a long history, also accumulated rich experience with Chinese herbal medicine prevention and treatment disease aspect, especially treating both the principal and secondary aspects of a disease is played in the comprehensive function of many target spots of Chinese medicine compound multicomponent, it is developed fully develop talents, to have great prospect, therefore the development and use of Chinese medicine are valid approach, also are the places of the advantage of the Chinese drug development of performance.

Inventor's preliminary study is found: the Radix Achyranthis Bidentatae ethanol extraction is to the effect of osteoblast and osteoclast, can promote formation and the differentiation and the anti-tartaic acid phosphatase activity of the propagation of osteoblast in neonatal calvaria cultures and alkaline phosphatase activities, inhibition osteoclast, show certain osteoporosis disease effect; Further three wonderful balls have been carried out screening active ingredients, confirmed that also three wonderful balls have the effect of osteoporosis disease, and have the effect of spleen invigorating, dampness concurrently, may potentiation also be arranged for geratic period, involutional osteoporosis disease, and reduce the scorching side effect that it brings.Osteoporosis is old people and postmenopausal women's commonly encountered diseases and a frequently-occurring disease, though there are medicines such as estrogen, calcium preparation and bis phosphoric acid salt available at present clinically, but because side effect is serious, late result is not good enough, restricted its extensive use to a great extent, therefore three wonderful balls are carried out prescription optimization, and extract active site and prepare osteoporosis disease product and have prospect preferably.

According to the theory of the traditional Chinese medical science " kidney governing bones ", the traditional Chinese medical science thinks that the key of osteoporosis morbidity is to suffer from a deficiency of the kidney, and suffering from a deficiency of the kidney makes hormonal system imbalance, hypothalamic pituitary gonadal axis dysfunction.In view of the above, the inventor by for many years clinical research and analyze a large amount of clinical datas after, think that the morbidity of osteoporosis is because deficiency of kidney-QI, deficiency of the kidney, bone marrow is not normal, and is dark weak unable, causes osteoporosis and fracture." interior warp " said: " the kidney person, main stinging, the dominator of storing essence, smart part also, its Hua Zaifa, it fills at bone ", " kidney producing bone marrow ", kidney and bone have confidential relation.Marrow is by the life of essence in kidney instituteization, and bone depends on the nourishing of marrow.The essence in kidney abundance, then skeleton development is normal, eburnation.If the vital essence deficiency of kidney, marrow dry bones flaccidity then, osteoporosis.Suffering from a deficiency of the kidney makes the hormonal system imbalance, the hypothalamic pituitary gonadal axis dysfunction, and the relative equilibrium of the various kinds of cell factor is destroyed in the bone microenvironment, causes bone metabolism disturbance.Especially particularly common woman woman postmenopausal osteoporosis (is called for short: PMOP), by the osteoclast mediation, be the quick bone loss of high conversion type.The women will occur bone loss rapidly after menopause, cause bone strength to descend, and risk of bone fracture increases greatly.A large amount of clinical and basic research are thought, this disease pathogenesis mainly is because the postmenopausal estrogen hyposecretion, estrogen has the effect that promotes the calcitonin secretion and suppress the osteoclast stimulating osteoblast, the estrogen secretion deficiency makes osteoclast too active, the bone conversion increases, be that bone formation and bone resorption increase, bone resorption influences maturation and the conversion and the bone mineralising of ossein greater than bone formation.Therefore can use estrogen control PMOP clinically, the estrogen commonly used at present or the derivant of semi-synthetic estrogen all have certain side effect.

Kidney-nourishing tcm drug can be adjusted ovarian function and estrogen level, can effectively control the Woman climacteric symptom.Kidney-nourishing tcm drug can recover the functional activity of hypothalamic pituitary gonadal axis, suppresses bone resorption, accelerates bone formation, delays bone loss, improves bone mineral content, reaches the purpose of treatment osteoporosis.The mechanism of its effect may be the function by excited pituitary adrenal axis or gonad axis, improves the estrogen level in the blood, and the quantity of the sensitivity of estrogen receptor or estrogen receptor realizes on the raising gonad.A large amount of experiments show the effect that kidney-nourishing tcm drug has the promotion bone formation and suppresses bone resorption, reduces bone turnover rate, improves bone mass; Can suppress the osteoclast proliferation and differentiation, regulate the trace element balance, cerebral tissue T-CHOL, total phospholipids content and the two ratio are reduced, improve the noradrenaline cellulose content, by delaying the aging central neurotransmitter level that changes of cerebral tissue, reach the purpose of protect against osteoporosis.

The inventor investigates the Chinese medicine compound of wide clinical application, and in conjunction with the working foundation of early-stage Study, has found the Chinese medicine compound three wonderful balls with protect against osteoporosis effect, and prescription Chinese medicine only has three Chinese medicines, and is fairly simple, is easy to research and development.Long from modern applicating history from Gu, safety can be protected.Not as the record of protect against osteoporosis, the inventor had found its new purposes, and its proportion of composing is optimized in the past.

The inventor studies the back and finds, the compound recipe that this three flavors Chinese medicine is formed has the double effects of the kidney invigorating and essence nourishing, nourishing YIN to lower pathogenic fire.Wherein, the principal agent Radix Achyranthis Bidentatae has the function of the kidney invigorating and essence nourishing, and modern pharmacology of the present invention studies show that to have adjusts ovarian function and estrogen level, delays and suppresses bone resorption and reduction bone turnover rate, and can promote osteoblast differentiation, promotes osteoplastic effect; The Rhizoma Atractylodis the spleen strengthening and damp drying as a means of " the foundation of acquired constitution ", helps source of generating QI and blood, constantly to replenish essence and blood in the kidney; The Cortex Phellodendri pathogenic fire purging is appointed to coordinate to dash.Full side based on the kidney invigorating and essence nourishing, be in harmonious proportion dash appoint, bone and muscle strengthening, to keep the normal level of body gonadal hormone, especially estrogen level.Estrogen can suppress bone resorption, stimulates bone formation, reduces losing of bone amount, reaches the purpose of protect against osteoporosis.

According to the opinion more than the inventor, the inventor is taken stopgap measures with the kidney invigorating bone strengthening, the spleen strengthening and damp drying, clearing away heat-fire, focus on and adjust the equilibrated theory of Chinese medical science of body yang blood and qi for basic, selection has the compound recipe that the Chinese herbal medicine that promotes Oesteoblast growth, keep the coordinated balance of bone formation and bone resorption is made into through modern pharmacological research, treat osteoporosis, and obtain clinical efficacy preferably.We to be with the kidney invigorating and essence nourishing, the heat clearing and damp drying spleen invigorating of holding concurrently, and suffer from a deficiency of the kidney deficiency, apoplexy due to deficiency of more suitable postmenopausal women has the physiological and pathological feature of heat.

The inventor has retrieved " newly organized national Chinese patent medicine ", " books such as clinical prescription handbook commonly used, " China's Chinese patent medicine complete works commonly used ", and the hospital administration and the commercially available medicine of osteoporosis carried out the investigation statistics analysis, find because not deeply its chemical constitution study, the pharmacological screening deficiency, cause active site and active component indeterminate and do not have feasible quality standard, having limited with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae is the further development and utilization of feedstock production extract.

Through chemical constitution study, prove with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae to be that main component in the feedstock production extract active site is volatile oil, saponin, sterol and total alkaloids compounds.And according to literature search, the inventor finds that the total diterpene compounds has the number of significant pharmacologically active mostly, it is the clinical drug effect of the protect against osteoporosis effect of feedstock production extract that thereby the inventor infers with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae, should mainly be to be that the extract drug effect of feedstock production is brought into play by active site with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae, result of study also proves and has confirmed that a kind of Chinese medicine extract has significant pharmacologically active.

That is to say that the present invention is by to comprising that with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae be activity and the applied research that the Chinese medicine extract of feedstock production carries out system, and screening and prove the composition and the purposes of this compound recipe.

(2) a kind of composition of Chinese medicine extract and preparation method

The inventor is by to being experimentation and the theory study that feedstock production extract etc. carries out system with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae, comprises that chemical constituent separation, purification and structure identify, pharmacology, and medicament, experimentation and analyses such as drug matching, find:

The prescription of this Chinese medicine extract is filled a prescription by monarch, minister, relation of helping, making etc., and each key component is respectively: with Radix Achyranthis Bidentatae, Cortex Phellodendri, Rhizoma Atractylodis is the feedstock production extract.

Wherein, be the kidney invigorating bone strengthening class medicine with the Radix Achyranthis Bidentatae for the feedstock production extract; Can be used in the medical material that substitutes Radix Achyranthis Bidentatae and be and comprise in Radix Cyathulae, Herba Epimedii, Fructus Psoraleae, Radix Dipsaci, Fructus Ligustri Lucidi, Semen Allii Tuberosi or the Rhizoma Cibotii etc. one or more, preferred Herba Epimedii.

Wherein, Cortex Phellodendri is the antipyretic and antidotal type medicine, can be used in the medical material that substitutes Cortex Phellodendri and be to comprise in Rhizoma Coptidis, Flos Lonicerae, Herba Ecliptae, Herba Taraxaci, Radix Isatidis, Herba Scutellariae Barbatae or the Folium Isatidis etc. one or more, preferred Rhizoma Coptidis;

Wherein, Rhizoma Atractylodis are the spleen strengthening and damp drying class medicine, can be used in the medical material that substitutes Rhizoma Atractylodis and be to comprise in the Rhizoma Atractylodis Macrocephalae or the Rhizoma Dioscoreae etc. one or more, the preferred Rhizoma Atractylodis Macrocephalae;

Monarch drug in the above-mentioned Chinese medicine extract and ministerial drug, adjuvant drug, messenger drug mutually 5, the medicine of getting its kidney invigorating bone strengthening is a monarch drug, is accessory drugs with the medicine of the spleen strengthening and damp drying, and the medicine of getting its heat-clearing and toxic substances removing is an adjuvant, and each medicine synergism becomes the effect of the kidney invigorating bone strengthening altogether.

The present invention is that the extract that primary raw material prepares has tangible regulating action to osteoporosis with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae, can be used in preparation osteoporosis disease product, especially anti-osteoporosis agents.Its extract can adopt three flavor medicines to close to carry or single medicinal material is singly carried earlier, the refining decomposite method preparation in back, proves that through pharmacological evaluation it has significant preventive and therapeutic effect to osteoporosis.

Consisting of of this Chinese medicine extract:

Rhzoma Atractylodis Lanceae volatile oil accounts for 10～25% (percentage by weights, down together), preferred 10～20%;

Cortex Phellodendri total alkaloids accounts for 25～45%, and preferred 30～40%;

The Radix Achyranthis Bidentatae total saponins accounts for 10～40%, and preferred 20～30%;

The Radix Achyranthis Bidentatae total sterone accounts for 5～15%, and preferred 10～15%;

Other compositions account for 15～45%.

In essence, the preparation method of this Chinese medicine extract of the present invention is all the operable methods well known in the art that comprise solvent extraction method, the preferred solvent extraction method.

Described solvent extraction method is to comprise hot reflux extraction commonly used, Continuous Countercurrent Extraction, ultrasonic extraction, dipping extraction method, percolation extraction method, decoct in extraction method or the continuous backflow extraction method one or more, and extraction time is an one or many; A kind of in preferred ultrasonic extraction, percolation extraction method, Continuous Countercurrent Extraction or the heating and refluxing extraction method, extraction time is repeatedly; Further preferred heating and refluxing extraction method, extraction time is repeatedly.

The employed extraction solvent of described heating and refluxing extraction method is to comprise that common water reagent, hydrophilic organic solvent or lipophilic organic solvent three classes extract one or more in the solvent; Described water reagent is to comprise a kind of in water, sour water or the aqueous alkali; Described hydrophilic organic solvent is to comprise in ethanol, ethanol water or the methanol one or more; Described lipophilic organic solvent is to comprise in petroleum ether, chloroform, ether, ethyl acetate, dichloromethane or the dichloroethanes one or more.

Below, just the adoptable two kinds of preparation methoies of extract of the present invention are described in detail:

Method one, close lifting manipulation

(1) prescription and proportioning

1～5 part of Rhizoma Atractylodis, 1～5 part of Cortex Phellodendri, 1～5 part of Radix Achyranthis Bidentatae; 1～3 part of preferred Rhizoma Atractylodis, 1～3 part of Cortex Phellodendri, 3～5 parts of examples of Radix Achyranthis Bidentatae; 1～1.5 part of further preferred Rhizoma Atractylodis, 2～3 parts of Cortex Phellodendris, 4～5 parts of Radix Achyranthis Bidentataes;

It is emphasized that when Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae proportioning and its effect and effect bigger relation is arranged: when three's ratio was 3: 2: 1, this prescription then was three wonderful balls; When three's ratio was 3: 2: 0, this prescription then was two wonderful balls; When three's ratio was 1: 2: 3, amount caused its effect that great variety has taken place than changing, and that is to say that its osteoporosis disease effect is optimum.

(2) preparation extracting solution

Take by weighing each crude drug coarse powder respectively in proportion, it is standby that Rhizoma Atractylodis extract volatile oil earlier by the pharmacopeia method, residue dries, the above-mentioned raw materials coarse powder is soaked in an amount of extraction solvent, extracting solvent is 0～95% ethanol or methanol aqueous solution, till percolation to saponins, alkaloids assay reactions is negative routinely, collect percolate, get extracting solution.This step also can adopt 5～10 times to extract solvent refluxing extraction one to repeatedly, gets extracting solution.

Preferred 30～95% ethanol of the used extraction solvent of this step or methanol, preferred especially 70～95% ethanol.Preferred 8 times are extracted solvent during reflux, extract,, reflux each 2 hours 3 times.

(3) refining concentrate drying

Said extracted liquid is concentrated into nothing alcohol flavor, and distilled water is regulated proportion to 1.05g/ml (25 ℃), and the macroporous resin column that filtrate is crossed by activation processing (every Kg medical material is with activating wet resin 1Kg) makes the active component in the filtrate be adsorbed by resin column, and discards effluent.Water eluting macroporous resin column discards eluent; Afterwards, adopt moisture lower alcohol as eluting adsorption columns such as 30～70% ethanol, and collect this lower alcohol eluent, concentrate this lower alcohol eluent to there not being alcohol, collect eluent, eluent is evaporated to dried for 60 ℃, crushed after being dried adds the Rhzoma Atractylodis Lanceae volatile oil mixing and promptly gets powder of the present invention.

Behind the adsorption column adsorbing and extracting liquid, preferably use the water washing of 1～5BV (bed volume), eluent flow rate is preferably 0.5～2.5 BV/ hour; Described moisture lower alcohol is the lower alcohol of moisture C1～C5, preferred aquiferous ethanol; This moisture lower alcohol eluent concentration is 20～70%, preferred 20～70% ethanol waters, and eluent circulation speed is preferably 0.5～2.5BV/ hour.

Described macroporous resin is generally polystyrene type porous adsorbent resin, the middle polarity adsorbent resin that contains ester group or cyano group of optimization styrene type copolymer, its structure is the macroporous netlike structure, for example D-101 macroporous resin, SIPI-40 macroporous resin, AB-8 macroporous resin, ZTC-1 macroporous resin, SP905 macroporous resin, SIPI-2 1 macroporous resin or SIPI-8 macroporous resin etc., preferred ZTC-1 macroporous resin and SP-905 macroporous resin.

Before last sample, should carry out pre-treatment to macroporous resin, run off from waste liquid to prevent effective ingredient.When the resin pre-treatment, use earlier the 5%HCl eluting usually, the back is washed till neutrality with purified water, reuse 2%NaOH eluting, the back is washed till neutrality with purified water, during continuous production, washes post with 95% ethanol, and it is zero degree that the back is washed till honeybee with purified water, can reuse.

(4) tabletting

Adjuvant mixes, tabletting if extract obtained powder and tablet used always, promptly gets tablet, also can make different dosage forms such as capsule, drop pill, pill, injection, unguentum, mixture routinely.

Method two, singly carry the back and mix

(1) prescription and proportioning

1～5 part of Rhizoma Atractylodis, 1～5 part of Cortex Phellodendri, 1～5 part of Radix Achyranthis Bidentatae; 1～3 part of preferred Rhizoma Atractylodis, 1～3 part of Cortex Phellodendri, 3～5 parts of examples of Radix Achyranthis Bidentatae; 1～1.5 part of further preferred Rhizoma Atractylodis, 2～3 parts of Cortex Phellodendris, 4～5 parts of Radix Achyranthis Bidentataes.

(2) preparation extracting solution

Take by weighing each crude drug coarse powder respectively in proportion, after Rhizoma Atractylodis extracted volatile oil by the pharmacopeia method, residue added 0～95% ethanol or the conventional percolation of methanol aqueous solution.Respectively Cortex Phellodendri, Radix Achyranthis Bidentatae raw material coarse powder are soaked in an amount of extraction solvent, extracting solvent is 0～95% ethanol or methanol aqueous solution, and difference is percolation routinely, till percolation to alkaloids, saponin component assay reactions is negative respectively, collect each percolate, get each single medicinal material extracting solution; This step also can adopt 5～10 times to extract solvent refluxing extraction one to repeatedly respectively, gets each single medicinal material extracting solution.

