CN108524685A - A kind of composition that treating children's herpangina and its application - Google Patents
A kind of composition that treating children's herpangina and its application Download PDFInfo
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- CN108524685A CN108524685A CN201810711558.9A CN201810711558A CN108524685A CN 108524685 A CN108524685 A CN 108524685A CN 201810711558 A CN201810711558 A CN 201810711558A CN 108524685 A CN108524685 A CN 108524685A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/85—Verbenaceae (Verbena family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/04—Drugs for disorders of the respiratory system for throat disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention belongs to the field of Chinese medicines, a kind of composition with treatment children's herpangina effect is specifically provided, the composition in parts by weight, is made of 40 50 parts of callicarpa nudiflora general flavones, 10 20 parts of callicarpa nudiflora total phenolics, the total benzyl carbinol glycosides of 20 30 parts of nakedflower purple strains and 26 parts of callicarpa nudiflora polysaccharide.The invention also discloses the applications of the composition.The present invention extracts from callicarpa nudiflora, definite ingredients using four kinds of components, cooperates with and plays a role between each component, has the effect of good treatment and/or pre- child-resistant herpangina.
Description
Technical field
The present invention relates to a kind of beautyberry extract composition and its applications, and in particular to one kind having treatment children's blister
The beautyberry extract composition of rash angina effect and its application.
Background technology
Herpangina is acute characterized by febris acuta and isthmus faucium portion bleb ulcer caused by enterovirus
Infectiousness angina, using excrement-mouth or respiratory tract as major transmission path, infectiousness is very strong, propagates soon, various regions all over the world are in
It distributes or popular, summer and autumn is season occurred frequently, mainly invades 1~7 years old children.
Clinic is to generate heat, pharyngalgia, isthmus faucium portion mucous membrane exanthema vesiculosum and superficial ulcer are main performance.Herpangina patient
And subclinical infection person is the major source of infection of this disease.Can directly via enteron aisle, respiratory infectious, also can indirectly the hand through pollution,
The propagation such as food, clothes, apparatus.Each age group crowd can be infected, but multiple with 1~7 years old.Because of infant's respiratory tract screen
It is insufficient to hinder function, respiratory mucosa is tender, and respiratory tract secretion resistance bacterium, the immune substance of virus is insufficient, respiratory tract " self-cleaning "
Function is poor, therefore susceptible disease.Incubation period is 2~4 days.Often drastically generate heat, heat be mostly it is low or in it is isocratic, accidental up to 40 DEG C with
On, or even cause to faint from fear.Hot journey mostly 2~4 days.Older infant can tell that pharyngalgia, pharyngalgia severe one can influence to swallow.Baby children
Youngster then shows as salivation, food refusal, dysphoria.Sometimes companioned with headache, abdominal pain or myalgia, 5 years old or less children have 1/4 can occur together to vomit
It spits.
Human enterovirus virus belongs to pico+ribonucleic acid+virus section enterovirus genus, including following four is viral：Spinal cord ash
Matter scorching viral (P), echovirus (E), Coxsackie virus (CV) and newtype enteroviru (EV).It can lead to the herpetic isthmus faucium of children
Scorching pathogen is mostly caused by Coxsack A group viruses, and B group viruses can also cause a disease, but more rare.In addition, echovirus 3,
6,9,16,17,25 types and Inhibition Tests with Enterovirus Type 70 can also cause this disease.Coxsackie virus A class (CVA), including CVA2, CVA4,
CVA5, CVA6, CVA7, CVA9, CVA10, CVA16 type, Coxsackie virus B class (CVB), wherein CVB classes have CVB1, CVB2,
CVB3, CVB4, CVB5 type, echovirus, Enterovirus 71 (EV71) type etc..
Treatment herpangina is nothing more than two kinds of Chinese medicine and Western medicine, western medicine quick result, but pharmacological dependence at present
Property it is strong, it is palliative, and also certain side effect, and Chinese medicine treatment is substantially without side-effects.Callicarpa nudiflora is Verenaceae
Callicarpa bodinieri Levl. platymiscium, medicinal part are ground drying nest, are a kind of large property genunie medicinal materials in Hainan, while being also Nationality in Hainan Province
One of doctor's medicinal herbs most in use.Active ingredient therein has iridoids, flavonoids, quinones, volatile oil, xylan class, organic
Acids etc., pharmacological activity callicarpa nudiflora at present are broadly divided into following five aspects：(1) hemostasis (2) anti-inflammatory (3) antibacterial (4) is thin
Born of the same parents' cytotoxic activity (5) enhancing is immune.
