CN109303791A - The new function and purposes of pill of Eight Treasures - Google Patents
The new function and purposes of pill of Eight Treasures Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
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Abstract
The invention belongs to medical research and development technology fields, and in particular to the new function and purposes of pill of Eight Treasures.The present invention is studies have shown that oral pill of Eight Treasures can promote the recovery and regeneration of liver after damage as the treatment means of a kind of promotion liver hepatocyte growth and adjusting immunocyte to fight related liver acute injury caused by hepatotoxicity substance.
Description
Technical field
The invention belongs to medical research and development technology fields, and in particular to the new function and purposes of pill of Eight Treasures.
Background technique
Liver is mostly important one of the organ of human body, is responsible for human body energy and is metabolized, the important function that stable state maintains, together
When also assisted in internal carbohydrate, fat, amino acid, synthesis, storage, decomposition of the important compounds such as vitamin etc. are important
Process.Liver is also the main place for decomposing bodily waste and poisonous and harmful substance simultaneously.The damage of liver, functional disturbance and
The failure of liver function may result in a variety of serious diseases.Studies have reported that, the liver cell of adult has potential according to existing
Splitting ability；However, liver cell generally remains quiescent condition under normal environment in vivo, and without cell division.When
Liver organization can restore rapidly cell division capacity under virus infection or other diseases state by physical damnification, to impaired
Tissue repaired.In mouse partially hepatectomized experimental model, after the liver organization operation excision of 2/3rds volumes,
Liver cell can make liver organization be restored to approximate normal volume size in week age by quick cell Proliferation.According to
Report, some growth factors such as such as HGF, IL-6 and cell factor can the hepatotoxic compounds such as carbon tetrachloride induce or
It plays a protective role during a variety of hepar damnifications that Fas correlation mediates.And liver stem cells are also reported and have participated in liver damage
After wound in the repair process of liver cell.However, recent is some studies have shown that liver stem cells and progenitor cells are in liver regeneration
Not necessarily factor, most of new life liver cell are not originating from stem cell or progenitor cells in the process, but by original position
Adult hepatic cell restore splitting ability expanded.
Pill of Eight Treasures is a kind of time-honored famous Chinese medicine, caused by south China and south east asia are widely used in damp-heat accumulation
Fever, jaundice and spreading venereal diseases toxicity hepatitis, acute cholecystitis etc..Pill of Eight Treasures belongs to national Chinese medicine protection kind,
It include: eight kinds of rare traditional Chinese medicines such as cow-bezoar, snake gall, antelope's horn, pearl, Radix Notoginseng, Moschus in the main component of announcement.Although eight
Baodan has had over one hundred year applicating history, however the curative mechanism of its cellular level is but and not well known so far.
Summary of the invention
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide the new function of pill of Eight Treasures and use
On the way.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
The first aspect of the present invention provides pill of Eight Treasures in preparation treatment hepatic injury and/or the drug for promoting liver reparation
New application.
Further, the drug at least has effects that any one of following or multinomial:
(1) glutamic-pyruvic transaminase is reduced；(2) glutamic-oxalacetic transaminease is reduced；(3) liver cell number downright bad in liver organization is reduced
Amount；(4) promote hepatocyte growth；(5) inhibit the growth of liver cancer cells；(6) Th1 cell is promoted to generate.
The drug necessarily includes pill of Eight Treasures, and take pill of Eight Treasures as the effective component of aforementioned effect.
Further, pill of Eight Treasures is one of effective component of the drug or sole active ingredient.
The form of the drug can be the various substance shapes such as solid, liquid, gel, semi-fluid, aerosol without specifically limited
Formula.
Further, the drug can be oral drugs.
The drug mainly for object be mammal, such as rodent, primate.
The second aspect of the present invention, provide pill of Eight Treasures has effects that in any one of following or multinomial drug in preparation
Purposes:
(1) glutamic-pyruvic transaminase is reduced；(2) glutamic-oxalacetic transaminease is reduced；(3) liver cell number downright bad in liver organization is reduced
Amount；(4) promote hepatocyte growth；(5) inhibit the growth of liver cancer cells；(6) Th1 cell is promoted to generate.
The drug necessarily includes pill of Eight Treasures, and take pill of Eight Treasures as the effective component of aforementioned effect.
Further, pill of Eight Treasures is one of effective component of the drug or sole active ingredient.
The form of the drug can be the various substance shapes such as solid, liquid, gel, semi-fluid, aerosol without specifically limited
Formula.
Further, the drug can be oral drugs.
The drug mainly for object be mammal, such as rodent, primate.
The third aspect of the present invention provides a kind of method treated hepatic injury and/or promote liver reparation, comprising steps of
Pill of Eight Treasures is applied to effective object.
The object is mammal, and the mammal is preferably that rodent, artiodactylous animals, Perissodactyla are dynamic
Object, Lagomorph, primate, rodent etc..The primate is preferably monkey, ape or people.It is described to nibble
Tooth class animal can be mouse.
