CN101057876A - Patrinias cabra aglycon extraction and its preparation method and anti colon cancer application - Google Patents
Patrinias cabra aglycon extraction and its preparation method and anti colon cancer application Download PDFInfo
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Abstract
The invention relates to a medicine treating carcinoma of colon and the method for preparing the same, which in detail is aglycone extract from root of Patrinia scabra Bge and the preparation method. The effective component is iridoid glycosides aglycone, the content is above 40%. The invention also provides the medicine agent and preparation. It is proved by pharmacologia test that the medicine is characterized by safe utilization, function of preventing the weight of tumor in transplanted carcinoma of colon. The extract can be produced into preparations of tablet, capsule, drop pill, granular or oral.
Description
Technical field
The present invention relates to medicine, food, technical field of beverage, specifically relate to Chinese medicine extract and its production and use, more particularly relate to Chinese medicine Radix Patriniae Scabrae aglycon extract and preparation method thereof and the purposes that is used to prepare resistive connection intestinal cancer product.
Background technology
(1) research overview of Radix Patriniae Scabrae
1, plant resources and application scenario
Radix Patriniae Scabrae (Patrinia scabra Bunge.) is a Valerianaceae Patrinia plant, and perennial herb is high 20～40 centimetres.Stem is grown thickly, stem top multi-branched, and bifurcation has the joint stricture of vagina.Leaf is to life; Keratin, pinnation, sliver falls lanceolar, narrow lanceolar or Long Circle, and tooth is arranged, and the top sliver is bigger than the side sliver, and leaf margin and blade face are by hair.Give birth on the cyme top, is umbrella room shape and arranges; Give birth to fine, soft fur on floral axis and the bennet; Bract is narrow, and residence is given birth to; Spend little, yellow, corolla is sympetalous, 5 split; Stamen 4; Ovary Room 3, the stigma head.The fruit aliform, avette or subcircular, flat thin as a piece of paper, about 6 millimeters of diameter has reticulate pattern, and seed is positioned at central authorities.Originate in ground such as the Chinese Henan that distributes, Hebei, Shanxi and northeast, wild in the graveyard and waste rand.
Beginning is stated from Shennong's Herbal.Original name " Herba Patriniae ".TAO Hong-Jing is annotated: " give birth to Jiangxia Chuan Gu, adopt root August, sudden and violent doing.Gas is as losing bean sauce, so to run after fame." Tang Xin book on Chinese herbal medicine " carries: Herba Patriniae is given birth between the mountain range, ridge more.Leaf is grown thickly like water Liang and common vetch rank, and brightly yellowish purple made old caramel." hence one can see that, ancient herbal contained Herba Patriniae root, autumn, plane was adopted, and cleaned, and dried, and gave birth to and used.It is irregular cylindrical, different in size that dry root is, and root is thicker, diameter 4～12mm; Crust sepia or brownish black, shrinkage is easily peeled off, and is khaki after peelling off crust.Body lightweight pine, the root outward appearance: section is radial crack, and skin is the circular whorl of yellowish-brown; The cross section of root: the group of xylem vessel is radial arrangement, and cluster crystal is bigger less in cortex and the medullary ray parenchyma, diameter 17～28～45 μ m., color brown person long, fat with bar is good.
" Guangxi Chinese medicinal herbal ": acrid in the mouth, warm in nature; Go into the heart, liver two warps.Relieve internal heat anticancer, be coated with wet blood stasis dispelling, among the peoplely be used for the treatment of typhoid fever, warm disease, traumatic injury, women's metrorrhagia, the inferior disease of red and white leukorrhea, the Radix Patriniae Scabrae crude extract is used to treat acute and chronic leukemia, uterus carcinoma, cervical cancer, colorectal cancer etc. clinically.
Now traditional Chinese medical science evolution is used Herba Patriniae more, market supply also be herb, its kind is complexity comparatively; Herba Patriniae is many in use among the people.
For with for the purpose of the Herba Patriniae difference, Herba Patriniae rhizome and root are called Radix Patriniae, and the title of " Herba Patriniae " in need not ancient book on Chinese herbal medicine.The commodity Radix Patriniae is detected in Chinese Chengde area medical material company.
Radix Patriniae Scabrae Chang Yuqi congener Radix Patriniae Scabrae (P.heterophylla Bunge.) is used with, be commonly called as Radix Patriniae heterophyllae (different name: tomb head ash, see " Shanxi Chinese medicinal herbal "; Arrow wind is seen " Guangxi Chinese medicinal herbal ", and also claim: wind catching arrow, sweaty foot are drawn), expression has the effect of coming back to life.Radix Patriniae Scabrae (Patrinia heterophylla Bge.) rhizome is thin, diameter 1～4mm, not starch-containing grain in the rhizome.
Other congener still has: patrima villosa (Patrinia villosa Juss.) rhizome is elongated, diameter 2～4mm, the long 3～6cm (Herba Patriniae is no more than 2cm) of internode.The rhizome cross section: cortex is narrow; Xylem does not have the parenchyma endless belt; Marrow is wide, and is rounded; Calcium oxalate cluster crystal is few, be present between the phloem and parenchyma cell that marrow has in.
2, chemical constituent
The countries in the world scholar has carried out broad research to the chemical constituent of Radix Patriniae Scabrae, find its contain the lignanoids chemical constituent (referring to Li Yanzhao, Zhang Weidong, Gu Zhengbing etc. Chinese patent medicine, 2005.36 (3): 338～340; Li Yanzhao, Zhang Weidong, Gu Zhengbing etc. Acta Pharmaceutica Sinica, 2003.38 (7): 520～522), the iridoid composition (referring to Kouno, I.; Yasuda, I.; Mizoshiri, H.et al.Phytochemistry, 1994,37 (2), 467～472; Kouno, I.; Yasuda, I.; Jiang ZH.et al.Phytochemistry, 1995,40 (5), 1567～1568; ), polysaccharide component (referring to Yang Jianping, He Fujiang, Li Honggang. Chinese herbal medicine, 1996,660～661), Saponin and volatile oil (main constituent is nopinene, δ-elemene in the volatile oil) 27 (11):, also contain in addition alkaloid, curvularin, flavone, cupreol, daucosterol, oleanolic acid, hexadecanoic acid α-Dan Ganzhi etc. (referring to Gu Zhengbing, Yang Genjin, Cong Haiying. Chinese herbal medicine, 2002,33 (9): 781～782; Gu Zhengbing, Yang Genjin, Cong Haiying. Acta Pharmaceutica Sinica, 2002,37 (11): 867～869).But the active component of its concrete resistive connection intestinal cancer is also indeterminate.
3, pharmacologically active
China has carried out than extensive studies the pharmacological action of Radix Patriniae Scabrae, by pharmacological research and clinical observation, proves that Radix Patriniae has tangible sedation, has now produced the extractum sheet of rhizome of Herba Patriniae and root and has used, and records in Chinese Pharmacopoeia version in 1977.The volatile oil of also finding Radix Patriniae Scabrae root and rhizome has calmness, analgesic activity, and its ethanol extract has anastalsis; Study its antitumor and immunoregulation effect and find that the ethanol extraction of Radix Patriniae Scabrae can increase the phagocytosis and the cellulotoxic effect of mouse macrophage, (be called for short: ANAE) positive lymphocyte percent and Ea rosettes form percentage rate to improve acidity-acetic acid naphthols esterase, and murine sarcoma S180 growth there is inhibitory action, to improve non-specific immunity relevant (referring to Wang Yintang with it, Wang Xuejiang, Yan Yuxia etc. Lanzhou medical college journal, 1988; 4 (1): 11-14); Its water extract lumbar injection can make mouse macrophage engulf and cytotoxicity all obviously improves, and the function that promotes active immunity is arranged; Its water extract intratumor injection reaches 62.5% to mice S180 sarcoma suppression ratio, and observe be direct killing effect (referring to Zhang Yuanxing, Wang Huifen, Huang Zesong etc. combination of Chinese and Western medicine magazine, 1984; 4 (2): 109-110).