The callicarpa nudiflora preparation of Chinese patent drug being made of the taste medicinal material medicinal extract, such as callicarpa nudiflora, callicarpa nudiflora capsule, nakedflower
Callicarpa bodinieri Levl. particle etc., clinically apply it is wide, be mainly used for treat bacterium infection caused by inflammation, acute infectious hepatitis,
The diseases such as the digestive tract bleedings such as respiratory tract and alimentary canal.Part is research shows that callicarpa nudiflora also have certain treatment in anti-virus aspect
It imitates, a kind of Chinese and Western medicine oral solution for treating herpangina, patent application is disclosed in patent application CN106177381A
CN104721634A discloses the Chinese medicine preparation for herpangina.But the active ingredient of above-mentioned drug in the prior art is multiple
It is miscellaneous, the shortcomings of active ingredient is indefinite, quality standard controllability is poor, it is difficult to meet drug " safely, effectively, controllably, stablize "
It is required that.
Invention content
Based on the defect of the above-mentioned prior art, the present invention carries out the extraction purification of active ingredient to callicarpa nudiflora, and passes through
Rational combination provides a kind of specific beautyberry extract composition of component, mutually plays and make between the composition each component
With, can significantly inhibit that CVA2 and CVB2 etc. is viral, for children's herpangina clinical diagnosis and treatment provide science according to
According to.
The present invention provides a kind of composition for treating children's herpangina, which is characterized in that the composition according to
Parts by weight meter, by 40-50 parts of callicarpa nudiflora general flavones, 10-20 parts of callicarpa nudiflora total phenolics, the 20-30 parts of total benzene of nakedflower purple strain
Ethyl alcohol glycosides and 2-6 parts of callicarpa nudiflora polysaccharide compositions.
Further, the composition in parts by weight, by 44-46 parts of callicarpa nudiflora general flavones, 15-17 parts of nakedflowers
The total benzyl carbinol glycosides of Callicarpa bodinieri Levl. total phenolics, 23-25 parts of nakedflower purple strains and 3-5 parts of callicarpa nudiflora polysaccharide compositions.
Further, the composition in parts by weight, by 45 parts of callicarpa nudiflora general flavones, 16 parts it is callicarpa nudiflora total
Phenolic acid, the total benzyl carbinol glycosides of 24 parts of nakedflower purple strains and 4 parts of callicarpa nudiflora polysaccharide compositions.
Further, component purifies to obtain by callicarpa nudiflora water extract in the composition.
Further, the preparation method of the callicarpa nudiflora water extract is：It is callicarpa nudiflora to add water to cook 1-3 times, merge filter
Liquid is concentrated into medicinal extract shape to get callicarpa nudiflora water extract.
Further, the preparation method of the callicarpa nudiflora water extraction is：Callicarpa nudiflora plus 100-110 DEG C of extraction 1-3 of water
Secondary, every time plus 8-12 times of water, first time extract 1-2.5h, extract 0.5-3h for the second time, merging filtrate, filtering, 60-90 DEG C depressurizes
It is concentrated into the medicinal extract shape of relative density 1.30~1.35, dry extract is dried in 60-75 DEG C to get callicarpa nudiflora water extract.
Further, the preparation method of the callicarpa nudiflora general flavone includes the following steps：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and obtains callicarpa nudiflora ethanol solution；
(2) for callicarpa nudiflora ethanol solution by AB-8 resins, ethyl alcohol is eluent, collects eluent, is dried in vacuo to obtain the final product
Callicarpa nudiflora general flavone.
Further, the preparation method of the callicarpa nudiflora general flavone is：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and is formulated as the callicarpa nudiflora ethanol solution of 0.5-1mg/mL；
(2) callicarpa nudiflora ethanol solution passes through AB-8 resins (blade diameter length ratio 1:8-1:15), 85-95% ethyl alcohol is elution
Liquid, sample rate 1.5-3mL/min, elution rate are 20-30 drops/min, collect eluent, eluent is through general flavone content
After measurement, the highest fraction of content is determined, the zinc chloride of callicarpa nudiflora 1-1.5 times of water extract quality is added, pH6.0-7.0's
Under the conditions of precipitate, filter, sediment is dried in vacuo up to callicarpa nudiflora general flavone.
