CN100509023C - Nutrient for cell growth, preparation method, and application of finished product thereof - Google Patents

Abstract

A cell growth nutrient in the any possible forms including oral liquid, syrup, medical wine, injection, solid, ointment, etc for preventing and treating tumor, cardiovascular and cerebrovascular diseases, bone fracture, osteoporosis, etc is prepared from 18 Chinese-medicinal materials including saffron, Chuan-xiong rhizome, cinnamon twig, notoginseng, etc. Its preparing process is also disclosed.

Description

A kind of nutrient for cell growth, preparation method, and the purposes of finished product

Technical field

The present invention relates to a kind of nutrient for cell growth, its preparation method and finished product thereof mends the living marrow of bone, improves serum HGH (GH), treats the purposes on the medicines such as fracture, traumatic injury, osteoporosis in preparation.

Background technology

Along with the progress in epoch, human average life all prolongs, and China is also for having striden into the country of aging.

The progress and development of science has also consumed limited natural resource and nutrient unintentionally, produces a large amount of chemistry and physical contamination, and human beings'health has been produced negative effect.Wherein the part pollution is the main inducing of condition of illness such as human body cardiovascular and cerebrovascular disease, tumor and osteoporosis.Mostly past is 50 years old generation disease later on, has also shifted to an earlier date greatly now.Therefore, the life sciences technology becomes the important subject of countries in the world.

British Koliker had found osteoclast first in 1873.(multinuclear giant cell MNGC) forms multinuclear giant cell, and diameter 100 μ m contain 2～50 nuclears, mainly is distributed in around bony surface, the interior blood vessel access of bone.The negligible amounts of osteoclast, it is merged by a plurality of mononuclear cells and forms, endochylema basophilia but aging along with cell fades to the acidophilia.Osteoclast has special absorption function, and during some local inflammation focus absorbed, macrophage also participated in the bone resorption process.

Absorb in the process of the Organic substance of bone matrix and mineral nitrogen in osteoclast, cause stromal surface irregular, form the lacuna of approximate cell shape, be called the Howship lacuna.Facing to the one side of sclerotin, cell stretches out many galley proof projections, resembles very much the longitudinal grin edge and the brush border of surface epithelial cell in lacuna.Under the Electronic Speculum, a side of pressing close to sclerotin has many irregular microvilluss, and promptly cell process is called ruffled border (ruffled border).Periphery in the ruffled border district has an annular cytoplasmic region, contains the volume microfilament, but lacks other organelle, is called clear zone (clear zone), and cell membrane herein is smooth and be close to the surface of sclerotin.The enclosure wall of clear zone just as constituting with kytoplasm together forms a microenvironment with institute's area surrounded.Osteoclast discharges lactic acid and citric acid etc. to the part, and under acid condition, inorganic mineral gulps down drink from ruffled border in the bone, forms some pinosomes or phagocytic vacuole in ruffled border substrate.In osteoclast, inanimate matter is degraded, and enters in the blood flow with the form of calcium ion.The collagen fiber that make in the bone matrix of losing of inanimate matter expose, and osteoclast is secreted multiple lysosomal enzyme, particularly cathepsin B and collagenolysis cathepsin.After osteoclast was left bone surface, its ruffled border disappeared, and changes in the cell, enters resting stage.

Phagocyte in mononuclear cell in the blood or the tissue can not be transformed into osteoclast, because that all these cells only contain is sophisticated, can not be splitted, the mononuclear phagocyte in late period, have only early stage immature proliferative mononuclear phagocyte to be only the precursor of osteoclast.

In vitro study to osteoclast over more than 100 year is made slow progress, and up to initial stage 1980's, British Chambers has at first set up the osteoclast extracorporeal culture-ing and succeedd, and the in vitro study of osteoclast just is able to develop rapidly.Osteocyte regeneration is promptly regarded as emphasis scientific research problem by World Health Organization (WHO) as far back as the twentieth century middle period, and each developed country of west is also subsidized by government or financial group's your kind effort one after another, conducts a research.Can use the regulation and control factor of several different methods research osteoclast at present, but research concentrates on various hormones and somatomedin mostly, and Chinese medicine fewer to research aspect the influence of osteoclast growth in vitro.

Chinese patent ZL93104876.1 discloses a kind of medicine pellet of curing osteoporosis, is with Lignum Aquilariae Resinatum 800～900, Stigma Croci 860～900, the Radix Aucklandiae 600～700, Rhizoma Chuanxiong 1500～1600, Ramulus Cinnamomi 1500～1600, the Radix Angelicae Dahuricae 600～700, Radix Notoginseng 450～550, Radix Dipsaci 1500～1600, Eupolyphaga Seu Steleophaga 1500～1600, Rhizoma Drynariae 800～900, Semen Strychni (processed) 450～550, Radix Achyranthis Bidentatae 600～700, Semen Cucumidis sativi 3300～3400, Os Gallus domesticus 1600～1700, Radix Et Rhizoma Rhei 300～400, Pyritum 650～700, Borneolum Syntheticum 450～550, Sanguis Draxonis 3300～3400, the above-mentioned 18 flavor Chinese herbal medicine dryings of weighing earlier, pulverize, sieve, be mixed in proportion.It is remarkable that this pellet medicine is used to treat the osteoporosis effect.But, because this Chinese medicine is red medicine, do not extract the effective ingredient in the Chinese medicine, absorption is restricted in vivo, influences its curative effect and otherwise purposes.

Summary of the invention

The object of the present invention is to provide a kind of nutrient for cell growth, this nutrient contains essential multiple negative and positive material and the trace element of cell growth, can effectively suppress osteoclast, promotes the effect of Oesteoblast growth.It can improve human serum GH concentration, increases the osteocyte growth trace element in the serum albumin, thereby reaches the effect that promotes blood microcirculation, blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain, dredge the meridian passage, the living marrow of benefit bone.

Another object of the present invention is to provide the preparation method of above-mentioned nutrient for cell growth, this method is effectively extracted the effective ingredient of needed by human in the raw material, and technology is simple, production cost is low.

A further object of the present invention is to provide nutrient for cell growth to give birth to marrow, improve the purposes on serum HGH (GH), cholesterol reducing, treatment traumatic injury, the osteoporosis diseases medicine in preparation treatment and prophylaxis of tumours, cardiovascular and cerebrovascular disease, benefit bone.

To achieve these goals, the technical solution used in the present invention is: a kind of nutrient for cell growth, be by Lignum Aquilariae Resinatum 800～900 weight portions, Stigma Croci 860～900 weight portions, the Radix Aucklandiae 600～700 weight portions, Rhizoma Chuanxiong 1500～1600 weight portions, Ramulus Cinnamomi 1500～1600 weight portions, the Radix Angelicae Dahuricae 600～700 weight portions, Radix Notoginseng 450～550 weight portions, Radix Dipsaci 1500～1600 weight portions, Eupolyphaga Seu Steleophaga 1500～1600 weight portions, Rhizoma Drynariae 800～900 weight portions, Radix Achyranthis Bidentatae 600～700 weight portions, Os Gallus domesticus 1600～1700 weight portions, Radix Et Rhizoma Rhei 300～400 weight portions, Pyritum 650～700 weight portions, Borneolum Syntheticum 450～550 weight portions, the effective ingredient that Sanguis Draxonis 3300～3400 weight portion Chinese crude drugs extract is characterized in that relative density is that the extract thick paste mixture of 1.00---1.40 contains following composition:

Na (sodium) 660～7000mg/L

K (potassium) 3750～5160mg/L

Ca (calcium) 830～1350mg/L

Mg (magnesium) 815～1400mg/L

Cu (copper) is greater than 0,＜0.1mg/L

Mn (manganese) 0.4312～0.7mg/L

Zn (zinc) 0.0244～0.5mg/L

Fe (ferrum) 22～33mg/L

P (phosphorus) 550～1020mg/L

Co (cobalt) 0.138～0.5mg/L

S (sulfur) 100～267.2mg/L

Mo (molybdenum) is greater than 0,＜0.1mg/L

B (boron) 6.3～10.256mg/L

Cr (chromium) 0.1～0.998mg/L

Total protein: 15000～40000mg/L

Aminoacid: 1～50mg/L

Vitamin: 100～250mg/L

Total sugar: 0.05～0.5mg/L

Nutrient for cell growth of the present invention is the additive of pharmaceutical preparation or nutriment, described pharmaceutical preparation is pharmaceutically acceptable dosage form or oral nutritional formulation, as oral liquid, syrup, soft extract, medicated wine and tincture, injection, capsule, tablet, granule, pill and externally applied ointment; Also can add in the Foods or drinks.

The content of nutrient for cell growth of the present invention in pharmaceutical preparation is 60-90wt%, and in nutriment, the content of nutrient for cell growth is 3.5～6.0wt%.

The preparation method of nutrient for cell growth of the present invention is: with Radix Angelicae Dahuricae percolation in ethanol, it is standby that percolate removes the thick paste that obtains behind the ethanol; With Lignum Aquilariae Resinatum, Stigma Croci, the Radix Aucklandiae, Rhizoma Chuanxiong, Ramulus Cinnamomi add 2～5 times of water and decoct altogether; With Radix Notoginseng, Radix Dipsaci, Eupolyphaga Seu Steleophaga, Rhizoma Drynariae, Radix Achyranthis Bidentatae, Os Gallus domesticus, Pyritum and Radix Et Rhizoma Rhei decoct 2～5 times in 5～8 times of water altogether, and each 2～8 hours, collect decoction liquor, filter, concentrate; Use ethanol precipitation, abandon precipitate, reclaim ethanol, obtain concentrated solution; Merge said extracted liquid, Borneolum Syntheticum, Sanguis Draxonis are pulverized, the back of sieving adds in the extracting solution.According to purposes above-mentioned thick paste and semi-solid the mixing are made as pharmaceutically acceptable dosage form or oral nutritional formulation, as oral liquid, syrup, soft extract, medicated wine and tincture, injection, capsule, tablet, granule, pill and externally applied ointment; Also can add in the Foods or drinks.

Chinese medicine and pharmacy is thought: Semen Strychni can be opened meridians, reach the joint thoroughly, prove the excited spinal cord of this medicine energy, oblongata, cerebral cortex according to modern pharmacological research, enhances skeletal muscle tonus degree, but Semen Strychni is again virose medicine, the expert thinks owing to contain the medicine of heat production in this medicine, make telangiectasis, blood circulation is fast, so drug absorption is more, easily produce irritated and other side reactions easily, human body is worked the mischief.Though toxicity is lowered,, be difficult to be effective if control badly.In the raw material prescription therefore of the present invention, do not select Semen Strychni for use, the prescription drug of the present invention through science is extracted can have better therapeutic effect.

Semen Cucumidis sativi also is one of conventional Chinese medicine composition that is used in the Chinese medicine relaxing muscles and tendons and activating QI and blood in the collateral, bone-setting pain stopping, treatment fracture contusion, the inventor of this patent finds through a large amount of experiments, in the prescription of the Chinese medicine of the present invention that process is extracted, add and do not add Semen Cucumidis sativi, not influence of curative effect for medicine, therefore, in the visit of the present invention, do not adopt Semen Cucumidis sativi.

The present invention has simplified prescription than prior art, extract through scientific technology, test shows, above-mentioned nutrient for cell growth effectively treatment and prophylaxis of tumours, cardiovascular and cerebrovascular disease, benefit bone give birth to marrow, improve serum HGH (GH), cholesterol reducing, treatment traumatic injury, fracture, osteoporosis, etc. disease.

The present invention's (nutrient for cell growth) has can suppress osteoclast effectively, promotes the effect of Oesteoblast growth; Improve human serum GH concentration, increase the osteocyte growth trace element in the serum albumin, thereby reach promotion blood microcirculation, blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain, dredge the meridian passage, the living marrow of benefit bone, make skeleton not degenerate, not wear out, childhood development can reach normal value; Suppress tumor; Life lengthening.

The specific embodiment

Describe the present invention in detail below in conjunction with drawings and Examples, described enforcement is used in understands the present invention rather than restriction the present invention.