Radix Patriniae Scabrae energy enhancing body antineoplastic immune function, in vitro tests proves, no matter water extract is oral or lumbar injection, to transplantability murine sarcoma S-180 and ehrlich carcinoma, inhibitory action is arranged all; Wherein Chinese mugwort formula ehrlich ascites carcinoma there are obvious inhibitory action and therapeutical effect, more obvious with the intraperitoneal administration curative effect especially; Local injection can make the hardening exsiccation gradually of mouse entity type ehrlich ascites carcinoma local tumor, comes off from root, and ulcer surface is repaired gradually and cured.
Have report Radix Patriniae heterophyllae general glycoside sheet to Emhorn ascitic type tumor and S180 sarcoma have stronger inhibitory action (referring to Chen Jinxiu. Chinese Chinese medicine science and technology 1999,6 (5): 299; Zhang Liwei, Zhao Chungui, Yang Pin. University Of Shanxi's journal (natural science edition) 1996,19 (2): 161-164).Clinical research shows: Radix Patriniae heterophyllae general glycoside sheet is in conjunction with operation, chemotherapeutic treatment 50 routine PATIENTS WITH LARGE BOWEL, and the survival rate in 1 year, 2 years, 3 years of patient is respectively 79%, 61%, 40%; Can obviously improve PATIENTS WITH LARGE BOWEL quality of life, effect duration, life cycle in the recent period, said preparation can obviously strengthen patient's immunologic function, significantly reduces carcinoembryonic antigen (CEA) level, can improve the T cellular immune function, strengthens the activity of NK killer cell in the body; With 5-fluorouracil share synergism and attenuation obviously (referring to Chen Jinxiu, Ma Peizhi, Wang Huaizhang. Chinese herbal medicine, 1999; 30 (7): 528-529; Wang Huaizhang, Wang Yinghong, Chen Jinxiu, Wang Shouzhang. Chinese clinical tumor and rehabilitation, 2001; 8 (1): 37-39).But the Radix Patriniae heterophyllae general glycoside remains crude extract, complicated component, and the active component of concrete resistive connection intestinal cancer does not appear in the newspapers as yet.
Show that according to Beijing medical science department pharmacological evaluation the volatile oil of this product also has sedation.The mouse sleep time that volatile oil energy significant prolongation pentobarbital sodium causes in the Radix Patriniae Scabrae root and rhizome, but be weaker than Patrinia scabiosaefolia Fisch volatile oil; P-450 there is activation.
Up-to-date achievement in research shows, the iridoid aglycon PS-I that contains in the Radix Patriniae Scabrae can significantly suppress Human Prostate Cancer Cells strain DU145 and PC3 growth, and it is relevant to be time, dosage, be one of Radix Patriniae Scabrae anti-tumor active ingredient (referring to Li Tiejun, Qiu Yan, Rui Yaocheng etc. PLA's Acta Pharmaceutica Sinica, 2004,20 (2): 101～102).But the activity whether PS-I also has resistive connection intestinal cancer is not illustrated in this research.
4, the clinical practice of folk prescription and compound recipe
" book on Chinese herbal medicine is original ": it is cold to cure the wound, pyrexial malaria." Henan Chinese medicine handbook: foot bath, control the foot pain." Shanxi Chinese medicinal herbal ": hold back liver dampness, hemostasis.Control married woman's hip cellulitis, leucorrhea with red and white discharge." Guangxi Chinese medicinal herbal ": blood stasis dispelling, detumescence.Control traumatic injury.
(1) " the bright collection proved recipe of Dong " choosing side
Control metrorrhagia, leucorrhea with red and white discharge: one of Radix Patriniae heterophyllae, half and half of drinks, new Flos Carthami one is twisted with the fingers, and fries in shallow oil decoction being taken warmly when crouching seven fens.Day nearly person's one clothes, three clothes then for a long time.For oral administration: as to fry in shallow oil soup, 2～3 money.External: fry in shallow oil washing.
(2) relieve internal heat anticancerly, be used for the long-pending poison of carcinoma
Hepatocarcinoma Radix Patriniae heterophyllae 15 grams, Rhizoma Paridis, each 30 gram of Herba Scutellariae Barbatae; Chopping, 3 sub-services of decocting, 1 dose of day.Can make clinical symptom remission, lump is soft to contract, and prolongs life cycle.
Leukemia Radix Patriniae heterophyllae 15 grams, Radix Rumicis Crispi 30 grams are decocted in water for oral dose 1 dose of day.Can make that rule is hemorrhage to be eliminated with heating, liver, the splenomegaly piece is soft contracts remission.
Cervical cancer Radix Patriniae heterophyllae, Semen Coicis, Cortex Moutan, Cortex Phellodendri, each 9 gram of Rhizoma Paridis, Radix Gentianae 6 grams, Rhizoma Smilacis Glabrae 15 grams, Herba Solani Lyrati, Herba Scutellariae Barbatae, each 30 gram of Herba Hedyotidis Diffusae.Chopping, 3 sub-services of decocting.Can make leucorrhea folder blood, lower abdomen weigh down remissions such as pain, cancerous protuberance is dwindled, and calms down gradually.
Gastric cancer Radix Patriniae heterophyllae, each 30 gram of brown sugar, 3 in Rhizoma Zingiberis Recens.Fry in shallow oil Tang Daicha, 1 dose of day.Symptoms such as pain with distension, hematemesis are reduced, and cancerous protuberance is dwindled, and diet takes a turn for the better.
Esophageal carcinoma Radix Patriniae heterophyllae 30 grams, Fructus Trichosanthis 15 grams, Pseudobulbus cremastrae seu pleiones, Rhizoma Curcumae, each 9 gram of Fructus Aurantii, Flos Carthami 6 grams.Chopping, dense frying in shallow oil, oral several times towards simple syrup, 1 dose of day.Clinical symptoms is significantly improved, and cancerous protuberance fades away, and swallows smoothly.
Colorectal cancer Radix Patriniae heterophyllae, Herba Houttuyniae, each 15 gram of Radix Sophorae Tonkinensis, Rhizoma Smilacis Glabrae, each 12 gram of Serissa foetida, Flos Hibisci, Auricularia, Flos Sophorae, each 9 gram of Cortex Ailanthi skin, Spina Gleditsiae 6 grams.Chopping, decocting 2 times, early, late sub-service, and day 1 dose, ball 1 ball that disappears of waking up of at every turn taking after mixing it with water: Olibanum (deoiling), each 30 gram of Myrrha (deoiling), Realgar 15 grams, Moschus 45 grams grind to the powder altogether, with about 30 grams of Semen setariae meal, smash and be the ball that disappears of waking up, and ball weighs 9 and restrains.Adhere to taking, can make the stool hematochezia, all kinds of remission that intestinal obstruction causes, cancerous protuberance is dwindled gradually, the ulcer reparation.Also can cooperate other Comprehensive Treatment.Be suitable for colon cancer and rectal cancer.
(3) the dehumidifying blood stasis dispelling is used for damp and hot blood stasis
Damp and hot leucorrhea Radix Patriniae heterophyllae 15 grams, Flos Carthami 1.5 grams are decocted in water for oral dose.
Rheumatic ostalgia Radix Patriniae heterophyllae 9 grams are decocted in water for oral dose.Use simultaneously in right amount, fry in shallow oil the hot soup fumigation and wash method.Bloody dysentery stasis Radix Patriniae heterophyllae 30 grams, Herba Portulacae, each 15 gram of Flos Lonicerae, Radix Glycyrrhizae 9 gram choppings are decocted in water for oral dose.