Further, the preparation method of the callicarpa nudiflora total phenolics includes the following steps：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and obtains callicarpa nudiflora ethanol solution；
(2) by callicarpa nudiflora ethanol solution, petroleum ether, dichloromethane, ethyl acetate extraction is sequentially added, is concentrated and dried
Afterwards, ethyl acetate phase is obtained；
(3) it by D101 macroreticular resins after concentrating ethyl acetate phase, is eluted with ethanol solution, collects eluent, vacuum
It is drying to obtain callicarpa nudiflora total phenolics.
Further, the preparation method of callicarpa nudiflora total phenolics is：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and is formulated as the callicarpa nudiflora ethanol solution of 0.5-1g/mL；
(2) by callicarpa nudiflora ethanol solution, petroleum ether, dichloromethane, the ethyl acetate extraction of 2-3 times of volume are sequentially added
Each reagent extracts 2-3 times, after concentrate drying, retains ethyl acetate phase；
(3) after ethyl acetate is added water redissolution, it is formulated as the solution of 0.5-1mg/mL, passing through D101 macroreticular resins, (diameter is high
Than being 1:8-1:20), with 50-80% ethanol solutions elute, sample rate 1.5-3mL/min, elution rate be 20-30 drops/
Min collects eluent, and eluent determines the highest fraction of content after total phenolics assay, is dried in vacuo purple up to nakedflower
Pearl total phenolics.
Further, the preparation method of callicarpa nudiflora total benzyl carbinol glycosides includes the following steps：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and obtains callicarpa nudiflora ethanol solution；
(2) by callicarpa nudiflora ethanol solution, petroleum ether, ethyl acetate, extracting n-butyl alcohol are sequentially added, after concentrate drying,
Obtain n-butanol phase；
(3) after mutually redissolving n-butanol, silicagel column and HPD100 resin columns is passed sequentially through, is freeze-dried after eluent concentration
To obtain the final product.
Further, the preparation method of callicarpa nudiflora total benzyl carbinol glycosides is：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and is formulated as the callicarpa nudiflora ethanol solution of 0.5-1g/mL；
(2) by callicarpa nudiflora ethanol solution, the petroleum ether, ethyl acetate, extracting n-butyl alcohol for sequentially adding 2-3 times of volume are every
Kind reagent extracts 2-3 times, after concentrate drying, retains n-butanol phase；
(3) n-butanol is added ethyl alcohol and redissolves, and is formulated as the solution of 0.5-1mg/mL, passes through silicagel column (blade diameter length ratio 1:10-
1:15, silica gel grain size is 200-300 μm), it is eluted with methanol dichloromethane solvent system, methanol dichloromethane volume ratio
It is 0:100、20:80、40:60、60:40、80:20、100:0 is eluted successively, sample rate 1.2-3mL/min, elution
Rate is 8-15 drops/min, and eluent determines the highest fraction of content through benzyl carbinol glycosides assay, after concentrate drying, must evaporate
Divide 1；
(4) fraction 1 plus water redissolve, and are formulated as the solution of 0.5-1mg/mL, pass through HPD100 resins (blade diameter length ratio 1:3-1:
9), water elution, sample rate 1.2-3mL/min, elution rate are 8-15 drops/min, and eluent is through benzyl carbinol glycosides containing measurement
It is fixed, it determines the highest fraction of content, is freeze-dried after concentration up to callicarpa nudiflora total benzyl carbinol glycosides.
Further, the preparation method of the callicarpa nudiflora polysaccharide includes the following steps：
(1) trichloroacetic acid, centrifugation, supernatant concentration is added in callicarpa nudiflora water extract；
(2) ethanol precipitation 24-48h is added, sediment is washed with absolute ethyl alcohol, acetone, ether successively, is dried in vacuo naked
Flower Callicarpa bodinieri Levl. Thick many candies；
(4) after callicarpa nudiflora Thick many candies redissolve, by HPD100 resins, water elution, eluent is freeze-dried after concentrating is
.