This step is extracted preferred 30～95% ethanol of solvent or methanol.Preferred especially: it is 85% ethanol that Rhizoma Atractylodis extract solvent; It is 85% ethanol that Cortex Phellodendri is extracted solvent; The Radix Acanthopanacis Bidentatae extraction solvent is 85% ethanol.

(3) concentrate drying

Respectively above-mentioned Rhizoma Atractylodis, Cortex Phellodendri, each single medicinal material extracting solution reclaim under reduced pressure of Radix Achyranthis Bidentatae are not distinguished the flavor of to there being alcohol, distilled water is regulated proportion to 1.05g/ml (25 ℃), the macroporous resin column that filtrate is crossed by activation processing (every Kg medical material is with activating wet resin 1Kg), active component in the filtrate is adsorbed by resin column, and discard effluent.Water eluting macroporous resin column discards eluent; Afterwards, adopt moisture lower alcohol as eluting macroporous resin column such as 30～70% ethanol, and collect this lower alcohol eluent, it is dried that 60 ℃ of eluents are evaporated to, and crushed after being dried gets powders A, B, C.

Behind the adsorption column adsorbing and extracting liquid, preferably use the water washing of 1～5BV (bed volume), eluent flow rate is preferably 0.5～2.5 BV/ hour; Described moisture lower alcohol is the lower alcohol of moisture C1～C5, preferred aquiferous ethanol; This moisture lower alcohol eluent concentration is 20～70%, preferred 20～70% ethanol waters, and eluent circulation speed is preferably 0.5～2.5BV/ hour.

Described macroporous resin is generally polystyrene type porous adsorbent resin, the middle polarity adsorbent resin that contains ester group or cyano group of optimization styrene type copolymer, its structure is the macroporous netlike structure, for example D-101 macroporous resin, SIPI-40 macroporous resin, AB-8 macroporous resin, ZTC-1 macroporous resin, SP905 macroporous resin, SIPI-21 macroporous resin or SIPI-8 macroporous resin etc., preferred ZTC-1 macroporous resin and SP-905 macroporous resin.

Before last sample, should carry out pre-treatment to macroporous resin, run off from waste liquid to prevent effective ingredient.When the resin pre-treatment, use earlier the 5%HCl eluting usually, the back is washed till neutrality with purified water, reuse 2%NaOH eluting, the back is washed till neutrality with purified water, during continuous production, washes post with 95% ethanol, and it is zero degree that the back is washed till honeybee with purified water, can reuse.

(4) mix

Gained powders A, B, C are mixed with Rhzoma Atractylodis Lanceae volatile oil, promptly get extract of the present invention.

(5) tabletting

With extract obtained powder and tablet adjuvant commonly used mix, tabletting, can get tablet, also can make different dosage forms such as capsule, drop pill, pill, injection, unguentum, mixture routinely.

Chinese medicine extract of the present invention control suffer from a deficiency of the kidney with spleen decline, insufficiency of vital energy and blood, adjust immunity of organism and be, take the heresy of wind and cold again into account, the mark of attacking, laying equal stress on both the incidental and fundamental, and based on principal agent, all medicines share the effect of playing treatment altogether outward.Therefore, extract of the present invention has the effect of significant protect against osteoporosis through the zoopery proof, so can be used for preparing the medicine or the food of protect against osteoporosis, for example it can be prepared into any common drug dosage form with acceptable accessories.

(3) the composition verification method and the result thereof of Chinese medicine extract

1, Rhzoma Atractylodis Lanceae volatile oil checking

Get extract 0.1g, add n-hexane 2ml, supersound process product 5min filters, and filtrate is as test sample liquid.Other gets Rhizoma Atractylodis control medicinal material 0.5g, shines medical material solution in pairs with legal system.Test according to thin layer chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), draw each 2～6 μ l of above-mentioned freshly prepd two kinds of solution, put respectively on same silica gel g thin-layer plate, with petroleum ether (60～90 ℃)-ethyl acetate (20: 1) is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid of 5% paradime thylaminobenzaldehyde, and it is clear to be heated to the speckle colour developing.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color; And should show an identical dirty green principal spot (atisine chloride atractydin) is arranged.

2, the Radix Achyranthis Bidentatae total saponins is identified verification method

Get extract 1g, add ethanol 20ml, reflux 40 minutes leaves standstill.Get supernatant 10ml, add hydrochloric acid 1ml, reflux is concentrated into about 5ml after 1 hour, add water 10ml, extracts with petroleum ether (60～90 ℃) 20ml, and extracting solution evaporate to dryness, residue add ethanol 2ml makes dissolving, as need testing solution.Other evens up pier fruit acid reference substance, adds ethanol and makes the solution that every 1ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (2005 editions appendix VIB of Chinese Pharmacopoeia), draw need testing solution 10～20 μ l, reference substance solution 10 μ l, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with chloroform-methanol (40: 1) is developing solvent, launch, take out, dry, spray is with the phosphomolybdic acid test solution, and it is clear to be heated to the speckle colour developing at 110 ℃.In the test sample chromatograph, with the reference substance chromatograph on the corresponding position, show identical blue spot.

3, the Cortex Phellodendri berberine is identified verification method

Get extract 0.1g, add methanol 5ml, reflux 15 minutes filters, and filtrate is replenished methanol to 5ml, as need testing solution.Other gets Cortex Phellodendri control medicinal material 0.1g, shines medical material solution in pairs with legal system.Get the berberine hydrochloride reference substance again, add methanol and make the solution that every 1ml contains 0.5mg, in contrast product solution.Test according to thin layer chromatography (2005 editions one appendix VI B of Chinese Pharmacopoeia), draw each 1 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with benzene-ethyl acetate-isopropyl alcohol-methanol-strong ammonia solution (6: 3: 1.5: 1.5: 0.5) is developing solvent, put in the expansion cylinder of ammonia saturated with vapor, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color; With the corresponding position of reference substance chromatograph on, show an identical yellow fluorescence speckle.

Each constituent content assay method and result

1. the mensuration of total alkaloids

1.1 the configuration of the preparation of standard curve (1) standard solution: precision takes by weighing berberine hydrochloride reference substance 5.0mg, puts in the 25ml volumetric flask, adds dissolve with methanol and is diluted to scale, shakes up.Accurate this liquid 5.0ml that draws puts in the 25ml volumetric flask, adds 95% ethanol dilution to scale, shakes up, in contrast the product mother solution.(2) drafting of standard curve: accurate reference substance mother solution 1.0,2.0,3.0,4.0,5.0, the 6.0ml of drawing, place the 10ml volumetric flask respectively, the hydrochloric acid solution that adds 0.1mol/L is diluted to scale, shakes up.With 95% ethanol is blank, measures absorption value under ultraviolet maximum absorption wavelength 345nm, is vertical coordinate with absorption value A, and concentration C is an abscissa, draws regression equation A=6.5234C, R=0.9997.

Take by weighing extract 50.0mg 1.2 sample solution prepares precision, place the 50ml volumetric flask, add small amount of methanol earlier, the supersonic oscillations dissolving, reuse methanol is diluted to scale, shakes up standby.

1.3 sample size is measured the accurate test liquid 5.0ml that draws, and places neutral alumina post (5g, wet method dress post, ethanol prewashing), 95% ethanol elution is collected eluent, places the 25ml volumetric flask,, shakes up to scale with 95% ethanol dilution.Accurate this liquid of 2.0ml of drawing places the 10ml volumetric flask, adds the 0.1moL/L hydrochloric acid solution and is diluted to scale, and ultraviolet 345nm measures trap down, calculates content according to standard curve.

1.4 average recovery is measured precision and taken by weighing known content sample 25.0mg, places the 25ml volumetric flask, adds berberine hydrochloride reference substance 2.5mg simultaneously, the dissolving of methanol sonic oscillation is diluted to scale, shakes up.Accurate this liquid 5.0ml that draws goes up the neutral alumina post, by 1.3 following method operations.

1.5 the method repeatability is investigated same sample is pressed sample treatment and assay method mensuration respectively.

The result shows that this assay method accuracy is good, stability, favorable reproducibility.Be applicable to total alkaloid contents control in this Chinese medicine extract suitability for industrialized production.Measurement result counts 26.1～42.3% with berberine hydrochloride.

2. volatile oil content testing

Method first method according to a regulation of Chinese Pharmacopoeia is measured volatile oil, get Chinese medicine extract and (be equivalent to contain volatile oil 0.5～1.0ml) approximately in right amount, claim to decide weight (accurately to 0.01g), put in the flask, add water 300～500ml (or an amount of) and bead number, after jolting mixes, connect volatile oil determination apparatus and reflux condensing tube.Autocondensation pipe upper end adds water makes the scale part that is full of volatile oil determination apparatus, and overflow when going into flask till.Put in the electric jacket or slowly be heated to other proper method and boil, and keep little and boiled about 5 hours, oil mass no longer increases to the determinator, stop heating, place a moment, open the piston of determinator lower end, water is slowly emitted, above oil reservoir upper end arrives scale 0 line till the 5mm place.Place more than 1 hour, opening piston again, to make oil reservoir drop to its upper end just concordant with scale 0 line, reads the volatilization oil mass, and volatile oil contents (%) in the calculating test sample.The measurement result volatile oil content is 11.3～21.5%.

3. the Radix Achyranthis Bidentatae total saponin content is measured

3.1 the drafting of standard curve: accurate oleanolic acid reference substance (0.1mg/ml) 0.1ml, 0.2ml, 0.3ml, 0.4ml, the 0.5ml of drawing, place tool plug test tube respectively, volatilize, the 5% vanillin glacial acetic acid solution 0.4ml that adds new system, perchloric acid 1.6ml, in 60 ℃ of heating in water bath 15min, ice-water bath cooling 2～3min measures absorption value in 527nm wavelength place.Concentration (C) with oleanolic acid returns absorbance (A), gets regression equation C=0.1097A-0.0419 (r=0.9975).As a result, the oleanolic acid detectable concentration is good in 0.01mg～0.05mg scope internal linear relation.

3.2 precision test: accurate totally 5 parts of the reference substance solution 0.3ml that draw, according to measuring trap behind 3.1 below Faxian colors, RSD=1.16% shows that precision is good as a result.

3.3 test sample liquid preparation: get Chinese medicine extract 1g, with 70% ethanol 25ml, supersound extraction 30min, the extracting solution water-bath is concentrated into does not have the alcohol flavor, adds distilled water and stirs and be settled to 50ml, and cooling back sucking filtration gets clear liquor and is test sample liquid.

3.4 recovery test: get 5 parts of concentration known extracts, every part of 0.9ml adds oleanolic acid reference substance solution 0.1ml respectively, makes need testing solution according to 3.4 following operational approach, measures total saponin content.As a result, average recovery is respectively 97.17%, 99.13%, 98.14%, 93.14%, 92.15%, and average average recovery is 96.13%, RSD=3.1%.

Measurement result is 17～36% by total saponin content in the regression equation calculation Chinese medicine extract.

4. Radix Achyranthis Bidentatae total sterone assay

4.1 the preparation of reference substance solution

Precision takes by weighing the ecdysterone reference substance that is dried to constant weight, and methanol is made the solution of ρ (ecdysterone)=5.02mg/ml, product solution in contrast.

4.2 the preparation of sample solution

Precision takes by weighing that extract is an amount of, and methanol is made solution, as sample solution.

4.3 measure the selection of wavelength

Get reference substance solution in right amount in test tube, solvent is removed in 80 ℃ of volatilizations, the glacial acetic acid solution that adds the freshly prepared vanillin 50mg/ml of 0.12ml, perchloric acid 0.18ml puts 15min in 60 ℃ of water-baths, takes out to put in the ice-water bath and cools off, add glacial acetic acid 5ml, shake up the back and make blank with reagent, with ultraviolet-uisible spectrophotometer in 400～800nm scanning, the sample maximum absorption band at 550nm as measuring wavelength.

4.4 the foundation of standard curve and methodological study

Accurate respectively reference substance solution 20,30,40,50, the 60 μ l that pipette ecdysterone are in test tube, and by 4.3 following method operations, A carries out linear regression to its concentration C with ecdysterone reference substance trap, gets linear equation to be:

A=0.00327.1C-0.085, r=0.9970, the range of linearity is 100.4～301.2 μ g.Investigate through methodology, in 80min, sample stability RSD is 0.42%, and instrument precision RSD is 0.54%, and sample repeatability RSD is 3.43%, and average average recovery is 101.1%, and RSD is 2.02%.

4.5 Radix Achyranthis Bidentatae total sterone Determination on content

Precision pipettes sample solution 100 μ l respectively, measures trap, and the content that calculates the Radix Achyranthis Bidentatae total sterone by standard curve is 10%～15%.

(4) effect of this Chinese medicine extract and relevant experiment

1. extract is to the influence of rat osteoblast propagation and mineralization function

1.1 reagent and material

Newborn Wistar rat provides for the The 2nd Army Medical College Experimental Animal Center, trypsin and RPMI-1640 culture medium are Gibco company product, the type i collagen enzyme is a Worthington company product, calf serum is a Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biological engineering company limited product, 3H-TdR is available from Institute for Atomic Research, Chinese Academy of Sciences Shanghai, and tetramethyl azo tetrazolium bromide (is called for short: MTT) available from Shanghai biological engineering company limited.All available from Shanghai biochemical reagents company, other are homemade analytical reagent for paranitrophenol disodium hydrogen phosphate, paranitrophenol, diethanolamine.Medicinal raw material is made extract and (is called for short: SMW) by expensive the providing of Haozhou, Anhui Liu Jin.

1.2 method

Cell culture is got newborn Wistar rat, under aseptic condition, separate calvarium lid bone, reject periosteum and hetero-organization thereof,, osteocomma is cut into the fritter of 1mm * 1mm with eye scissors with D-Hank ' s liquid flushing 3 times, the trypsin of adding 0.25%, in 37 ℃ of digestion 30 minutes, abandon supernatant, D-Hank ' s flushing 3 times, the Digestive system that adds the type i collagen enzyme that contains 0.05% trypsin and 1mg/ml, 37 ℃ digested 2 hours, drew Digestive system, through 140 order nylon net filters, the residual collection of bone of reuse RPMI-1640 culture fluid flushing digestion, collect flushing liquor, filter the back and mixes, centrifugal 10 minutes with 1000r/min with Digestive system, collecting cell, be fresh osteoblast, add the RPMI-1640 culture medium that contains 10% calf serum, adjusting density is 1 * 10 ⁵Cell/ml is inoculated in the culture bottle of 100ml, puts 37 ℃, 5%CO ₂Incubator in cultivate.Get the second subculture cell and carry out following test.

Proliferation assay is got the osteoblast of cultivating second filial generation end, digests with 0.25% trypsin, and in containing the RPMI-1640 culture medium of 10% calf serum, adjusting cell concentration is 1 * 10 with cell suspension ⁵Cell/ml is inoculated in 96 well culture plates, every hole 100 μ l, and in 37 ℃, 5%CO ₂Incubator in cultivate, after 24 hours, change the culture fluid of the Fructus Broussonetiae extracting solution that contains variable concentrations, continue to cultivate 48 hours and 72 hours, cultivate and finish preceding 4 hours, every hole adds the MTT (5mg/ml) of 20 μ l, and cultivation is abandoned supernatant after finishing, every hole adds the DMSO of 150 μ l, place, formation De Jia Za granule is dissolved fully, survey absorption value in the 550nm place.

Alkali phosphatase (be called for short: determination of activity ALP) is got the second filial generation and is cultivated the osteoblast that finishes the end, the trypsinization with 0.25%, and in containing the RPMI-1640 culture medium of 10% calf serum, adjusting cell concentration is 1 * 10 with cell suspension ⁵Cell/ml is inoculated in 24 well culture plates, and every hole 1ml cultivated 24 hours, and behind the cell attachment, the RPMI-1640 culture medium of changing 10% calf serum that contains variable concentrations Fructus Broussonetiae extracting solution continues to cultivate.With PBS washed cell 3 times, the diethanolamine 200 μ l that add 50mmol/L, 2.5mmol/L paranitrophenol disodium hydrogen phosphate 100 μ l, 37 ℃ were reacted 30 minutes, 0.3mol/L NaOH count 100 μ l cessation reactions, get 150 μ l and add in 96 orifice plates, on microplate reader, survey its absorbance in the 405nm place.Standard curve is the standard curve that the p-nitrophenyl phenol solution of variable concentrations is done in the absorption value and the concentration at 405nm place.

The mineralising tuberosity forms measures cell with 2.2 * 10 ⁴/ ml density is inoculated in 12 well culture plates, culture fluid is to add in the RPMI-1640 culture medium of 10% calf serum of 50ug/ml vitamin C and 10mmol/L sodium glycerophosphate, 2d changes liquid 1 time, fix with 95% ethyl alcohol in situ in 14d, the dyeing of 0.1% alizarin red (is called for short: ARS) 30min, with there being the lattice mylar to be attached at the culture plate bottom, make mineralising tuberosity counting under the low power light microscopic.

1.3 result

Chinese medicine extract to the influence of osteoblastic proliferation from table 1-1 (Tab 1-1 Effect of SMW on proliferation ofosteoblasts Cultured in vitro) as can be seen, Chinese medicine extract is at the 48h time point, in 0.25～25mg/L concentration range osteoblastic propagation being concentration dependent increases, the strongest with the 25mg/L mass action, increase by 58.3% than negative control.At the 72h time point, SMW has significant facilitation to osteoblastic propagation in 0.0025～25mg/L concentration range, and is the strongest with the 2.5mg/L mass action, increases by 46.7% than negative control.