Metrorrhagia stasis Radix Patriniae heterophyllae 45 grams, Radix Rubiae 9 grams are decocted in water for oral dose.Leukorrhagia folder blood Radix Patriniae heterophyllae, each 30 gram of Herba Salviae Chinensis.3 sub-services of decocting serve on for 1 week.
Just defecation with blood and pus Radix Patriniae heterophyllae 15 restrains, fresh rehmannia charcoal, Radix Sanguisorbae, each 9 gram of the Fructus Sophorae, Rhizoma Atractylodis Macrocephalae preparata, each 6 gram of Radix Saposhnikoviae.Be decocted in water for oral dose 1 dose of day.
(2) 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 gas chromatogram-mass spectral 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.
(3) progress of colon cancer
Colorectal cancer comprises rectal cancer and colon cancer, is one of common cancer, and is the highest with 40～50 years old postadolescent sickness rate.West Europe and North America developed country are the highest countries of sickness rate, and sickness rate is up to 35,/10 ten thousand～50,/10 ten thousand, and its sickness rate and case fatality rate occupy the 2nd in malignant tumor; In the Orient, colorectal cancer is quite a few to be seen, the interrelated data of China shows that incidence of colorectal accounts for the 4th～6 of whole malignant tumor, and annual nearly 140,000 people's new trouble colorectal cancer account for whole cancer patients' 6.8%; The colorectal cancer mortality rate is 3.54/10 ten thousand, accounts for 5.29% of cancer mortality, occupies the 6th.The East China is the district occurred frequently of colorectal cancer, and wherein Shanghai is again the highest city of M ﹠ M.Colorectal cancer thick sickness rate 19.6/10 ten thousand in Shanghai City in the time of 1979 is the 4th common cancer (order is: gastric cancer, pulmonary carcinoma, hepatocarcinoma, colorectal cancer); Played thick sickness rate in 1989 and risen to more than 28.2/10 ten thousand, be only second to pulmonary carcinoma, gastric cancer, and become the 3rd common cancer; The sickness rate male of Shanghai colorectal cancer in 1997 is up to 37.2/10 ten thousand, and the women has also reached 35.5/10 ten thousand.It is disturbing that the statistics of various countries all shows: colorectal cancer colon cancer sickness rate especially wherein has the trend that increases, and incidence rate also obviously raises in adolescence in recent years, 5% patient's age is arranged approximately below 30 years old.So colorectal cancer especially colon cancer is to be worth everybody a special disease of paying attention to.The treatment of colon cancer mainly is excision and chemotherapy.Chemotherapeutics comprises fluorouracil, mitomycin, nitrosourea etc., has bigger toxic and side effects, brings the human body and spiritual misery to the patient, therefore seeks the colon cancer medicine of preventing and treating more safe and effective, that toxic and side effects is little and is extremely important.
By literature search etc., up to the present, still find no the Radix Patriniae Scabrae aglycon extract and have the report of preventing and treating colon cancer effect aspect.
Summary of the invention
The technical problem that will solve required for the present invention is an especially medicine of aglycon extract that to disclose a kind of product of preventing and treating colon cancer be the Chinese medicine Radix Patriniae Scabrae, this product is an effective ingredient with the total iridoid aglycon that is contained in the Radix Patriniae Scabrae, and the preparation method and the purposes of this product are disclosed, to overcome the above-mentioned defective that prior art exists.
It is the Radix Patriniae Scabrae aglycon extract that one of purpose of the present invention is to provide a kind of product that can prevent and treat colon cancer, and effective ingredient wherein is the total iridoid aglycon in the Radix Patriniae Scabrae, most preferably medicine and pharmaceutical composition.
Another object of the present invention is to be provided for prevent and treat the preparation method, the particularly preparation method of Radix Patriniae Scabrae aglycon extract of this product of colon cancer.
The present invention further provides with described Radix Patriniae Scabrae aglycon extract is active component, is used to prevent and treat the preparation and the corresponding dosage forms of colon cancer.
That is to say, the invention is intended to clear and definite a kind of medicine of preventing and treating colon cancer and preparation method thereof, be i.e. the active site of Chinese medicine Radix Patriniae Scabrae and active component and preparation method thereof; And then the aglycon extract of Radix Patriniae Scabrae is used to prepare resistive connection intestinal cancer product, with the total iridoid aglycon that is contained in the Radix Patriniae Scabrae is that effective ingredient prepares resistive connection intestinal cancer product especially medicine and pharmaceutical composition thereof, and this effective ingredient has good efficacy to the control of colon cancer.In other words, the present invention relates to Chinese medicine Radix Patriniae Scabrae aglycon extract and preparation method thereof, and this aglycon extract and the application in preparation resistive connection intestinal cancer product such as compositions such as pharmaceutical composition thereof.
Described resistive connection intestinal cancer product is meant the product that is used to diagnose, detect, prevent, treat or study colon cancer and associated conditions; Wherein, described resistive connection intestinal cancer 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 resistive connection bowelcancer medicine, resistive connection intestinal cancer reagent, resistive connection intestinal cancer food or the resistive connection intestinal cancer beverage etc. one or more, most preferably resistive connection bowelcancer medicine.
(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, and has also accumulated rich experience with Chinese herbal medicine prevention and treatment disease aspect, and therefore seeking effective active component from Chinese medicine is a valid approach, also is the place of the advantage of Chinese original new drug development.
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 " are inferred very likely to have active material of resistive connection intestinal cancer or material group in Radix Patriniae Scabrae.But because to its chemical constitution study not deeply, the pharmacological screening deficiency causes active site and active component indeterminate and do not have feasible quality standard, has limited the further development and utilization of Radix Patriniae Scabrae.
Therefore, the present invention carries out the chemical constitution study of system by the aglycon extract to single Radix Patriniae Scabrae medical material (root and herb), and screens and prove the activity and the purposes of this aglycon extract.
Through chemical constitution study, prove that the main component in the Radix Patriniae Scabrae active site is the total iridoid aglycon.And according to literature search, the inventor finds that the total iridoid aglycon has the number of significant pharmacologically active mostly, thereby infer the clinical drug effect of Radix Patriniae Scabrae treatment colon cancer, should mainly be to bring into play by the drug effect of active site Radix Patriniae Scabrae aglycon extract, result of study proves also and has confirmed that the Radix Patriniae Scabrae aglycon extract has more significant pharmacologically active that experiment confirm Radix Patriniae Scabrae aglycon extract is better than the monomer effect in the total iridoid aglycon.
(2) Radix Patriniae Scabrae aglycon extract
1, the composition of Radix Patriniae Scabrae aglycon extract
The inventor by the chemical constituent of Radix Patriniae Scabrae medical material (root and herb) being carried out system separate, experimentatioies such as purification and structure evaluation, find mainly to contain multiple chemical compounds such as a large amount of volatile oil, total iridoid aglycon in the Radix Patriniae Scabrae.
Radix Patriniae Scabrae aglycon extract of the present invention is meant the mixture that comprises multiple iridoid that gone out by extraction separation in the root of Radix Patriniae Scabrae (Patrinia scabra Bunge.) or congener or the herb, and this mixture contains one or more in 8 kinds of iridoid glycoside metamembers (PS-I, G7-C, G7-E, G7-Z, BJ3401, G9-A, G9-C and G9-D).
This mixture also has following feature: this mixture and Godin reagent (alcoholic solution of 1% vanillin and 3% high chloro acid solution) reaction, in 281nm place colorimetric, measure trap.This trap is converted in the standard curve of 281nm place colorimetric by monomers B J3401 and Godin reagent reacting, and the monomers B J3401 content of converting out should account for more than 40% of this amount of the mixture.