Further, the preparation method of the callicarpa nudiflora polysaccharide includes the following steps：
(1) callicarpa nudiflora water extract, is added 2-5 times of 8-15% trichloroacetic acid (w/v), and 4-8 DEG C of standing 20-30h repeats 2-
3 times, 15-20min is centrifuged, the 1/5-1/10 of supernatant volume is concentrated under reduced pressure into；
(2) 90%-100% ethyl alcohol, 4-8 DEG C of precipitation 24-48h is added in the concentrate into step (1), and sediment is used successively
Absolute ethyl alcohol, acetone, ether wash 2-3 times, are dried in vacuo to obtain callicarpa nudiflora Thick many candies；
(3) after callicarpa nudiflora Thick many candies add water to redissolve, it is formulated as the solution of 0.5-1mg/mL, passes through HPD100 resin (diameters
Height is than being 1:3-1:9), water elution, sample rate 1.2-3mL/min, elution rate is 8-15 drops/min, after eluent concentration
It is freeze-dried to obtain the final product.
The present invention also provides a kind of pharmaceutical preparations for treating children's herpangina, by combinations of the above object and pharmaceutically
Acceptable auxiliary material composition.
Further, the preparation is oral solution, particle electuary, capsule, tablet, dispersant.
The present invention further provides treatment children's herpangina compositions to prepare treatment and/or pre- child-resistant
Application in herpangina.
Further, the composition is herpetic in preparation treatment and/or prevention children caused by CVA2 and/or CVB2
Application in angina drug.
Beneficial effects of the present invention are：
(1) active ingredient in callicarpa nudiflora is extracted and is purified, choose callicarpa nudiflora general flavone, callicarpa nudiflora total
Phenolic acid, callicarpa nudiflora total benzyl carbinol glycosides and callicarpa nudiflora polysaccharide are reasonably combined, and the composition of preparation is by influencing virus
Replicative cycle, show as that there is better effect compared to the extraction of callicarpa nudiflora water, alcohol extracting in terms of inhibiting enterovirus, have
There is the effect of good treatment children's herpangina.
(2) four kinds of callicarpa nudiflora general flavone, callicarpa nudiflora total phenolics, callicarpa nudiflora total benzyl carbinol glycosides and callicarpa nudiflora polysaccharide
The effect of component is mutually cooperateed with and played a role, and is adjusted to its content, treatment children's herpangina is substantially better than
One-component or beautyberry extract.
(3) beautyberry extract composition provided by the invention, ingredient and content are clear, and significant in efficacy, quality is steady
Fixed, preparation method is simple, overcomes the problem that the components such as single Aqua pure extract, alcohol extract are indefinite, and curative mechanism is not known.
Specific implementation mode
The extraction of embodiment 1 callicarpa nudiflora general flavone, total phenolics, benzyl carbinol glycosides and callicarpa nudiflora polysaccharide
(1) preparation of callicarpa nudiflora water extract：
Callicarpa nudiflora plus 100 DEG C of water extracts 3 times, and every time plus 8 times of water, first time extract 2.5h, the 2nd and 3 extraction 0.5h,
Merging filtrate, filtering, 60 DEG C of medicinal extract shapes for being concentrated under reduced pressure into relative density 1.30~1.35 are dried to dry extract in 60 DEG C, standby
With；
(2) preparation of callicarpa nudiflora general flavone：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 0.5mg/mL；
2. callicarpa nudiflora ethanol solution passes through AB-8 resins (blade diameter length ratio 1:15), 95% ethyl alcohol is eluent, sample introduction speed
Rate is 1.5mL/min, and elution rate is 20 drops/min, collects eluent, eluent determines content after determination of total flavonoids
Highest fraction is added the zinc chloride of 1.5 times of callicarpa nudiflora water extract quality, is precipitated under conditions of pH7.0, filters, precipitation
Object is dried in vacuo up to callicarpa nudiflora general flavone.
(3) preparation of callicarpa nudiflora total phenolics：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 0.5-1g/mL；
2. by callicarpa nudiflora ethanol solution, petroleum ether, dichloromethane, the ethyl acetate extraction for sequentially adding 2 times of volumes are every
Kind reagent extracts 2 times, after concentrate drying, retains ethyl acetate phase；
3. ethyl acetate is added after water redissolves, it is formulated as the solution of 0.5mg/mL, (blade diameter length ratio is by D101 macroreticular resins
1:8) it, is eluted with 50% ethanol solution, sample rate 1.5mL/min, elution rate is 20 drops/min, collects eluent, washes
De- liquid determines the highest fraction of content after total phenolics assay, is dried in vacuo up to callicarpa nudiflora total phenolics.