Table 1-1, Chinese medicine extract are to the influence of In vitro culture osteoblastic proliferation (n=4, x ± SD)

Concentration	OD value (550nm)
			48h	72h
	Controle SMW(mg/L) 0.0025 0.025 0.25 2.5 25	0.784±0.137 0.898±0.141 *0.849±0.050 1.051±0.038 *1.113±0.064 *1.198±0.114 **			1.168±0.039 1.605±0.124 *** 1.424±0.102 * 1.543±0.070 *** 1.6940.045 *** 1.536±0.078 **

Remarks: ^*P＜0.05 ^*P＜0.01 ^{* *}P＜0.001 vs control

The Concentration indicated concentration, Controle represents reference substance.

SMW to the influence of osteoblast alkaline phosphatase activities from table 1-2 (Tab 1-2 Effect of SMW on the alkalinephosphatase activity ofosteoblasts Cultured in vitro) as can be seen, the osteoblast secreting alkaline phosphorus phytase, along with the prolongation activity of incubation time strengthens gradually, but behind the 10d, activity begins to descend.After adding SMW, during 6d, in the concentration range of 0.0025mg/L～0.25mg/L, to the remarkable facilitation of osteoblast alkaline phosphatase activity, the strongest with the 0.025mg/L mass action, compare with negative control, increase by 31.2%, during 10d, 0.0025 and the SMW of 0.025mg/L can increase the osteoblast alkaline phosphatase activity in various degree, difference is significance not.

Table 1-2, SMW are to the influence of In vitro culture osteoblast alkaline phosphatase activities (n=4, x ± SD)

Concentration	ALP activity(nmol/well)
				2d	6d	10d
	Control SMW(mg/L) 0.0025 0.025 0.25 2.5 25	17.21±1.87 17.08±1.41 16.94±0.26 13.87±0.35 13.02 ±0.58 14.07±0.86	62.64 ±2.81 74.15±3.11 82.18±4.09 ** 73.1 8±3.90 70.85±2.34 * 72.74±1.16				73.88±4.38 80.00±5.93 81.64±7.15 63.19±6.86 57.48±5.84 56.72±4.82

Remarks: ^*P＜0.05 ^*P＜0.01 vs control

ALP activity represents the ALP activity.

SMW sees Table 1-3 (Tab 1-3 Effect of SMW on the number ofmineral nodes of osteoblasts ultured in vitro) to the influence that the mineralising tuberosity forms, osteoblast is cultivated the visible mineralising tuberosity of 14d and is formed, present the red positive tuberosity that size, form differ after the alizarin red dyeing, the tuberosity of 0.025～2.5mg/L concentration group of SMW forms several comparing with negative control group and is significantly increased, and wherein 2.5mg/L concentration group is compared with matched group and improved 18.2%.

The influence that table 1-3, SMW form In vitro culture osteoblast mineralising tuberosity (n=4, x ± SD)

Concentration	number of mineral nodes
		Control SMW(mg/L) 0.0025 0.025 0.25 2.5 25	287.7±22.3 327.9±32.6 330.5±29.7 * 312.8±34.3 340.2±28.4 ** 327.0±35.2 *

Remarks: P＜0.05 ^*P＜0.01 vs control

Number of mineral nodes represents mineralising tuberosity quantity.

2.SMW influence to osteoclasts cultured in vitro

Osteoclast is the apocyte of regulation and control bone resorption, and its precursor is the cell in courageous and upright source, and as myelomonocyte, spleen cell etc., these cells are transferred under the effect of hormone and local factors at bone, can be differentiated to form multinucleated osteoclast.Participate in the hormone of bone resorption, as 1,25 (OH) ₂D ₃, PTH and PGE etc., in medullary cell is cultivated, the coenocytic formation of obvious stimulation osteoclast sample.Osteoblast participates in osteoclast and forms and atomization, and osteoblast is secreted formation and the differentiation that some factors promote osteoclast.This experiment adopts osteoblast and myelomonocyte to unite cultivation, at 1,25 (OH) ₂D ₃Under induced by dexamethasone, make myelomonocyte be divided into osteoclast, investigate the effect of SMW to osteoclast.

2.1 material and reagent

The Wistar rat of 3～4d is available from the The 2nd Army Medical College Experimental Animal Center.It is the same that osteoblast is cultivated agents useful for same, α-MEM culture medium, and newborn hyclone is Gibco company product, and 1, the 25-dihydroxyvitamin D ₃(1,25-(OH) ₂VitaminD ₃), dexamethasone, naphthols AS-BI phosphate, six azo paramagenta are Sigma company product.Glycol monoethyl ether, sodium potassium tartrate tetrahydrate etc. are homemade analytical reagent.

2.2 method

Osteoclast is cultivated the newborn Wistar rat choose 3～4d, and cervical vertebra is broken execution, is 75% soak with ethanol 5 minutes with volume fraction, separates femur under aseptic condition, contains 10 with 5ml ^-81 of M, the 25-dihydroxyvitamin D ₃With 10 ^-7The α of M dexamethasone and 10% hyclone-MEM culture medium flushing medullary cavity goes out the cell in the bone marrow.Wash cell 2 times with the PBS buffer.Promptly get fresh medullary cell.Medullary cell and osteoblast be suspended in jointly contain 10 ^-81 of M, the 25-dihydroxyvitamin D ₃, 10 ^-7In the α of the dexamethasone of M and 10% hyclone-MEM culture medium, making osteoblastic concentration is 10 ⁵Cell/ml, the concentration of myelomonocyte is 10 ⁶Cell/ml is inoculated in 6 orifice plates, every hole 1.5ml, the built-in coverslip of plate.In 37 ℃, 5%CO ₂Incubator in cultivate.

Anti-tartaic acid phosphatase activity is measured as previously mentioned, osteoblast and myelomonocyte is suspended in contain 10 ^-81 of M, the 25-dihydroxyvitamin D ₃, 10 ^-7In the α of M dexamethasone and 10% hyclone-MEM culture medium, making osteoblastic concentration is 10 ⁵Cell/ml, myelomonocyte are 10 ⁶Cell/ml, with cell suspension inoculation in 96 well culture plates, every hole 100 μ l, in 37 ℃, 5%CO ₂Incubator in cultivate, after 8 days, the osteoclast differentiation and maturation, change the α-MEM culture medium 100 μ l that contain variable concentrations SMW, continue to cultivate 24,48 and 72 hours, abandon supernatant, use the PBS washed twice, draw logical smudge cells with 0.1% the district of 10 μ l, add the reactant liquor (preparation of reactant liquor: take by weighing p-nitrophenyl disodium hydrogen phosphate 0.4g after 15 minutes, use deionized water dissolving, add Soluble tartar. 2.0g, be dissolved in water to 150ml, HCl with 1N regulates PH to 3.5, adds deionized water again to 200ml.) 100 μ l, in 37 ℃ of reactions 30 minutes, the sodium hydroxide solution cessation reaction that adds the 1N of 100 μ l rapidly, measure its absorption value in wavelength 410nm place, make standard curve with the p-nitrophenyl phenol solution, the nanomole numerical table of the paranitrophenol that generates with every hole shows the activity of anti-tartaic acid phosphatase.

The mensuration of bone resorption lacuna area is suspended in osteoblast suspension and myelomonocyte jointly and contains 10 ^-81 of M, the 25-dihydroxyvitamin D ₃, 10 ^-7In the α of the dexamethasone of M and 10% hyclone-MEM culture medium, adjust cell concentration, making osteoblastic concentration is 10 ⁵Cell/ml, the concentration of myelomonocyte is 10 ⁶Cell/ml gets 96 well culture plates, puts an aseptic cattle cortical bone osteocomma in every hole, and 100 μ l are inoculated in 96 well culture plates with cell suspension, cultivate after 24 hours, change the SMW and 10 that contains variable concentrations ^-81 of M, the 25-dihydroxyvitamin D ₃, 10 ^-7The α of the dexamethasone of M and 10% hyclone-MEM culture medium continues to cultivate 10 days (changing liquid once in per 2 days), abandons culture medium, takes out osteocomma, and is fixing in 70% ethanol, spends the night Yihong dyeing, neutral gum mounting.Calculate the area of bone resorption lacuna on the osteocomma with Computerized image processing system.

2.3 result

SMW produces anti-tartaic acid phosphatase to osteoclast influence from table 1-4 (Tab 1-4 Effects of SMWon TRAP activity ofosteoclasts in cultures) as can be known, osteoclast is secreted anti-tartaic acid phosphatase and is strengthened gradually along with the prolongation activity of time.At the 24h time point, SMW is in the concentration range of 0.25～25mg/L, and concentration relies on the generation that ground suppresses the anti-tartaic acid phosphatase of osteoclast; At the 48h time point, 2.5 and after the SMW of 25mg/L acts on osteoclast, the activity of its anti-tartaic acid phosphoric acid prunus mume (sieb.) sieb.et zucc. descends 16.27%, 25.44% respectively with comparing; At the 72h time point, the SMW concentration of 2.5～25mg/L relies on the generation that ground suppresses the anti-tartaic acid phosphatase of osteoclast, with matched group relatively, descended 16.88% and 24.87% respectively, and had significant difference with comparing.

Table 1-4, SMW are to the influence of the anti-tartaic acid phosphatase activity of osteoclast (n=3, x ± SD)

Concentration	TRAP activity (nmol/well)
				24h	48h	72h
	Control SMW(mg/L) 0.0025 0.025 0.25 2.5 25	16.67±0.33 16.68±0.33 16.07±0.77 16.04±0.54 * 15.62±0.39 * 15.48±0.14 *	22.25±1.67 18.04±1.71 21.81±2.99 21.75±2.72 18.63±0.97 * 16.59±0.31 **				25.53±1.11 20.95±2.790 24.66±2.24 24.08±1.79 21.22±1.05 * 19.18±1.38 **

Remarks: ^*P＜0.05 ^*P＜0.01 vs control

The influence of the bone resorption lacuna area that table 1-5, SMW form osteoclast (n=3, x ± SD)

Concentration	Area(um 2)	Inhibitary rate
			Control SMW(mg/L) 0.0025 0.025 0.25 2.5 25	2013.1±207.2 1713.0±69.5 * 1598.1±202.3 * 1759.8±114.0 * 1800.7±237.9 1704.5±38.3 *	14.91％ 20.62％ 12.58％ 10.55％ 15.33％

Remarks: ^*P＜0.05 ^*P＜0.01 vs control

The influence of the bone resorption lacuna area that SMW forms osteoclast from table 1-5 (Tab 1-5 Effect of SMW onthe pit area on bone slices formed by osteoclast in cultures) as can be seen, with cattle cortical bone osteocomma and osteoblast in neonatal calvaria cultures and myelomonocyte co-cultivation, 1, the 25-dihydroxyvitamin D ₃Under the inducing of dexamethasone, myelomonocyte is differentiated to form osteoclast, and forms absorption lacuna on cattle cortical bone osteocomma.SMW can suppress the formation of osteoclast bone resorption lacuna.SMW significantly reduces the area of osteoclast bone resorption lacuna under the concentration of 0.0025～0.25mg/L and 25mg/L, suppression ratio is respectively 14.91%, 20.62%, 12.58% and 15.33%.

2.4 discuss

Osteoclast (osteoclast) is a kind of multinuclear giant cell, in the heavily absorption and reconstruction process of bone, mainly is to carry out bone resorption.Lysosomal enzyme in the osteoclast forms a tart microenvironment in part in osteoclast.Dissolve the inorganic salt and the organic matter of bone again by different acid hydrolases, and be absorbed in the osteoclast, be discharged to extracellular fluid then, thereby finish its heavy absorption process, make the part of bone form lacuna.SMW can suppress 1, the 25-dihydroxyvitamin D ₃Inductive myelomonocyte is differentiated to form osteoclast, thereby has suppressed the bone resorption in the bone process of reconstruction, has reduced losing of bone amount.

Anti-tartaic acid phosphatase is one of marker enzyme of osteoclast, and it is active directly related with the bone resorption of osteoclast.SMW produces anti-tartaic acid phosphatase by suppressing mature osteoclast, suppresses bone resorption, and can suppress the formation of osteoclast bone resorption lacuna on the osteocomma.

According to " specification requirement of study of tcm new drug " pertinent regulations, this Chinese medicine extract (dry extract) has been carried out the maximum dosage-feeding test of acute toxicity.

Choose 20 of health kunming mices in 6 age in week, male and female half and half.With this Chinese medicine extract Cmax, maximum volume gastric infusion 2 times, 3 hours at interval, this maximum dosage-feeding was 660g crude drug/kg/ day, observes continuously 7 days in 24 hours.Animal does not see and is poisoned to death as a result, the serious toxicity reaction do not occur yet.This dosage is more than 180 times of clinical people's consumption.

This experimental observation is given repeatedly continuously and given Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae is the feedstock production extract capsule to toxic reaction that rat produced.With Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae is that feedstock production extract (dry extract) is made capsule.The SD rat, in 120 of 6 all ages, be divided into heavy dose of group at random, 30,8g crude drug/kgd (be equivalent to approximately clinical dosage 60 times): middle dosage group, 30,4g crude drug/kgd (be equivalent to approximately clinical dosage 30 times): small dose group, 30,1.33g crude drug/kgd (experiment effective dose, be equivalent to 10 times of clinical dosage approximately): the distilled water group, gavages isometric(al) distilled water 1ml/100g body weight by 30.Successive administration six months continues after the drug withdrawal to observe 1 month.

The result: rat general state from start to finish is good, and is movable normal, do not see any symptom and death; Hematology, blood biochemical are learned, pathological examination finishes (6 months) in administration mid-term (3 months), administration and there is no and the obvious relevant toxic reaction of medicine with convalescent period (drug withdrawal 1 month).

Conclusion: with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae is that 6 months gastric infusion long term toxicity tests of feedstock production extract capsule rat are not found overt toxicity.

(5) purposes of this Chinese medicine extract

1, general introduction

Osteoporosis be with the bone amount reduce, the microstructure degeneration of bone is feature, a kind of general skeletal diseases that causes the fragility of bone to increase and fracture easily takes place.China has stepped into aging society, has the people more than 100,000,000 to suffer from degrees of osteoporotic disease, estimates that every year also will be more than double figures because of the percentage rate of the concurrent fracture number of osteoporosis.

Above experimentation proof is that main component in the feedstock production extract active site is volatile oil, saponin, sterol and total alkaloids compounds with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae, this Chinese medicine extract has significant pharmacologically active, therefore the inventor assert with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae is the clinical drug effect of the protect against osteoporosis effect that extract was had of feedstock production, should mainly be to be that the extract drug effect of feedstock production is brought into play by active site with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae, activity be the strongest when the ratio of three's composition is 1: 2: 3.

This Chinese medicine extract of indication of the present invention is that feedstock production extract etc. is formed by Chinese medicine with Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae mainly, can heat-clearing and toxic substances removing, dispel the wind, QI invigorating, invigorate blood circulation, can be used in diseases such as treatment osteoporosis, experimental result of the present invention shows that this Chinese medicine extract has better curative effect, above-mentioned experiment is the clinical osteoporosis that is used for the treatment of of this Chinese medicine extract, and more pharmacology's foundation is provided.This shows the effect that this Chinese medicine extract has, is this Chinese medicine extract to one of mechanism of osteoporosis treatment.

2, the using method of this Chinese medicine extract and requirement

This Chinese medicine extract of the present invention can be united use separately or with other active component, comprises being used to prepare the product that is used to diagnose, detect, prevent, protect, treat or study relevant disease, comprises medicine, reagent, Foods or drinks etc., especially medicine.

Aspect concrete use, this Chinese medicine extract of the present invention can use separately, can also use with other many chemical substances.These chemical substances biologically active or have the function of treatment disease whether no matter, comprise miscellaneous function as collaborative amplification, antagonism or alleviate the side effect etc. of this Chinese medicine extract, these chemical substances are to comprise in pharmaceutically acceptable carrier, food, natural product, chemical synthetic drug or the human medication etc. one or more; Preferably include in pharmaceutically acceptable carrier or the food etc. one or more; Further preferred pharmaceutically acceptable carrier.

Described activated chemical substance is to comprise medicative chemical substance, or treatment is had in the chemical substance etc. of assosting effect one or more.Medicative chemical substance is to comprise in estrogen, the bis phosphoric acid salt etc. one or more, has the chemical substance of assosting effect to comprise in the materials such as calcium preparation, phytoestrogen one or more to treatment.

" pharmaceutically acceptable carrier " used herein comprises one or more in any He all physiology suitable solvent, disperse medium, afterbirth, antibacterial and antifungal, isotonic agent or the absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more in one or more water, saline, phosphate-buffered saline, glucose, glycerol or ethanol or the like and the compositions thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as in the polyhydric alcohol of mannitol, sorbitol, sorbitol or the sodium chloride etc. one or more.Pharmaceutically acceptable carrier can also comprise a spot of auxiliary substance, one or more in wetting agent or emulsifying agent, antiseptic or the buffer etc. for example, and they have strengthened the effect duration or the effectiveness of this Chinese medicine extract.