That is to say that the composition in the Radix Patriniae Scabrae aglycon extract mainly comprises the total iridoid aglycon, also comprise other compositions.
Wherein, the content range of total iridoid aglycon in this aglycon extract is 40%～99.5% (W/W, i.e. percentage by weight), preferred 40%～90%, further preferred 50%～60%; The content range of other compositions in this aglycon extract is below 0.5%, and other compositions are to comprise in tannin, monosaccharide or polysaccharide, xanthene ketone (xanthones) or the carotenoid etc. one or more.
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.
2, each monomeric chemical constitution of the total iridoid aglycon in the Radix Patriniae Scabrae aglycon extract
The PS-I structural formula
PS-I Chinese: 9 α-1,7-dihydro-1-β-O-(3-methyl-butyryl)-4-[(3-methyl-butyryl)-O-] methyl-6-ketone-8-hydroxyl-8-alpha-hydroxymethyl-cyclopenta [c] pyrans
The PS-I English name: (9 α-1,7-dihydro-1-β-O-(3-methylbutanoyl)-4-[(3-methylbutanoyl)-O-] methyl-6-one-8-α-hydroxymethyl-8-β-hydroxycyclopenta[c] pyron)
Work as R=H, be 1R, when 3R and 1S, the racemic modification of 3S, expression G9-C;
Work as R=H, be 1R, when 3S and 1S, the racemic modification of 3R, expression G9-A;
Work as R=COCH
2CH (CH
3)
2, be 1R, when 3S and 1S, the racemic modification of 3R, expression G7-Z.
The structural formula of the structural formula G9-D of G7-E
The structural formula of the structural formula G7-C of BJ3401
3, the activity of Radix Patriniae Scabrae aglycon extract
Have in the active skull cap components process of resistive connection intestinal cancer effect in screening, the inventor finds that the methanol extractum of Chinese medicine Radix Patriniae Scabrae (Patriniascabra Bunge.) root or herb has the effect of resistive connection intestinal cancer, by it is carried out with the activity is the separation of instructing, the chloroform extraction part activity of finding methanol extractum is the strongest, continue to separate and find, the chloroform extraction part is through silica gel column chromatography, and eluent ethyl acetate partly is an active site.This active site is more repeatedly by silica gel column chromatography, HPLC, SephdexLH-20 chromatographic separation and purification, find that main component is a several iridoid glucosides unit, isolate 8 kinds of iridoid glycoside metamembers (PS-I, G7-C, G7-E, G7-Z, BJ3401, G9-A, G9-C and G9-D) altogether, wherein PS-I, G7-C, G7-E, G7-Z, G9-A, G9-C noval chemical compound for from this plant, finding first.
Radix Patriniae Scabrae aglycon extract of the present invention shows good inhibition effect to mouse junction cancer (C26) transplanted tumor, and the oral inhibitory rate of its high dose group (800mg/kg) is specifically seen embodiment more than 40%.
PS-I and BJ3401 are from the isolated monomer of Radix Patriniae Scabrae aglycon extract, prove that by experiment in vitro it has resistive connection intestinal cancer activity, but the two activity all is lower than the Radix Patriniae Scabrae aglycon extract, specifically see embodiment, illustrate that the Radix Patriniae Scabrae aglycon extract comprehensively brings into play the effect of resistive connection intestinal cancer by multiple composition, other iridoid composition also has activity except that PS-I and BJ3401.
(3) preparation method of Radix Patriniae Scabrae aglycon extract
The preparation method of the said Radix Patriniae Scabrae aglycon extract of the present invention comprises the steps:
(1) extract: it is some to get the Radix Patriniae Scabrae raw material, extracts, and gets extracting solution, i.e. Radix Patriniae Scabrae aglycon extract;
(2) separation and purification:, promptly get the Radix Patriniae Scabrae aglycon extract with the extracting solution separation and purification.
Described Radix Patriniae Scabrae raw material comprises the root or the rhizome of Radix Patriniae Scabrae (Patrinia scabra Bunge.) or congener.
Described extracting method comprises all operable methods well known in the art such as solvent extraction method.
(4) purposes of Radix Patriniae Scabrae aglycon extract
1, general introduction
Further purpose of the present invention provides the new purposes in preparation resistive connection intestinal cancer product of Radix Patriniae Scabrae aglycon extract, and product is to comprise in medicine, reagent, the Foods or drinks etc. one or more, preferred agents.
Completed acute toxicity testing proves that the mouse stomach administration surpasses 2.0g/kg to the maximum tolerated dose of Radix Patriniae Scabrae aglycon extract, is equivalent to 440 times of clinical recommended drug dosage, shows that this effective site is safe and reliable.
Above-mentioned experimental result shows that the Radix Patriniae Scabrae aglycon extract of indication of the present invention has better curative effect, and being used for the treatment of colon cancer and associated conditions thereof for this Chinese medicine extract is clinical provides more pharmacology's foundation.This Radix Patriniae Scabrae aglycon extract and compositions thereof are a kind of safe treatment colon cancer and the medicine of associated conditions thereof, the aerial parts of the root of its raw material, rhizome, herb, herb or other positions etc., or its raw material combination thing, also can directly or indirectly be used to prepare resistive connection intestinal cancer product.
2, the using method of Radix Patriniae Scabrae aglycon extract and compositions thereof and requirement
Radix Patriniae Scabrae aglycon extract of the present invention can be united use separately or with other active component; comprise and be used to prepare the product that is used to diagnose, detect, prevent, protect, treat or study relevant disease; comprise medicine, reagent, Foods or drinks etc., especially medicine.
Aspect concrete use, Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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.
" 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 water, saline, phosphate-buffered saline, glucose, glycerol or ethanol etc. and in the compositions one or more 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 Radix Patriniae Scabrae aglycon 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, agar, calcium carbonate or sodium bicarbonate 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.; 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, active component Radix Patriniae Scabrae aglycon extract is dissolved, 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 the Radix Patriniae Scabrae aglycon 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, Radix Patriniae Scabrae aglycon extract of the present invention can be added be suitable for to curee's Pharmaceutical composition in.Usually, this Pharmaceutical composition comprises Radix Patriniae Scabrae aglycon extract of the present invention and pharmaceutically acceptable carrier.
The compositions of Radix Patriniae Scabrae aglycon 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 the Radix Patriniae Scabrae aglycon extract for the treatment of effective dose is an active component, and one or more pharmaceutically acceptable carriers.
The pharmaceutical composition of Radix Patriniae Scabrae aglycon 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 and diagnosis, detection, treatment or the scientific research of colon cancer and associated conditions.
It is 0.5%～99% active component Radix Patriniae Scabrae aglycon extract that pharmaceutical composition preferably contains weight ratio, further preferably contain weight ratio and be 1%～95% active component Radix Patriniae Scabrae aglycon extract, most preferably contain weight ratio and be 5%～90% active component Radix Patriniae Scabrae aglycon extract.
The pharmaceutical composition of Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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, Radix Patriniae Scabrae aglycon extract dosage of the present invention is 1mg～500mg/kg days (d), 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, preferred 5mg～40mg/kgd.
Radix Patriniae Scabrae aglycon extract of the present invention and Pharmaceutical composition thereof can comprise the Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon extract such as individuality.The treatment effective dose also refers to that the useful therapeutic effect of this Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon extract of the present invention or prevention effective dose is 5mg～40mg/kg, more preferably 5mg～20mg/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 Radix Patriniae Scabrae aglycon 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 for the treatment of and the situation etc. of treatment, change Radix Patriniae Scabrae aglycon extract of 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 Radix Patriniae Scabrae aglycon extract is about 50mg～200mg, preferably about 100mg, 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 the active matter Radix Patriniae Scabrae aglycon extract 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 Radix Patriniae Scabrae aglycon 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 individual sensitivity Radix Patriniae Scabrae aglycon extract by following in restriction.