(4) preparation of callicarpa nudiflora total benzyl carbinol glycosides：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 1g/mL；
2. by callicarpa nudiflora ethanol solution, sequentially add 2 times of volumes petroleum ether, ethyl acetate, extracting n-butyl alcohol each
Reagent extracts 2 times, after concentrate drying, retains n-butanol phase；
It is redissolved 3. n-butanol is added ethyl alcohol, is formulated as the solution of 0.5mg/mL, passes through silicagel column (blade diameter length ratio 1:10, silicon
Micelle diameter is 200-300 μm), it is eluted with methanol dichloromethane solvent system, methanol dichloromethane volume ratio is 0:
100、20:80、40:60、60:40、80:20、100:0 is eluted successively, sample rate 1.2mL/min, elution rate 8
Drop/min, eluent determine the highest fraction of content through benzyl carbinol glycosides assay, after concentrate drying, obtain fraction 1；
4. fraction 1 plus water redissolve, it is formulated as the solution of 0.5mg/mL, passes through HPD100 resins (blade diameter length ratio 1:3) it, washes
De-, sample rate 1.2mL/min, elution rate is 8 drops/min, and eluent determines content most through benzyl carbinol glycosides assay
High fraction is freeze-dried after concentration up to callicarpa nudiflora total benzyl carbinol glycosides.
(5) preparation of callicarpa nudiflora polysaccharide
1. callicarpa nudiflora water extract, is added 2 times of 15% trichloroacetic acid (w/v), 4 DEG C of standing 20h are repeated 3 times, centrifuge
15min is concentrated under reduced pressure into the 1/5 of supernatant volume；
2. 90% ethyl alcohol is added in the concentrate into step (1), for 24 hours, sediment uses absolute ethyl alcohol, third to 4 DEG C of precipitations successively
Ketone, ether wash 2 times, are dried in vacuo to obtain callicarpa nudiflora Thick many candies；
After 3. callicarpa nudiflora Thick many candies add water to redissolve, it is formulated as the solution of 0.5mg/mL, passes through HPD100 resin (blade diameter length ratios
It is 1:3), water elution, sample rate 1.2mL/min, elution rate are 8 drops/min, are freeze-dried after eluent concentration to obtain the final product.
The extraction of embodiment 2 callicarpa nudiflora general flavone, total phenolics, benzyl carbinol glycosides and callicarpa nudiflora polysaccharide
(1) preparation of callicarpa nudiflora water extract：
Callicarpa nudiflora plus 105 DEG C of water extracts 2 times, each plus 10 times of water, extracts 2h for the first time, and second of extraction 1h merges
Filtrate, filtering, 80 DEG C of medicinal extract shapes for being concentrated under reduced pressure into relative density 1.30~1.35 are dried to dry extract in 70 DEG C, spare；
(2) preparation of callicarpa nudiflora general flavone：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 0.8mg/mL；
2. callicarpa nudiflora ethanol solution passes through AB-8 resins (blade diameter length ratio 1:10), 90% ethyl alcohol is eluent, sample introduction speed
Rate is 2mL/min, and elution rate is 25 drops/min, collects eluent, eluent determines content most after determination of total flavonoids
High fraction is added the zinc chloride of 1.5 times of callicarpa nudiflora water extract quality, is precipitated under conditions of pH6.5, filtering, sediment
It is dried in vacuo up to callicarpa nudiflora general flavone.
(3) preparation of callicarpa nudiflora total phenolics：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 0.5-1g/mL；
2. by callicarpa nudiflora ethanol solution, petroleum ether, dichloromethane, the ethyl acetate extraction for sequentially adding 3 times of volumes are every
Kind reagent extracts 3 times, after concentrate drying, retains ethyl acetate phase；
After 3. ethyl acetate is added water redissolution, it is formulated as the solution of 1mg/mL, passes through D101 macroreticular resins (blade diameter length ratio 1:
15) it, is eluted with 65% ethanol solution, sample rate 2mL/min, elution rate is 25 drops/min, collects eluent, eluent
After total phenolics assay, the highest fraction of content is determined, be dried in vacuo up to callicarpa nudiflora total phenolics.