From concrete classification, said pharmaceutically acceptable carrier is meant the pharmaceutical carrier of medicine and pharmacology field routine, comprises excipient, as in starch or the water etc. one or more; Lubricant is as in glycerol or the magnesium stearate etc. one or more; Disintegrating agent is as microcrystalline Cellulose etc.; Filler is as in starch or the lactose etc. one or more; Bonding agent is as in pregelatinized Starch, dextrin, cellulose derivative, alginate, gelatin or the polyvinylpyrrolidone etc. one or more; Osmotic pressure regulator is as in glucose, sucrose, sorbitol or the mannitol etc. one or more; Diluent is as water etc.; Disintegrating agent is as in agar, calcium carbonate or the sodium bicarbonate etc. one or more; Absorption enhancer is as quaternary ammonium compound etc.; Surfactant is as hexadecanol etc.; Absorption carrier is as in Kaolin or the soap clay etc. one or more; Lubricant is as in Pulvis Talci, calcium stearate, magnesium stearate or the Polyethylene Glycol etc. one or more; In addition, can also in compositions, add other adjuvant, as in flavouring agent or the sweeting agent etc. one or more.

For example, with this Chinese medicine extract dissolving of active component, suspendible or (for example be emulsifiable in the suitable aqueous solvent, distilled water, in normal saline or the Green's solution etc. one or more) or in the oil-based solvent (for example, vegetable oil is olive oil for example, Oleum sesami, Oleum Gossypii semen, in Semen Maydis oil or the propylene glycol etc. one or more) in, can make ejection preparation, wherein (for example can contain dispersant in the solvent, polyoxyethylene sorbitan monoleate, polyoxyethylene hardened castor oil 60, Polyethylene Glycol, benzyl alcohol, in chlorobutanol or the phenol etc. one or more), osmotic pressure regulator (for example, sodium chloride, glycerol, the D9-mannose, in D-sorbitol or the glucose etc. one or more).In this case, if necessary, can add additive, for example solubilizing agent (for example, one or more in sodium salicylate or the sodium acetate etc.), stabilizing agent (for example, human serum albumin etc.), analgesic (for example, benzyl alcohol etc.) etc.

Of the present invention and this Chinese medicine extract can also unite use with the form of compositions, particularly with other chemical substance such as medicine animal especially mammal is comprised that people or other animals treat compositions for use or similar compositions.Described mammal comprises in people, mice, rat, sheep, monkey, cattle, pig, horse, rabbit, dog, chimpanzee, baboon, Adeps seu carnis Rhiopithecus roxellanae, macaque or the Rhesus Macacus etc. one or more.For example, this Chinese medicine extract of the present invention can be added be suitable for to curee's Pharmaceutical composition in.Usually, this Pharmaceutical composition comprises this Chinese medicine extract of the present invention and pharmaceutically acceptable carrier.

The compositions of this Chinese medicine extract particularly pharmaceutical composition can have various forms, comprises in the dosage forms such as liquid, semisolid and solid for example one or more; Wherein said pharmaceutical composition comprises that this Chinese medicine extract for the treatment of effective dose is an active component, and one or more pharmaceutically acceptable carriers.

The pharmaceutical composition of this Chinese medicine extract can adopt conventional production method well known in the art to make various dosage forms, and active component is mixed with one or more carriers, is made into required dosage form then.Described dosage form comprises one or more in tablet, capsule, granule, suspensoid, Emulsion, solution, syrup or the injection etc., takes one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out diagnosis, detection, treatment or the scientific research of osteoporosis disease and associated conditions.

It is this Chinese medicine extract of active component of 0.5～99% that pharmaceutical composition preferably contains weight ratio, further preferably contains weight ratio and be this Chinese medicine extract of active component of 1～95%, most preferably contains weight ratio and be this Chinese medicine extract of active component of 5～90%.

The pharmaceutical composition of this Chinese medicine extract generally must be aseptic and stable under the production condition of storage.Said composition can be mixed with solution, microemulsion, dispersion liquid, liposome or other is suitable for the ordered structure of high drug level.By with a kind of of this Chinese medicine extract of aequum and required mentioned component or combine to add in the appropriate solvent and then carry out aseptic filtration and prepare aseptic parenteral solution.Generally speaking, prepare dispersion liquid by this Chinese medicine extract being added in the aseptic solvent that contains basic disperse medium and required above-mentioned other composition.Under the situation of the sterile powder that is used to prepare aseptic parenteral solution, the preparation method of recommendation is vacuum drying and lyophilized preparation.For example, by passing through to keep required granular size such as the coating of lecithin, under the situation of dispersion liquid and, can keeping the adequate liquidity of solution by using surfactant.By comprising that in said composition the medicament (for example Monostearate or gelatin) that postpones to absorb can reach the prolongation absorption of injectable composition.

When being used for the patient, this Chinese medicine extract dosage of the present invention is 5～20mg/kgd, and this dosage or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.

This Chinese medicine extract of the present invention and Pharmaceutical composition thereof can comprise this Chinese medicine extract of the present invention of " treatment effective dose " or " prevention effective dose "." treatment effective dose " is meant at the dosage of necessity and effectively reaches the amount of required therapeutic effect under the time.The treatment effective dose of this Chinese medicine extract can cause that at this individuality the factors such as ability of required reaction change according to the patient's condition, age, sex and body weight and this Chinese medicine extract such as individuality.The treatment effective dose also refers to that the useful therapeutic effect of this Chinese medicine extract surpasses the amount of its any toxicity or harmful effect." prevention effective dose " is meant the amount that effectively reaches required preventive effect under necessary dosage and time.Because preventive dose is used for the ill preceding or early stage curee of disease, the prevention effective dose is usually less than the treatment effective dose.The typical non-limiting scope of the treatment of this Chinese medicine extract of the present invention or prevention effective dose is 5～20mg/kg, more preferably 5～10mg/kg.Should note, dose value will change according to disease type of desiring to alleviate and seriousness, that is to say when being used for the patient that the dosage of this Chinese medicine extract of the present invention or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.In addition; should understand; for any specific curee; should along with the time according to individual need and give with or supervision give with the people's of described compositions professional judgement and adjust the given dose system; and the dosage range that this paper sets only be illustrative, the scope or the practice of the compositions of can't requirement for restriction protecting.

That is to say, need be according to object, route of administration, institute's disease of treat and the situation etc. of treatment, this Chinese medicine extract of variation the present invention at every turn and/or dosage or the consumption of every day.For example, give mammal through vein, adult (as body weight 60kg) especially, the single dose of described this Chinese medicine extract is about 5～10mg, preferably about 10mg, preferred administration every day 1～3 time.Can adjust dosage unit, to propose the best required reaction of arch (for example treatment or prevention are replied).For example, can single heavy dose of administration can give several divided doses or reduce or increase dosage in proportion according to the urgency of treatment situation in a period of time.The non-intestinal compositions that preparation is easy to the unified dosage unit form of administration and dosage is especially favourable.Dosage unit form used herein refers to be suitable for the physical separation unit of dosage unit of the mammalian subject of desire treatment; The calculating that each unit contains scheduled volume is used for together producing with required pharmaceutical carrier this Chinese medicine extract of active matter of required therapeutic effect.The specification of dosage unit form of the present invention, determine and directly depend on the specific characteristic of following (a) this Chinese medicine extract and the particular treatment of desiring to reach or preventive effect and (b) interior in mixing this technology that is used for the treatment of this Chinese medicine extract of individual sensitivity by following in restriction.

3, the pharmaceutical dosage form of this Chinese medicine extract and route of administration

This Chinese medicine extract of the present invention prepares osteoporosis disease product, wherein can diagnose, detects, prevents, protects, treats or study osteoporosis and complication thereof according to the product of the requirement of beverage, food technology field preparation; Can be used in patient's treatment or health care according to the product of the requirement of medical technical field preparation, can either be directly used in the medicine of preparation treatment or health care separately, also can mix with many chemical substances or make up, directly or indirectly be used to prepare the medicine of treatment or health care.Chemical substance described here is above described identical with this section.

In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should adopt the material of food stage or pharmaceutical grade according to practical situation and needs.

This Chinese medicine extract of the present invention can be with the whole bag of tricks administration known in the art, although route of administration/administering mode of recommending in many therapeutic use is an oral administration.But the technical staff will appreciate that route of administration/administering mode changes with required result.In some concrete enforcement, the carrier that this reactive compound can avoid rapid release with this chemical compound of protection is preparation example such as empty release formulation together, comprises that graft transmission system, transdermal paste one or more in transmission system or the microcapsule transmission system etc.In addition, can also use biodegradable, biocompatible polymer, for example one or more in ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or the polylactic acid etc.Prepare the equal patent applied for of many methods of this preparation or generally known to those skilled in the art (referring to for example Sustained and Controlled Release DrugDelivery Systems, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978).

This Chinese medicine extract of the present invention usually by one or more modes in oral, rectum or the parenteral etc., is applied to the patient who needs this treatment.

Be used for when oral, can be made into conventional solid preparation such as in tablet, granule or the capsule etc. one or more.When implementing, this Chinese medicine extract of the present invention can be together oral with for example inert diluent or assimilable edible carrier.This Chinese medicine extract (with its composition altogether, if desired) can also be wrapped in hard or soft shell gelatin capsules, is pressed into tablet or directly adds in curee's the meals.About oral therapeutic administration, described this Chinese medicine extract can be added with excipient and use with one or more forms in edible tablet, buccal tablet agent, tincture, capsule, suspension, syrup or wafer or the like.

For to give the present invention this Chinese medicine extract outside the parenterai administration, may need with preventing that the material of its inactivation from together giving to this Chinese medicine extract coating or with this Chinese medicine extract.The reactive compound that replenishes can also be added in the said composition.In the specific implementation, this Chinese medicine extract of the present invention and one or more other medicines that can be used for the treatment of disease are prepared altogether and/or given altogether.Thisly unite use, can utilize this medicine that gives primely, therefore avoid possible toxicity or the complication relevant with various monotherapies than low dosage.

Make in liquid preparation such as water preparation, oil-suspending agent or other liquid preparation one or more, as in syrup or the elixir etc. one or more; When being used for parenteral, can be made in solution, water preparation or the oiliness suspending agent etc. of injection one or more.

In the above-described type of service, preferred form is one or more in granule, tablet, coated tablet, capsule, suppository or the injection etc., in further preferred particulates agent, tablet, coated tablet or the capsule etc. one or more, special preferred particulates agent, coated tablet.

In addition, the employed medicinal raw material of this Chinese medicine extract also can be directly used in preparation osteoporosis disease product in some cases separately, also can mix with many chemical substances or make up, directly or indirectly be used to prepare osteoporosis disease product with the form of compositions.Chemical substance described here is above described identical with this section.

For example, the powder of the employed medicinal raw material of this Chinese medicine extract is used to prepare the particularly various dosage forms of medicine of osteoporosis disease product, or the powder of the employed medicinal raw material of this Chinese medicine extract is used to prepare the especially various dosage forms of medicine of osteoporosis disease product with relevant adjuvant, or the powder of the employed medicinal raw material of this Chinese medicine extract and relevant preparation osteoporosis disease product such as medicine one are used from the various dosage forms of preparation osteoporosis disease product such as medicine, or the powder of the employed medicinal raw material of this Chinese medicine extract and relevant ancillary drug one are used from the various dosage forms of preparation osteoporosis disease product such as medicine, as the preferred particulates agent, tablet, coated tablet, capsule, in suppository or the injection etc. one or more, preferred especially coated tablet.

One of described method is that the powder fill with the employed medicinal raw material of this Chinese medicine extract is a capsule, two of method is to be capsule with the powder of the employed medicinal raw material of this Chinese medicine extract and relevant preparation osteoporosis disease product such as medicine fill together, and three of method is to be capsule with the powder of the employed medicinal raw material of this Chinese medicine extract with relevant ancillary drug fill together; Four of method is the powder of the employed medicinal raw material of this Chinese medicine extract directly to be pressed together according to a conventional method with relevant adjuvant be tablet or granule, five of method is the powder of the employed medicinal raw material of this Chinese medicine extract, relevant preparation osteoporosis disease product such as medicine directly to be pressed according to a conventional method with relevant adjuvant together be that tablet or granule, six of method are the powder of the employed medicinal raw material of this Chinese medicine extract, relevant ancillary drug directly to be pressed according to a conventional method with relevant adjuvant together be tablet or granule etc.

Except that six kinds of above-mentioned basic skills, can also select other forms of the employed medicinal raw material of this Chinese medicine extract or the employed medicinal raw material of this Chinese medicine extract is carried out after method well known in the art handles, prepare the product such as the medicine that contain the employed medicinal raw material of this Chinese medicine extract of various dosage forms.But, it should be noted that, in the employed medicinal raw material of above-mentioned this Chinese medicine extract of direct use, should be earlier according to the dosage requirement of employed this Chinese medicine extract, conversion obtains the consumption of the employed medicinal raw material of this Chinese medicine extract of required use.

In sum, this Chinese medicine extract of the present invention can be used for preparing osteoporosis disease product, preferred agents and food, further preferred agents.

(6) technology speciality

The present invention studies a kind of Chinese medicine extract targetedly, and this raw material is safe in utilization, takes into account each other, one-object-many-purposes has been brought into play effect to greatest extent, and the scope of application is wide especially, therefore apply easily, can have a tremendous social and economic benefits in the short period of time.

That is to say that result of study of the present invention can further deeply be developed as the protect against osteoporosis product new drug especially with potential applicability in clinical practice.This product has the advantage that determined curative effect, effective dose are low, have no side effect, and has significant advantage compared with similar products.Osteoporosis is common clinical, frequently-occurring disease, along with the aggravation of aging, estimates that the sickness rate of diseases such as osteoporosis will constantly rise.Therefore, the present invention also can be follow-up new drug research and lays a good foundation, and further produces bigger economic benefit and social benefit.

Through secular pharmacology test, a kind of Chinese medicine extract has the activity of osteoporosis disease and complication thereof, directly uses a kind of Chinese medicine extract, act on more direct, thereby overcome the unmanageable problem of Chinese medicine dosage, and improved bioavailability; And this Chinese medicine extract pharmacological action is stronger, and its raw material sources are abundant, and preparation technology is simple.

The present invention selects three Chinese medicines of classics side for use, forms through optimizing, and reaches optimum activity of resisting osteoporosis, and has reduced side effect.

In a word, active adaption of the present invention modern medical service and the job demand of scientific research field and the needs of human nature service, be the safe raw material that is used for osteoporosis disease and complication thereof.

The specific embodiment

The present invention has studied a kind of new Chinese medicine compound and extract and purposes, a kind of raw material that can be used in preparation osteoporosis disease and complication product such as medicine is provided, be convenient to the safe handling in medical industry and fields such as relevant industries such as food, beverage, comprise disease treatment, scientific research etc.

Several representative formulas with this Chinese medicine extract are example below, elaborate the concrete prescription and the method thereof of this Chinese medicine extract.

Method one: Radix Achyranthis Bidentatae 600 grams, Cortex Phellodendri 400 grams, Rhizoma Atractylodis 200 grams.

Method two: Radix Achyranthis Bidentatae 500 grams, Cortex Phellodendri 400 grams, Rhizoma Atractylodis 300 grams.

Method two: Radix Achyranthis Bidentatae 400 grams, Cortex Phellodendri 400 grams, Rhizoma Atractylodis 400 grams.

The extracting method of this Chinese medicine extract is as follows:

Method one:

(1) above-mentioned medical material is cleaned, soaked to insert after 6～24 hours and extract the interior decoction of molten device 2～6 times, each time that decocts is 0.5～3 hour.

(2) medicinal liquid that will extract for several times merges, and is condensed into extractum simultaneously.

(3) extractum is left standstill after, add ethanol and advance precipitate with ethanol, reclaim ethanol and go precipitation.

(4) get supernatant behind the precipitate with ethanol, be condensed into pure extractum, this pure extractum proportion 1.0～1.5 (yield 8～16%)

(5) add dry 200～400 mesh sieves of crossing of adjuvant granulation.

(6) be packaged into 10～15g/ bag, finished particle.

Method two:

(1) above-mentioned medical material was soaked 6～24 hours, insert in the extraction vessel, decoct 2～6 times, each time that decocts is 0.5～3 hour.

(2) will extract medicinal liquid for several times and merge, and carry out negative pressure simultaneously and be condensed into thick paste, this thick paste proportion 1.0～1.5.

(3) with thick paste negative-pressure vacuum drying (0.085 ± 0.005mpa), 70 ℃ ± 5 ℃ get dry extract (moisture＜3%); Pulverized 50～200 mesh sieves fine powder that gets dry extract, yield is 6.8%.

Existing studies show that, other compositions do not have negative influence at least to the aspects such as use of drug effect or medicine, and it is higher to separate these composition costs, therefore the necessity that is not further purified at present.

Be example with several representative dosage forms below, elaborate the concrete preparation method of the various dosage forms of this Chinese medicine extract.

The present invention prepares injectable powder and generally adopts conventional freeze-drying, as solvent, the steps include: to get this Chinese medicine extract with water, adds excipient, is dissolved in water, and adds active carbon, filtration sterilization, and plug is partly rolled in fill, and lyophilization, tamponade are rolled lid and are got final product.Used excipient is selected from one or more in mannitol, gelatin hydrolysate, glucose, lactose or the dextran etc.Every bottle contains this Chinese medicine extract 10～100mg.