3, the pharmaceutical dosage form of Radix Patriniae Scabrae aglycon extract and compositions thereof and route of administration
Colon cancer and associated conditions thereof can be diagnosed, detect, prevent, protect, treat or be studied to Radix Patriniae Scabrae aglycon extract of the present invention and preparation of compositions resistive connection intestinal cancer product thereof wherein 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.
Radix Patriniae Scabrae aglycon extract of the present invention and compositions thereof 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 spray or 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 Drug Delivery Systems, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978).
Radix Patriniae Scabrae aglycon extract of the present invention and compositions thereof, usually by oral, snuffing is gone into, one or more modes in rectum or the parenteral etc., is applied to the patient who needs this treatment.
That is to say, the medicine and the pharmaceutical composition thereof of the preparation of Radix Patriniae Scabrae aglycon extract can adopt method well known in the art to make various dosage forms, as tablet, capsule, granule, suspensoid, Emulsion, solution, syrup, injection etc., take oral or route of administration such as injection (comprising intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection etc.), mucosa dialysis are carried out the control of colon cancer.
Be used for when oral, can be made into conventional solid preparation such as in tablet, powder, granule or the capsule etc. one or more.When implementing, Radix Patriniae Scabrae aglycon extract of the present invention can be together oral with for example inert diluent or assimilable edible carrier.This Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon extract of the present invention outside the parenterai administration, may need with preventing that the material of its inactivation from together giving to this Radix Patriniae Scabrae aglycon extract coating or with this Radix Patriniae Scabrae aglycon extract.The reactive compound that replenishes can also be added in the said composition.In the specific implementation, Radix Patriniae Scabrae aglycon 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 tablet, coated tablet, capsule, granule, suppository, suspensoid, Emulsion, solution, syrup or the injection etc., in further preferred capsule, granule, suspensoid, Emulsion, solution or the injection etc. one or more, one or more in preferred especially suspensoid, Emulsion, solution or the injection etc.
In addition, the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract also can be directly used in preparation resistive connection intestinal cancer product in some cases separately, also can mix with many chemical substances or make up, directly or indirectly be used to prepare resistive connection intestinal cancer 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 Radix Patriniae Scabrae aglycon extract is used to prepare the particularly various dosage forms of medicine of resistive connection intestinal cancer product, or the powder of the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract is used to prepare the especially various dosage forms of medicine of resistive connection intestinal cancer product with relevant adjuvant, or the powder of the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract and relevant preparation resistive connection intestinal cancer product such as medicine one are used from the various dosage forms of preparation resistive connection intestinal cancer product such as medicine, or the powder of the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract and relevant ancillary drug one are used from the various dosage forms of preparation resistive connection intestinal cancer product such as medicine, as tablet, capsule, in granule or the suspensoid etc. one or more, preferred capsule.
One of described method is that the powder fill with the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract is a capsule, two of method is to be capsule with the powder of the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract and relevant preparation resistive connection intestinal cancer product such as medicine fill together, and three of method is to be capsule with the powder of the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract with relevant ancillary drug fill together; Four of method is the powder of the employed medicinal raw material of Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon extract, relevant preparation resistive connection intestinal cancer 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 Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon extract or the employed medicinal raw material of Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon extract of various dosage forms.But, it should be noted that, in the employed medicinal raw material of above-mentioned direct use Radix Patriniae Scabrae aglycon extract, should be earlier according to the dosage requirement of employed Radix Patriniae Scabrae aglycon extract, conversion obtains the consumption of the employed medicinal raw material of Radix Patriniae Scabrae aglycon extract of required use.
In sum, Radix Patriniae Scabrae aglycon extract of the present invention and compositions thereof can be used for preparing resistive connection intestinal cancer product, preferred agents and food, further preferred agents.
(5) technology speciality
The present invention studies the Radix Patriniae Scabrae aglycon extract targetedly, has expanded new medical usage, also provides a kind of new medicament sources for prevention, diagnosis, detection, protection, treatment and research colon cancer.This materials safety low toxicity, pharmacological action is stronger, and its raw material sources are extensive, inexpensive, and preparation technology is simple, yield is high, can be used for preparing the product of prevention, diagnosis, detection, protection, treatment and research colon cancer, is easy to apply.
That is to say that result of study of the present invention can further deeply be developed as the product of the preventing and treating colon cancer 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 domestic with China, international like product is compared has significant advantage.Colon cancer is common clinical, frequently-occurring disease, along with going from bad to worse of global environment, estimates that the sickness rate of diseases such as colon cancer 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, the Radix Patriniae Scabrae aglycon extract has the activity of treatment colon cancer and associated conditions thereof, directly use the Radix Patriniae Scabrae aglycon extract, need not be through the metabolism of gastrointestinal tract antibacterial, make effect more direct, avoid that gastrointestinal bacterial flora between the individuality is active variantly to be caused the difference of metabolite and produce difference on the drug effect, thereby overcome the unmanageable problem of Chinese medicine dosage, and improved bioavailability.
Experiment showed, of the present invention and the Radix Patriniae Scabrae aglycon extract and compositions particularly pharmaceutical composition be a kind of safe, efficient, stable, inexpensive medicine of preventing and treating colon cancer.Its preparation cost is cheap, and active component is clear and definite; Active high, the oral tumour inhibiting rate of Radix Patriniae Scabrae aglycon extract is greater than 40%; Toxicity is lower, the maximum tolerated dose＞2g/kg of Radix Patriniae Scabrae aglycon extract.
Directly use the Radix Patriniae Scabrae aglycon extract, the bioavailability of medicament height can accurately be controlled dosage.Radix Patriniae Scabrae aglycon extract stable in properties, the effect of treatment colon cancer and associated conditions thereof is obvious, uses the quality of the pharmaceutical preparations of Radix Patriniae Scabrae aglycon extract preparation stable, so it is more suitable for treating the suitability for industrialized production of colon cancer and associated conditions medicine thereof.
In a word; active adaption of the present invention the job demand of modern medical service, food, beverage and scientific research field and the needs of human nature service; be the safe raw material that is used to prepare resistive connection intestinal cancer product, can directly or indirectly and diagnose, detect, prevent, protect, treat or study colon cancer and associated conditions thereof.
Description of drawings
Fig. 1 represents: the ultraviolet absorpting spectrum of reference substance
Abscissa is represented: wavelength (Wavelength); Unit: nanometer (nm);
Vertical coordinate is represented: trap (Abs);
Samplle Name：bzp-2
Collection Time 2005-6-20 16:10:57
Peak Table
Peak Style Peaks
Peak Threshold 0.0100
Range 500.00nm to 200.00nm
Wavelength(nm) Abs
281.00 0.8176
209.00 0.4317
Translator of Chinese is as follows:
Sample title: bzp-2
Acquisition time 2005-6-20 16:10:57
The peak table
Peak shape formula peak
Peak threshold value 0.0100
Scope 500.00 nanometers are to 200 nanometers
Wavelength (nanometer) trap
281.00 0.8176
209.00 0.4317
Fig. 2 represents: the ultraviolet absorpting spectrum of Radix Patriniae Scabrae medical material aglycon
Abscissa is represented: wavelength (Wavelength); Unit: nanometer (nm);
Vertical coordinate is represented: trap (Abs);
Sample Name：sample2
Collection Time 2005-6-20 16:36:49
Peak Table
Peak Style Peaks
Peak Threshold 0.0100
Range 500.00nm to 200.00nm
Wavelength(nm) Abs
282.00 1.2420
208.00 2.7341
Translator of Chinese is as follows:
Sample title: sample 2
Acquisition time 2005-6-20 16:36:49
The peak table
Peak shape formula peak
Peak threshold value 0.0100
Scope 500.00 nanometers are to 200 nanometers
Wavelength (nanometer) trap
282.00 1.2420
208.00 2.7341
The specific embodiment
The present invention has studied the purposes of Radix Patriniae Scabrae aglycon extract, a kind of particularly raw material of medicine of preparation resistive connection intestinal cancer product such as medicine etc. or resistive connection intestinal cancer product that can be used in is provided, be convenient to the safe handling in medical industry and fields such as relevant industries such as food, beverage, comprise diagnosis, detection, treatment or the scientific research etc. of disease.