It is described to impose on the effective object and impose on work specifically by oral mode in some embodiments of the present invention
Use object.
The administration dosage of the pill of Eight Treasures is in the range of those skilled in the art and known doctor.
The fourth aspect of the present invention provides a kind of drug treated hepatic injury and/or promote liver reparation, the drug packet
Include a effective amount of pill of Eight Treasures.
Further, the pill of Eight Treasures is one of effective component of the drug or sole active ingredient.
Further, the drug further includes pharmaceutically acceptable carrier.
Further, the drug can be oral drugs.
As described above, the pill of Eight Treasures in the present invention is the same as pill of Eight Treasures (pastille) documented in Chinese Pharmacopoeia or pill of Eight Treasures
Capsule.
Further, the composition of the pill of Eight Treasures includes cow-bezoar, snake gall, antelope's horn, pearl, Radix Notoginseng, Moschus.
Further, the dosage form of the pill of Eight Treasures is pastille or capsule.
That is: the pill of Eight Treasures be include cow-bezoar, snake gall, antelope's horn, pearl, Radix Notoginseng, Moschus flavour of a drug processed be made
Pastille or capsule.
Further, the pill of Eight Treasures (pastille) is that class flat ellipse is blocky；Surface yellowish-brown or grey sepia, section are in
Sepia.Gas micro-perfume, bitter, micro-sweet.
Further, in pill of Eight Treasures (pastille) described in every 1g, containing Radix Notoginseng with ginsenoside Rg1 (C42H72O14), ginsenoside
Rb1(C54H92O23) and notoginsenoside R (C47H80O18) total amount meter, no less than 33.0mg.
It is listed in some embodiments of the present invention, the dosage form of the pill of Eight Treasures can be pastille, the eight treasures (choice ingredients of certain special dishes)
Pellet can be the pill of Eight Treasures (pastille) of Xiamen Chinese Medicine Co., Ltd's production, and the authentication code of the pill of Eight Treasures (pastille) is traditional Chinese medicines
Quasi- word Z10940005.
Containing Radix Notoginseng with ginsenoside Rg1 (C42H72O14), ginsenoside Rb1 in pill of Eight Treasures described in every 1g (capsule)
(C54H92O23) and the total amount meter of notoginsenoside R (C47H80O18) 10.0mg must not, be less than.
It is listed in some embodiments of the present invention, the dosage form of the pill of Eight Treasures can be capsule, e.g. tall building
The eight treasures (choice ingredients of certain special dishes) capsule of door Chinese medicine Co., Ltd., Factory production, the national drug standard of the eight treasures (choice ingredients of certain special dishes) capsule is Z10940006.
For convenience in the experiment for carrying out cellular level and animal level in some embodiments of the present invention, using pill of Eight Treasures
The pill of Eight Treasures extract obtained after Extraction solvent extracts is tested, and the usage of oral pill of Eight Treasures is simulated.
The Extraction solvent is not particularly limited as long as not damaging effect of the invention, can be water or alcohol.
The extracting method can be conventional water extraction or alcohol extracting method.
The pill of Eight Treasures extract is listed in some embodiments of the present invention, can be pill of Eight Treasures water extract.
The pill of Eight Treasures water extract is that pill of Eight Treasures obtains after water is extracted as Extraction solvent.
Comprising steps of pill of Eight Treasures is added water, ultrasonic extraction, boiling water reflux mentions the extracting method of the pill of Eight Treasures water extract
It takes, merges Aqueous extracts, be separated by solid-liquid separation, obtain powder, as pill of Eight Treasures water extract.
Compared with prior art, the invention has the following beneficial effects:
It was found that oral pill of Eight Treasures can greatly alleviate the chmice acute hepar damnification by tetrachloro-methane induction, add
Liver organization regeneration after speed damage.In vitro and in vivo in experiment, pill of Eight Treasures can promote the proliferation of source of people inducing hepatocyte,
Especially under the cachexia similar with after damage, pill of Eight Treasures promotees the effect that stem cell increases and becomes apparent；And to liver cancer phase
Closing cell does not have proliferation.Simultaneously it has been found that pill of Eight Treasures being capable of specific lymphatic organ in a degree of promotion Mice Body
The generation of Th1 T helper cell subset in official, and the differentiation efficiency of source of people Th1 T helper cell is improved in vitro.Ours grinds
Study carefully show oral pill of Eight Treasures can as it is a kind of promotion liver hepatocyte growth and adjust immunocyte treatment means, come pair
Related liver acute injury caused by anti-hepatotoxicity substance promotes the recovery and regeneration of liver after damage.
Detailed description of the invention
Figure 1A: HPLC 6 different batches pill of Eight Treasures finger-prints of detection compare the similarity of chemical composition between different batches.