The present invention prepares injectable powder also can adopt spray drying method, as solvent, the steps include: to get this Chinese medicine extract with water, adds or do not add excipient (excipient is the same), be dissolved in water, add active carbon, filtration sterilization, spray drying, aseptic subpackaged, tamponade is rolled lid and is got final product.Every bottle contains this Chinese medicine extract 10～100mg.

When the present invention prepared small-volume injection, preparation got final product as solvent with water for injection, also can add appropriate amount of auxiliary materials, and adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzyl benzoate or the dimethyl acetylamide etc.Every contains this Chinese medicine extract 10～100mg.

The present invention prepares glucose infusion liquid or sodium chloride transfusion, with water for injection as solvent, add the preparation of an amount of glucose or sodium chloride and get final product, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzyl benzoate or the dimethyl acetylamide etc.Every bottle contains this Chinese medicine extract 10～100mg.

The present invention prepares oral formulations such as tablet, capsule, granule, oral liquid, and adjuvant can be lactose, starch, dextrin, stearate etc., technology preparation routinely.

This Chinese medicine extract acute toxicity test

One, test objective

Observe a large amount of toxic actions after giving mice oral in this Chinese medicine extract 24 hours.

Two, material

1, be subjected to the reagent thing: this Chinese medicine extract is extracted dry extract and is made capsule by the expensive crude drug that provides of Haozhou city, Anhui, Anhui Province Liu Jin.

2, animal: Kunming mouse, 6 ages in week, male and female half and half, the The 2nd Army Medical College Experimental Animal Center provides, production licence number: Shanghai SCXK2002-2006.

Three, method

1, experimental situation: the Experimental Animal Center SPF of The 2nd Army Medical College level laboratory.

Ambient temperature: 23 ℃.

2, route of administration: irritate stomach.

3, tried the thing compound method: claim that 20g is subjected to the reagent thing, adding distil water reaches 80ml (this concentration is the maximum concentration under the animal permissive condition).Every milliliter includes and is tried thing 0.25g.

4, dosage and method: choose 20 of health Kunming mouses in 6 age in week, male and female half and half, overnight fasting (can't help water).The back weighed in second day by 0.4ml/10g body weight gastric infusion, press above dosage repeat administration once behind the 3h.Each dosage is: 10g/kg/ day, the secondary dosage reaches 20g/kg/ day.

5, laboratory observation: the according to the form below content is tightly observed animal performance, continuous 7 days after the administration.

Four, result

The spontaneous activity of administration about half animal on the same day obviously reduces, and loose stool occurs.Played activity freely in second day, the basic recovery normally.The others no abnormality seen does not have dead.Body weight gain is good, by increasing to 29.1 ± 1.0g for preceding 21.2 ± 0.66g.The sign observed result sees Table 2.

Table 2, this Chinese medicine extract acute toxicity test sign

The toxic reaction degree	Spontaneous activity		The abnormal muscle activity			Reaction to external world		Pupil		Secretions		Stool			The colour of skin			Eye		Breathe		Other
																							Increase	Reduce	Twitch	Paralysis	Abnormal gait	Irritated	Indifferently	Enlarge	Dwindle	Sialorrhea	Shed tears	Loose stool	Constipation	Color and luster is unusual	Pale	Cyanosis	Congested	Eyeball protrudes	Blepharoptosis	Excited	Suppress
	-	√		√	√	√	√	√	√	√	√	√		√	√	√	√	√	√	√	√	√																							√
±
	+		√										√
+ +

Five, conclusion

This Chinese medicine extract gives mice by maximum dosage-feeding, 20g/kg/ day, be equivalent to crude drug amount 580g/kg/ day, and the serious toxicity reaction does not appear in animal.This dosage is clinical people's consumption 218g (crude drug)/60kg/ day) more than 159 times.

This Chinese medicine extract is to the long term toxicity test of rat

One, test objective

Observe and give this Chinese medicine extract repeatedly to the toxic reaction that rat produced continuously, determine the dosage of nontoxic reaction.

Two, material

1, be subjected to the reagent thing: this Chinese medicine extract is by expensive the supplying raw materials of Haozhou, Anhui city Liu Jin, and experimental group is made granule according to a conventional method, be the special preparation of pharmacodynamics test and long term toxicity test tried thing, lot number 050920.

2, animal: the SD rat, 6 ages in week, male and female half and half, the The 2nd Army Medical College Experimental Animal Center provides, production licence

Number: Shanghai SCXK2002-2006.

Three, method

1, grouping: (National Drug Administration: the regulation specification requirement 1999:22 of study of tcm new drug), this test are established three dosage groups and a matched group with " specification requirement of study of tcm new drug " according to provisions for new drugs approval.Get 6 age in week the SD rat, male and female half and half are raised earlier before the experiment and are adapted to a week, get 120 of healthy normal animals, are divided into four groups at random:

Heavy dose of group, 30,8g crude drug/kgd (be equivalent to approximately clinical dosage 60 times)

In the dosage group, 30,4g crude drug/kgd (be equivalent to approximately clinical dosage 30 times)

Small dose group, 30,1.33g crude drug/kgd (test effective dose, be equivalent to 10 times of clinical dosage approximately)

The distilled water group, gavages isometric(al) distilled water 1ml/100g body weight by 30.

2, administration commencement date and deadline: October 09～2006 in 2005 years 04 month 11 days, six totally months.

3, experimental arrangement: behind the rat packet numbering, male and female are divided foster, 5 in every cage.Successive administration six months.Observe its behavior performance every day, feces character and other symptoms, sign.Survey body weight and food-intake weekly, and, adjust dose according to body weight change.Administration was put to death 10 animals (male and female half and half) for every group and is carried out gross anatomy, histopathologic examination and hematological examination, blood biochemical inspection in the time of three months.Remain 80 animals (20 every group, male and female half and half) and continue administration three months, administration is put to death 10 animals (male and female half and half) again for every group and is carried out gross anatomy, histopathologic examination and hematological examination, blood biochemistry checking after finishing.Continue to observe one month after remaining 40 animals (10 every group, male and female half and half) drug withdrawal, carry out gross anatomy, histopathologic examination and hematological examination, blood biochemical inspection.

4, test item

(1) body weight, food-intake: measure once weekly.

(2) hematological examination: blood counting instrument is surveyed erythrocyte, and (be called for short: RBC) counting, hemoglobin (are called for short: Hb), (be called for short: WBC) counting, leukocyte differential count, platelet (are called for short: Plt) counting leukocyte.

(3) blood biochemical is learned and is checked: use biochemistry analyzer respectively, measuring alanine aminotransferase with the lactic acid dehydrogenase KINETIC METHOD (is called for short: ALT), the malic dehydrogenase KINETIC METHOD is measured aspartic transaminase and (is called for short: AST), phosphoric acid is surveyed alkali phosphatase to nitro matter KINETIC METHOD and (is called for short: ALP), the picric acid KINETIC METHOD is measured creatinine and (is called for short: Crea), single agents enzymatic determination method is measured T-CHOL and (is called for short: Tch), glucose oxidase method is surveyed blood glucose and (is called for short: Glu), dimethyl sulfoxide method is measured total bilirubin and (is called for short: Tbil), biuret method is measured total protein and (is called for short: TP), the bromocresol green method is measured albumin, and (be called for short: Alb), urase-Berthelot colorimetric method for determining blood urea nitrogen (is called for short: BUN).

(4) pathological examination

Organ index: the heart, liver, spleen, lung, kidney, adrenal gland, brain, thymus, testis and epididymis or ovary and uterus.Internal organs are inhaled with filter paper and are removed surplus blood, and scales/electronic balance weighing multiply by 100.

Histopathologic examination: the heart, liver, spleen, lung, kidney, testis and epididymis or ovary and uterus.Internal organs are through 10% formalin fixed, and routine is drawn materials, and ethanol, dimethylbenzene are handled, paraffin Bao Li, HE dyeing, om observation.

Four, result of the test

1, in body weight and when feed: begin to end from administration, each organizes activities in rats, and feces etc. there is no unusually.Administration tailend in mid-term and the variation of convalescent the weight of animals and each administration group of food-intake and matched group be no significant difference relatively.Body weight, food-intake change see Table 3, table 4, table 5 and table 6.

Table 3, the long malicious administration of this Chinese medicine extract 6 months and convalescent period male rat body weight change (X=SD)

Cycle	Normal group (g)	Heavy dose of group (g)	Middle dosage group (g)	Small dose group (g)
					0 1 2 3 4 5 6 7 8	120.6±6.2 168.9±9.2 202.3±8.5 229.6±20.6 245.6±43.2 255.8±33.9 287.3±28.6 305.5±24.1 317.9±20.2	119.3±5.9 168.6±12.8 198.5±13.8 217.5±21.6 238.7±13.3 263.3±15.0 285.9±13.9 306.4±13.6 310.8±14.6	117.5±8.5 164.8±11.7 195.9±14.8 224.1±20.4 234.7±20.6 270.1±29.6 284.8±33.8 308.7±27.3 32 1.0±24.5	117.1±7.3 166.7±7.6 193.2±12.1 223.5±22.6 233.6±17.8 270.1±23.4 296.0±23.2 311.5±21.4 326.5±23.8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

330.7±17.9 340.1±16.9 351.5±17.3 361.1±20.0 368.3±21.8 376.6±39.0 392.2±19.7 395.4±19.6 401.0±24.4 400.2±19.5 408.8±20.7 410.8±22.8 409.6±23.9 405.8±36.7 416.8±30.2 418.6±31.3 435.4±33.0 410.0±61.4 414.0±61.4 434.8±42.6 456.4±36.3 463.6±37.8

320.8±18.7 33.6±19.4 349.6±20.9 361.2±23.4 373.2±28.2 391.2±20.0 395.6±17.6 398.4±17.5 397.2±22.3 400.0±20.1 402.2±21.1 403.8±23.1 405.1±19.8 404.8±19.8 411.4±19.8 427.4±23.4 449.2±66.5 435.2±23.0 438.4±23.9 429.6±26.6 43 8.8±29.4 441.6±28.9

337.9±20.5 341.2±33.6 359.2±22.3 368.7±24.2 376.5±25.6 400.8±25.2 406.2±23.4 409.0±22.7 417.6±23.2 410.4±23.9 413.2±25.8 415.2±29.3 424.2±27.4 424.2±27.8 428.2±32.6 428.6±32.8 435.4±33.0 439.2±19.5 443.6±21.3 450.0±20.4 458.0±20.6 462.4±19.6

338.0±26.7 340.1±30.8 356.7±24.6 365.1±27.5 374.3±35.0 400.6±45.8 410.4±45.6 413.0±45.6 414.6±44.5 422.8±50.8 424.4±55.4 426.2±58.1 436.4±60.8 436.4±60.9 445.4±65.4 445.4±65.3 449.2±66.7 450.2±92.7 440.6±101.6 452.1±93.1 448.3±89.5 455.1±113.6

Annotate: 1, during 3 months (0～13 week) of administration, matched group, heavy dose of group, middle dosage group and small dose group n=15;

2, administration after 3 months during 6 months (14～15 week), every group of n=10; Convalescent period (26～30 week) every group of n=5.

Table 4, the long malicious administration of this Chinese medicine extract 6 months and convalescent period female rats body weight change (X=SD)

Cycle	Normal group (g)	Heavy dose of group (g)	Middle dosage group (g)	Small dose group (g)
					0 1 2 3 4 5 6 7 8 9 10 11 12 13	11 8.7±6.8 144.3±5.7 1 64.8±8.0 179.2±11.8 191.3±13.6 205.7±13.4 213.9±10.5 219.1±8.8 224.0±9.8 228.4±10.9 235.7±9.2 241.5±9.2 247.1±10.1 253.1±11.6	116.3±6.9 141.9±8.7 163.5±12.3 175.5±14.3 187.1±12.4 199.1±14.0 208.9±14.5 215.2±13.6 220.3±13.1 224.8±12.4 229.3±12.2 233.5±12.9 238.5±14.4 242.8±15.5	114.7±7.2 144.0±4.8 165.1±6.2 185.5±8.1 200.1±7.9 208.8±9.3 218.4±8.9 221.9±7.7 226.9±7.0 231.7±6.1 234.1±7.8 236.9±7.4 239.7±8.3 246.3±17.7	110.9±6.1 144.0±5.0 164.9±8.4 179.7±15.5 180.5±14.6 195.7±15.7 214.1±13.8 217.3±13.4 222.9±11.8 232.5±11.5 237.7±11.3 243.2±10.9 250.9±10.7 253.2±14.5

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

258.4±12.4 262.2±9.8 264.4±10.6 267.6±10.6 267.0±9.0 268.6±11.5 270.4±14.6 271.2±18.6 271.6±17.7 275.2±18.8 278.4±18.7 280.4±19.8 270.4±13.7 272.8±13.8 269.6±13.2 272.4±12.9 276.9±13.1

254.0±14.6 258.8±16.8 262.7±16.3 266.6±15.6 263.8±15.2 267.0±15.7 269.4±16.2 258.2±25.6 262.2±19.4 268.0±22.6 274.2±26.0 278.4±28.6 275.2±21.0 277.6±21.5 275.2±21.4 282.0±29.5 285.6±30.1

251.0±11.2 255.0±9.8 257.0±9.9 253.2±9.6 258.0±8.3 259.6±7.8 258.6±12.2 266.2±14.7 262.4±14.8 264.2±14.9 266.2±15.4 267.2±15.3 265.6±17.5 268.0±16.7 268.0±16.8 270.4±15.8 273.9±17.6

250.8±10.8 264.4±15.7 266.6±15.5 269.8±11.6 267.4±10.5 269.8±8.8 273.2±9.5 271.2±8.2 270.8±8.6 277.6±11.4 283.0±13.0 286.4±14.3 283.2±15.8 285.2±15.4 285.2±15.5 297.6±21.6 300.5±22.3

Table 5, the long malicious administration of this Chinese medicine extract 6 months and convalescent period male rat appetite variation (X=SD)

Cycle	Normal group (g)	Heavy dose of group (g)	Middle dosage group (g)	Small dose group (g)
					0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22	77.9±12.3 80.0±10.6 81.3±9.0 84.0±8.6 88.0±7.2 93.3±3.1 96.7±3.2 101.6±3.1 104.0±2.0 107.3±1.4 109.3±1.3 110.7±1.6 112.7±1.4 114.7±2.1 116.0±3.0 117.0±2.9 118.0±3.2 119.0±1.4 120.0±1.2 121.0±1.4 122.0±2.8 123.0±1.4 124.0±1.6	80.9±8.1 82.7±6.4 84.0±5.4 86.0±3.5 89.3±2.3 90.7±3.1 96.1±3.1 102.3±2.3 105.3±1.6 107.3±1.4 108.7±1.7 110.0±0.9 112.0±2.0 114.7±1.2 116.0±0.0 117.0±1.4 118.0±0.9 119.0±1.4 120.0±1.8 122.0±1.3 123.0±1.4 124.0±1.9 125.0±1.4	70.9±4.9 72.0±5.0 74.0±4.1 76.0±3.9 81.3±2.5 90.0±5.0 97.3±2.7 101.3±3.1 106.7±1.8 108.7±1.2 110.0±0.0 111.3±1.2 112.7±1.3 113.3±2.1 114.0±0.9 115.0±1.4 118.0±1.3 120.0±3.1 121.0±1.9 121.0±1.4 122.0±2.8 123.0±1.4 124.0±2.8	76.5±4.5 78.7±4.0 80.7±4.2 83.3±3.0 87.3±3.1 91.3±1.6 97.3±1.2 102.0±2.0 107.3±2.9 109.3±3.2 110.7±2.8 112.0±0.0 113.3±1.2 114.7±2.3 115.0±1.4 116.0±1.2 117.0±1.5 119.0±1.6 120.0±3.0 121.0±2.1 122.0±2.8 123.0±1.4 124.0±1.9

23 24 25 26 27 28 29 30

125.0±1.4 126.0±2.8 128.0±3.2 128.3±4.1 130.1±1.8 130.2±1.5 131.4±1.6 131.6±2.9

126.0±5.0 127.0±3.2 127.0±2.7 126.1±3.1 127.1±0.9 130.3±2.1 130.7±1.9 131.5±3.1

125.0±1.9 126.0±0.9 127.0±2.8 126.0±2.8 127.1±1.5 126.1±1.9 128.5±0.7 130.7±2.0

125.0±2.1 126.0±0.0 127.0±3.2 128.1±2.8 128.0±1.9 129.3±1.6 129.6±0.9 130.5±3.0

Annotate: 1, matched group, heavy dose of group, middle dosage group and small dose group n-3 cage during three months (0～13 week) of administration:

2, during 6 months (14～15 week), each organizes the n-2 cage after 3 months in administration: convalescent period (26～30 week) every group of n-1 cage.