Elaborate the concrete operation method of preparation Radix Patriniae Scabrae aglycon extract below.
(1) extract: it is some to get exsiccant Radix Patriniae Scabrae raw material, pulverize, add the extraction solvent and mix, extract (with low first pure or low first alcohol-water solution percolation or reflux, extract), particular methanol, ethanol, 95～70% alcoholic solution, with extracting liquid filtering, concentrated, promptly get Radix Patriniae Scabrae medical material aglycon extracting solution.
(2) separation and purification: Radix Patriniae Scabrae aglycon extracting solution through purification procedures, is obtained the Radix Patriniae Scabrae aglycon extract.
Here said extraction, isolation and purification method comprise extraction well known in the art, isolation and purification methods such as solvent extraction method, solvent extraction, column chromatography.
For example, Radix Patriniae Scabrae aglycon extracting solution is added the water suspendible,, obtain petroleum ether or hexane layer and water layer with petroleum ether or hexane extraction.Water layer is with ether or chloroform or ethyl acetate extraction, reclaim under reduced pressure ether or chloroform or ethyl acetate, obtain extractum, the extractum silica gel mixed sample, evaporate to dryness, dress silicagel column, use petroleum ether successively, ethyl acetate is eluting successively, and eluent ethyl acetate position evaporate to dryness obtains the Radix Patriniae Scabrae aglycon extract, specifically sees embodiment.
Specifically:
In step (1):
1. in the solvent extraction method that is adopted, extracting solvent is to comprise water, in alcohols or other organic solvents etc. one or more, described alcohols extracts solvent and comprises methanol, ethanol, ethanol water, in propanol or the butanols etc. one or more, described other organic solvents comprise ethyl acetate, acetone etc., preferred water, methanol, ethanol, ethanol water, propanol, butanols, ethyl acetate, in acetone and other organic solvent or its mixed solvent one or more, further preferably include a kind of in ethanol or the ethanol water etc., preferred especially 60%～90% ethanol water.
2. in the solvent extraction method that is adopted, one or more in the preferred ultrasonic extraction of extracting method, percolation extraction method or the heating and refluxing extraction method etc., further preferred heating and refluxing extraction method; Extraction time can be one or many.
In step (2):
The isolation and purification method preferred solvent extraction that is adopted is about to the aqueous dispersion of Radix Patriniae Scabrae medical material aglycon extract, behind defat with petroleum ether, adopts suitable organic solvent extraction with the weeding of grease soluble components again, obtains extraction leftover; Or directly extract with the weeding of grease soluble components with suitable organic solvent, obtain extraction leftover; Described suitable organic solvent can select for use in chloroform, dichloromethane, ether or the ethyl acetate etc. one or more to extract; Extraction times can be one or many.Divide and to get organic solvent layer, be evaporated to driedly, promptly get the Radix Patriniae Scabrae aglycon extract.At this moment, the yield of Radix Patriniae Scabrae aglycon extract is 1%～10%, and promptly the Radix Patriniae Scabrae aglycon extract accounts for 1%～10% of Radix Patriniae Scabrae medical material gross weight.
Generally speaking, in the preparation method of Radix Patriniae Scabrae aglycon extract, be to be raw material based on fresh medical material of Radix Patriniae Scabrae or commercially available medical material; After the raw material pulverizing, adopt in solvent extraction method, solvent extraction or the macroporous adsorbent resin method etc. one or more in turn, promptly get the crude product of Radix Patriniae Scabrae aglycon extract; If in conjunction with in the conventional drying method such as concentrating under reduced pressure drying, spray drying, lyophilization one or more, then can prepare the extractum or the dry thing of Radix Patriniae Scabrae aglycon extract crude product.
If further adopt separation purification process such as column chromatography, then can access the Radix Patriniae Scabrae aglycon extract; At this moment, the total iridoid aglycon that obtains accounts for the content of Radix Patriniae Scabrae aglycon extract at 40%～60% (W/W).
The dry extract of this extract or powder if desired, can further carry out drying:
(3) drying: drying obtains the dry extract or the powder of extract as spray drying or drying under reduced pressure, is the dry extract or the powder of this extract of the present invention.
In step (3):
This extract that step 2 obtains carries out dried, obtains the extract of dried forms, and used drying means comprises vacuum drying, spray drying and lyophilization etc.
Be example with several representative dosage forms below, elaborate the concrete preparation method of the various dosage forms of Radix Patriniae Scabrae aglycon extract.
The present invention prepares injectable powder and generally adopts conventional freeze-drying, as solvent, the steps include: to get the Radix Patriniae Scabrae aglycon extract with water, adds excipient, be dissolved in water, add active carbon, filtration sterilization, fill, partly roll plug, 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 Radix Patriniae Scabrae aglycon extract 10～100mg.
The present invention prepares injectable powder also can adopt spray drying method, as solvent, the steps include: to get the Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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 Radix Patriniae Scabrae aglycon 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.
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 method of embodiment 1, Radix Patriniae Scabrae aglycon extract
(1) preparation of Radix Patriniae Scabrae aglycon extract
50 kilograms on exsiccant Radix Patriniae Scabrae rhizome after the pulverizing, after wetted with methanol, in the percolation bucket of packing into, is used the methanol percolation.50 ℃ of concentrating under reduced pressure add about 50 ℃ hot water, suspendible.Extract with petroleum ether (60～90).Obtain petroleum ether layer and water layer.Petroleum ether layer obtains petroleum ether part extractum after concentrating.Get the 200ml water layer and use ethyl acetate and n-butanol extraction successively, reclaim under reduced pressure obtains ethyl acetate part and n-butyl alcohol part respectively.
Get ethyl acetate extractum 250g, with chloroform-methanol dissolving in 10: 1, silica gel (100-200 order) 500g mixes sample, 60 ℃ of evaporates to dryness.The dress silicagel column, dress post silica gel is 2000g.Use petroleum ether successively, ethyl acetate, ethyl acetate-methanol 4: 1 is eluting successively, and every kind of polar solvent load is 20L.The petroleum ether eluting obtains 1.6g, and eluent ethyl acetate obtains 97.4g, and 4: 1 eluting of ethyl acetate-methanol obtain 53.8g..Thin layer detects, and aglycon is present in the eluent ethyl acetate position.
(2) assay of Radix Patriniae Scabrae aglycon extract
The content of determined by ultraviolet spectrophotometry Radix Patriniae Scabrae aglycon extract.
(1) instrument and reagent: Varian Cary 100 uv-spectrophotometric instrument;
Water is distilled water, and all the other are analytical pure.Balance: 100,000/.
(2) reference substance: aglycon BJ3401.
(3) preparation of reference substance solution: precision weighs reference substance 5.14mg, in the 25ml volumetric flask, adds dissolve with methanol, is settled to scale.Get in 5ml to the 50ml volumetric flask, be diluted to scale with methanol.