Figure 1B: intragastric administration on mice gives pill of Eight Treasures third turn of ammonia of paddy in different time detection mice serum after carbon tetrachloride modeling
Enzyme, only, quantitative result is expressed as average value ± standard error to every group of n=5；*P<0.05。
Fig. 1 C: after carbon tetrachloride modeling intragastric administration on mice give pill of Eight Treasures different time detection mice serum in millet straw turn ammonia
Enzyme, only, quantitative result is expressed as average value ± standard error to every group of n=5；*P<0.05。
Different dosing processing mouse liver degree of impairment is compared in Fig. 1 D:HE dyeing；20 times of amplification picture scales are 100 microns
40 times of amplification picture scales are 50 microns.
Fig. 2A: to people's inducing hepatocyte, proliferative effect detects the processing of pill of Eight Treasures water extract in complete medium for 24 hours, fixed
Amount result is expressed as average value ± standard error；*P < 0.05,**P<0.01。
Fig. 2 B: pill of Eight Treasures water extract handles 48h, and to people's inducing hepatocyte, the proliferative effect in complete medium is detected, fixed
Amount result is expressed as average value ± standard error；*P < 0.05,**P<0.01。
Fig. 2 C: pill of Eight Treasures water extract handles 24 hours and is proliferated detection under cachexia to people's inducing hepatocyte, fixed
Amount result is expressed as average value ± standard error；*P < 0.05,**P<0.01。
Fig. 2 D: pill of Eight Treasures water extract handles 48 hours and is proliferated detection under cachexia to people's inducing hepatocyte, fixed
Amount result is expressed as average value ± standard error；*P < 0.05,**P<0.01。
Fig. 3 A: (1% fetal calf serum) pill of Eight Treasures influences MHCC97 cell Proliferation under low nutrition condition of culture, quantitative result
It is expressed as average value ± standard error；*P < 0.05,**P < 0.01,***P<0.01。
Fig. 3 B: (10% fetal calf serum) pill of Eight Treasures is influenced on MHCC97 cell Proliferation under regular culture conditions, quantitative to tie
Fruit is expressed as average value ± standard error；*P < 0.05,**P < 0.01,***P<0.01。
Fig. 3 C: (1% fetal calf serum) pill of Eight Treasures influences PLC-PRF cell Proliferation under low nutrition condition of culture, quantitative to tie
Fruit is expressed as average value ± standard error；*P < 0.05,**P < 0.01,***P<0.01。
Fig. 3 D: (10% fetal calf serum) pill of Eight Treasures influences PLC-PRF cell Proliferation under normal nutrition condition of culture, quantitative
As a result it is expressed as average value ± standard error；*P < 0.05,**P < 0.01,***P<0.01。
Fig. 4 A: wild type C57BL/6 mouse gives pill of Eight Treasures extract 1 week by stomach-filling, detects in spleen and respectively T is assisted to drench
The variation of bar cell subsets, quantitative result are expressed as average value ± standard error；*P<0.05。
Fig. 4 B: wild type C57BL/6 mouse gives pill of Eight Treasures extract 1 week by stomach-filling, detects in lymph node and respectively assists T
Lymphocyte subgroup variation, quantitative result are expressed as average value ± standard error；*P<0.05。
Fig. 4 C: source of people CD4 detects Th1 helper T lymphocyte ratio after the positive CD25 negative T cell vitro differentiation 3 days, fixed
Amount result is expressed as average value ± standard error；*P<0.05。
Fig. 4 D: source of people CD4 detects Th1 helper T lymphocyte statistics after the positive CD25 negative T cell vitro differentiation 3 days, fixed
Amount result is expressed as average value ± standard error；*P<0.05。
Specific embodiment
Pill of Eight Treasures
The dosage form of pill of Eight Treasures can be pastille or capsule etc..
According to the record of Chinese Pharmacopoeia, pill of Eight Treasures (pastille) is the medicines such as cow-bezoar, snake gall, antelope's horn, pearl, Radix Notoginseng, Moschus
Pastille made of taste is processed.
Character: the pill of Eight Treasures (pastille) is that class flat ellipse is blocky；Surface yellowish-brown or grey sepia, section is in brown
Color.Gas micro-perfume, bitter, micro-sweet.
Identify:
(1) take this product finely ground, set microscopically observation: irregular fragment is colourless, translucent, glossy, sometimes visible thin
Close wavy grain (pearl).
(2) take this product appropriate, it is finely ground, 0.5g is taken, is set in tool plug centrifuge tube, is added water about 5 to drip, stir evenly, add water saturation
N-butanol 3ml, close plug shake 10 minutes, place 2 hours, and centrifugation takes supernatant, and 3 times of water amounts for adding n-butanol to be saturated shake up,
Placement makes to be layered, and takes n-butanol liquid, recycling design is to doing, and residue adds methanol 0.5ml to make to dissolve, as test solution.Separately take
Ginsenoside Rg1's reference substance, ginsenoside Rb1's reference substance and notoginsenoside R reference substance add methanol that every 1ml is made and contain 1mg
Mixed solution, as reference substance solution.It tests, draws above-mentioned according to thin-layered chromatography (Chinese Pharmacopoeia version general rule 0502 in 2015)
Each 2 μ l of two kinds of solution is put respectively on same high-efficient silica gel G lamellae, with n-butanol-ethyl acetate-water (4:1:5) upper layer
Solution is solvent, is unfolded, and takes out, dries, and with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C for spray,
It is inspected under daylight.In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, the spot of same color is shown.