Table 6, the long malicious administration of this Chinese medicine extract 6 months and convalescent period female rats appetite variation (X=SD)

Cycle	Normal group (g)	Heavy dose of group (g)	Middle dosage group (g)	Small dose group (g)
					0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27	63.9±7.9 65.0±8.1 69.3±8.8 70.7±5.8 78.0±3.5 81.3±1.4 83.3±1.3 84.7±2.1 86.0±0.0 88.7±3.2 90.7±1.6 92.0±0.0 92.7±1.2 94.7±5.0 97.0±1.4 99.0±2.8 100.0±7.1 101.0±1.4 102.0±6.2 103.0±1.4 104.4±3.2 104.3±5.0 106.0±1.4 106.6±0.0 108.8±2.7 109.9±1.9 112.9±3.1 114.3±5.0	72.8±12.1 74.7±12.2 84.0±5.3 78.7±10.3 82.0±7.2 84.6±5.3 85.3±4.2 87.8±3.1 89.3±1.2 90.7±1.9 91.3±3.1 92.7±2.5 94.0±2.0 95.3±1.2 97.0±1.4 98.0±1.6 99.0±3.5 100.0±6.1 101.0±1.4 103.0±5.8 104.0±1.6 105.0±1.4 106.0±1.7 107.0±1.4 108.0±2.5 108.0±0.0 109.1±3.0 112.3±1.9	69.1±6.0 68.7±4.2 74.0±5.3 72.7±4.1 74.7±2.3 78.0±3.2 80.7±1.2 82.0±0.0 85.3±2.3 87.3±1.6 88.7±1.2 90.0±0.0 91.3±2.1 92.7±1.2 93.0±2.1 99.0±1.4 118.0±1.6 101.0±2.7 102.0±0.8 102.0±3.2 103.4±1.4 104.0±0.0 105.6±1.4 106.8±0.0 107.6±1.4 108.9±2.8 108.9±3.6 110.7±4.1	72.9±3.5 75.3±3.1 80.7±4.5 80.1±3.2 80.7±1.2 82.7±1.8 84.7±1.2 86.0±2.1 88.7±1.2 90.0±0.0 90.7±1.2 92.0±1.6 93.3±1.2 93.3±1.9 94.0±2.7 97.0±1.4 117.0±0.6 99.0±1.6 101.0±1.6 102.0±1.7 103.0±1.4 104.0±3.1 105.0±1.5 106.0±1.2 107.0±1.4 108.0±2.6 108.7±2.6 110.9±1.7

29 30 31

114.7±1.6 115.2±0.9 116.2±3.5

112.7±0.9 114.2±3.7 114.5±5.1

110.6±3.7 112.1±2.9 114.0±1.9

112.3±2.6 112.4±3.5 114.6±1.9

2, hematological examination

Rat aorta is got blood, does hematological examination, measures Hb content, RBC counting, WBC counting and classification, platelet count.The result shows: medication is after three months, raises from cell number, neutrophilic granulocyte percentage in heavy dose of group blood, and the lymphocyte percentage reduces (p＜0.05), but all in normal range, in, small dose group compares no significant difference with normal group; Other respectively organize index administration group and matched group does not more all have significant difference.After the medication six months, middle dosage group blood hemoglobin reduces (p＜0.05), but still in normal range, large and small dosage group and normal group be there was no significant difference relatively also; Other each index administration groups and matched group more all do not have significant difference.Convalescent period each index administration group and matched group more all do not have significant difference.Administration the results are shown in Table 7 after three months, administration the results are shown in Table 8 after six months, and convalescent period the results are shown in Table 9.

Table 7, three months rat bloods of the long malicious administration of this Chinese medicine extract are learned and are checked (X=SD)

Project	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					WBC(10 9/ L) neutral (%) lymph (%) RBC (10 12/L) Hb(g/L) P1t(10 9/L)	4.6±1.7 2.12±1.8 75.8±2.0 8.6±0.7 150.4±8.1 1010.9±60.5	7.6±3.2 * 27.9±8.6 * 68.3±8.5 * 8.8±0.9 148.6±10.8 1090.8±106.3	7.6±3.0 21.0±5.1 74.6±4.9 8.6±±1.4 148.4±15.0 971.8±148.0	4.6±1.0 22.3±3.4 75.8±3.0 9.0±0.8 158.5±12 994.8±84.6

Annotate: 1, owing to blood coagulation, normal group n=8; Heavy dose of group n=9.

2, all the other respectively organize n=10.

Table 8, six months rat bloods of the long malicious administration of this Chinese medicine extract are learned and are checked (X=SD)

Project	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					WBC(10 9/ L) neutral (%) lymph (%) RBC (10 12/L) Hb(g/L) Plt(10 9/L)	5.6±1.6 39.6±15.4 56.9±15.5 8.4±0.7 155.0±9.3 68.4±38.4	7.2±3.2 35.9±12.0 59.5±11.8 * 8.5±0.8 156.8±8.5 136.7±140.0	5.5±1.8 33.5±8.6 61.9±8.8 7.9±0.7 146.2±8.0 180.4±236.6	5.9±2.8 39.8±13.3 56.2±13.0 8.7±0.6 155.0±6.4 62.7±15.9

Table 9, this Chinese medicine extract convalescent period rat blood are learned and are checked (X=SD)

Project	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					WBC(10 9/L) RBC(10 12/L) Plt(10 9/L)	5.3±2.3 13.6±7.2 488.5±116.3	5.4±2.1 13.4±1.8 506.7±195.3	5.2±1.8 12.8±2.6 308.3±115.8	5.5±2.0 13.2±2.4 410.1±87.9

3, blood biochemical is learned and is checked:

Rat aorta is got blood, and is centrifugal, gets serum and makes blood biochemistry checking, measures ALT, AST, ALP, BUN, Crea, Chol, Clu, TP, Alb, Tbil.The result shows: administration is after three months, and the BUN of heavy dose of group is higher than normal group (p＜0.05), in, small dose group and normal group relatively there is no significant difference.The Tbil of small dose group is lower than normal group (p＜0.05), but in normal range; Administration life in six months, the Tbil of large, medium and small dosage group is lower than normal group, is the fluctuation in normal range, no pathology meaning.Convalescent period, the Glu of large, medium and small dosage group was lower than normal group, and the TP of small dose group is lower than normal group, was the fluctuation in normal range, no pathology meaning.Administration the results are shown in Table 10 after three months, six months results of administration see Table 11, and convalescent period the results are shown in Table 12.

Table 10, three months rat blood biochemical analysises (X=SD) of the long malicious administration of this Chinese medicine extract

Project	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					TP(g/L) Alb(g/L) ALT(U/L) AST(U/L) ALP(U/L) Tbil(μmol/L) Crea(μmol/L) BUN(mmol/L) Glu(mmol/L) Chol(mg/dl)	61.6±13.4 33.2±7.7 21.0±11.9 19.4±8.6 62.3±17.1 0.159±0.038 50.8±10.4 6.0±0.5 8.4±3.0 1.7±0.3	67.7±14.9 33.5±4.0 28.7±11.3 21.8±12.0 45.6±20.6 0.180±0.046 49.1±8.5 7.0±1.0 * 5.8±3.6 1.4±0.2	63.6±12.4 8.4±3.9 21.8±5.8 23.6±10.2 53.9±21.4 0.169±0.080 47.9±12.6 6.4±0.9 6.6±1.5 1.4±0.2	63.9±6.9 34.7±4.5 31.4±14.1 24.6±10.2 52.3±9.8 0.119±0.035 48.9±11.0 6.4±1.4 8.0±1.7 1.4±0.3

Table 11, six months rat blood biochemical analysises (X=SD) of the long malicious administration of this Chinese medicine extract

Project	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					TP(g/L) Alb(g/L) ALT(U/L) AST(U/L) ALP(U/L) Tbil(μmol/L) Crea(μmol/L) BUN(mmol/L) Glu(mmol/L) Chol(mg/dl)	66.8±2.4 27.1±5.6 21.8±9.9 26.3±10.4 60.3±17.3 29.5±16.1 84.9±15.7 10.6±1.3 3.2±0.8 1.9±0.4	70.3±9.5 25.3±7.1 27.1±9.6 21.4±8.2 46.6±17.6 26.7±16.5 77.2±17.1 9.7±1.5 2.6±0.5 1.8±0.3	73.9±5.7 25.8±6.1 27.6±9.2 20.3±6.3 51.9±20.4 28.0±13.0 69.3±1 7.0 10.0±1.3 2.8±0.5 1.8±0.2	71.6±4.2 29.2±5.9 25.0±10.4 20.3±7.6 52.3±8.5 24.4±9.5 74.0±12.7 9.6±1.1 3.2±0.6 1.9±0.3

Table 12, the long poison of this Chinese medicine extract convalescent period rat blood biochemical analysis (X=SD)

Project	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					TP(g/L) Alb(g/L) ALT(U/L) AST(U/L)	67.7±4.1 31.9±2.0 46.1±20.6 208.7±41.6	67.7±4.9 30.6±2.8 44.0±16.0 212.9±39.4	70.1±3.5 29.6±3.0 54.2±27.8 242.7±40.5	63.8±3.8 28.6±3.2 43.1±22.4 184.7±42.7

ALP(U/L) Tbil(μmol/L) Crea(μ mol/L) BUN(mmol/L) Glu(mmol/L) Chol(mg/dl)

78.7±32.8 ＜0.18 64.6±11.0 7.2±0.9 6.3±1.4 1.9±0.4

78.4±19.3 ＜0.18 67.5±30.4 9.0±4.8 4.5±1.1 * 1.9±0.4

96.7±19.8 ＜0.18 60.1±8.3 8.1±0.9 3.8±1.2 ** 1.9±0.3

88.2±70.0 ＜0.18 59.5±9.1 7.1±0.7 4.5±0.7 ** 1.6±0.2

Annotate: 1. ^*P＞0.05, ^*P＜0.01, n=10.

4. pathologic finding

(1) becomes celestial substantially: behind the sacrifice of animal, dissect immediately, win main organs, take by weighing its weight in wet base, obtain organ index, to compare between T check work group.The result shows: administration is after three months, and heavy dose of group kidney heavily is higher than matched group, in, small dose group and normal group comparison no significant difference.Convalescent period after six months, each dosage group rat main organs index and normal group be no significant difference relatively.Administration the results are shown in Table 13 after three months, administration the results are shown in Table 14 after six months, and convalescent period the results are shown in Table 15.

Table 13, three months rat main organs indexes (X=SD) of the long poison of this Chinese medicine extract

Cycle	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					Conscience spleen lung kidney adrenal gland brain thymus gland appendix testis ovary and uterus	30.3±2.5 248.6±17.3 18.6±2.5 50.9±8.8 59.8±3.4 1.6±0.5 53.5±13.6 7.8±1.8 91.3±2.6 33.1±2.4 18.2±3.2 4.8±0.6	33.0±3.7 268.0±30.6 22.7±5.4 * 56.1±10.4 64.7±3.2 1.8±0.5 59.3±16.7 8.8±3.5 91.5±11.2 32.5±3.6 21.4±3.9 5.2±0.6	32.0±2.8 259.2±23.5 31.0±2.9 22.6±5.1 * 61.7±3.5 2.0±0.6 58.8±12.8 9.4±2.3 89.2±3.5 33.4±2.1 18.0±3.3 5.1±0.7	32.8±3.0 262.4±15.3 31.4±3.1 18.4±2.9 61.3±5.1 1.8±0.4 58.3±13.5 8.2±2.0 91.9±3.2 33.6±1.0 19.7±2.3 5.3±1.4

Annotate: testis, uterus, ovary, n=5, other internal organs n=10

Table 14, six months rat main organs indexes (X=SD) of the long poison of this Chinese medicine extract

Cycle	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					Conscience spleen lung kidney adrenal gland brain thymus gland appendix testis uterus	31.6±1.6 235.8±16.7 18.1±2.6 58.4±13.0 61.6±7.7 1.5±0.6 56.2±9.2 5.4±1.4 92.8±5.8 34.5±2.8 21.8±5.4	32.5±2.8 238.9±23.1 18.7±3.4 61.5±12.7 61.4±4.8 1.6±0.7 56.3±14.5 5.0±1.6 84.9±4.3 33.2±3.5 23.0±5.6	31.8±3.4 234.8±17.6 17.1±2.7 54.9±9.6 63.5±3.8 1.8±0.7 57.4±14.2 5.6±1.2 86.2±14.2 33.8±3.8 24.8±4.1	31.8±2.9 250.5±11.5 17.4±1.7 53.3±6.0 58.7±4.8 1.7±0.6 53.8±9.9 5.3±1.2 85.3±4.6 32.9±1.5 22.3±6.6

Ovary

5.2±1.4

5.6±0.8

5.1±0.8

5.4±1.4

Annotate: testis, uterus, ovary, n=5, other internal organs n=10

Table 15, the long poison of this Chinese medicine extract convalescent period rat main organs index (X=SD)

Cycle	Normal group	Heavy dose of group	Middle dosage group	Small dose group
					Conscience spleen lung kidney adrenal gland brain thymus gland appendix testis ovary and uterus	32.9±3.1 224.7±24.8 17.4±3.0 51.4±14.9 58.5±8.0 1.4±0.4 53.9±13.6 5.2±2.0 77.2±10.6 32.4±2.5 28.3±5.6 4.6±1.2	32.4±2.6 226.4±14.7 17.7±2.2 46.3±8.3 56.9±3.6 1.5±0.8 54.4±12.3 5.2±1.4 72.0±14.9 31.7±3.5 23.2±6.7 4.8±0.5	31.8±4.6 230.8±23.5 17.8±4.3 50.3±10.5 58.8±2.8 1.6±0.7 56.5±14.6 5.9±1.4 85.2±6.4 32.3±2.5 27.7±5.1 5.4±1.4	29.9±5.3 224.2±28.4 16.0±3.1 49.0±8.2 57.9±9.0 1.5±0.4 52.7±9.6 6.8±2.9 78.5±12.3 33.2±6.5 24.5±5.3 4.7±0.7

Annotate: testis, uterus, ovary, n=5, other internal organs n=10

(2) pathologic finding: behind the sacrifice of animal, win internal organs such as the heart, liver, spleen, lung, kidney, brain, through 10% formalin fixed, routine is drawn materials, and ethanol, dimethylbenzene are handled, paraffin Bao Li, HE dyeing, pathologic finding under the light microscopic.

The result shows:

Administration three months: each group of lungs all degree of taking a favourable turn interstitial pneumonia changes, and each group of liver all has individual animal point-like inflammatory infiltration kitchen range to occur, all sporadically appears in each group (containing normal group), does not have obvious pass property with medicine.

There is no abnormal change under the heart, liver, kidney, adrenal gland, thymus, uterus, ovary, testis, the adnexa histopathology light microscopic.

Administration six months: have two examples to see the special mess cell infiltration in the hepatic tissue section, this variation and medicine do not have obvious dependency.Each group of lungs all has matter pneumonia pathological changes, wherein #1 (normal group) and #29 (middle dosage group) and the pathological change of degree of taking a favourable turn bronchiolitis between the appearance slightly of minority tissue.These change with medicine does not have obvious dependency.There is no abnormal change under the heart, spleen, kidney, adrenal gland, thymus, uterus, ovary, testis, the epididymis histopathology light microscopic.

Convalescent period: slight cellular fat change appears in the accidental example of liver organization.Other has 7 routine accidental point-like cell infiltration kitchen ranges, does not have obvious dependency with medicine.All seeing in each treated animal of lungs has lighter interstitial pneumonia pathological changes, in, small dose group sees respectively to have in the 1 routine minority alveolar space and oozes out that above-mentioned change and medicine do not have obvious dependency.

Five, discussion and conclusion

This tests 6 months gastric infusions of this Chinese medicine extract rat, and from start to end, each is organized activities in rats, feces etc. and there is no unusually, generally in order, does not see any sings and symptoms.Each administration group of body weight food-intake and matched group be no significant difference relatively also.

Hematological examination, administration WBC in mid-term and neutrophilic granulocyte percent are higher than normal group, lymphocyte percentage is lower than normal group, but all test (Chen Changxun in normal range at the long over the years poison of this laboratory, Jin Ruomin, Li Yikui. etc.: the part biological data determination of normal SD rats, zoology magazine 1996; 31 (4): 19).And these variations did not appear in (6 months) when administration finished, and it is irrelevant to analyze its reason and medicine factor.Administration is in the time of 6 months, and middle dosage group Hb reduces, but heavy dose and normal group are not relatively seen significant change on the contrary, and the variation of dosage group and medicine are irrelevant in the analysis.

Blood biochemical learn to check that administration heavy dose of group in mid-term BUN is higher than normal group, but not overrun (Chen Changxun, Jin Ruomin, Li Yikui. etc.: the part biological data determination of normal SD rats, zoology magazine 1996; 31 (4): 19), and administration when finishing (6 months) this index and normal group relatively there is no significant difference.Histopathologic examination there is no administration mid-term and tailend the administration group and show obvious pathological change aspect kidney, so think that the variation of administration BUN in the time of 3 months and medicine factor have nothing to do.

Pathomorphology checks that all seeing when administration mid-term and 6 months has the minority liver point-like focal inflammation disease to occur, but these variations can occur under normal condition, and sporadically appears in normal group and each group of administration, so think and have nothing to do with medicine; Lung lesion also sporadically appears in normal group and each group of administration, so also think irrelevant with medicine.

In sum, general state is good from start to finish for this Chinese medicine extract long term toxicity test rat, and is movable normal, do not see any symptom and death; Hematology, blood biochemical are learned, pathological examination finishes (6 months) in administration mid-term (3 months), administration and there is no the toxic reaction relevant with medicine with convalescent period (drug withdrawal 1 month).

Conclusion: 6 months gastric infusion long term toxicity tests of this Chinese medicine extract rat are not found overt toxicity.

In the present invention, the example of the above-described specific embodiment and the following stated all is in order to set forth the present invention better, is not to be used for limiting scope of invention.

Below by embodiment the present invention is described in detail.