Description of drawings:

Fig. 1, different times are respectively organized the positive apocyte counting of TRAP;

Fig. 2, osteocyte growth nutrient solution dosing group and the contrast of control group A LP content, wherein series 1 is that the plain group of osteocyte growing nutrient, series 2 are matched group;

Fig. 3, the plain dosing group of osteocyte growing nutrient and the contrast of matched group average A LP content, wherein 1 is that the plain group 2 of osteocyte growing nutrient is matched group;

Fig. 4, the plain dosing group of osteocyte growing nutrient and the contrast of matched group OGN content, wherein series 1 is that the plain group of osteocyte growing nutrient, series 2 are matched group;

Fig. 5. the average OGN content contrast of plain dosing group of osteocyte growing nutrient and matched group, wherein 1 is that the plain group of osteocyte growing nutrient, 2 is matched group.

Embodiment 1

Be prepared as follows nutrient for cell growth of the present invention:

With 620 gram Radixs Angelicae Dahuricae percolation in 2 times ethanol, extremely effective ingredient wherein fully extracts, and waste, extracting solution reclaim ethanol, and it is standby to obtain the Radix Angelicae Dahuricae extract thick paste.Lignum Aquilariae Resinatum, each 820 gram of Stigma Croci, the Radix Aucklandiae 620 grams, Rhizoma Chuanxiong, each 1520 gram of Ramulus Cinnamomi add 3 times of water and decoct altogether, collect decoction liquor, filter, concentrate; Use ethanol precipitation, abandon precipitate, reclaim ethanol, obtain concentrated extracting solution; With Radix Notoginseng 550 grams, Radix Dipsaci, each 1500 gram of Eupolyphaga Seu Steleophaga, Rhizoma Drynariae 800 grams, Radix Achyranthis Bidentatae 600 grams, Os Gallus domesticus 1600 grams, Pyritum 650 grams and Radix Et Rhizoma Rhei 300 grams decoct 3 times in 7 times of water altogether, and 2 hours for the first time, 5 hours for the second time, 8 hours for the third time, collect decoction liquor, filter, concentrate; Use ethanol precipitation, abandon precipitate, reclaim ethanol, obtain concentrating thick paste; Merge said extracted and concentrated solution, with Borneolum Syntheticum 400 grams, Sanguis Draxonis 3300 restrains pulverizing, crosses the extraction of No. 6 sieve back adding merging and concentrates thick paste, and obtaining relative density is extraction and the concentrated thick paste of 1.00---1.20.

As Chinese medicine, can be prepared as acceptable forms on the pharmaceutics according to Chinese medicine preparation excipient commonly used or adjuvant.For example:

(1) granule: above-mentioned Chinese medicine extract 300 grams, add sucrose 90 grams, magnesium stearate 1.5 grams, the consequence that stirs 14～16 mesh sieves are made granule, drying, splitting, every bag 4 gram.Boiled water is taken after mixing it with water, and each 1 bag, or follow the doctor's advice.

(2) tablet: above-mentioned Chinese medicine extract 300 grams, add microcrystalline Cellulose 50 grams, starch 15 grams, magnesium stearate 5 grams are granulated behind the mix homogeneously, cross sieve No. 3, tabletting, every heavy 0.4～0.5 gram.

(3), can add in drinks, beverage or other food as nutrient.General addition is 3.5～6%.

(4) powder: above-mentioned Chinese medicine extract 300 grams, add the powder preparation adjuvant commonly used or the excipient that contain liquid medicine, prepare powder according to common process.

Embodiment 2

The 700 gram Radixs Angelicae Dahuricae are pulverized, percolation in 5 times ethanol, extremely effective ingredient wherein fully extracts, and reclaims ethanol, and it is standby to obtain the Radix Angelicae Dahuricae extract thick paste.Lignum Aquilariae Resinatum, each 800 gram of Stigma Croci, the Radix Aucklandiae 700 grams, Rhizoma Chuanxiong, each 1600 gram of Ramulus Cinnamomi add 3 times of water and decoct altogether, filter and obtain first decoction liquor; With Radix Notoginseng 450 grams, Radix Dipsaci, each 1550 gram of Eupolyphaga Seu Steleophaga, Rhizoma Drynariae 900 grams, Radix Achyranthis Bidentatae 700 grams, Os Gallus domesticus 1700 grams, Pyritum 700 grams and Radix Et Rhizoma Rhei 400 grams decoct 2 times 4 hours for the first time altogether in 8 times of water, 6 hours for the second time, collect and and each time decoction liquor, filtration obtains second decoction liquor, and first and second decoction liquor are merged; Use ethanol precipitation, abandon precipitate, reclaim ethanol, obtain concentrated solution; Merge said extracted liquid and concentrated solution, with Borneolum Syntheticum 550 grams, Sanguis Draxonis 3350 grams are pulverized, are crossed No. 5 sieve backs and add in the extracting solution, and to obtain relative density be the extraction of 1.30---1.40 and concentrate thick paste.

As Chinese medicine, can be prepared as acceptable forms on the pharmaceutics according to Chinese medicine preparation excipient commonly used or adjuvant.For example:

(1) pill: above-mentioned Chinese medicine extract 100 grams, add starch 5 grams, general ball, cold drying, every ball weighs 0.6 gram.Oral, one time 2～5 ball, every day 2 this or follow the doctor's advice;

(2) capsule: above-mentioned Chinese medicine extract 300 grams, add Chinese medicine and prepare capsule adjuvant commonly used, prepare capsule according to common process.

(3) oral liquid: relative density is that the extraction of 1.30---1.40 and the pure water that concentrates thick paste adding 80% are 20% to extracting and concentrating thick paste concentration.Prepare concentration according to the method described above and be 2%, 10%, 30%, 40%, 50% oral liquid.

If necessary, can also add an amount of flavoring agent and other adjuvant in the oral liquid.

Embodiment 3

The 650 gram Radixs Angelicae Dahuricae are pulverized, percolation in 3 times ethanol, extremely effective ingredient wherein fully extracts, and reclaims ethanol, and it is standby to obtain the Radix Angelicae Dahuricae extract thick paste.Lignum Aquilariae Resinatum, each 850 gram of Stigma Croci, the Radix Aucklandiae 650 grams, Rhizoma Chuanxiong, each 1550 gram of Ramulus Cinnamomi add 5 times of water and decoct altogether, filter and obtain first decoction liquor; With Radix Notoginseng 500 grams, Radix Dipsaci, each 1660 gram of Eupolyphaga Seu Steleophaga, Rhizoma Drynariae 850 grams, Radix Achyranthis Bidentatae 650 grams, Os Gallus domesticus 1650 grams, Pyritum 680 grams and Radix Et Rhizoma Rhei 350 grams decoct 5 times in 3 times of water altogether, each 4 hours, collect each time of merging decoction liquor, filtration obtains second decoction liquor, and first and second decoction liquor are merged; Use ethanol precipitation, abandon precipitate, reclaim ethanol, obtain concentrated solution; Merge said extracted liquid and concentrated solution, with Borneolum Syntheticum 500 grams, Sanguis Draxonis 3400 grams are pulverized, are crossed No. 5 sieve backs and add in the mixed liquor of extracting solution and concentrated solution, and to obtain relative density be the extraction of 1.20---1.30 and concentrate thick paste.

With reference to composition and the method for embodiment 1,2, or, will extract and concentrate thick paste and be prepared as oral liquid, syrup, soft extract, medicated wine and tincture, capsule, tablet, granule, pill and externally applied ointment according to the general preparation method of Chinese medicine preparation.

Experimental example 1

The composition check of nutrient for cell growth of the present invention

(1) adopt absorption flame photometry method (AAS), plasma spectrometry mass spectrum (ICP-MS) method, and colorimetric method for determining trace element:

It is testing result after 20% the oral liquid following (do not contain in this oral liquid other any adjuvants and) that the extract of embodiment 1-3 is mixed with concentration:

(2) according to aminoacid, the total protein content of GB7649-87, GB2905-82 standard detection product of the present invention.

Adopt amino-acid analyzer, nucleic acid-protein analyser to detect aminoacid, the total protein content of cell growth medium of the present invention.Testing result is as follows:

(3) the present invention's (nutrient for cell growth) vitamin content

Adopt high-pressure liquid phase-multi-stage ms instrument to detect the present invention's's (nutrient for cell growth) vitamin content, the result is as follows:

Test item	Content (mg/L)
		Vitamin B1	2.24—3.60
Vitamin B2	5.86—8.00
		Vitamin B6	4.72—6.47
Vitamin PP	17.61—21.00

(4) the present invention's (nutrient for cell growth) total sugar content

Adopt the nucleic acid-protein analyser, anthrone method detects the present invention's's (nutrient for cell growth) total sugar content.Testing result is as follows:

From The above results as can be seen, the overwhelming majority of vitamin and 18 kinds of trace element of needed by human body is rich in the present invention (nutrient for cell growth).

Albumen, sugar, fat and water in the six big nutrients of needed by human body, the people sound for physiological function can satisfy by the normal diet picked-up.But vitamin and inorganic salt (trace element) only rely on the picked-up of diet but can not reach the level of keeping normal human's physiological activity as other two kinds of nutrients.Must replenish by alternate manner.Vitamin and trace element absorption in vivo and utilization are complementary, lack trace element, and protease, the vitamin that can cause regulating human body normal physiological biochemical function can't operate as normal.

Vitamin and trace element not only to organism growing development, improve immunity, disease-resistantly have important physiological function, but also can promote nucleic acid synthetic, cell regeneration prevents that histoorgan is aging, slow down aging; Nervous system regulation, analgesic activity.Therefore, in human development's stage, vitimin supplement and trace element are vital in good time.Overwhelming majority's (content is all in normal range) of vitamin and 18 kinds of trace element of needed by human body is rich in the present invention's (nutrient for cell growth).Therefore, visual this nutritional solution by this arbitrary way, reaches the physiological and biochemical index of needed by human body vitamin and trace element for to supply with needed by human body vitamin and trace element storehouse except that food.

Test example 2

This experimental example relates to the influence of the present invention's (nutrient for cell growth) to Mus osteoclast growth in vitro

One, the preparation of bone abrasive disc

Fresh adult cattle cortical bone is worn into the thick bone abrasive disc of 100 μ m, ultrasonic cleaning 30 minutes, and-30 ℃ are frozen standby.Time spent thaws, 70% alcohol-pickled 30min, super-clean bench middle-ultraviolet lamp irradiation 1h.Take out to add and to contain in 24 well culture plates of α-MEM culture medium, 4 4mm in every hole * 4mm osteocomma, 37 ℃ to hatch 1h standby.

Two, the separation and Culture of osteoclast

Get 24 hours newborn Wistar rats, after disconnected neck was put to death, 75% alcohol disinfecting, aseptic condition separated long bone of limbs down, the flushing of PBS liquid is key, longitudinal incision backbone in α-MEM culture fluid gently scrapes medullary cavity with scalpel, blows and beats bone cips repeatedly with the round end suction pipe, precipitate 30 seconds, absorption cell suspension is centrifugal, and (250g * 5min), α-MEM culture fluid cleans twice, adjusts 1 * 106/ml of cell concentration and evenly is inoculated on 24 well culture plates that preset the bone abrasive disc.

Three, the evaluation of osteoclast

1, morphological observation: the Olympus inverted phase contrast microscope is observed adherent situation of osteoclast and form thereof.

Osteoclast promptly begins adherent growth after cultivating 30min, and clear, rounded, oval, the decocting egg type of the osteoclast form of growth etc. is polymorphic after about 2 hours.

2, anti-tartaic acid phosphatase (TRAP): this enzyme is the enzyme-specific of osteoclast, and naphthols AS-BI phosphate can produce special insoluble claret precipitation with this enzyme.As seen TRAP dyes: the TRAP zymophore is dyed claret, and nuclear is negative, contains a plurality of nuclears, can see the ruffled border district of osteoclast, does not see the apocyte of TRAP feminine gender.