(3) take this product appropriate, it is finely ground, 3g is taken, add diethyl ether 15ml, soaked overnight, and filtration, filtrate volatilizes, and residue adds anhydrous
Ethyl alcohol 1ml makes to dissolve, and takes supernatant as test solution.Muskone reference substance separately is taken, adds dehydrated alcohol that every 1ml is made and contains
The solution of 0.5mg, as reference substance solution.It is tested according to gas chromatography (Chinese Pharmacopoeia version general rule 0521 in 2015), with phenyl
(50%) methyl silicone (OV-17) is stationary phase, coating concentration 1.5%；160 DEG C of column temperature.Draw each 2 μ of above two solution
L injects gas chromatograph.Chromatographic peak corresponding with chromatographic peak retention time of reference substance should be presented in sample chromatogram.
It checks:
Loss on drying: 12.0% (Chinese Pharmacopoeia version general rule 0831 in 2015) must not be crossed.
Other should meet related every regulation (Chinese Pharmacopoeia version general rule 0182 in 2015) under pastille item.
Assay:
It is measured according to high performance liquid chromatography (Chinese Pharmacopoeia version general rule 0512 in 2015).
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase
A, using water as Mobile phase B, the regulation according to the form below carries out gradient elution；Detection wavelength is 203nm.Number of theoretical plate presses notoginsenoside
The peak R1, which calculates, should be not less than 4000.
Time (minute) mobile phase A (%) Mobile phase B (%)
0~12 19 81
12~60 19 → 36 81 → 64
The preparation of reference substance solution: ginsenoside Rg1's reference substance, ginsenoside Rb1's reference substance and notoginsenoside R pair are taken
It is appropriate according to product, it is accurately weighed, add methanol that every lml 0.4mg containing ginsenoside Rg1, ginsenoside Rb1 0.4mg, Radix Notoginseng soap is made
The mixed solution of glycosides R1 0.1mg to get.
The preparation of test solution: taking this product appropriate, finely ground, takes about 0.4g, accurately weighed, sets in stuffed conical flask, essence
Close addition methanol 25ml, close plug, weighed weight are stood overnight, and ultrasonic treatment (power 500W, frequency 45kHz) 1 hour is let cool,
Weighed weight again is supplied the weight of less loss with methanol, is shaken up, filtration, take subsequent filtrate to get.
Measuring method: accurate absorption reference substance solution and each 10 μ l of test solution respectively, injection liquid chromatograph, measurement,
To obtain the final product.The every 1g of this product is containing Radix Notoginseng with ginsenoside Rg1 (C42H72O14), ginsenoside Rb1 (C54H92O23) and notoginsenoside R
(C47H80O18) total amount meter, 33.0mg must not be less than.
It is listed in some embodiments of the present invention, the dosage form of the pill of Eight Treasures can be with pastille, e.g. Xiamen
The pill of Eight Treasures (pastille) of Chinese medicine Co., Ltd., Factory production, the authentication code of the pill of Eight Treasures (pastille) is national drug standard
Z10940005。
According to the record of Chinese Pharmacopoeia, eight treasures (choice ingredients of certain special dishes) capsule is the flavour of a drug such as cow-bezoar, snake gall, antelope's horn, pearl, Radix Notoginseng, Moschus
Capsule made of processed.
Character: this product is hard capsule, and content is the powder of yellowish-brown or grey sepia；Gas is fragrant, bitter, micro-sweet.
Identify:
(1) take this product finely ground, set microscopically observation: irregular fragment is colourless, translucent, glossy, sometimes visible thin
Close wavy grain (pearl).
(2) this product 0.5g is taken, sets in tool plug centrifuge tube, adds water about 5 to drip, stir evenly, add water the n-butanol 3ml of saturation, it is close
Plug shakes 10 minutes, places 2 hours, and centrifugation takes supernatant, and 3 times of water amounts for adding n-butanol to be saturated shake up, and placement makes to be layered,
N-butanol liquid is taken, recycling design is to doing, and residue adds methanol 0.5ml to make to dissolve, as test solution.Separately take ginsenoside Rg1
Reference substance, ginsenoside Rb1's reference substance and notoginsenoside R reference substance add methanol every 1ml to be made respectively containing the mixed solution of 1mg,
As reference substance solution.It is tested according to thin-layered chromatography (Chinese Pharmacopoeia version general rule 0502 in 2015), draws above two solution each 2
μ l puts respectively on same high-efficient silica gel G lamellae, is expansion with n-butanol-ethyl acetate-water (4:1:5) upper solution
Agent is unfolded, and takes out, dries, and with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C for spray, examines in the sunlight
Depending on.In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, the spot of same color is shown.