The preparation of embodiment 1, this Chinese medicine extract capsule

(1) prescription

This Chinese medicine extract 1000.0g

Microcrystalline Cellulose 1000g

Carboxymethyl starch sodium 140g

Dehydrated alcohol is an amount of

Pulvis Talci 80g

Make 10000 capsules altogether

(2) preparation technology gets in this Chinese medicine extract of crude drug and the prescription other adjuvant respectively by above prescription and crosses 100 mesh sieves respectively, put 60 ℃ of oven dry, take by weighing this Chinese medicine extract of recipe quantity and microcrystalline Cellulose, the carboxymethyl starch sodium equivalent method mix homogeneously that progressively increases, with an amount of dehydrated alcohol system soft material, 30 mesh sieves are granulated, 50～60 ℃ of dryings 2 hours with 30 mesh sieve granulate, add the Pulvis Talci and the carboxymethyl starch sodium mix homogeneously of recipe quantity.

The preparation of embodiment 2, this Chinese medicine extract tablet

Get this Chinese medicine extract meter 80g, microcrystalline Cellulose 80g, lactose 15g, pregelatinized Starch 60g sieve, and mix homogeneously with an amount of 10%PVP alcoholic solution system soft material, is granulated, and drying adds magnesium stearate 3g, granulate, and tabletting is made 1000.

The preparation of embodiment 3, this Chinese medicine extract tablet

(1) prescription

This Chinese medicine extract 1000.0g

Microcrystalline Cellulose 1170.0g

Pregelatinized Starch 690.0g

Lactose 125.0g

The 5%PVP dehydrated alcohol is an amount of

Magnesium stearate 15.0g

Make 10000 altogether

(2) preparation technology

Take by weighing the principal agent and the adjuvant of recipe quantity respectively by above prescription, progressively increase behind the method mix homogeneously according to making soft material, system granule under the formulation and technology item by equivalent, dry, processes such as granulate, the heavy back of sheet single punch tablet machine and 10.5mm shallow concave punch tabletting have been calculated, control nude film hardness 5～7kg makes 9698 in tablet altogether, and yield rate is 96.98%.Adopt lift-over nebulization coating, art for coating is as follows:

The preparation of coating solution: gastric solubleness thin film dress material: 85G61235 is provided by Shanghai Colorcon Coating Technology Co., Ltd

Art for coating: will treat that (hardness 5～7kg) is put into coating pan to the coating nude film, start agitating device and air blast heater, when treating that the nude film temperature rises to 40 ℃, last 1/3 place that begins to open spray gun alignment tab bed sprays into the coating solution coating, 38～42 ℃ of control strip bed tempertaures, gas pound pressure 6kg, the coating solution flow velocity is 50ml/min, coating membrane heavily account for coated tablet heavy 3%.

The preparation method of embodiment 4, this Chinese medicine extract

(1) each medical material of this Chinese medicine extract is cleaned, soaked to insert after 12 hours and extract the interior decoction of molten device three times, the time is: 1 hour, 1 hour, 0.5 hour.

(2) extract the medicinal liquid merging with three times and be condensed into extractum simultaneously.

(3) extractum is left standstill after, add ethanol and advance precipitate with ethanol, reclaim ethanol and go precipitation.

(4) supernatant concentration of getting behind the precipitate with ethanol becomes pure extractum, this pure extractum proportion 1.2～1.3 (yield 12.5%).

(5) add dry 300 mesh sieves of crossing of adjuvant granulation.

(6) be packaged into 10g/ bag, finished particle.

The preparation method of embodiment 5, this Chinese medicine extract: close one of lifting manipulation

(1) the preparation extracting solution is got Rhizoma Atractylodis coarse powder 1kg (extracting volatile oil earlier), Cortex Phellodendri coarse powder 2kg, Radix Achyranthis Bidentatae coarse powder 3kg, add 95% ethanol 3000ml, soaked 24 hours, percolation routinely, control percolation speed 3～5ml/ minute is added solvent, till percolation to alkaloid, saponin component assay reactions is negative at any time in the percolation process, collect percolate, be extracting solution.

(2) the preparation extract is not distinguished the flavor of said extracted liquid decompression and solvent recovery to there being alcohol, remove by filter float and insoluble impurities, distilled water is regulated proportion to 1.05g/ml (25 ℃), the macroporous resin column that filtrate is crossed by activation processing (every Kg medical material is with activating wet resin 1Kg), active component in the filtrate is adsorbed by resin column, and discard effluent.Water eluting macroporous resin column discards eluent; Continue to collect eluent with 50% ethanol elution macroporous resin column, it is dried that 60 ℃ of eluents are evaporated to, and mixes with Rhzoma Atractylodis Lanceae volatile oil, must extract powder 121.0g.The TLC calibrating contains alkaloid and saponin component, is index with the total alkaloid content, measures total alkaloid content 13.55% with the HPLC method.

(3) preparation preparation

Prescription and proportioning:

Extract powder 100g

Starch 40g

Lactose 30g

Microcrystalline Cellulose 30g

7% starch slurry is an amount of

Magnesium stearate 1%

By said ratio extract powder, starch, lactose, microcrystalline Cellulose are crossed 80 mesh sieves respectively, mixing, after 40 mesh sieves 3 times, above-mentioned mixed powder with 7% starch slurry system soft material, is crossed 24 mesh sieve grains, 50 ℃ of dryings 2 hours, dried particles is crossed 30 mesh sieve granulate, adds magnesium stearate, mixing, tabletting gets 1000 in tablet.

The preparation method of embodiment 6, this Chinese medicine extract: close two of lifting manipulation

Get Rhizoma Atractylodis coarse powder 1kg (extracting volatile oil earlier), Cortex Phellodendri coarse powder 2kg, Radix Achyranthis Bidentatae coarse powder 3kg, be soaked in the 15000ml distilled water, heating decocts 2 times, each 1 hour, filter, concentrated filtrate is to 1.05g/ml (25 ℃), the macroporous resin column that filtrate is crossed by activation processing (every Kg medical material with activation wet resin 1Kg), active component in the filtrate is adsorbed by resin column, and discard effluent.Water eluting macroporous resin column discards eluent; Continue to collect eluent with 50% ethanol elution macroporous resin column, it is dried that 60 ℃ of eluents are evaporated to, crushed after being dried, extract powder 131.40g, the TLC calibrating contains alkaloids, saponin component, HPLC method mensuration total alkaloid content 11.10%.

The existing berberine constituents that studies show that has the effect of osteoporosis disease, embodiment 2 and embodiment 1 compare, and the extract powder yield is high slightly, and total alkaloids constituents content is similar, solvent for use is cheap free of contamination water, so embodiment 2 is for being fit to the large-scale production preparation method.

The preparation method of embodiment 7, this Chinese medicine extract: close three of lifting manipulation

(1) the preparation extracting solution is got Rhizoma Atractylodis coarse powder 1kg (extract earlier volatile oil), Cortex Phellodendri coarse powder 2kg, Radix Achyranthis Bidentatae coarse powder 3kg, adds 95% ethanol 3000ml, soaks 24 hours, and reflux 3 times each 2 hours, merges the extracting solution of 3 backflows, filtration.

(2) the preparation extract is not distinguished the flavor of said extracted liquid decompression and solvent recovery to there being alcohol, remove by filter float and insoluble impurities, distilled water is regulated proportion to 1.05g/ml (25 ℃), the macroporous resin column that filtrate is crossed by activation processing (every Kg medical material is with activating wet resin 1Kg), active component in the filtrate is adsorbed by resin column, and discard effluent.Water eluting macroporous resin column discards eluent; Continue to collect eluent with 50% ethanol elution macroporous resin column, it is dried that 60 ℃ of eluents are evaporated to, and mixes with Rhzoma Atractylodis Lanceae volatile oil, must extract powder 113.0g.The TLC calibrating contains alkaloid and saponin component, is index with the total alkaloid content, measures total alkaloid content 12.3% with the HPLC method.

(3) preparation preparation

Prescription and proportioning:

Extract powder 100g

Starch 40g

Lactose 30g

Microcrystalline Cellulose 30g

7% starch slurry is an amount of

Magnesium stearate 1%

By said ratio extract powder, starch, lactose, microcrystalline Cellulose are crossed 80 mesh sieves respectively, mixing, after 40 mesh sieves 3 times, above-mentioned mixed powder with 7% starch slurry system soft material, is crossed 24 mesh sieve grains, 50 ℃ of dryings 2 hours, dried particles is crossed 30 mesh sieve granulate, adds magnesium stearate, mixing, tabletting gets 1000 in tablet.

The preparation method of embodiment 8, this Chinese medicine extract

(1) each medical material of this Chinese medicine extract was measured distilled water immersions 12 hours with 8 times, insert in the extraction vessel, decoct three times, the time is: 1 hour, 1 hour, 0.5 hour.

(2) negative pressure is carried out in the merging of three extraction medicinal liquids simultaneously and be condensed into thick paste, this thick paste proportion 1.3.

(3) with thick paste negative-pressure vacuum drying-0.085 ± 0.005mpa, 70 ℃ ± 5 ℃ get dry extract (moisture＜3%); Pulverized the 80 mesh sieves fine powder that gets dry extract, yield is 6.8%.

The preparation method of embodiment 9, this Chinese medicine extract: single lifting manipulation

Take by weighing Rhizoma Atractylodis coarse powder 1kg (carrying volatile oil earlier), Cortex Phellodendri coarse powder 2kg, Radix Achyranthis Bidentatae coarse powder 3kg respectively, respectively above-mentioned each raw material coarse powder being added 95% ethanol 1000ml soaks, difference is percolation routinely, Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae are till percolation to alkaloids, saponin component assay reactions is negative respectively, collect percolate, get each single medicinal material extracting solution; Respectively each single medicinal material extracting solution reclaim under reduced pressure is not distinguished the flavor of to there being alcohol, filter to get filtrate, distilled water is regulated proportion to 1.05g/ml (25 ℃), the macroporous resin column that filtrate is crossed by activation processing (every Kg medical material is with activating wet resin 1Kg), active component in the filtrate is adsorbed by resin column, and discard effluent.Water eluting macroporous resin column discards eluent; Continue to collect eluent with 50% ethanol elution macroporous resin column, it is dried that 60 ℃ of eluents are evaporated to, and crushed after being dried respectively must 33.2g Rhizoma Atractylodis powders A, 47.1g Cortex Phellodendri powder B, 32.40g twotoothed achyranthes root C.A, B, C are mixed with Rhzoma Atractylodis Lanceae volatile oil, promptly get extract powder of the present invention, the TLC calibrating contains alkaloid, saponin component, and the HPLC method is measured total alkaloid content 11.43%.

Embodiment 10, pharmacological evaluation

Retinoic acid can cause osteoporosis, and the ethoxy Alendronate is the medicine that is used for protect against osteoporosis at present clinically.By pharmacological evaluation, observe Chinese medicine extract protect against osteoporosis of the present invention effect.

1 experiment material

1.1 animal

SD rat The 2nd Army Medical College Experimental Animal Center provides the laboratory animal production licence number: the SCXK of The 2nd Army Medical College (Shanghai) 2002-0006.

1.2 medicine

Pharmaceutical college of ethoxy Alendronate The 2nd Army Medical College new drug assessment centers provides, and is made into 2.0% suspension with 0.5%CMC-Na

Retinoic acid Shanghai No.6 Pharmaceutical Factory lot number 200106012 usefulness 0.5%CMC-Na are made into 1.5% suspension

Extract A is made into 0.5%CMC-Na by embodiment 1 preparation and is equivalent to crude drug 2g/ml suspension

Extract B is made into 0.5%CMC-Na by embodiment 2 preparations and is equivalent to crude drug 2g/ml suspension

CMC-Na Shanghai chemical reagents corporation

1.3 instrument

QCT-bone density analytical system U.S. LACA company

2 methods and result

2.1 to experimental osteoporosis prevention effect

50 of SD rats, be divided into 5 groups at random, every group 10, be normal control group (group of solvents), model control group, positive controls (ethoxy Alendronate group), experimental group A (example 1 is extract obtained, divides high low dosage), (example 2 is extract obtained for experimental group B, divide high low dosage), except that the normal control group, all the other are respectively organized rat and all irritate stomach and give retinoic acid 0.5ml/kg, every day 1 time, continuous 14 days, when giving retinoic acid, each medication group is pressed dosage gastric infusion every day 1 time of table 1, continuous 28 days, normal control group and model group give 0.5%CMC, dose is per kilogram of body weight 0.5ml, weighs weekly therebetween 1 time, and adjusts the administration consumption in view of the above.The 29th day sacrificed by decapitation, get rat bilateral femur, peel meat and other tissue off, wherein a side femur is done rat femur scanning on dual intensity X line borne densitometers, measures bone density (g/cm2), then in 120 ℃ of oven dry one hour, place interior 200 ℃, 400 ℃, 600 ℃, 800 ℃ each ashing of mnffer stove 2 hours again, after ashing finishes cooling, claim ash heavy (Washg), measure bone ash calcium, bone ash phosphorus content after extracting calcium, phosphorus with the HCL of 6N.

The opposite side femur is made the decalcification paraffin section, and microscopically is observed bone structure.The results are shown in Table 16.By table 16 as seen, the femur thigh density of extract group rat of the present invention, bone ash weight, calcium content of bone and bone phosphorus content are all apparently higher than model group, near normal control group and positive controls, show that extract of the present invention has significant preventive effect to osteoporosis.

From pathological section as seen, the cortical bone of model group presents the pathological change that loose, attenuation and bone trabecula loosen, attenuate.Each administration group and positive controls pathological change significantly alleviate, and also show that extract of the present invention has significant preventive effect to osteoporosis.

Table 16, Chinese medicine extract of the present invention are to experimental osteoporosis prevention effect

Group	Dosage	Femoral bmd g/cm 2	The heavy wash (g) of femur bone ash	Femur bone ash calcium g/100g bone ash	Femur bone ash phosphorus g/100g bone ash
						Normal group model control group ethoxy Alendronate group extract A group extract A group extract B group extract B group	0.5％ CMC-Na 0.5％ CMC-Na 50mg/kg 1.0g/kg 2.0g/kg 1.0g/kg 2.0g/kg	0.223 ± 0.008 0.209 ± 0.007 *0.220 ± 0.020 *0.215 ± 0.012 *0.222 ± 0.010 *0.211 ± 0.013 *0.220 ± 0.012 *	0.248 ± 0.023 0.211 ± 0.009 *0.260 ± 0.013 *0.237 ± 0.016 *0.244 ± 0.011 *0.241 ± 0.020 *0.250 ± 0.013 *	30.98 ± 1.72 29.28 ± 1.10 *31.09 ± 2.32 *30.21 ± 1.67 *30.34 ± 1.37 *31.10 ± 0.90 *31.42 ± 0.68 *	18.16 ± 1.05 17.43 ± 0.90 *18.17 ± 1.30 *17.67 ± 0.88 *17.86 ± 0.47 *18.01 ± 1.09 *18.12 ± 0.88 *
Annotate: 1. x ± SD; 2. compare with the normal control group *P＜0.05; 3. compare with model control group *P＜0.05.

Table 17, Chinese medicine extract of the present invention are to the therapeutical effect of experimental osteoporosis

Group	Dosage	Femoral bmd g/cm 2	Femur bone ash heavy (g)	Femur bone ash calcium g/100g bone ash	Femur bone ash phosphorus g/100g bone ash
						Normal group model control group ethoxy Alendronate group extract A group extract A group extract B group extract B group	0.5％CMC 0.5％CMC 50mg/kg 1.0g/kg 2.0g/kg 1.0g/kg 2.0g/kg	0.213±0.007 0.189±0.009 *0.212±0.013 **0.214±0.009 **0.216±0.014 **0.216±0.010 **0.228±0.011 **	0.239±0.007 0.210±0.004 *0.242±0.007 **0.245±0.010 **0.251±0.013 **0.250±0.008 **0.264±0.012 **	31.89±0.90 30.23±0.33 * 30.91±0.42 ** 30.78±1.50 ** 31.40±0.89 ** 31.30±1.48 ** 31.53±2.40 **	18.06±0.56 17.23±0.33 * 17.67±0.30 ** 17.68±1.25 ** 17.76±0.90 ** 17.88±1.32 ** 17.90±2.47 **
Annotate: 1. 2. x ± SD compares with the normal control group *3. compare with model control group P＜0.05 **P＜0.05

2.2 therapeutical effect to experimental osteoporosis

50 of rats, be divided into 5 groups at random, every group 10, be normal control group (group of solvents), model control group, positive controls (ethoxy Alendronate group), experimental group A (example 1 is extract obtained, divides high low dosage), experimental group B (example 2 is extract obtained, divides high low dosage), except that the normal control group, each is organized rat and all irritates stomach retinoic acid 0.5m1/kg, every day 1 time, continuous 14 days, the 15th day each medication group pressed in the table 2 dosage gastric infusion every day 1 time, continuous 28 days, normal control group and model group gave 0.5%CMC, and dose is 0.5ml/kg, weigh weekly therebetween 1 time, and adjust the administration consumption in view of the above.Observation index the results are shown in Table 17 with experiment 2.1.