3, transmission electron microscope observing: isolating osteoclast suspension is centrifugal, get precipitation, behind fixing, the serial dehydration of alcohol, embedded section, electron staining, JEM-1220 transmission electron microscope observing.Through the JEM-1220 transmission electron microscope observing, the osteoclast of being cultivated contains a plurality of nuclears, can obviously distinguish 4 functional areas of osteoclast: ruffled border district, vesicle district, clear zone, basal area.Obviously be different from multinucleated giant cell.

The TRAP enzyme is a kind of significant enzyme of osteoclast, and naphthols AS-BI phosphate can combine with it and generate insoluble red precipitate.Osteoclast contains abundant mitochondria, lysosome and a large amount of rough endoplasmic reticulums under the Electronic Speculum.The ruffled border structure of all visible osteoclast under light microscopic and the Electronic Speculum, this is that multinucleated giant cell is not available.

Four, serum drug detects

A, blank serum: get adult Wistar rats and feed 5% arabic gum, blood sampling after 2 days gets serum 1ml.

B, take the Mus serum of nutrient for cell growth of the present invention: the oral liquid of the embodiment of the invention 2, press the 1g/kg.d amount for same rat and irritate stomach, 1 disappears, takes a blood sample after 2 hours, 3 hours, 4 hours, each gets serum 1ml.In the experiment we with before same the rat medicine feed and the serum behind the medicine feed make comparisons, eliminated interindividual variation.Experimental result has confirmed really to contain in the rat blood serum nutrient for cell growth of the present invention.The dosage difference of rat oral gavage in the experiment, the drug level in the serum are also different.Our evidence on the basis of a large amount of preliminary experiments is irritated stomach and is taken a blood sample after 2 hours, and a high relatively absorption peak is arranged.

The medicine of C, extracting: nutrient for cell growth 5g of the present invention is dissolved in the 50ml trifluoroacetic acid aqueous solution, and supersound extraction 30min gets supernatant after centrifugal.

More than three kinds of sample treatment after U.S. waters series liquid chromatograph instrument is analyzed, draw three picture groups spectrum.From three-dimensional collection of illustrative plates as can be known, 200nm～600nm scope has all compositions of uv absorption, drug serum figure and blank serum figure are relatively, many new chromatographic peaks have been produced, illustrated that novel substance is absorbed into blood, identical chromatographic peak has relatively been arranged, illustrated that the material that absorbs is a pharmaceutical compositions with medicine figure, the drug serum chromatographic peak is few than the medicine chromatograph, illustrates that not all pharmaceutical compositions all is absorbed into blood.

Five, pharmaceutical intervention and grouping

Experiment divides six groups, four periods, takes out the bone abrasive disc respectively at 24,48,72,96 hours and does TRAP dyeing, the male apocyte number of counting TRAP.

Took out osteocomma in per 24 hours, 2.5% glutaraldehyde fixedly added behind the 7min in the TRAP Incubating Solution 1 hour, to whole the male apocyte counting of osteocomma TRAP, experiment repeats twice at least, therefore the positive apocyte counting of each some TRAP is at least 8 times, adopt two people counting, everyone repeats twice, and the result represents with MEAN ± SEM.

Referring to accompanying drawing 1, M-CSF (M-CSF, M-CSF group) group can stimulate the propagation of osteoclast can promote the differentiation of its precursor again, and having confirmed at present has the M-CSF receptor in the sophisticated osteoclast.The osteoclast of this group nearly doubles in 48 hours at culture period.Calcitonin (calcitonin, CT group) can act on a plurality of stages that osteoclast forms, and comprises the fusion and the bone resorption ability that suppress osteoclast.This experiment calcitonin " escape phenomenon " occurs after cultivating 72 hours, its reason thinks that at present calcitonin has suppressed the expression of the mRNA of calcitonin receptor.

In the culture period 48 hours, the male osteoclast of nutrient for cell growth of the present invention (nutrient group) TRAP is lacked than matched group, but does not have significant difference.Behind the culture period 72 hours, 20% nutrient for cell growth group osteoclast number of the present invention be starkly lower than matched group (P＜0.01 but be inferior to the calcitonin group=, 10% nutrient for cell growth of the present invention after 96 hours) group the osteoclast number compared significant difference (P＜0.05) with matched group.Wherein matched group is blank serum, and the result proves that the blank serum of rat is little to the growth in vitro influence of osteoclast.

Osteoclast is the main cell that causes bone absorption, and osteoclasts cultured in vitro is research bone resorption and the basic skills that suppresses the bone resorption medicine.The present invention's (nutrient for cell growth) is made up of the effective ingredient that plurality of Chinese such as Radix Notoginseng, Stigma Croci, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Pyritum are extracted, and has activating blood circulation to dissipate blood stasis is arranged, the effect of reunion of fractured tendons and bones.Rat is taken the change that causes hormone in vivo behind the present invention's (nutrient for cell growth), calcitonin, 1.25 (OH) in the serum ₂D ₃The variation of content can directly influence the quantity of osteoclast.Various trace elements that the present invention's (nutrient for cell growth) comprises such as copper, zinc, manganese etc. are gone into blood with ionic species, these trace element can suppress the growth of osteoclast, promote the apoptosis of osteoclast, indicating that the present invention's (nutrient for cell growth) can exert an influence to the growth of osteoclast to a certain extent.

Simultaneously, the present invention (nutrient for cell growth) has inhibitory action to the fusion of the rat osteoclast of In vitro culture.

Nutrient for cell growth and preparation thereof present embodiment of the present invention and that preparation and other embodiment obtain also have essentially identical effect.

Test example 3

Present embodiment relates to the influence of the present invention's (nutrient for cell growth) to adult's osteoblastic proliferation, differentiation and the apoptosis of In vitro culture.

Method: get adult's ilium spongy bone, adopt collagen-trypsinization method, obtain the osteoblast in the spongy bone, carry out purification and cultivation.On this basis, add the serum that contains the plain medicine of osteocyte growing nutrient, continuous dosing 3 days, CO ₂Cultivate in the incubator, make the blank of not dosing simultaneously.By inverted phase contrast microscope and electron microscopic observation propagation and differentiation situation, detect alkali phosphatase and BGP content in the supernatant, and osteoblastic apoptosis situation is measured.

Experiment shows that adult's Oesteoblast growth that collagen-trypsinization method obtains is all right, and biochemical indicator is reliable and stable, and the cell purification effect is good, can satisfy the needs of correlational study.Further result of study shows, in osteoblast density is under the condition of 10000/ml, after adding contains the serum of nutrient for cell growth, supernatant neutral and alkali phosphatase and BGP content are obviously increased, the bone tuberosity forms to be accelerated, with the matched group of not dosing is more variant the significance meaning is arranged, but osteoblastic propagation and apoptosis situation there is not significant change.

The ilium that the adult is directly drawn materials in this experiment adopts collagen-trypsinization method, discharges a large amount of osteoblast in the spongy bone, and carries out purification and the cultivation of going down to posterity.On this basis, add the serum that contains osteocyte growing nutrient element, observe its influence, provide experimental basis for screening promotes the research of bone formation medicine to osteoblastic proliferation, differentiation and apoptosis.

As can be seen: osteocyte growing nutrient element has obvious facilitation to the osteoblastic differentiation of the adult of In vitro culture, but osteoblastic propagation and apoptosis are not had obvious inducing action, can be used as to promote osteoplastic drug use.

Materials and methods

1, draw materials: experiment material is taken from the patient who implements the cervical vertebra bone graft fusion, checks no metabolic bone disease, wherein male 6 examples, women 2 examples, 32～46 years old age, average 38.8 years old before patient's art.When the row ilium was got the bone bone graft fusion in the operation, the unnecessary spongy bone of excision was inserted in the aseptic bottle that the DMEM-F12 culture fluid is housed when getting its plant bone mass finishing, brings to laboratory.

Used culture fluid, hyclone are purchased the company in Gibco in the experiment, and animal is Wistar pure lines rats, and used nutrient for cell growth in the experiment, product compound concentration are 90% oral liquid.

2, osteoblast is cultivated

The GUSUIPIAN that obtains in the operation is washed 3 times the broken 1～2mm that is about of bone shears earlier with aseptic Hanks liquid ³Size, Hanks liquid repeatedly washes, and bleaches until GUSUIPIAN, inserts in bottle ware, pours collagen-trypsin cell dissociation buffer into, at room temperature digests 45min in the magnetic force oscillator.Remove the suspension that digests gained first, add an amount of cell dissociation buffer again, room temperature lower magnetic force concussion digestion 30min.Leave standstill 5min, collect Digestive system, filter screen filtering collagen impurity is removed GUSUIPIAN, and the centrifugal 10min of 2200r/min abandons supernatant under the room temperature, adds 5ml culture fluid suspension cell again, inserts in the 25ml culture bottle and cultivates, the cell numeration.After treating that cell is adherent fully, liquid is changed in flushing.After, the next day change liquid 1 time.7d, trypsinization, cell counting, be inoculated in respectively in 8 culture bottles with 10000/ml, every bottle of 5ml culture fluid (contains antibiotic, 12% hyclone, DMEM-F12,2mmol/L β sodium glycerophosphate and 100ug/ml L-ascorbic acid), and with corresponding fibroblast in contrast, identify.

3, osteoblastic evaluation

By inverted phase contrast microscope and transmission electron microscope observing osteoblast being carried out morphology identifies; Detect by ALP in the supernatant and OGN, osteoblast is carried out biochemical indicator identify; And in contrast with fibroblast.

4, nutrient for cell growth pharmaceutical intervention

4.1, the producing of nutrient for cell growth serum

Get 8 adult Wistar rats, female 4, male 4, average weight 340 grams.At first the nutrient for cell growth of the embodiment of the invention 1 is concentrated thick paste and is dissolved in 5% the arabic gum, divide two groups at random with rat then, one group by every day per kilogram of body weight 1 gram nutrient for cell growth irritate stomach; Another group is irritated 5% arabic gum, and each every Mus is irritated the no more than 4ml of bone, continuous irrigation bone 10 days, the fasting in preceding 24 hours of taking a blood sample is taken a blood sample and was irritated stomach more once in preceding 2 hours, every Mus blood sampling 4ml, in aseptic centrifuge tube centrifugal 10 minutes (3000rpm), respectively getting 2ml serum, to put into aseptic bottle standby.

Get in the DMEM-F12 culture fluid that contains nutrient for cell growth drug serum 2ml adding 8ml, make the culture fluid that contains 20% nutrient for cell growth drug serum.

Equally, get the blank serum of 2ml and add in the DMEM-F12 culture fluid of 8ml, make the culture fluid that contains blank serum.Stand-by.

4.2, the evaluation of serum Chinese medicine composition

4.2.1, the processing of laboratory animal:

Get adult Wistar rats, feed 5% arabic gum, after 2 days as above-mentioned method blood sampling 4ml, get 1ml (blank serum) after centrifugal, afterwards osteocyte growing nutrient element is dissolved in 5% arabic gum, same Wistar rat pressed the plain stomach of irritating of 1g/kg.d osteocyte growing nutrient

4.2.2, the processing method before the sample feeding:

Get blank serum, each 0.5ml of drug serum adds the 0.5ml trifluoroacetic acid aqueous solution, vortex concussion 5min, centrifugal 10 minutes (3000rpm) gets supernatant, with sample introduction 50nl behind the 0.45nm filtering with microporous membrane, the plain 5g of osteocyte growing nutrient adds 50ml trifluoroacetic acid aqueous solution, supersound extraction 30min, get supernatant after centrifugal (3000rpm), sample introduction 50nl behind the filtering with microporous membrane (this experiment repeats 4 times).

4.2.3, the serum after handling and the detection of medicine:

Serum after the processing and medicine are analyzed through U.S. waters series liquid chromatograph instrument respectively, draw three picture groups spectrum.