(4) preparation of sample solution: precision weighs Radix Patriniae Scabrae aglycon extract 10.83mg, in the 25ml volumetric flask, adds dissolve with methanol, is settled to scale.Get in 5ml to the 50ml volumetric flask, be diluted to scale with methanol.
(5) absorbing wavelength is investigated: measure reference substance solution ultraviolet absorption curve in 200nm～500nm scope, there is maximum absorption band at the place at the 281nm wavelength, therefore adopts 281nm to detect.The results are shown in Figure 1.
The assay of (6) three batch samples is got three crowdes of each 8mg of this product, accurate claims surely, in the 25ml volumetric flask, accurately pipettes 5ml to 50ml volumetric flask with methanol constant volume, with methanol constant volume to scale.Getting reference substance 5mg prepares with method.Measure uv absorption respectively, external standard method is calculated content.The results are shown in following table 1.
Table 1, three batches of raw material Radix Patriniae Scabrae aglycon extract assays
| Lot number | Content (%) | Meansigma methods (%) |
| 050802 | 62.47 | 62.86 |
| 63.25 | ||
| 050828 | 68.72 | 68.44 |
| 68.15 | ||
| 050925 | 59.39 | 58.94 |
| 58.48 |
Embodiment 2, Radix Patriniae Scabrae aglycon extract and monomer (PS-I, BJ3401) are to the influence of mouse colonic cell strain C26
1 material and method
1.1 medicine: the Radix Patriniae Scabrae aglycon extract extracts with reference to embodiment 1, and PS-I, the BJ3401 monomer for wherein containing separates preparation by HPLC from the Radix Patriniae Scabrae aglycon extract.
1.2 tumor strain: mouse colonic cell strain C26 is provided by Shanghai City medical professionals institute
1.3 reagent and instrument: MTT (purchasing company) in Sigma; DMEM, trypsin, calf serum (purchasing company) in GIBCO.Multiskan MK3 type microplate reader (Labsystems Dragon company)
Iridoid aglycon in table 2, the Radix Patriniae Scabrae (PS-I, BJ3401) is to the growth effect of mouse colonic cell strain C26
( x±S，n＝3)
| Group | Concentration (mg/ml) | C26 | |
| A | Suppression ratio (%) | ||
| Matched group | - | 1.132±0.063 | - |
| Iridoid aglycon (PS-I) | 10 -7 | 0.707±0.050 ** | 37.59 |
| Iridoid aglycon (PS-I) | 10 -6 | 0.552±0.063 ** | 51.23 |
| Iridoid aglycon (PS-I) | 10 -5 | 0.448±0.061 ** | 60.44 |
| Iridoid aglycon (BJ3401) | 10 -7 | 0.763±0.040 ** | 32.67 |
| Iridoid aglycon (BJ3401) | 10 -6 | 0.671±0.049 ** | 40.77 |
| Iridoid aglycon (BJ3401) | 10 -5 | 0.528±0.034 ** | 53.34 |
| The iridoid total aglycon | 10 -7 | 0.792±0.056 ** | 30.04 |
| The iridoid total aglycon | 10 -6 | 0.713±0.067 ** | 37.01 |
| The iridoid total aglycon | 10 -5 | 0.547±0.048 ** | 51.68 |
With the matched group ratio
*P＜0.05,
*P＜0.01
1.4MTT method is surveyed cell proliferation inhibition rate: collect respectively exponential phase mouse colonic cell strain C26 (2 * 104/ml), plant 96 well culture plates, every hole 100 μ l add medicinal liquid by design respectively, negative control for it, is put 5%CO with corresponding culture fluid
2, cultivate 24h in 37 ℃ the incubator.Cell after the processing is removed the DMEM culture medium, and D-Hank ' s liquid is washed 2 times, and every hole adds 100 μ lDMEM culture medium and 10 μ lMTT (5mg/ml), hatches 4h for 37 ℃.Discard liquid, every hole adds 100 μ lDMSO, places several minutes, makes MTT crystallization dissolving, and the 540nm place surveys absorption value on microplate reader.
1.5 statistical procedures: data represent that with ± S significant difference adopts student ' s-t test evaluation.
2 results
The growth of PS-I, BJ3401 mouse colonic cell strain C26 all has inhibitory action, and has certain concentration dependence.When concentration is 10-5mg/ml, the suppression ratio of C26 is respectively 60.44% and 53.34%.The results are shown in Table 2.
The influence of embodiment 3, Radix Patriniae Scabrae aglycon extract mouse junction cancer (C26) transplanted tumor
1 material and method
1.1 laboratory animal: mouse colonic cell strain C26 is provided by Shanghai City medical professionals institute; The BABL/C+ICR=F1 mice, 19～22 grams are available from credit number is provided by Shanghai Slac Experimental Animal Co., Ltd.: SCXK (Shanghai) 2003-0003.
1.2 reagent and instrument: cyclophosphamide (specification: the 200mg/ bottle, lot number: 050206, Hualian Pharmaceutical Co., Ltd., Shanghai); Balance (JA2003A, Shanghai Jingtian Electronic Instrument Co., Ltd.)
1.3 mouse junction cancer (C26) transplanted tumor modelling and drug effect: mouse junction cancer (C26) the tumor source that the choosing growth is vigorous is prepared into cell suspension with homogenate method (1: 8), every the right axil subcutaneous vaccination of mice 0.2ml.Irritate stomach Radix Patriniae Scabrae aglycon extract (200mg/kg, 800mg/kg), positive control drug intraperitoneal injection of cyclophosphamide (25mg/kg), every day 1 time every day simultaneously.Inoculate back 10 days and put to death animal, dissect, mouse tumor under the bark fetching claims tumor heavy.
1.4 statistical procedures: data represent that with ± S significant difference adopts student ' s-t test evaluation.
Table 3, to the influence of transplanting mouse junction cancer (C26) tumor-bearing mice tumor weight (x ± S, n=10)
| Group | Dosage | Route of administration | Tumor heavy (g) | Tumour inhibiting rate (%) |
| The normal saline group | - | Irritate stomach | 1.555±0.257 | 0 |
| The cyclophosphamide group | 25mg/kg | Lumbar injection | 0.423±0.145 ** | 72.8％ |
| Low dose group | 200mg/kg | Irritate stomach | 1.129±0.236 ** | 27.4％ |
| High dose group | 800mg/kg | Irritate stomach | 0.912±0.231 ** | 41.4％ |
Compare with the normal saline group,
*P＜0.01
2 results
The normal saline group is transplanted mouse junction cancer (C26) lotus struma tumor weight can reach 1.555 ± 0.258g, the iridoid aglycon can significantly reduce to transplant mouse junction cancer (C26) tumor-bearing mice tumor in the Radix Patriniae Scabrae, highly significant ameliorate tumor weight (P＜0.01), low, high dose group (200mg/kg, 800mg/kg) suppression ratio is respectively 27.4% and 41.4%, shows that the iridoid aglycon has inhibitory action to transplanting mouse junction cancer (C26) tumor-bearing mice tumor in the Radix Patriniae Scabrae.The results are shown in Table 3.
Conclusion: show through external and in vivo test: the iridoid aglycon has the effect of resistive connection intestinal cancer in the Radix Patriniae Scabrae.
The preparation of embodiment 4, Radix Patriniae Scabrae aglycon extract tablet
Prescription:
Radix Patriniae Scabrae aglycon extract 500g
Starch 30g
Lactose 40g
Carboxymethyl starch sodium 5g
Starch slurry (7%) is an amount of
Magnesium stearate 1% (1.45g)
Make 1000
Technology:
Get recipe quantity Radix Patriniae Scabrae aglycon extract, pulverized 100 mesh sieves, again starch, the lactose of recipe quantity were pulverized 100 mesh sieves, mixing.The Fructus Akebiae total saponins is added in the starch and lactose of mixing mixing.Add an amount of 50% ethanol and mix thoroughly, granulate through 16 order ferrum silk screen nets, dry below 60 ℃, granulate adds an amount of magnesium stearate mixing, tabletting behind the analysis content.Can be made into 1000 every tablet tablet that contains 500mg Radix Patriniae Scabrae aglycon extract.