(3) take this product appropriate, it is finely ground, 3g is taken, add diethyl ether 15ml, soaked overnight, and filtration, filtrate volatilizes, and residue adds anhydrous
Ethyl alcohol 1ml makes to dissolve, and takes supernatant as test solution.Muskone reference substance separately is taken, adds dehydrated alcohol that every 1ml is made and contains
The solution of 0.5mg, as reference substance solution.It is tested according to gas chromatography (Chinese Pharmacopoeia version general rule 0521 in 2015), with phenyl
(50%) methyl silicone (OV-17) is stationary phase, coating concentration 1.5%；160 DEG C of column temperature.Draw each 2 μ of above two solution
L injects gas chromatograph.Chromatographic peak corresponding with chromatographic peak retention time of reference substance should be presented in sample chromatogram.
It checks:
Loss on drying: 12.0% (Chinese Pharmacopoeia version general rule 0831 in 2015) must not be crossed.
Other should meet related every regulation (Chinese Pharmacopoeia version general rule 0103 in 2015) under pastille item.
Assay:
It is measured according to high performance liquid chromatography (Chinese Pharmacopoeia version general rule 0512 in 2015).
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase
A, using water as Mobile phase B, the regulation according to the form below carries out gradient elution；Detection wavelength is 203nm.Number of theoretical plate presses notoginsenoside
The peak R1, which calculates, should be not less than 4000.
Time (minute) mobile phase A (%) Mobile phase B (%)
0~12 19 81
12~60 19 → 36 81 → 64
The preparation of reference substance solution: ginsenoside Rg1's reference substance, ginsenoside Rb1's reference substance and notoginsenoside R pair are taken
It is appropriate according to product, it is accurately weighed, add methanol that every lml 0.4mg containing ginsenoside Rg1, ginsenoside Rb1 0.4mg, Radix Notoginseng soap is made
The mixed solution of glycosides R1 0.1mg to get.
The preparation of test solution: taking this product appropriate, finely ground, takes about 0.4g, accurately weighed, sets in stuffed conical flask, essence
Close addition methanol 25ml, close plug, weighed weight are stood overnight, and ultrasonic treatment (power 500W, frequency 45kHz) 1 hour is let cool,
Weighed weight again is supplied the weight of less loss with methanol, is shaken up, filtration, take subsequent filtrate to get.
Measuring method: accurate absorption reference substance solution and each 10 μ l of test solution respectively, injection liquid chromatograph, measurement,
To obtain the final product.
The every 1g of this product is containing Radix Notoginseng with ginsenoside Rg1 (C42H72O14), ginsenoside Rb1 (C54H92O23) and Radix Notoginseng soap
The total amount meter of glycosides R1 (C47H80O18) must not be less than 10.0mg.
It is listed in some embodiments of the present invention, the dosage form of the pill of Eight Treasures can be capsule, e.g. tall building
The eight treasures (choice ingredients of certain special dishes) capsule of door Chinese medicine Co., Ltd., Factory production, the national drug standard of the eight treasures (choice ingredients of certain special dishes) capsule is Z10940006.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or according to condition proposed by each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Domain conventional technique.These technologies have perfect explanation in the prior art.
Embodiment 1
We are convenient for the experiment of cellular level and animal level in present invention implementation, are extracted using pill of Eight Treasures by water
Pill of Eight Treasures water extract is obtained afterwards to be tested, and the usage of oral pill of Eight Treasures is simulated.
One, the preparation of pill of Eight Treasures water extract
The powder 5.067g of the pill of Eight Treasures (pastille) or pill of Eight Treasures capsule 's content that take Xiamen Chinese Medicine Co., Ltd to produce,
Add 10 times of volume of water, ultrasonic extraction 30 minutes, boiling water refluxing extraction 1 hour, be repeated once, merges Aqueous extracts, after concentration,
6000rpm is centrifuged 10 minutes, takes supernatant to be freeze-dried to obtain 2.739 grams of powder, yield about 54.1%.
Two, acute liver modeling and administration
8 week old Male wild-type C57BL/6 mouse are bought by Shanghai Experimental Animal Center, and follow Chinese Academy of Sciences Shanghai
Biochemistry and the raising of Institute of Cell Biology relevant regulations and instruction are in specific pathogen free microbial environment.By four
Chlorination carbon is uniformly mixed by 5% concentration with olive oil, by 10ml/kg mouse weight, by being injected intraperitoneally in Mice Body；Control
Olive oil is injected intraperitoneally by 10ml/kg mouse weight in group.Mouse is given by 0.3g/kg dosage by stomach-filling immediately after injection modeling
Give pill of Eight Treasures water extract or water, every 24 hours later by same dose by stomach-filling give corresponding pill of Eight Treasures water extract or
Water, and be administered continuously three days.Respectively 24 hours after mouse modeling, acquire corresponding mouse peripheral blood within 48 hours, 72 hours, pass through
Centrifugation obtains serum and detects the enzymatic activity of glutamic-pyruvic transaminase therein (ALT) and glutamic-oxalacetic transaminease (AST).