4.3, the interpolation of medicine

After osteoblast is cultivated and gone down to posterity for the 1st time,, add the 1ml0.25% trypsin, digested 10 minutes, observe to cell separation under the inverted microscope, take off wall with four times culture dishs of D-Hank liquid flushing.Collect digestion gained cell suspension to centrifuge tube, add the equivalent culture fluid, the centrifugal 5min of 2200r/min.Abandon supernatant, cell is suspended in the culture fluid again, mix homogeneously, and counting is inoculated into culture bottle with 10000/ml, inserts CO ₂Cultivate in the incubator.

Postvaccinal cell culture 12 hours (cell attachment) is inhaled and is abandoned original fluid, adds the drug serum that contains osteocyte growing nutrient element in the new culture fluid, makes the not blank group of adding medicine simultaneously, continuous dosing 3d like this, and put CO ₂Cultivate in the incubator.After stopping dosing, continue to cultivate with culture fluid, 2d changes a culture fluid.

5, morphological observation and biochemical indicator detect

5.1, morphological observation

After stopping dosing, continue to cultivate with culture fluid (containing 2mmol/L β sodium glycerophosphate and 100ug/ml L-ascorbic acid etc.), 2d changes a culture fluid.Before and after dosing, under inverted phase contrast microscope, dynamic observe cell growing state, tetracycline marker fluorescence staining, photomicrography.To the sample cell, to fix, embedding, the thin layer section is by Phillips CM10 transmission electron microscope observing.

5.2, osteoblast supernatant content of alkaline phosphatase detects

Be inoculated in the culture bottle every bottle of 5ml culture fluid with 10000/ml.14d gets supernatant, and adopting the p-nitrophenyl phosphate disodium salt is substrate, and through 405nm wavelength colorimetric, (optical density, OD) value is converted into iu, obtains alkali phosphatase (alkaline phosphatase, ALP) content to record absorbance.

5.3, osteoblast supernatant BGP content detects

Be inoculated in the culture bottle every bottle of 5ml culture fluid with 10000/ml. 14d, get supernatant, adopt the BGP radioimmunological kit to detect.The T enumerator is measured precipitate count per minute value, and (counts per minute cpm), obtains sample Bone Gla protein (osteocalcin, OGN) content according to the standard substance curve.

5.4, statistical procedures

To every routine sample, measure and respectively organize corresponding ALP and OGN numerical value, obtain meansigma methods and standard error, calculate the t value then, carry out the t check, adopt statpal software, carry out statistical analysis.

6, TNF-a Induced Apoptosis in Osteoblasts detects and analyzes

6.1, apoptosis detects

(terminal deoxynuc leotidyl transferase, TdT) (TdT-mediated Dutp nick end labeling, TUNEL) technology detects the apoptosis situation to mediated dUTP nick end labeling breach end labelling to adopt terminal deoxynucleotidyl transferase.Specifically pass through R﹠amp; (Minneapolis, MN) TdT original position apoptosis detects and finishes d system.

Key step comprises:

1. at first use the neutral formalin buffer and volume fraction 70% ethanol of volume fraction 10%, fixedly 10min and 5min of pair cell does further detection behind the dry 24h of cell respectively;

2. use E.C. 3.4.21.64 at 37 ℃, carry out the cell permeability step under the 10min condition;

3. adopt the horseradish peroxidase of chain avidin labelling to position detection, the location product strengthens;

4. the closed box medium chain avidin incubated cell 20min of humidifying at room temperature;

5. the 10min that develops the color in DAB liquid that cuts into slices redyes 10min with mass concentration 1% methyl blue then;

Adopt the standard recording parameter of human fibroblasts to be optimized processing when 6. detecting cell earlier.

6.2, quantitative analysis

(Cool Camera Co GA) carries out the inspection and the counting of cell to use Cool Cam 2000 image systems.Behind the cell counting, apoptotic percentage rate is calculated divided by apoptosis and normal cell sum by apoptosis cell.Obtain the percentage rate meansigma methods that each sample counting obtains.

The result

1, osteoblast is identified

The Olympus inverted phase contrast microscope is observed continuously, the 1d of cultivation, and the visible cell quantity that obtains is more, rounded, and nucleus is justified greatly, is positioned at an end of cell space.Behind the 72h, cell is all adherent.After one week, cell is fusiformis.

Biochemical indicator detects, and the ALP average content is 3.12 ± 0.26U/ml in the osteoblast supernatant; Fibroblast is 1.86 ± 0.28U/ml; P＜0.01, difference has significance.The OGN average content is 2.218 ± 0.246ng/ml in the osteoblast supernatant; Fibroblast is Ong/ml.

2, morphological observation

The osteoblastic time-to-live of cultivating can reach more than 10 weeks, if converge back and time-division bottle, added the growth of can going down to posterity of the plain group of osteocyte growing nutrient.

Compare with matched group, add the plain osteocyte of forming of osteocyte growing nutrient and be flat fusiformis very soon, cell volume increases.But the propagation situation of cell does not have significant difference, converges the time and does not have significant change; The multilamellar growth all appears in medicine group and cellular control unit, and medicine group bone tuberosity generates to be accelerated.Observe under the Electronic Speculum and see that medicine group cytoplasm is abundant, have a large amount of rough endoplasmic reticulums to occur, be vigorous secretion phase.

3, osteoblast supernatant A LP content detection

Experimental data shows, the 8 routine specimen of drawing materials add in the culture fluid of the plain group of osteocyte growing nutrient, and average A LP content is 4.06 ± 0.18U/mlU/ml; The average A LP content that does not add the group of being used as medicine is 2.58 ± 0.16U/ml.The ALP content of dosing group is apparently higher than the blank group, P＜0.01, and difference has the significance meaning.Referring to accompanying drawing 2,3.

4, osteoblast supernatant OGN content detection

Experimental data shows, the 8 routine specimen of drawing materials add in the plain medicine group of the osteocyte growing nutrient culture fluid, and average OGN content is .016 ± 0.225ng/ml; The average OGN content of not dosing group is 1.682 ± 0.257ng/ml.The OGN content of dosing group is apparently higher than the blank group, P＜0.01, and difference has the significance meaning, referring to Fig. 4.

5, TNF-a Induced Apoptosis in Osteoblasts detects and analyzes

The apoptosis testing result, the TNF-a Induced Apoptosis in Osteoblasts percentage rate meansigma methods that adds the plain drug serum of osteocyte growing nutrient in the sample is 3.2% ± 0.5, the TNF-a Induced Apoptosis in Osteoblasts percentage rate meansigma methods that does not add osteocyte growing nutrient element is 2.8% ± 0.3.Data result shows, add the plain drug serum group of osteocyte growing nutrient and do not add the medicine group relatively, the percentile difference of TNF-a Induced Apoptosis in Osteoblasts does not have significance meaning, P〉0.05, promptly adding the plain drug serum of osteocyte growing nutrient does not have obvious influence to osteoblastic apoptosis.

Along with the aged's increase, caused that as the osteoporosis of one of Senile disease people pay close attention to greatly.Osteoporosis is to reduce with the bone amount, and feature is changed in the microstructure of bone, causes the fragility of bone to increase, so that a kind of general metabolism skeletal diseases that is easy to fracture.The adult is along with the increase at age, and bone resorption causes the minimizing of bone amount greater than bone formation, can cause osteoporosis.Seek determined curative effect, side effect is little, and the osteosporosis resistant medicament that the mechanism of action is clear and definite is urgent day by day.

Cultivate and the pharmaceutical intervention experiment by external one-tenth human osteoblast cell, find to promote that osteoplastic material is one of important means of exploitation osteoporosis remedy thing.The present invention adopts external one-tenth human osteoblast cell cultured method, observe of the influence of osteocyte growing nutrient element to external adult's osteoblastic proliferation, differentiation and apoptosis, filter out the material that osteoblast is had facilitation, so that a kind of new drug of osteoporosis disease to be provided.

In the sixties in this century, Peck etc. bring into use the collagenase digesting osteocomma, and the success of In vitro culture osteoblast.1975, use collagenases such as Wong repeatedly digested the cranium of Mus, made osteoblast obtain to a certain degree purification.1985, Robey etc. adopted low Ca ²⁺Culture fluid is cultivated the osteoblast that osteocomma obtains the people, and the cell that obtains is further purified.At present, the relevant medicine of China is to the research of external Rat Osteoblast Induced be mostly to draw materials tire Mus or ripe rat, and it is few directly to draw materials into the report that the human osteoblast cell studies.This is that toleration is poor owing to become the human osteoblast cell low than the osteoblast activity of tire Mus, and In vitro culture is difficult for surviving, and must obtain the cell of sufficient amount, could keep its activity.In the present invention, the hipbone spongy bone that we directly draw materials and are grown up adopts collagen-trypsinization method to obtain into the human osteoblast cell.During experiment, at first collagenase and trypsin are mixed with Digestive system according to a certain percentage, twice digestion cancellous bone tissue, and with the osteoblast of gained with no Ca ²⁺The DMEM/F-12 culture fluid cultivate.Observe by inverted phase contrast microscope, the visible cell quantity that obtains is more, and form is normal, and nucleus is justified greatly, is positioned at an end of cell space, and membrane structure is good.Dynamically observe continuously and find that cell attachment is normal, growth is stable, and purification effect is good, shows osteoblastic various feature.As seen, cell volume is bigger under the Electronic Speculum, rectangle, and about 40 μ m, cytoplasm is abundant, includes a large amount of rough endoplasmic reticulums, is vigorous secretion phase; Biochemical indicator detects and shows, the osteoblast secretion ALP and the OGN of cultivation show vigorous secretory function.Every experiment shows, adopts this collagen-trypsinization method, and the osteoblast that obtains is good.The present invention's (nutrient for cell growth) has the effect that promotes union of fracture.In experiment of the present invention, observe by inverted phase contrast microscope, the 3d of the plain group of osteocyte growing nutrient dosing, the osteoblast volume obviously increases, and engenders many pseudopodium growths, accelerates but the bone tuberosity generates.Biochemical indicator detects and shows that in the supernatant of cell culture, (4.06 ± 0.18U/ml) apparently higher than blank group content (2.58 ± 0.16U/ml) for dosing group ALP content; (3.016 ± 0.225ng/ml) also apparently higher than not dosing group content (1.682 ± 0.257ng/ml) for dosing group BGP content.We know that the self replication of cell and cell differentiation are the main vital movements in the cells survival.Cell differentiation is the specialization of cellularity and function.For osteoblast, ALP is the early stage index of osteoblast differentiation, its expression strengthens along with the development of cell differentiation, its effect is that the hydrolysis organic phosphoric acid discharges Phos and acts on the formation of hydroxyapatite, it is the necessary enzyme of bone formation, osteoplastic situation is being represented in its expression, shows the beginning of cell differentiation.And the present invention's (nutrient for cell growth) is considered to work in the calcification of keeping normal bone calcium rate and inhibition cartilage (OGN) as the index in mid-term of osteoblast differentiation.When bone formation and bone resorption coupling, OGN is that the reflection bone is pass on the index of changing; When bone formation and bone resorption uncoupling, it is the osteoplastic specific parameters of reflection.

The nodular formation of bone, osteoblastic propagation and differentiation and osteoblastic apoptosis situation are closely related.Apoptosis (apoptosis) claims apoptosis again, and (programmed cell death PCD), plays an important role to growth, homeostasis, the development of pathological process of body.Its feature shows as nucleus and cytoplasmic pyknosis, goes out bleb, the formation of apoptosis body and the fracture of DNA etc.Apoptosis is a kind of important model of cell death, is to rely on environment or developmental stimulation, activates a program of a series of special incidents, causes the death of cell.All mammalian cells are all had the ability to start and are carried out the passage of apoptosis, and many point of adjustment are arranged in the cascade of apoptosis.About osteocyte growing nutrient element the research that external osteoblastic proliferation, differentiation and apoptosis influence be yet there are no report at present.We pass through the most accurate apoptosis detection technique (TUNEL) at present, and osteoblastic apoptosis situation after the effect of Chinese medicine nutrient for cell growth is studied.The result shows, the osteoblastic apoptosis percentage rate of its adult to In vitro culture did not influence.Whole experimental result shows that osteocyte growing nutrient element does not have obvious influence to external osteoblastic propagation and apoptosis, but osteoblastic differentiation is but had significant facilitation, shows as ALP and OGN secretion increasing, and rough endoplasmic reticulum increases or the like.By this experiment, confirmed that the present invention's (nutrient for cell growth) can promote the osteoblastic differentiation of external adult, thereby accelerated bone formation, and can carry out preliminary quantificational expression.The result of study prompting, the plain hypophysis auxin cell synthetic auxin that increases of osteocyte growing nutrient is one of mechanism of promotion union of fracture.It promotes that the excretory amount of auxin is limited, can not cause the hypophysis endocrine regulation, causes the misgrowth of body.