By table 17 as seen, extract A group, extract B group rat femur bone density, bone ash weight, calcium content of bone and bone phosphorus content are all apparently higher than model group, more also be in same level with the normal control group, show that extract of the present invention has significant therapeutical effect to osteoporosis

From pathological section as seen, the cortical bone of model group all has the pathological change that loose, attenuation and bone trabecula loosen, attenuate and disappear.Each administration group and positive controls only have the cortical bone attenuation of individual animal and the change that bone trabecula attenuates, and pathological changes is lighter, also show that extract of the present invention has significant therapeutical effect to osteoporosis.

Preparation method of extract of the present invention is simple, and is with low cost, and the protect against osteoporosis effect is remarkable, has no side effect, thereby can be used for preparing the medicine or the food of protect against osteoporosis.

One of embodiment 11, clinical case

The king * *, woman, 71 years old, the first visit on the 5th of calendar year 2001 December.The patient has diabetic history, informal dress antidiabetic drug disease controlling.Frequent lumbago over nearly 5,6 years, it is unfavorable to change one's position, and pain is unbearably, influence daily life, checked through the lumbar vertebra sheet last year: L4, the two-sided depression of L5 vertebral body are fish tail shape and change, and the sparse gap of vertebral body bone trabecula is widened, intervertebral space broadening is fusiformis, and is neither significantly through integrative therapy, physical therapy, acupuncture effect.Examine quarter, aches and can not stand in the waist mansion, and nearly January almost can not stride, and the fear of lower limb knee is cold, often wraps up in it with cotton.Between larynx as stalk, the time sputum thickness is arranged, it is frequently many to urinate night so that wet bed, constipation with dry stool.The white thick pulse condition of tongue is heavy thin, and card subordinate unit is not enough, the Liver and kidney damage of essence.Dispose: in order to Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae is the feedstock production extractive compound preparation, each 0.5g, every day 3 times, take medicine a course of treatment (three months), clinical symptoms is clearly better, take medicine again and check after consolidating a course of treatment, check the disappearance of all diseases, the lumbar vertebra sheet shows with contrast last time: bone density increases, transparency lowers, osteoporosis takes an evident turn for the better.Advise and continue to take this medicine and consolidate curative effect a course of treatment, follow up a case by regular visits to so far (in December, 2006) stable disease.

Two of embodiment 12, clinical case

Wu * *, woman, 68 years old, the patient examines on November calendar year 2001 5, and the patient is because of surplus the rheumatoid arthritis 10 year, long-term oral glucocorticoid, because of lumbago was once made CT examination: the attenuation of lumbar vertebra cortex, bone trabecula reduces and attenuates, and bone density lowers, transparency strengthens, L2, L3 annuler bulge, L4, L5 intervertebral disk hernia (central type), the lumbar vertebra right lateral bend.Bone density measurement (X line two-fold energy absorption surveying) is diagnosed as osteoporosis.Oral Calcium Preparations, estrogen, the row physical therapy is massaged still pain unbearably, symptom is that the waist lower limb is miserable, and pain is unable, and the acupuncture pain is arranged during the waist lower limb, lower limb muscles is very thin, and movable function is limited, recently because of the weightening finish of the cold weather state of an illness, the right side pain as being stabbed that has a pain in the leg, day and night unbearably, appetite still can, two is just as usual, thin white fur of tongue, pulse condition is heavy tight.Dispose: in order to Rhizoma Atractylodis, Cortex Phellodendri, Radix Achyranthis Bidentatae is the each 0.5g of feedstock production extractive compound preparation, every day 3 times, takes medicine a course of treatment (three months), check the disappearance of all diseases, bone densitometry more obviously increased, and contrast increases by 8%, and CT sheet lumbal vertebra is greatly improved.Advise and continue to take this medicine and consolidate curative effect a course of treatment, follow up a case by regular visits to stable disease so far.

Embodiment 13, clinical statistics case

Physical data: MethodsThe cases enrolled Total Test case 285 examples, male's 150 examples, women's 135 examples.Case derive from January, 2002 to 2004 year December go to a doctor in first, second, third, fourth, penta, oneself etc. the patient of 6 tame hospitals.The patient measures bone density according to Chinese medical discrimination diagnosis employing dual energy X line borne densitometers and (is called for short: BMD), be less than or equal and organized 215 standard deviations in 30～35 years old with the sex normal healthy people, and divide into groups according to the result of dependent diagnostic standard.

The case inclusion criteria: with reference to the relevant diagnostic criteria of Liu Zhonghou chief editor " osteoporosis " [Liu Zhonghou. osteoporosis, Beijing: Beijing science tech publishing house, 1998:142～162,529～534].Selected condition is: 1. readme has spontaneous lumbago or/and heavy burden pain; 2. use dual energy X line borne densitometers and measure bone density (abbreviation: BMD), be less than or equal and organized 215 standard deviations in 30～35 years old with the sex normal healthy people; 3. blood calcium, phosphorus, alkali phosphatase are normal; 4. get rid of secondary osteoporosis due to hyperparathyroidism, multiple myeloma, the prolonged application glucocorticoid etc.; 5. not having in nearly 3 months to use influences the bone metabolism medicine, as vitamin D, estrogen, calcium preparation, diphosphate etc.; 6. there are not severe cardiac, liver, nephropathy change.45～75 years old age.

Therapeutic Method: comprise Primary Care (1. Diet Therapy; 2. conventional prevention of osteoporosis disease treatment, 3. oral medicine such as calcium preparation etc. give this Chinese medicine compound extract glue to assist, each 0.5g, every day 3 times, one month is a course of treatment.

The therapeutic evaluation standard: two groups of patients before treatment, treatment adopted dual energy X line borne densitometers to measure lumbar vertebra (L2～4) and non-advantage side near end of thighbone (Total) bone density in back 3 months, and mensuration blood, routine urinalysis, blood fat, liver function, renal function etc.Produce effects: lumbago and backache, malaise and muscle spasm pain disappear, and breast or lumbar X line are taken the photograph the sheet prompting: the vertebral body density lowers not progress again, and bone trabecula attenuates not obvious, double concave deformity that vertebral body is slight.Take a turn for the better: lumbago and backache obviously alleviates, and malaise, muscle spasm pain disappear, and breast or lumbar X line are taken the photograph the sheet prompting: the vertebral body density slightly lowers, and bone trabecula attenuates, and vertebral body is the moderate double concave or slight wedge shape changes.Invalid: symptom and sign all do not have improvement, and bone trabecula fogs, the vertebral body double concave is obvious, and one or more vertebral body wedge shapes change.

Statistical method: adopt SAS 8.0 statistical analysis systems to carry out, measurement data is checked with variance analysis, t; Ranked data with Ridit analyze, the rank test of Kruskal-Wallis method; Enumeration data is relatively used X ²Check.

The clinical case statistics

Select 112 routine senile osteoporosis patients, be divided into treatment group and matched group at random, 58 examples are organized in treatment, wherein male 11 examples, women 47 examples; 4 example and compression fracture of lumbar vertebra are arranged, 32 example companion necks, breast, lumbar vertebra hyperosteogeny in various degree.Matched group 54 examples, wherein male 13 examples, women 41 examples; 2 example and compression fracture of lumbar vertebra are arranged, 28 example companion necks, breast, lumbar vertebra hyperosteogeny in various degree, two groups of ordinary circumstances compare, and difference does not have significance meaning (P＞0.05), has comparability.Matched group gives Gaierqi D (vitamin D3 and calcium carbonate) (production of Suzhou Rieter pharmaceutical Co. Ltd), and 1/time, every day 2 times, the treatment group gives herbal mixture extract glue and assists (according to the raw material of embodiment 8 preparations and according to the preparation of embodiment 1 method), observes two groups curative effect after 3 months.

The result: treatment group total effective rate is 87.93% (produce effects 34 examples, effective 17 examples, invalid 7 examples), and matched group is 64.81% (produce effects 13 examples, effective 22 examples, invalid 19 examples), and treatment group and matched group compare, P＜0.01, and difference has the significance meaning.

Claims (29)

1. Chinese medicine extract that is used for protect against osteoporosis, it is characterized in that, this Chinese medicine extract is to be the extract of feedstock production with Radix Achyranthis Bidentatae, Cortex Phellodendri, Rhizoma Atractylodis, comprise Rhzoma Atractylodis Lanceae volatile oil, Cortex Phellodendri total alkaloids, Radix Achyranthis Bidentatae total saponins, Radix Achyranthis Bidentatae total sterone and other compositions, wherein Rhzoma Atractylodis Lanceae volatile oil account for 10～25% (percentage by weights, down with), Cortex Phellodendri total alkaloids accounts for 25～45%, the Radix Achyranthis Bidentatae total saponins accounts for 10～40%, the Radix Achyranthis Bidentatae total sterone account for 5～15% and other compositions account for 15～45%.

2. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 1, it is characterized in that the Rhzoma Atractylodis Lanceae volatile oil in described this Chinese medicine extract accounts for 10～20%, Cortex Phellodendri total alkaloids accounts for 30～40%, the Radix Achyranthis Bidentatae total saponins accounts for 20～30%, the Radix Achyranthis Bidentatae total sterone accounts for 10～15%.

3. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 1 and 2, it is characterized in that, described Radix Achyranthis Bidentatae is a kidney invigorating bone strengthening class medicine, can be used in the medical material that substitutes Radix Achyranthis Bidentatae and be to comprise in Radix Cyathulae, Herba Epimedii, Fructus Psoraleae, Radix Dipsaci, Fructus Ligustri Lucidi, Semen Allii Tuberosi or the Rhizoma Cibotii one or more.

4. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 3 is characterized in that, the described medical material that can be used in alternative Radix Achyranthis Bidentatae is a Herba Epimedii.

5. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 1 and 2, it is characterized in that, described Cortex Phellodendri is the antipyretic and antidotal type medicine, can be used in the medical material that substitutes Cortex Phellodendri and be to comprise in Rhizoma Coptidis, Flos Lonicerae, Herba Ecliptae, Herba Taraxaci, Radix Isatidis, Herba Scutellariae Barbatae or the Folium Isatidis one or more.

6. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 5 is characterized in that, the described medical material that can be used in alternative Cortex Phellodendri is a Rhizoma Coptidis.

7. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 1 and 2 is characterized in that described Rhizoma Atractylodis are the spleen strengthening and damp drying class medicine, can be used in the medical material that substitutes Rhizoma Atractylodis and be to comprise a kind of in the Rhizoma Atractylodis Macrocephalae or the Rhizoma Dioscoreae.

8. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 1 is characterized in that, the described raw material that is used to prepare this Chinese medicine extract is to comprise 1～5 part of Rhizoma Atractylodis, 1～5 part of Cortex Phellodendri, 1～5 part of Radix Achyranthis Bidentatae.

9. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 8 is characterized in that, the described raw material that is used to prepare this Chinese medicine extract is to comprise 1～1.5 part of Rhizoma Atractylodis, 2～3 parts of Cortex Phellodendris, 4～5 parts of Radix Achyranthis Bidentataes.

10. a kind of Chinese medicine extract that is used for protect against osteoporosis according to claim 9 is characterized in that, the described raw material Rhizoma Atractylodis that are used to prepare this Chinese medicine extract, Cortex Phellodendri, Radix Achyranthis Bidentatae three's ratio is 1: 2: 3.

11. according to claim 1,2,8～10 each described a kind of preparation methoies that are used for the Chinese medicine extract of protect against osteoporosis, it is characterized in that described extracting method is all the operable methods well known in the art that comprise solvent extraction method.

12. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 11 is characterized in that described extracting method is a solvent extraction method.

13. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 12, it is characterized in that described solvent extraction method is to comprise hot reflux extraction commonly used, Continuous Countercurrent Extraction, ultrasonic extraction, dipping extraction method, percolation extraction method, decoct in extraction method or the continuous backflow extraction method one or more; Extraction time is an one or many.

14. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 13, it is characterized in that described solvent extraction method is to comprise a kind of in ultrasonic extraction, percolation extraction method, Continuous Countercurrent Extraction or the heating and refluxing extraction method; Extraction time is repeatedly.

15. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 14 is characterized in that described solvent extraction method is the heating and refluxing extraction method; Extraction time is repeatedly.

16. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 15, it is characterized in that the employed extraction solvent of described heating and refluxing extraction method is to comprise that common water reagent, hydrophilic organic solvent or lipophilic organic solvent three classes extract one or more in the solvent.

17. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 16 is characterized in that, described water reagent is to comprise a kind of in water, sour water or the aqueous alkali; Described hydrophilic organic solvent is to comprise in ethanol, ethanol water or the methanol one or more; Described lipophilic organic solvent is to comprise in petroleum ether, chloroform, ether, ethyl acetate, dichloromethane or the dichloroethanes one or more.

18., it is characterized in that this method comprises the steps: according to claim 1,2,8～10 each described a kind of preparation methoies that are used for the Chinese medicine extract of protect against osteoporosis

(1) preparation extracting solution

Take by weighing each crude drug coarse powder respectively in proportion, it is standby that Rhizoma Atractylodis extract volatile oil earlier by the pharmacopeia method, residue dries, the above-mentioned raw materials coarse powder is soaked in an amount of extraction solvent, extracting solvent is 0～95% ethanol or methanol aqueous solution, till percolation to saponins, alkaloids assay reactions is negative routinely, collect percolate, get extracting solution;

Perhaps: this step adopts 5～10 times to extract solvent refluxing extraction one to repeatedly, gets extracting solution;

(2) refining concentrate drying

Said extracted liquid is concentrated into nothing alcohol flavor, and distilled water is regulated proportion to 1.05g/ml, and the macroporous resin column that filtrate is crossed by activation processing makes the active component in the filtrate be adsorbed by resin column, and discards effluent.

Water eluting macroporous resin column discards eluent;

Continue to collect eluent with 30～70% ethanol elution macroporous resin column;

It is dried that 60 ℃ of eluents are evaporated to, and crushed after being dried adds Rhzoma Atractylodis Lanceae volatile oil and mixes and promptly get powder of the present invention.

19. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 18 is characterized in that used extraction solvent is 30～95% ethanol or methanol in the step of this method (1).

20. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 19, it is characterized in that used extraction solvent is 70～95% ethanol in the step of this method (1), 8 times are extracted solvent during reflux, extract,, reflux each 2 hours 3 times.

21., it is characterized in that this method comprises the steps: according to claim 1,2,8～10 each described a kind of preparation methoies that are used for the Chinese medicine extract of protect against osteoporosis

(1) preparation extracting solution

Take by weighing each crude drug coarse powder respectively in proportion, after Rhizoma Atractylodis extracted volatile oil by the pharmacopeia method, residue added 0～95% ethanol or the conventional percolation of methanol aqueous solution.Respectively Cortex Phellodendri, Radix Achyranthis Bidentatae raw material coarse powder are soaked in an amount of extraction solvent, extracting solvent is 0～95% ethanol or methanol aqueous solution, and difference is percolation routinely, till percolation to alkaloids, saponin component assay reactions is negative respectively, collect each percolate, get each single medicinal material extracting solution;

Perhaps: this step adopts 5～10 times to extract solvent refluxing extraction one to repeatedly, gets each single medicinal material extracting solution;

(2) concentrate drying

Respectively above-mentioned Rhizoma Atractylodis, Cortex Phellodendri, each single medicinal material extracting solution reclaim under reduced pressure of Radix Achyranthis Bidentatae are not distinguished the flavor of to there being alcohol, distilled water is regulated proportion to 1.05g/ml, and the macroporous resin column that filtrate is crossed by activation processing makes the active component in the filtrate be adsorbed by resin column, and discards effluent.Water eluting macroporous resin column discards eluent; Continue with 30～70% ethanol elution macroporous resin column, collection eluent, 60 ℃ of eluents are evaporated to dried, and crushed after being dried gets powders A, B, C.

(3) mix

Gained powders A, B, C are mixed with Rhzoma Atractylodis Lanceae volatile oil, promptly get extract of the present invention.

22. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 21 is characterized in that used extraction solvent is 30～95% ethanol or methanol in the step of this method (1).

23. a kind of preparation method that is used for the Chinese medicine extract of protect against osteoporosis according to claim 22, it is characterized in that used extraction solvent is respectively in the step of this method (1): it is that 85% ethanol, Cortex Phellodendri extraction solvent are that 85% ethanol, Radix Acanthopanacis Bidentatae extraction solvent are 85% ethanol that Rhizoma Atractylodis extract solvent.

24. application that is used for the Chinese medicine extract of protect against osteoporosis at preparation osteoporosis disease product.

25. the compositions of a Chinese medicine extract that is used for protect against osteoporosis is in the application of preparation osteoporosis disease product.

26. according to claim 24 or 25 described a kind of application that are used for the Chinese medicine extract of protect against osteoporosis; it is characterized in that; described osteoporosis disease product is meant the product that is used for osteoporosis disease and associated conditions, the product of diagnosis, detection, prevention, protection, treatment or research osteoporosis and associated conditions.

27. a kind of application that is used for the Chinese medicine extract of protect against osteoporosis according to claim 26 is characterized in that, described osteoporosis disease product is to comprise in medicine, reagent, the Foods or drinks one or more.

28. a kind of application that is used for the Chinese medicine extract of protect against osteoporosis according to claim 27, it is characterized in that described osteoporosis disease product is to comprise in medicine, reagent, food, health food, additive or the beverage one or more.

29. a kind of application that is used for the Chinese medicine extract of protect against osteoporosis according to claim 28, it is characterized in that described osteoporosis disease product is to comprise in anti-osteoporosis agents, osteoporosis disease reagent, osteoporosis disease food or the osteoporosis disease beverage one or more.