Mouse is put to death after carbon tetrachloride modeling 72 hours, wins corresponding mouse liver tissue, it is molten in 10% formalin
12 hours are fixed in liquid and enter organization embedding in paraffin.The histotomy of 5 mm of thickness is carried out to the liver organization of embedding, and
HE dyeing is carried out with hematoxylin, Yihong dyestuff.Microscopically observation each group mouse liver tissue damage and recovery situation are simultaneously taken pictures
Record.
Three, source of people inducing hepatocyte (hiHep) proliferation experiment
Type i collagen (CollagenI) aqueous solution for being 27ug/ml with concentration carries out coating processing to tissue culture plate, place
Culture plate after reason is spare after natural drying.Source of people inducing hepatocyte in containing HMMsupplement, B27supplement,
ITS, 40ug/ml EGF, 40ug/ml TNF-a are cultivated and are passed in the DMEM/F12 complete medium of dexamethasone.
Source of people inducing hepatocyte is digested and carries out cell count and is resuspended to every milliliter of 1*10^5 cell, by every hole 50ul
(5000 cells) is inoculated into 96 good porocyte culture plates of handle before happening, and 50ul is added and contains respective concentration (final concentration point
Wei 0.3,1,3,10,30,100ug/ml) in the culture solution of pill of Eight Treasures water extract in 37 DEG C, 5%CO2Under the conditions of cultivate.In
Agent-feeding treatment collects cell after 24 hours, 48 hours, and uses PromegaCellTiter-
GloLuminescentCellViability kit detects corresponding ATP content.It is complete in above-mentioned complete medium and 10% respectively
It is cultivated in the low nutrition culture medium that full culture medium adds 90%DMEM/F12 to prepare and carries out corresponding pill of Eight Treasures to proliferative effect reality
It tests.
Four, hepatoma cell proliferation is tested
This experiment detects pill of Eight Treasures water extract to source of people metastatic hepatic carcinoma MHCC97 cell line and source of people liver cancer PLC-PRF
The influence of cell line proliferation, method are as described below: MHCC97 and PLC-PRF cell is in the DMEM/ containing 10% fetal calf serum
It cultivates and passes in F12 culture medium.With respective concentration (final concentration is respectively 1,3,10,30,100,300,1000,3000ug/
Ml) pill of Eight Treasures extract-treated cell utilizes PromegaCellTiter- in 24 hours, 48 hours, 72 hours
GloLuminescent CellViability kit detects above two liver cancer cells respectively and ties up to containing 10% fetal calf serum
DMEM/F12 complete medium or containing the ATP content in 1% fetal calf serum DMEM/F12 low nutrition culture medium, and it is indicated with this
Proliferative conditions.
Five, mouse administration and Mice Body in CD4 positive helper T cells subsets counts
8 week old wild type C57BL/6 mouse press 0.25g/kg, 0.5g/kg, 1g/kg weight by the method for stomach-filling respectively
Dosage gives pill of Eight Treasures water extract, once a day, continues one week.After one week of dosing, put to death mouse and separating mouse spleen and
Inguinal lymph nodes.Mouse spleen and lymph node cell suspensions are prepared by 40 micron screen grindings, with 50ng/
Mlphorbol12-myristate 13-acetate (PMA), 1uMionomycin are in golgiosome blocking agent BrefeldinA
In the presence of stimulate 4 hours.Post-stimulatory cell is first marked with anti-CD 4 antibodies, uses BDFixation/ later
Permeabilization kit is fixed and permeabilized cells are marked with IFN-g antibody label Th1 cell with IL-17A antibody
Th17 cell marks regulatory T cells (Treg) with Foxp3 antibody.Cell after dyeing uses flow cytomery after being resuspended
Wherein Th1, Th17, Treg cell content.
Six, the separation of CD4 positive CD25 negative T-lymphocytes and the differentiation of Th1 helper T lymphocyte in human peripheral
Healthy adult human peripheral is acquired with EDTA anticoagulant tube, removes blood plasma after centrifugation.Haemocyte is 1.077g/ with density
The Hitopaque 1077 (Sigma) of ml separates mononuclearcell therein by density gradient centrifugation and is resuspended in RPMI1640
It is spare to train liquid.
With CD4TCellIsolation, human (Miltenyi, 130-096-533) kit yin selects isolated CD4
Positive t lymphocytes.CD25MicroBeadsII is used later, and human (Miltenyi, 130-092-983) kit separates
To CD4 positive CD25 negative T-lymphocytes.