Though nutrient for cell growth does not have obvious influence to external osteoblastic propagation and apoptosis, and osteoblastic differentiation is but had significant facilitation, show as ALP and OGN secretion increasing, rough endoplasmic reticulum increases or the like.By this experiment, confirmed that the Chinese medicine nutrient for cell growth can promote the osteoblastic differentiation of external adult, thereby accelerated bone formation.

Similar nutrient for cell growth and the preparation thereof that experiment showed, that other embodiments of the invention obtain also has essentially identical effect.

In sum, nutrient for cell growth of the present invention has obvious facilitation to the osteoblastic differentiation of the adult of In vitro culture, cause osteoplastic quickening, therefore can be used as the osteoplastic drug use of promotion, its definite influence mechanism and new purposes remain further to be studied.

Test example 4

Present embodiment relates to nutrient for cell growth of the present invention (OGN) to fracture Mus growth hormone of serum and pituitary growth hormone impact cell.This experiment promotes the union of fracture mechanism of action for inquiring into this Chinese medicine, has studied the union of fracture commitment emphatically, and nutrient for cell growth of the present invention (OGN) is to the excretory influence of rat growth hormone.Experiment is divided into group of taking medicine (obeying 2% osteocyte growing nutrient element (OGN) suspension 0.5ml/100g body weight of 2 quilts of the embodiment of the invention every day) and matched group (obeying 5% arabic gum every day) with the Wistar Mus; Every group of equal underwent operative is made the left tibia fracture model, gets blood respectively at 3,7, the 14 and 28 days broken ends in operation back and surveys Serum GH concentration, gets hypophysis check weighing amount simultaneously and observes pituitary growth hormone cell area and gray scale through immunohistochemical staining with image analyzer.By the observation of fracture Mus growth hormone of serum and pituitary growth hormone cell, research osteocyte growing nutrient element (OGN) promotes the mechanism of union of fracture.The result shows that Serum GH was taken medicine in the time of 14 days and there were significant differences for matched group, and the somatotroph gray scale also has same trend to change, but this variation can return to normal level after the union of fracture or after the drug withdrawal.Pointing out nutrient for cell growth of the present invention (OGN) to increase Serum GH concentration and increase pituitary growth hormone cell synthetic auxin may be one of its mechanism that promotes union of fracture.

Zoopery and clinical practice have confirmed that Chinese medicine nutrient for cell growth (OGN) has the effect that promotes union of fracture.The factor that influences union of fracture is many-sided.

Material and method

1, laboratory animal

Wistar adult rat (providing) by Institute of Experimental Animals, Chinese Academy of Medical Sciences, male totally 54, body weight 220～260g.12 every batch, same on the same group cage in the test (6 in every cage) is raised, and 20～24 ℃ of receptacle temperature give granule Mus feedstuff, and free drinking water and food are quantity-unlimiting in the test.

2, medicine

The nutrient for cell growth of embodiment 2 (OGN), sample medicinal liquid preparation: make the solution that contains 2% osteocyte growing nutrient element (OGN) with 5% Arabic glue.

3, fracture model is made

Rat is with 5% chloral hydrate (0.15ml/100g) intraperitoneal injection, after anaesthetizing successfully, the left hind shank is shaved the conventional iodine tincture ethanol of hair and is taken off the iodine sterilization, select outside longitudinal incision under the left tibia tuberosity, be about 2.5cm, cut skin to subcutaneous, do not hinder subcutaneous superficial vein as far as possible, cut tibialis, the blunt tibia that is separated to separates around week along medial tibial periosteal, inboard separately toe side extensor, note not hindering arteria saphena, anterior tibial artery and corresponding neural, saw cross-section tibia with Xiao Bao, traverse point is about 1cm place under tibial tuberosity.Subcutaneous and skin is sewed up in the reduction of the fracture before sewing up sarolemma, is above-knee 2cm with Gypsum Fibrosum (6 layers) gauze and fixes to toes point plaster cylinder, removes after 7 days.

4, grouping and medicine feed

Above-mentioned model mouse is divided at random:

A organizes～takes medicine the fracture group: by sample 0.5ml/100g (method of " pressing body surface area between humans and animals and converting dose,equivalent ratio table " defined in the pharmacological experimental methodology second edition is seen in the dosage measuring and calculating) for oral administration, with Mus irrigation stomach device medicine feed every day once.Medicine feed for the first time at once after anesthesia is waken up is fixed the date to institute continuously.

B group～simple fracture group (matched group): the 5% Arabic glue that feed respective amount with the Mus irrigation stomach device every day is once fixed the date to institute continuously.

The C group one of was organized in～28 days: be take medicine after the fracture 14 days, 14 days groups of drug withdrawal.This group is not got blood, only does the hypophysis pathologic finding.

5, get blood and survey Serum GH concentration

Above-mentioned three groups of test rats, broken end was got blood (Fig. 3) when clear-headed in four batches respectively at fracture back 3,7,14,28 days, got blood 10ml for every, natural coagulation, low-speed centrifugal under the room temperature (2000 rev/mins) 15 minutes is got the about 2ml of serum, be stored in-20 ℃ standby.Adopt Mus GH radio immunoassay (RIA) (test box is provided by U.S. NHPP) to measure Serum GH concentration.

6, dyeing of pituitary growth hormone cellular immunization group and mensuration

Take out hypophysis immediately behind the rat broken end and fix 24 hours, take out and blot fixative, claim hypophysis weight with 1/100,000 electronics Libra with filter paper with the Bouin fixative.The conventional dehydration of hypophysis, paraffin embedding, serial section, thickness 5 μ m, every example is got the middle part greatest cross-section, carry out the somatotroph immunohistochemical staining, the monkey Chinese People's Anti-Japanese Military and Political College Mus CH antibody that reagent is provided by U.S. NHPP is that I is anti-, replaces the anti-indirect method of II to dye with enzyme standard gold Staphylococcus aureus protein A (SPA).The anti-optimum dilution degree of I is 1:200, and golden Portugal globulin A optimum dilution degree is 1:40, the DAB colour developing.Every example is established negative control.Coloration result carries out GH content measuring in the antepituitary somatotroph with computer gray scale testing software (gray scale is divided 32 grades).Simultaneously with calculated for pixel values somatotroph area size.Every example is surveyed 20 somatotrophs, obtains mean.

7, statistical analysis

Above gained data are carried out t check, variance analysis and q check with PRIMER software.

The result:

1, the Serum GH concentration ratio was than (seeing Table 1) between rat fractured and organized in back 3 days.

Serum GH value (ng/ml) relatively between table 1, rat fractured and organized in back 3 days

Know that by table 1 A group and B group rat blood serum GH concentration B group are organized equal numerical value height than A, but the Serum GH value is learned (t check) no significant difference by statistics between two groups

2, the Serum GH concentration ratio was than (seeing Table 2) between rat fractured and organized in back 7 days

Serum GH value (ng/ml) relatively between table 2, rat fractured and organized in back 7 days

Know that by table 2 A group and B group rat blood serum GH concentration B organize equal numerical value obvious rising, handles no significant difference between two groups but learn (t check) by statistics.

3, the Serum GH concentration ratio saw Table 3 between rat fractured and organized in back 14 days.

Serum GH value (ng/ml) relatively between table 3 rat fractured and organized in back 14 days

Known that by table 3 A group and B group rat blood serum GH concentration are learned (t check) processing by statistics, t=2.45 has notable difference between P＜0.05, two group.A group rat blood serum GH concentration obviously raises.

4. the Serum GH concentration ratio was than (seeing Table 4) between rat fractured and organized in back 28 days

Serum GH value (ng/ml) relatively between table 4, rat fractured and organized in back 28 days

Know that by table 4 A group and B group rat blood serum GH concentration value are learned (t check) by statistics and handled no significant difference between two groups.

5. pituitary growth hormone cellular immunization group dyeing measurement result (seeing Table 5).

GH content gray value relatively in table 5, the GH cell

	3 days groups	7 days groups	14 days groups	28 days groups
					G	A	A	A	A
	B	B	B	B
					N	120	120	120	120
	120	120	120	120
					M±SD	5.93±1.11 *	3.13±1.78 *	2.73+1.45 *	2.35±1.16 *
	5.04±0.95	5.00±1.44	4.69±1.51	5.29±1.01

Annotate: the A group is clothes arabic gum group for the group B group of taking medicine

* the expression interior GH content of P＜0.001 cell of comparing with experimental group is high more, and gray value is low more.

Know that by table 5 group of taking medicine was low than matched group pituitary growth hormone cell GH content in 3 days, but 7,14, the 28 days interior GH content of group pituicyte all is higher than matched group.

In order to check clothes the present invention (nutrient for cell growth) (OGN) after the drug withdrawal, whether hypophysis GH cellular change can restore, and establishes the C group in addition in fracture experiment in 28 days, and A, B, C relatively see Table 6 for three groups.

Three groups of table 6,28 days hypophysis GH cell areas of fracture and gray scale A, B, C be (x ± s) relatively

Group	The example number	Area (pixel)	Gray scale (level)
				A G F value (P value)	120120120	921.39±152.80911.74±134.69925.62±133.17	2.35±1.16?5.27±1.01 *5.53±1.47 *

254.03(P<0.01)

0.37(P>0.05)

Annotate: * represents to compare P＜0.05 with the A group

The result shows that gray level B, C organize and the A group compares, and all is higher than the A group respectively, and there is significant difference P＜0.05, but B, C compare there was no significant difference for two groups.The GH cell area relatively, each group there are no significant difference.

Discuss:

1, the effect of growth hormone in osteogenesis and union of fracture

The back takes place by early stage inflammatory reaction, macrophage cleaning in fracture, promptly enters the important stage of union of fracture, and fibrous callus, primitiveness callus formation are finished union of fracture at last.This complicated process is regulated and control by multiple factor.Wherein growth hormone has play a part important to osteogenesis and union of fracture.

Growth hormone (GH) is produced by the antepituitary acidophil, secretes in the mode of pulsed, and tangible species specificity is arranged.

(1) growth hormone can stimulate insulin like growth factor (IGF-I and IGF-II) to form in liver, and this factor can stimulate mesenchymal cell propagation, forms fibrocyte and myofibroblast.Simultaneously GH is a kind of main regulator that bone matrix glue unit forms, and myofibroblast, fibroblast and various glue unit are in the local formation of fracture fibrous callus.After this in original and Secondary cases callus formation stage, playing two kinds of cell-skeletonization of most critical effect and osteoclast and GH also has substantial connection.Osteoblast can make fracture site that enough callus formation are arranged, and to recover the seriality and the toughness of bone, IGF-I and IGF-II have very strong stimulation as vehicle to osteoblast.

I type and II type IGF receptor that strong affinity is arranged on the osteoblast energy film, IGF-I and IGF-II can obviously promote the mitosis of osteocyte, influence osteoblastic differentiation, excited alkaline phosphatase activities promotes Bone Gla protein to synthesize and strengthen the gene expression of osteonectin.Fracture back Serum GH concentration increases, and pituitary growth hormone cell (being acidophil) also is in high functional status simultaneously.Above-mentioned reaction is strengthened, and promotes union of fracture.

(2) GH can promote growth plate cell differentiation and growth, and the growth plate endochondral ossification is depended in the growth of bone to the direct effect of cartilage, has confirmed that GH can cause new osteogenesis simultaneously.