The preparation of embodiment 5, Radix Patriniae Scabrae aglycon extract capsule
Prescription:
Radix Patriniae Scabrae aglycon extract 500g
Starch 140g
Micropowder silica gel 10g
Make 1000
Technology:
Get recipe quantity Radix Patriniae Scabrae aglycon extract, pulverized 100 mesh sieves, in the starch of adding recipe quantity, the micropowder silica gel, mixing directly incapsulates, and can be made into 1000 every capsule that contains 500mg Radix Patriniae Scabrae aglycon extract.
The preparation of embodiment 6, Radix Patriniae Scabrae aglycon extract injection
Prescription:
Radix Patriniae Scabrae aglycon extract 50g
Ethanol (95%) 4000g
Propylene glycol (C.P.) 1000g
Water for injection adds to 10000ml
Make 1000
Technology:
Get 20 ℃ of ethanol (95%) 5000g, add active carbon 5g and stir, through core group filtration under diminished pressure, standby.Getting recipe quantity Radix Patriniae Scabrae aglycon extract adds among the above-mentioned ethanol 4000g, be heated to about 50 ℃, fully stir, make it to dissolve clear and bright, add the recipe quantity propylene glycol then, add fresh water for injection again and make full dose become 10000ml, fully stir mixing, add active carbon 3g, clear and bright through core group filtration under diminished pressure, filtrate before and after merging is clear and bright with special-purpose core fine straining again.Check the qualified back embedding of content and clarity, 100 ℃ of circulation steam sterilization 30min promptly get the injection that every 10ml contains Radix Patriniae Scabrae aglycon extract 50mg.
Claims (20)
1. the Radix Patriniae Scabrae aglycon extract is characterized in that, this aglycon extract comprises total iridoid aglycon and other compositions, and wherein the content of total iridoid aglycon is 40%～99.5% (percentage by weight, down together), and the content of other compositions is below 0.5%; In this total iridoid aglycon, be to comprise among PS-I, G7-C, G7-E, G7-Z, BJ3401, G9-A, G9-C or the G9-D one or more.
2. Radix Patriniae Scabrae aglycon extract according to claim 1 is characterized in that, the content of the total iridoid aglycon of described this aglycon extract is 50%～60%.
3. Radix Patriniae Scabrae aglycon extract according to claim 1 is characterized in that, described other compositions are to comprise in tannin, monosaccharide or polysaccharide, xanthene ketone or the carotenoid etc. one or more.
4. according to the preparation method of each described Radix Patriniae Scabrae aglycon extract of claim 1～3, it is characterized in that this method comprises the steps:
(1) extract: it is some to get the Radix Patriniae Scabrae raw material, extracts, and gets extracting solution, i.e. Radix Patriniae Scabrae aglycon extract;
(2) separation and purification:, promptly get the Radix Patriniae Scabrae aglycon extract with the extracting solution separation and purification;
Described Radix Patriniae Scabrae raw material comprises the root or the rhizome of Radix Patriniae Scabrae (Patrinia scabra Bunge.) or congener;
Described extracting method comprises all operable methods well known in the art such as solvent extraction method;
Here said extraction, isolation and purification method are to comprise a kind of in solvent extraction method well known in the art, solvent extraction or the column chromatography.
5. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 4 is characterized in that, in the described extracting method, the dry extract of this extract or powder can further carry out drying if desired.
6. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 4 is characterized in that, described extracting method is all the operable methods well known in the art that comprise solvent extraction method.
7. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 6 is characterized in that, described extracting method is a solvent extraction method.
8. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 7, 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.
9. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 8 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.
10. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 9 is characterized in that, described solvent extraction method is the heating and refluxing extraction method; Extraction time is repeatedly.
11. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 10, 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.
12. the preparation method of Radix Patriniae Scabrae aglycon extract according to claim 11 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.
13. the application of Radix Patriniae Scabrae aglycon extract according to claim 1 in preparation resistive connection intestinal cancer product.
14. the application of the compositions of Radix Patriniae Scabrae aglycon extract according to claim 1 in preparation resistive connection intestinal cancer product.
15. the application according to claim 13 or 14 described Radix Patriniae Scabrae aglycon extracts is characterized in that, described resistive connection intestinal cancer product is to comprise in medicine, the Foods or drinks field product one or more.
16. the application of Radix Patriniae Scabrae aglycon extract according to claim 15 is characterized in that, described resistive connection intestinal cancer product is to comprise in medicine, reagent, food, health food, additive or the beverage one or more.
17. the application of Radix Patriniae Scabrae aglycon extract according to claim 16 is characterized in that, described resistive connection intestinal cancer product is one or more in resistive connection bowelcancer medicine, resistive connection intestinal cancer reagent, resistive connection intestinal cancer food or the resistive connection intestinal cancer beverage.
18. the application of the raw material of Radix Patriniae Scabrae aglycon extract according to claim 1 in preparation resistive connection intestinal cancer product.
19, the application of the raw material combination thing of Radix Patriniae Scabrae aglycon extract according to claim 1 in preparation resistive connection intestinal cancer product.
20, according to the application in preparation resistive connection intestinal cancer product of the raw material of claim 18 or 19 described Radix Patriniae Scabrae aglycon extracts, it is characterized in that the raw material of described Radix Patriniae Scabrae aglycon extract is to comprise a kind of in the aerial parts of root, rhizome, herb, herb or other positions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710038981 CN101057876A (en) | 2007-04-03 | 2007-04-03 | Patrinias cabra aglycon extraction and its preparation method and anti colon cancer application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710038981 CN101057876A (en) | 2007-04-03 | 2007-04-03 | Patrinias cabra aglycon extraction and its preparation method and anti colon cancer application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113223A (en) * | 2013-01-23 | 2013-05-22 | 安徽医科大学 | Patrinin heterophylla A and B as well as preparation method and application thereof in antineoplastic drugs |
| CN103951648A (en) * | 2014-04-30 | 2014-07-30 | 武汉联合药业有限责任公司 | Sesterterpene lactone compound |
| CN103965157A (en) * | 2014-04-30 | 2014-08-06 | 武汉联合药业有限责任公司 | Preparation method for sesquiterpene lactone compound |
-
2007
- 2007-04-03 CN CN 200710038981 patent/CN101057876A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103113223A (en) * | 2013-01-23 | 2013-05-22 | 安徽医科大学 | Patrinin heterophylla A and B as well as preparation method and application thereof in antineoplastic drugs |
| CN103113223B (en) * | 2013-01-23 | 2016-01-20 | 安徽医科大学 | Scabrous Patrinia Root A prime, B prime and preparation method thereof and the application on antitumor drug |
| CN103951648A (en) * | 2014-04-30 | 2014-07-30 | 武汉联合药业有限责任公司 | Sesterterpene lactone compound |
| CN103965157A (en) * | 2014-04-30 | 2014-08-06 | 武汉联合药业有限责任公司 | Preparation method for sesquiterpene lactone compound |
| CN103951648B (en) * | 2014-04-30 | 2015-08-26 | 武汉联合药业有限责任公司 | A kind of sesterterpene lactone compound |
| CN103965157B (en) * | 2014-04-30 | 2016-03-16 | 武汉联合药业有限责任公司 | A kind of preparation method of sesterterpene lactone compound |
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Open date: 20071024 |