CD4 positive CD25 negative T-lymphocytes are planted by 500,000 cell per wells in 1ug/ml anti-cd 3 antibodies and 1ug/ml
In processed 96 porocyte culture plates of anti-CD28 antibody packet, with interleukin 12 containing 40ng/ml (IL-12), 10% tire ox
The RPMI1640 training liquid of serum is cultivated under the conditions of 5%CO2 at 37 DEG C.And 0.03mg/ml is added simultaneously, 0.1mg/ml, 0.3mg/
The differentiation of ml pill of Eight Treasures water extract collects cell after culture 4 days.Cell after differentiation is with PMA, Ionomycin in BrefeldinA
In the presence of stimulate 4 hours；It is fixed with BDFixation/Permeabilization kit, permeabilized cells, with anti-CD 4 antibodies,
It wherein Th1 helper T lymphocyte quantity and is counted after anti-IFN-g antibody labeled cells with flow cytomery.
Seven, data statistics and analysis
All experimental datas are expressed as mean+/-standard error.It is compared between different disposal group using t inspection.It is more
It is analyzed between group result using one-wayANOVA, and carries out post-hoc tests with Bonferronitest.Recognize when P < 0.05
There is significant difference between group.
Eight, experimental result
1. the acute liver of pill of Eight Treasures alleviation tetrachloro-methane induction
Firstly, we have detected the similarity of compound composition between pill of Eight Treasures different batches by HPLC.By to 6 batches
Secondary pill of Eight Treasures finger-print compares, and similarity is very high between showing pill of Eight Treasures different batches, and chemical composition stablizes (Figure 1A).
Carbon tetrachloride is widely used in the hepatotoxicity compound of acute liver damage modeling.We are in tetrachloro-methane induction
By the characteristic index glutamic-pyruvic transaminase of detection hepatic injury and glutamic-oxalacetic transaminease activity on acute liver model, compare
The degree of pill of Eight Treasures administration group and control group hepar damnification and reparation.By detection we have found that control group paddy after modeling 24 hours
Pyruvic transaminase is sharply increased with glutamic-oxalacetic transaminease, shows that hepar damnification is serious；And pill of Eight Treasures administration group is compared with third turn of ammonia of control group paddy
Enzyme, glutamic-oxalacetic transaminease decreased significantly, and illustrate that administration group mouse hepar damnification after modeling 24 hours gentlier restores very fast (figure
1B and 1C).Meanwhile comparing administration group and control group liver organization HE coloration result, it is also the same to show pill of Eight Treasures administration group liver
Downright bad liver cell quantity is considerably less than non-administered group (Fig. 1 D) in tissue.The experimental results showed that, given after acute liver damage above
Therapeutic effect to damage can be played by giving pill of Eight Treasures, and the recovery of liver organization can be accelerated to regenerate.
2. pill of Eight Treasures promotes source of people induced liver cell (hiHep) proliferation
In above-mentioned experimental result, it has been found that pill of Eight Treasures can play therapeutic effect to acute hepatic.Source of people induces liver
Dirty cell is that reported reprogrammed by fibroblast conversion obtains people's liver cell, and it is thin that document before is proved this
Born of the same parents have the cell functions identical with normal liver cell such as cytochrome P 450 enzyme activity, removing toxic substances.And according to existing document
The proliferation of report liver cell is the most critical process restored after acute liver damage, therefore we utilize source of people induced liver cell
To have detected influence of the pill of Eight Treasures to hepatocyte growth.By experiment we have found that pill of Eight Treasures handles 24 hours, 48 hours equal energy
Enough it is obviously promoted the proliferation (Fig. 2A and 2B) of liver cell.Further we have found that under sub-nutrition environment, pill of Eight Treasures promotes liver
The ability of cell Proliferation becomes apparent (Fig. 2 C and 2D)；The result shows pill of Eight Treasures in the disease that is similarly low nutrition or toxic
Hepatic injury position caused by substance has extremely strong promotion proliferative capacity, can accelerate the regeneration and reparation of liver organization.
3. pill of Eight Treasures does not promote hepatoma cell proliferation
Next we carry out pill of Eight Treasures dosing by the cell line to the two plants of human liver cancer sources MHCC97-H and PLC-PRF
Processing, has detected the specificity of pill of Eight Treasures proliferation.By comparing cell after dosing 24 hours, 48 hours, 72 hours
Quantity, it has been found that: no matter (the 1% tire ox under (10% fetal calf serum) or low nutrition condition of culture under regular culture conditions
Serum) pill of Eight Treasures will not all promote liver cancer cell growth (Fig. 3 A, Fig. 3 B).The result shows pill of Eight Treasures to normal liver cell
Proliferation is specific.Further, it has been found that liver cancer cells, there are one for the pill of Eight Treasures processing of higher concentration
Determine the growth inhibition effect (Fig. 3 C, Fig. 3 D) of degree.Based on the above results, illustrate that pill of Eight Treasures is a kind of potential promotion well
Reparation and regenerated drug after hepar damnification, and potential cancer in regenerative process will not be caused to occur.