(3) GH can improve the intestinal calcium absorption, and excited kidney 1-α hydroxylase activity makes 25 (OH) ₂D ₃Be converted to 1.25 (OH) ₂D ₃Concentration increases, and the latter is active strong, and 1.25 (OH) are arranged on the osteoblast ₂D ₃Receptor, it increases the osteocyte number.Form bone mineral, effect is higher than the former 100 times.

Increase the bone amount, accelerating union of bone fracture and add the rupture strength of bone strengthening, 1.25 (OH) simultaneously ₂D ₃In the secondary callus formation stage, can stimulate the differentiation and the fusion of predecessor's mononuclear phagocyte of osteoclast, be beneficial to bone resorption, recover the form of collagen.

In addition, the effect of many situation explanation growth hormone in promoting fracture healing process still arranged.For example human since the period of maturation, Serum GH concentration begins to descend, and then most of later on or all disappear to 65 years old, the decline of old people GH level can be considered as a kind of form of GHD (being that GH lacks), outstanding behaviours be that bone density descends.It is late also to find in the while clinical position to heal, heal after the middle-aged and elderly people fracture, and higher child's stage of Serum GH concentration heals after the fracture soon, heals early.

In a word, GH has significant effects to each stage of union of fracture of osteogenesis, although the complicated mechanism of union of fracture, some problem is unclear fully as yet, also is absolutely not that GH works separately simultaneously, and the effect of GH is indubitable.Fracture back Serum GH concentration reduces will be unfavorable for union of fracture, and raising or keeping certain level to help or to promote union of fracture.

2. test result analysis

Because the Serum GH secretion has the pulsed characteristics, so the detected value fluctuation is bigger, when measuring every batch of blood serum sample simultaneously, calibration curve is also inequality, when therefore analyzing every batch of Serum GH concentration detected value, lateral comparison between this test is not done every batch is only done the group inner analysis relatively.For making every batch of measured value accurate, we have got rid of interference factor as far as possible, accomplish to organize interior term harmonization on blood time, environment, the method getting.

The result shows, the group of taking medicine after the fracture has certain difference with matched group Serum GH concentration, the back 3 days two groups of no significant differences of fracturing, the back Serum GH concentration of taking medicine in 7 days of fracturing raises, but learn to handle by statistics, the two no significant difference is to fracturing 14 days the time, take medicine group and matched group relatively, and both have evident difference.

And in fracture in the time of back 28 days, two groups of Serum GH concentration levels are got back to again and are equated level substantially.Illustrate that osteocyte growing nutrient element (OGN) has the effect that promotes that Serum GH concentration raises in the certain hour after fracture.

In addition, in order to reflect the secretion situation of GH more accurately, this experiment is also measured the volume and the GH content (representing with gray value) of pituitary growth hormone cell, the rat GH growth hormone immunoreagent that experiment provides with U.S. NHPP, specificity shows pituicyte, wherein cell volume shows apparently higher than matched group (P＜0.05) that the gray level of GH content cell in then took medicine from 7 days to organize in 7 days groups, the group of taking medicine and is higher than matched group always, and the P value is all less than 0.05.

Explanation removes fracture and injury extremely in early days (3 days) thus, after beginning the reparation phase (7 days), osteocyte growing nutrient element (OGN) all has the excretory effect of the hypophysis GH of increasing, and the time that the variation of morphocytology (it is big that volume becomes) is kept is shorter than the time that its function changes (GH content).

But in union of fracture the 7th～14 day, the secretion of GH is to be in peak phase, this variation is consistent with the result of previously histopathology variation [2], has supported that osteocyte growing nutrient element (OGN) may be the diagnosis of one of its mechanism of action by increasing GH promotion union of fracture.

The present invention's (nutrient for cell growth) (OGN) promotes that the excretory amount of GH is limited, tests each batch the weight of animals and hypophysis weight by this, and it is not enough to cause the supernormal growth of body the group and the equal no significant difference susceptible of proof of matched group of taking medicine.Special in 28 days three prescription differences and q assay, also explanation is taken medicine after fracture, and drug withdrawal was after 14 days again in 14 days, and the change level of hypophysis returns to the matched group level substantially.

This shows that osteocyte growing nutrient element (OGN) is that existing promotion GH secretion increases, the effect of accelerating union of bone fracture can not cause a kind of relatively safe drugs of hypophysis endocrine regulation again.

Use the Wistar rat to make the left tibia fracture model, be divided into take medicine group and matched group, the result shows that fracture took medicine group and matched group Serum GH concentration ratio in back 14 days, significant difference is arranged, pituitary growth hormone cell GH content has the change of identical trend, point out osteocyte growing nutrient element (OGN) to have in the certain hour after fracture and increase, the effect of Serum GH concentration and increase somatotroph content may be that nutrient for cell growth (OGN) promotes one of union of fracture mechanism.

Similar nutrient for cell growth and the preparation thereof that experiment showed, that other embodiments of the invention obtain also has essentially identical effect.

Test example 5

Present embodiment relates to the clinical and experimental study that the present invention's (nutrient for cell growth) promotes union of fracture.

In order further to prove its effect and the mechanism of action, estimate its curative effect, safety, adaptability and application notice.We use the present invention's (nutrient for cell growth) and (OGN) with control drug 450 routine fracture patients have been carried out the clinical observation research of system, now result of study are reported as follows.

This group is totally 450 examples, (A) test group 300 examples of the osteocyte growing nutrient element (OGN) of embodiment of the invention 1-3 wherein, powder for fracture and trauma (B) positive controls 100 examples, blank (C) group 50 examples.Patient age is between 18～60 years old, and concrete the distribution sees Table 1; The sex distribution situation sees Table 2; Fracture mostly occurs at positions such as distal radius, humerus, femur, tibiofibula and chi radius, and fracture site distributes and sees Table 3; The Drug therapy time was 2～4 weeks; Therapeutic Method accounts for 395 examples based on closed reduction plintlet or external plaster fixation, and the surgical incision internal fixation is auxilliary, accounts for 55 examples.

Clinical data

1, physical data

Table 1 age distribution situation (routine %)

Age (year)	Test group	Positive controls	The blank group	Add up to
					18～30	98(32.67)	31(31)	17(34)	146(32.45)
31～40	57(19.00)	30(30)	13(26)	100(22.22)
					41～50	72(24.00)	16(16)	12(24)	100(22.22)
51～60	73(24.33)	23(23)	8(16)	104(23.11)

Table 2 sex distribution situation (routine %)

Sex	Test group	Positive controls	The blank group	Add up to
					The man	157(52.33)	61(61)	28(56)	246(54.67)
The woman	143(47.67)	39(39)	22(44)	450(45.33)
					Add up to	300(100)	100(100)	50(100)	450(100)

Table 3 fracture site and Therapeutic Method distribute

2, study subject is selected

Diagnostic criteria: clear and definite trauma history is arranged.General symptom is a local pain, and swelling has ecchymosis, dysfunction.Special sign is local deformity, and bony crepitus or bone are wiped sense, abnormal movement.

The performance of X line: the prompting of X line has the integrity of bone or seriality to wreck, and palpus is just being clapped, lateral projection, comprises adjacent joint.

Traditional Chinese medical science disease: disease is seen swelling due to the traumatic injury, pain, the limited bones and muscles injury of limb activity, qi depression to blood stasis, and pale complexion, the skin ecchymosis, body of the tongue is light red or ecchymosis is arranged, yellow and thin fur, stringy pulse speed or heavy real and puckery.

2.2, test case standard

2.2.1, include the case standard in

Meet diagnostic criteria, with extremity tubular bone closure, fresh fracture (in three days), plintlet and (or) external plaster fixation is that the main object of observation, operative treatment are the auxiliary object of observation.

Get rid of the case standard:

(1) age is at under-18s or more than 60 years old; Gestation or women breast-feeding their children; This medicine there is allergy; Pathologisch Bruch;

(2) be associated with liver, kidney and cardiovascular disease; The psychotic; Dysgenic other diseases is arranged;

(3) fracture does not reach the functional reduction standard, effectively fixing person after resetting;

(4) all standards of including in that do not meet, not medication in accordance with regulations can't be judged that curative effect or data are not congruent to affect the treatment or safety judgement person.

Materials and methods

1 test specimen

Test group: the nutrient for cell growth of the embodiment of the invention 1 (OGN) granule or powder, dress Chinese medicine 5 grams in every bag, active constituent content 90%.

Positive controls: powder for fracture and trauma, Kiamusze City, the Heilongjiang Province pharmaceutical factory of traditional Chinese medicine produces, and black (82) No. 1172 powder and capsules of the accurate word of medicine of defending, every intragranular are adorned 0.4 gram medicated powder.

Blank group: for not containing any activity, toxicity contrast medicine.Get thick red sorghum end, slow fire is suitably fried and is processed, and after cooling, pulverizes, and crosses 80 mesh sieves, gets fine powder; With osteocyte growing nutrient element (OGN) empty package bag, the 5 gram fine powders of packing into, sealing.

Situations such as the outward appearance of each sample of clinical trial medication and character see Table 4.

Situations such as the outward appearance of table 4 clinical trial medicine and character

2, test method

This study subject to be being in hospital and special outpatient clinic combines, 289 examples of wherein being in hospital, outpatient service 161 examples, clinic case make regular check on or drug delivery to the people, the strict variable factor of controlling.

2.1, group technology

Adopt the double blind random pair principle, specifically be divided into test group, positive controls and blank group, each research unit's case allocation proportion such as table 5.

2.2, medication and the course of treatment

Test group: oral nutrient for cell growth of the present invention (OGN), each 1 bag (5 gram), 2 times on the 1st, 14 days is a course of treatment, can take two courses of treatment continuously.

Positive controls: oral powder for fracture and trauma, each 1 bag (4 gram), 3 times on the 1st, 14 days is a course of treatment, can take two courses of treatment continuously.

The blank group: oral, each 1 bag (5 gram), 2 times on the 1st, 14 days is a course of treatment, takes two continuously

The individual course of treatment.

2.3, observation index and project

2.3.1, safety observation comprises general health check-up project, blood, urine, just routine test, the heart, liver, kidney function test

2.3.2, health giving quality observation comprises related symptoms and sign: swelling, pain and fractures X ray examination.

Table 5 is respectively participated in research unit and is observed the case load situation

Unit	Test group	Positive controls	The blank group	Add up to (example)
					Hubei Prov. Inst. of Traditional Chinese Medicine	110	40	50	200

Fujian Institute of Traditional Chinese Medicine	70	30	0	100
					The BJ Medical University 1st Subsidiary Hospital	35	15	0	50
Beijing Hospital of Traditional Chinese Medicine	50	0	0	50
					General Hospital of Beijing Mine Bureau	35	15	?0	50
Add up to (example)	300	100	50	450

Therapeutic outcome

1, criterion of therapeutical effect and foundation thereof.

Clinical research guideline according to new Chinese medicine treatment traumatic fracture.

1.1, the fracture the clinical healing standard: the position of fracture does not have pain, tenderness and longitudinal axis percussion pain; Fracture site does not have bony crepitus and abnormal movement; The X line shows that fracture line is fuzzy, has the seriality callus to pass through fracture line; After removing external fixation, upper limb flat forward stretched and held 1 kilogram and weighed 1 minute, and lower limb are not held up and turned level land continuous walking 3 minutes, and are no less than 30 step persons.

Observed for 2 weeks continuously, the indeformable person of activity bone generally does exercises.The 1st day that then observes is the clinical healing date.

1.2, the common clinical union of bone time

3～5 weeks of clavicular fracture; 3～5 weeks of fracture of the surgical neek of the humerus; 4～6 weeks of humeral shaft fracture; 3～4 weeks of supracondylar fracture of the humerus; 4～6 weeks of double fracture of shafts of ulna and radius; 3～5 weeks of radius far-end fracture; 4～6 weeks of metacarpal fracture; 6～8 weeks of intertrochanteric fracture of the femur; 6～8 weeks of fracture of shaft of femur; 6～8 weeks of fracture of tibia and fibula; 4～8 weeks of fracture of the metatarsal bone.