4. pill of Eight Treasures promotes Th1 cell to generate
Th1 helper T lymphocyte and the reaction of its related inflammation and secreted cell factor such as IFN-g and TNF-a
Deng also playing a significant role in tissue regeneration processes.It has been reported that, group after these immune responses can promote liver acute to damage
The regenerative process knitted.We have carried out one-week pill of Eight Treasures extract gastric infusion to normal adult mice and have tested, experiment
We have detected the variation of each helper T lymphocyte subgroup in Mouse Spleen and lymph node afterwards.We can be with from result
See, although each helper T lymphocyte does not have significant difference between administration group and control group in spleen；But in lymph node
Th1 T helper cell has a degree of raising.
In order to further verify ours as a result, we utilize the CD4 positive CD25 negative T cell separated in human peripheral
External helper T lymphocyte Analytical Chemical Experiment is carried out, and gives pill of Eight Treasures extract-treated simultaneously and detects its effect.Experimental result card
Bright, pill of Eight Treasures extract can actually promote the differentiation of source of people Th1 helper T lymphocyte, promote the generation of Th1 cell.This
As a result illustrate, pill of Eight Treasures in addition to can isomorphism reinforce human liver cell proliferation other than treating acute liver damage, can also pass through adjusting
The intracorporal immunocyte of people to promote hepatic tissue reparation from another angle.
Nine, it discusses
We start with from the animal model that acute liver damage is treated, it was found that traditional Chinese medicine pill of Eight Treasures can effectively be alleviated
Acute liver damage simultaneously accelerates the hepatic tissue reparation after damage.We have found that pill of Eight Treasures can promote people liver thin in further research
The proliferation of born of the same parents, especially under low nutrition state；Without causing liver tumor cells to be proliferated；And can be immune from adjusting, promote
The repair process organized after the angle auxiliary hepatic injury that Th1 cell generates.To sum up, it is believed that pill of Eight Treasures can pass through hepatocyte
It is proliferated and multiple angles such as adjusting is immune treats hepatic injuries, one kind can be developed into and potentially treat hepatic injury and liver is promoted to repair
Multiple drug.
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that under the premise of not departing from the method for the present invention, can also be made for those skilled in the art
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are equivalent embodiment of the invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical solution of the present invention
It is interior.
Claims (10)
1. purposes of the pill of Eight Treasures in preparation treatment hepatic injury and/or the drug for promoting liver reparation.
2. purposes according to claim 1, which is characterized in that the drug at least has any one of following or multinomial function
Effect:
(1) glutamic-pyruvic transaminase is reduced；(2) glutamic-oxalacetic transaminease is reduced；(3) liver cell quantity downright bad in liver organization is reduced；(4)
Promote hepatocyte growth；(5) inhibit the growth of liver cancer cells；(6) Th1 cell is promoted to generate.
3. purposes according to claim 1, which is characterized in that pill of Eight Treasures is for one of effective component of the drug or uniquely
Effective component.
4. purposes according to claim 1, which is characterized in that the drug is oral drugs.
5. purposes according to claim 1, which is characterized in that the composition of the pill of Eight Treasures includes cow-bezoar, snake gall, antelope
Angle, pearl, Radix Notoginseng, Moschus.
6. purposes according to claim 1, which is characterized in that the dosage form of the pill of Eight Treasures is pastille or capsule.
7. pill of Eight Treasures has effects that the purposes in any one of following or multinomial drug in preparation:
(1) glutamic-pyruvic transaminase is reduced；(2) glutamic-oxalacetic transaminease is reduced；(3) liver cell quantity downright bad in liver organization is reduced；(4)
Promote hepatocyte growth；(5) inhibit the growth of liver cancer cells；(6) Th1 cell is promoted to generate.
8. a kind of method treated hepatic injury and/or promote liver reparation, comprising steps of pill of Eight Treasures is applied to effective object.
9. a kind of drug treated hepatic injury and/or promote liver reparation, the drug includes a effective amount of pill of Eight Treasures.
10. drug according to claim 9, which is characterized in that the pill of Eight Treasures is one of the effective component of the drug
Or sole active ingredient.
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| CN114252511A (en) * | 2020-09-21 | 2022-03-29 | 厦门中药厂有限公司 | Method for detecting fingerprint of Babaodan, standard fingerprint of Babaodan and application |
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| CN111728990A (en) * | 2019-12-23 | 2020-10-02 | 南京大学 | Application of Babaodan in preparing medicine for preventing and treating liver cancer |
| CN114252511A (en) * | 2020-09-21 | 2022-03-29 | 厦门中药厂有限公司 | Method for detecting fingerprint of Babaodan, standard fingerprint of Babaodan and application |
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