1.3, curative effect judging standard

(1) clinical cure: the similar healing time of clinical application time ratio reaches clinical healing standard person in advance more than 1/3.

(2) produce effects: the similar healing time of clinical application time ratio shifts to an earlier date 1/3～1/4, reaches clinical healing standard person.

(3) effective: the similar healing time of clinical application time ratio shifts to an earlier date 1/4～1/5, reaches clinical healing standard person.

(4) invalid: the clinical application time is identical with the normal healing time of similar fracture.

2, efficacy analysis

2.1, efficacy determination

Non-operation test group 265 routine curative effects of table 6 and sex situation are relatively

Sex	Clinical cure	Produce effects	Effectively	Invalid	R
						The man	74	41	19	3	0.511
The woman	74	37	15	2	0.488

Sex and therapeutic effect relationship are through Ridit statistical procedures x in test group nutrient for cell growth of the present invention (OGN) cooperation plintlet external fixation 265 examples ²=0.420, P＜0.05, difference not statistically significant.Through Ridit check u=0.648＜1.96, P〉0.05 also show the difference not statistically significant.Illustrate that the sex factor does not have influence to curative effect.

Table 7 test group 265 routine each age group and therapeutic effect relationship

Age	Clinical cure	Produce effects	Effectively	Invalid	R
						18—30	58	20	11	1	0.459
31—40	27	12	9	1	0.515
						41—50	32	20	11	1	0.533
51—60	31	26	3	2	0.512

Test group cooperates manual reduction, and in plintlet external fixation 265 examples, each age group and therapeutic effect relationship are through Ridit statistical procedures x ²=2.891＜7.815, P〉0.05 difference not statistically significant, illustrate that the age do not have influence to curative effect.Through the Ridit check, calculate u respectively again ₁=1.568 u ₂=0.328 u ₃=0.408 all＜1.96, P〉0.05 also show the difference not statistically significant.

Drug effect relatively between each group of table 8:395 example (through the non-operative treatment patient)

Group	Clinical cure	Produce effects	Effectively	Invalid	R
						Test group	148	78	34	5	0.370
Positive controls	10	14	8	56	0.727
						The blank group	0	2	5	35	0.839

In the fracture patient of 395 routine underwent operative treatment skills (manual reduction+small splints fixation), analyze through the Ridit statistical method, nutrient for cell growth of the present invention (OGN) medication group (test group), drug effect between powder for fracture and trauma medication group (positive drug matched group) and non-medication group (blank group), show between three groups and have significant differences, x ²=166.07, P＜0.01.

Again through Ridit method difference comparative test group and blank group u ₁=9.77, there is significant differences P＜0.01, drug effect between positive drug matched group and blank group, u ₂=2.074, there is significant difference P＜0.05.Test group and positive controls be u relatively ₃=10.048, P＜0.01 difference highly significant meaning.Illustrate that test group obviously is better than matched group and blank group.

Main fracture site of table 9 and therapeutic effect relationship analysis

The position	Clinical cure	Produce effects	Effectively	Invalid	R
						Clavicle	15	8	8	0	0.513
Humerus	21	11	9	1	0.504
						The chi radius	19	10	8	1	0.504
Distal radius	40	21	15	2	0.482
						Femur	23	12	9	1	0.496
Tibiofibula	18	9	7	1	0.497

Main fracture site and therapeutic effect relationship are through Ridit statistical procedures x ²=0.349, P〉0.05 difference not statistically significant.Again through the Ridit statistical method relatively each the group between curative effect u ₁=0.132, u ₂=0.129, u ₃=0.506, u ₄=0.252, u ₅=0.225, all＜1.96, the difference not statistically significant.Illustrate that using osteocyte growing nutrient element (OGN) treatment different parts fracture does not have influence to curative effect.

Curative effect relatively between each group of table 10 55 routine operative treatments

Group	Clinical cure	Produce effects	Effectively	Invalid	R
						Test group
	8	7	18	2	0.426
						Matched group	2	2	4	4	0.554
Blank group	0	1	2	5	0.742

Curative effect is relatively through Ridit statistical procedures x between 55 example cooperation operative treatment groups ²=27.30, meaning that there was a significant difference in P＜0.01.Through the Ridit check, test group compares u with blank group again ₁=2.796, P＜0.01 difference has non-significant difference, and positive controls and blank group be u relatively ₂=1.424, P＜0.05 difference not statistically significant.

Table 11: operative treatment group 265 routine curative effects and sex situation are relatively

Therapeutic Method	Clinical cure	Produce effects	Effectively	Invalid	R
						Plintlet	148	78	34	5	0.370
Operation	8	4	18	2	0.558

Use osteocyte growing nutrient element (OGN) in test group 300 examples and cooperate small splints fixation to treat 265 examples, cooperate operative treatment 35 examples, two kinds of methods influence Ridit statistical procedures x to curative effect ²=55.15, P＜0.01 difference has the highly significant meaning, again through Ridit check u=3.613〉2.58, P＜0.01, difference has the highly significant meaning, illustrates that using osteocyte growing nutrient element (OGN) cooperates the plintlet external fixation to be better than cooperating therapeutic method of surgery

Table 12: curative effect comprehensively compares between three groups

Group	Clinical cure	Produce effects	Effectively	Invalid	R
						Test group	156(52.00)	85(28.33)	52(17.33)	7(2.34)	0.375
Positive controls	12(12.00)	16(16.00)	12(12.00)	60(60.00)	0.711
						The blank group	0(0.00)	3(6.00)	7(14.00)	40(80.00)	0.829

In the table 12 through Ridit statistical procedures x ²=174.62, difference has the highly significant meaning, compares u through Ridit check test group and blank group again ₁=10.29, there is significant differences P＜0.01, and positive controls and blank group be u relatively ₂=2.632, meaning that there was a significant difference in P＜0.05.

Test group and positive controls be u relatively ₃=10.08, P＜0.01 difference has the highly significant meaning.Thereby illustrate that osteocyte growing nutrient element of the present invention (OGN) group is better than positive controls and blank group.

2.2, promote the union of fracture situation analysis

All clapped X-ray film in three group of 450 routine therapeutic process at 14 days or 21 days, 28 days; Found that as 14 days or 21 days union of fracture person just no longer makes film, fuzzy according to fracture line, the growth of spur situation is given a mark continuously.Fracture line remains unchanged even widens, and external callus was not made a call to 0 fen in not having, and fracture line fogs, and has inside and outside slight callus to make a call to 1 fen, and fracture line almost disappears, and has continuous growth of spur to make a call to 2 fens.It the results are shown in Table 13.

Table 13: the scoring of therapeutic outcome X line callus relatively

Nutrient for cell growth (OGN) obviously is better than powder for fracture and trauma group and blank group as seen from Table 13, has to promote the union of fracture effect preferably.

2.3, the detumescence situation analysis

By fracture swelling degree be divided into nothings, gently, in, weigh 4 grades, be respectively 0,1,2,3.Observed at 3,7,10,14 days respectively by administration time, it the results are shown in Table 14.

Table 14: the detumescence situation relatively

Time (my god)	Test group	Positive controls	The blank group
				3	2.323±0.873**	2.710±0.782△	2.98±0.707
?7	1.473±0.741**	1.840±0.996△	2.12±0.9233

10	1.200±0.605**	1.290±0.946△	1.600±0.95
				14	0.927±0.610**	1.140±0.828△	1.44±0.890

Annotate: * * test group and positive control and blank group be P＜0.01 relatively all, and the highly significant meaning is arranged.

* test group and positive controls compare P〉0.05 zero difference, relatively highly significant difference is arranged P＜0.01 with the blank group.

△ positive controls and blank group compare P＜0.05, and there were significant differences.Thereby illustrate that osteocyte growing nutrient element (OGN) has detumescence effect preferably.

2.4, the pain relieving situation analysis

The observation of pain mainly is patient main suit's situation marking, and the pain complete obiteration maybe can be ignored and makes a call to 1 fen, and slightly, accidental pain was made a call to 2 fens, and Chang Zijue has pain person to make a call to 3 fens.It the results are shown in Table 15.

Table 15: pain situation comparative analysis

Time (my god)	Test group	Positive controls	The blank group
				1	2.880±0.719▲	2.930±0.671▲	2.840±0.717
3	2.330±0.756*	2.880±0.763▲	2.780±0.729
				5	2.303±0.744▲	2.430±0.769▲	2.320±0.719
7	2.107±0.6489△	2.270±0.750▲	2.220±0..671

Annotate: * P＜0.01, △ P＜0.05, ▲ P 0.05

In analgesia effect in the 3rd day and positive controls highly significant difference is arranged relatively from table 15 explanation test group, be better than positive controls, there were significant differences in the 7th day, and the 1st, 5 day zero difference, no significant difference between positive controls and the blank group.

2.5, obvious adverse reaction do not take place at predetermined treatment in analysis of adverse reactions three groups as yet in the time, 5 routine female patients are arranged because of not tolerating osteocyte growing nutrient element (OGN) abnormal smells from the patient, vomiting is felt sick when taking medicine, refusal treatment, and use its medicine instead is not included in the checking.Most patient's reflections are taken osteocyte growing nutrient element (OGN) back and are defecated thinning.But there is not obvious diarrhoea situation.

3, OGN pain relieving and synthetism clinical observation and analysis

Select each fracture patient 30 example of limbs (except long bone in section fracture far away), after wound, take the embodiment of the invention? nutrient for cell growth (OGN).Select the same period equal case to take Chinese and western drugs controlled observations such as Anisodus carniolicoides C.Y.Wu et C.Chen, dolantin, bucinnazine after wound, its observed result is listed as follows:

From last table can/go out, the pain relieving intensity of osteocyte growing nutrient element (OGN) is similar to bucinnazine, is only second to dolantin, is better than Anisodus carniolicoides C.Y.Wu et C.Chen.The pain relieving of its osteocyte growing nutrient element (OGN) is held time longer than bucinnazine and dolantin.Its blood circulation promoting and blood stasis dispelling synthetism effect is better than the Anisodus carniolicoides C.Y.Wu et C.Chen medicine, osteocyte growing nutrient element (OGN) callus can occur in general about 18 days, on average shift to an earlier date 8～9 days than other invigorating blood circulation what Chinese patent medicines such as Anisodus carniolicoides C.Y.Wu et C.Chens, ratio is without drug for invigorating blood circulation and eliminating stasis, single callus with analgesic occurs about 17 days in advance, shortens nearly one times of time of the course of disease.

Find no significant discomfort in nutrient for cell growth of the present invention (OGN) the clinical observation process and close untoward reaction such as allergy, and do not produce addiction, this can give up the suffering of the easy addiction of narcotics such as dolantin and bucinnazine.From whole structure, " osteocyte growing nutrient element (OGN) " is a kind ofly can pain relieving can improve the little orthopedics department new drug of synthetism efficient and side effect again.

4, model case

Example 1 Pan, women, 46 years old, workman, admission number: 0084006.

Because of get injured by a fall right wrist, swell and ache, movable function is limited is admitted to hospital.Have a medical check-up: refreshing clear, vital sign is normal, right wrist swelling, and tenderness, movable function is limited, can lay one's hand on and bony crepitus.The film making check, para-position is good to line.Promptly take osteocyte growing nutrient element of the present invention (OGN), every day 2 times, each 5 grams, the patient takes nutrient for cell growth (OGN) back same day, local swelling is obviously disappeared, pain obviously alleviates, and through the 2 weeks film making check of taking medicine, the X line shows: fracture line is fuzzy, continuous growth of spur is arranged, remove external fixation, right hand movable function is normal, and recovery from illness is left hospital.

Example 2 Wang, man, 25 years old, the masses, admission number: 124576.

Swell and ache because of the motor-vehicle accident injury right leg causes, limitation of activity is admitted to hospital.A corpse or other object for laboratory examination and chemical testing: vital sign is normal, and the obvious swelling of right leg, livid purple, local tenderness can be laid one's hand on and bony crepitus and abnormal movement, and X-ray film shows: 1/3 comminuted fracture under the right tibiofibula, displacement.

Clinical diagnosis: right tibiofibula comminuted fracture, the treatment of the grace of performing the operation De Shi pin internal fixation external plaster fixation, be oral nutrient for cell growth of the present invention (OGN) same day, looked in second day and examine, patient's local swelling, pain obviously alleviate, the X ray examination shows after taking for 3 weeks: right tibia hypomere fracture fracture line is fuzzy, and continuous growth of spur is arranged.The back recovery from illness of 4 weeks is left hospital.

Discuss

1, fracture is clinical common multiple disease, and orthopedics and traumatology of Chinese medicine has accumulated rich experience in the history in existing several thousand of China aspect fractures." interior warp " said: " impairment of QI resulting in pain, injury of configuration resulting in swelling ".Wang Bing annotates: " so gas is invisible grieved, and tangible then wound of blood swollen ".Fracture and injury QI and blood, hyperamization arteries and veins are from through absurd row, and stagnant blood is detained, and form blood stasis, so that QI-blood circulation are not normal, and the stasis of blood is long-pending does not loose, and are pain for swollen." blood not reviver's stasis of blood does not go; the stasis of blood does not go then can not connect " event is controlled and is worked as blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain, reunion of bone, the present invention's (nutrient for cell growth) (OGN) has above effect, and wherein Radix Notoginseng is sweet little bitter warm, be the traumatology hemostasia and dissipation blood stasis, the panacea of subduing swelling and relieving pain has hemostasis not stay stasis of blood characteristics, is monarch drug; Rhizoma Chuanxiong, Radix Angelicae Sinensis, Stigma Croci circulation of qi promoting and blood, nourish blood; Ramulus Cinnamomi, Radix Et Rhizoma Rhei, Pheretima warming the meridian and promoting blood circulation, clearing away damp-heat, reducing swelling and alleviating pain; Sanguis Draxonis, Eupolyphaga Seu Steleophaga, nux vomica powder blood stimulate the menstrual flow, the knot pain relieving that disappears, and all medicines are minister.Assistant is with Radix Dipsaci, Pyritum liver-kidney tonifying, reunion of fractured tendons and bones; Borneolum Syntheticum, Rhizoma Zingiberis, the Radix Angelicae Dahuricae, the hot perfume (or spice) of Lignum Aquilariae Resinatum are walked to scurry, and except that promoting the circulation of QI to relieve pain, still can draw the institute that all medicines reach the disease wound, are messenger drug.Make a general survey of full side's monarch, bring out the best in each other, complement each other, be combined into blood stasis dispersing and fresh blood promoting, subduing swelling and relieving pain, the effect of reunion of fractured tendons and bones.

2. union of fracture (fracture healing) is a complex physical pathological process, osteoblastic regeneration is the basis of union of fracture in the periosteum, its agglutination can be divided into hematoma and form, fibrous callus forms, the several processes that replace evolution successively of the reconstruction of osseous callus formation and callus, union of fracture is divided into intermembranous ossification and endochondral ossification, fractured back 24 hours in, the periosteum at fracture end place begins the hypertrophy plumpness, and the internal layer hyperplasia of periosteum produces osteoid tissue, form area of new bone, be intermembranous ossification, its process is rapid, and is connected firmly.

Periosteum bone cortex and contiguous soft tissue were damaged when os endochondrale was fracture, angiorrhexis is hemorrhage, form hematoma at fractures, because blood cell destroys, fibrin oozes out, and granulation tissue forms, hematoma begins machineization, hematoma is surrounded in the proliferation of fibrous tissue that is positioned at around the hematoma, and stretches into wherein, and the effect by phagocyte and foreign-body giant cell is absorbed replacement.

The absorption of hematoma replaces, and often needs 2～3 weeks or longer time just can finish.After absorption of hematoma substitutes, inmature fibrous tissue between fracture site, major part changes cartilage into, and intercalation is between the external callus of two fracture sites, and chondrocyte ossify through hyper-proliferative, degeneration, calcification, and this is need 1～3 month often.

The present invention's (nutrient for cell growth) infrastest (OGN) proves: have blood circulation promoting and blood stasis dispelling, reducing swelling and alleviating pain, the effect of reunion of bone.The effect of antiinflammatory, analgesia, microcirculation improvement and promotion union of fracture is arranged again simultaneously.We think it may is that nutrient for cell growth (OGN) promotes the absorption of hematoma and reduce hematoma to form in early days, and the blocking-up endochondral ossification in addition, stimulates Oesteoblast growth in the periosteum, helps skeletonization in the periosteum, thereby promotes the immediate union of fracture.

3. osteocyte growing nutrient element (OGN) clinical experiment observed result all obviously is being better than powder for fracture and trauma aspect reducing swelling and alleviating pain, the promotion fracture heals in early days.It is the clinical desirable medication of orthopedics department.

Test example 6

Present embodiment relates to archusia of the present invention at treatment cancer, cardiovascular and cerebrovascular disease and suppress purposes aspect old and feeble.

Growth hormone HGH is secreted by anterior lobe of hypophysis, HGH can strengthen health immune system, smooth away wrinkles, make hair regeneration, prevention of cardiac and apoplexy, bring high blood pressure down.But growth hormone HGH secretory volume reduced along with the age, and from 21 after 31 years old, per ten years reduce 14%, therefore have only 60 years old the time youthful half; Have only in the time of 80 years old at an early age 1/5th.

Experimental example 4 is verified, the present invention's (nutrient for cell growth) (OGN) can increase serum HGH concentration and increase pituitary growth hormone cell synthetic auxin, therefore, the present invention (nutrient for cell growth) (OGN) can strengthen health immune system, smooth away wrinkles, make hair regeneration, prevention of cardiac and apoplexy, bring high blood pressure down, prevent and treat cancer.

Case:

One, tame hardwood academician: (man) age: 93 years old.The what is said or talked about at 93 year old advanced age is lived in the Shanghai Tung Wah hospital always because of serious osteoporosis and vertebral compression fracture, lies in bed 6 months, and the plain tablet of the cell regeneration through taking the embodiment of the invention 1 is after 20 days, and the beginning function exercise of can leaving the bed recovers normal after 30 days.The preparation that other similar case is taken various embodiments of the present invention all obtains similar effect.

Two, Han Changxi professor (man) is 67 years old, and the emergency treatment of apoplexy hypertension is admitted to hospital.Take the embodiment of the invention 1 cell regeneration and left hospital in plain 1 month, do not stay sequela.Nowadays all are normal, and can take care of oneself.The preparation that other similar case is taken various embodiments of the present invention all obtains similar effect.

Three, Li Siqi (woman) suffers from advanced uterine carcinoma, chemicotherapy causes sclerotin pine disease, seek medical advice respectively at Hong Kong, Shanghai, Germany, treatment does not all have positive effect through each side, after take the embodiment of the invention 2 cell regeneration elements, 1 is below the moon, takes care of oneself in 4 months, recover operate as normal half a year, and participate in taking 100 collection TV plays.The preparation that other similar case is taken various embodiments of the present invention all obtains similar effect.

Four, Mr. Zhu (man) is 57 years old, suffers from the cerebral tumor, and is pernicious.Take the cell regeneration element after the operation, check in three months, now all are normal.The preparation that other similar case is taken various embodiments of the present invention all obtains similar effect.

Five, Zhou Ye suffers from lumbar vertebra old disease because of (man) 63 years old, and lumbar vertebra is given prominence to disease, and hemorrhoid body constitution is very weak, and through taking nutrient for cell growth after 3 months, it is normal that body constitution is recovered.Look into now that all are normal, and get back to the preceding 20 years health status of treatment.The preparation that other similar case is taken various embodiments of the present invention all obtains similar effect.

Six, Tang Xiangzhen (woman) cardiovascular disease, heart disease.For many years, by rescuing the psychological treatment product, to hospital, the underwent operative treatment also fails to cure through regular incidence.After taking the cell regeneration element, recover normal, not recurrence over 2 years.The preparation that other similar case is taken various embodiments of the present invention all obtains similar effect.

Such as above-mentioned case, provable curative effect, and illustrate that cell regeneration of the present invention have clinical practice and the scientific value that is of great value.

Claims (8)

1, a kind of nutrient for cell growth, be by Lignum Aquilariae Resinatum 800～900 weight portions, Stigma Croci 860～900 weight portions, the Radix Aucklandiae 600～700 weight portions, Rhizoma Chuanxiong 1500～1600 weight portions, Ramulus Cinnamomi 1500～1600 weight portions, the Radix Angelicae Dahuricae 600～700 weight portions, Radix Notoginseng 450～550 weight portions, Radix Dipsaci 1500～1600 weight portions, Eupolyphaga Seu Steleophaga 1500～1600 weight portions, Rhizoma Drynariae 800～900 weight portions, Radix Achyranthis Bidentatae 600～700 weight portions, Os Gallus domesticus 1600～1700 weight portions, Radix Et Rhizoma Rhei 300～400 weight portions, Pyritum 650～700 weight portions, the effective ingredient that Borneolum Syntheticum 450～550 weight portions, Sanguis Draxonis 3300～3400 weight portion Chinese crude drugs extract is formed, and it is characterized in that relative density is that the extract thick paste mixture of 1.00-1.40 contains following composition:

Sodium 660～7000mg/L

Potassium 3750～5160mg/L

Calcium 830～1350mg/L

Magnesium 815～1400mg/L

Copper is greater than 0,＜0.1mg/L

Manganese 0.4312～0.7mg/L

Zinc 0.0244～0.5mg/L

Ferrum 22～33mg/L

Phosphorus 550～1020mg/L

Cobalt 0.138～0.5mg/L

Sulfur 100～267.2mg/L

Molybdenum is greater than 0,＜0.1mg/L

Boron 6.3～10.256mg/L

Chromium 0.1～0.998mg/L

Total protein: 15000～40000mg/L

Aminoacid: 1～50mg/L

Vitamin: 100～250mg/L

Total sugar: 0.05～0.5mg/L.

2, nutrient for cell growth according to claim 1 is characterized in that described nutrient for cell growth is an acceptable drug preparation on the pharmaceutics.

3, nutrient for cell growth according to claim 2 is characterized in that described pharmaceutical preparation is oral liquid, syrup, soft extract, medicated wine and tincture, capsule, tablet, granule, pill or externally applied ointment.

According to claim 2 or 3 described nutrient for cell growth, it is characterized in that in the described pharmaceutical preparation that 4, the content of nutrient for cell growth is 60-90wt%.

5, nutrient for cell growth according to claim 1 is characterized in that described nutrient for cell growth is the additive of nutriment.

6, nutrient for cell growth according to claim 5 is characterized in that in the described nutriment that the content of nutrient for cell growth is 3.5～6.0wt%.

7, the described nutrient for cell growth of claim 1, its preparation method is: with Radix Angelicae Dahuricae percolation in ethanol, it is standby that percolate removes the thick paste that obtains behind the ethanol; With Lignum Aquilariae Resinatum, Stigma Croci, the Radix Aucklandiae, Rhizoma Chuanxiong, Ramulus Cinnamomi add 2～5 times of water and decoct altogether; With Radix Notoginseng, Radix Dipsaci, Eupolyphaga Seu Steleophaga, Rhizoma Drynariae, Radix Achyranthis Bidentatae, Os Gallus domesticus, Pyritum and Radix Et Rhizoma Rhei decoct 2～5 times in 5～8 times of water altogether, and each 2～8 hours, collect decoction liquor, filter, concentrate; Use ethanol precipitation, abandon precipitate, reclaim ethanol, obtain concentrated solution; Merge said extracted liquid, with Borneolum Syntheticum, Sanguis Draxonis is pulverized, is sieved in the back adding extracting solution.

8, the described nutrient for cell growth of claim 1 is mended the living marrow of bone, improves the serum HGH, is treated the purposes on fracture, traumatic injury, medicine for treating osteoporosis or the health food in preparation.

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