(4) preparation of callicarpa nudiflora total benzyl carbinol glycosides：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 1g/mL；
2. by callicarpa nudiflora ethanol solution, sequentially add 3 times of volumes petroleum ether, ethyl acetate, extracting n-butyl alcohol each
Reagent extracts 3 times, after concentrate drying, retains n-butanol phase；
It is redissolved 3. n-butanol is added ethyl alcohol, is formulated as the solution of 1mg/mL, passes through silicagel column (blade diameter length ratio 1:12, silica gel
Grain size is 200-300 μm), it is eluted with methanol dichloromethane solvent system, methanol dichloromethane volume ratio is 0:100、
20:80、40:60、60:40、80:20、100:0 is eluted successively, sample rate 1.5mL/min, elution rate be 10 drop/
Min, eluent determine the highest fraction of content through benzyl carbinol glycosides assay, after concentrate drying, obtain fraction 1；
4. fraction 1 plus water redissolve, it is formulated as the solution of 1mg/mL, passes through HPD100 resins (blade diameter length ratio 1:6) it, washes
De-, sample rate 2mL/min, elution rate is 13 drops/min, and eluent determines content highest through benzyl carbinol glycosides assay
Fraction, be freeze-dried after concentration up to callicarpa nudiflora total benzyl carbinol glycosides.
(5) preparation of callicarpa nudiflora polysaccharide
1. callicarpa nudiflora water extract, is added 3 times of 10% trichloroacetic acid (w/v), 5 DEG C of standing 25h are repeated 3 times, centrifuge
15min is concentrated under reduced pressure into the 1/7 of supernatant volume；
2. 95% ethyl alcohol, 6 DEG C of precipitation 30h is added in the concentrate into step (1), sediment uses absolute ethyl alcohol, third successively
Ketone, ether wash 3 times, are dried in vacuo to obtain callicarpa nudiflora Thick many candies；
After 3. callicarpa nudiflora Thick many candies add water to redissolve, it is formulated as the solution of 0.8mg/mL, passes through HPD100 resin (blade diameter length ratios
It is 1:5), water elution, sample rate 2mL/min, elution rate are 12 drops/min, are freeze-dried after eluent concentration to obtain the final product.
The extraction of embodiment 3 callicarpa nudiflora general flavone, total phenolics, benzyl carbinol glycosides and callicarpa nudiflora polysaccharide
(1) preparation of callicarpa nudiflora water extract：
Callicarpa nudiflora plus 110 DEG C of water extracts 1 time, each plus 12 times of water, extracts 1h for the first time, and second of extraction 3h merges
Filtrate, filtering, 90 DEG C of medicinal extract shapes for being concentrated under reduced pressure into relative density 1.30~1.35 are dried to dry extract in 75 DEG C, spare；
(2) preparation of callicarpa nudiflora general flavone：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 1mg/mL；
2. callicarpa nudiflora ethanol solution passes through AB-8 resins (blade diameter length ratio 1:8), 85% ethyl alcohol is eluent, sample rate
For 3mL/min, elution rate is 30 drops/min, collects eluent, eluent determines content highest after determination of total flavonoids
Fraction, the zinc chloride of 1 times of callicarpa nudiflora water extract quality is added, is precipitated under conditions of pH6.0, filter, sediment vacuum
It is drying to obtain callicarpa nudiflora general flavone.
(3) preparation of callicarpa nudiflora total phenolics：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 1g/mL；
2. by callicarpa nudiflora ethanol solution, petroleum ether, dichloromethane, the ethyl acetate extraction for sequentially adding 3 times of volumes are every
Kind reagent extracts 3 times, after concentrate drying, retains ethyl acetate phase；
After 3. ethyl acetate is added water redissolution, it is formulated as the solution of 1mg/mL, passes through D101 macroreticular resins (blade diameter length ratio 1:
20) it, is eluted with 80% ethanol solution, sample rate 3mL/min, elution rate is 30 drops/min, collects eluent, eluent
After total phenolics assay, the highest fraction of content is determined, be dried in vacuo up to callicarpa nudiflora total phenolics.
(4) preparation of callicarpa nudiflora total benzyl carbinol glycosides：
1. callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, it is formulated as the callicarpa nudiflora ethanol solution of 0.5g/mL；
2. by callicarpa nudiflora ethanol solution, sequentially add 3 times of volumes petroleum ether, ethyl acetate, extracting n-butyl alcohol each
Reagent extracts 3 times, after concentrate drying, retains n-butanol phase；
It is redissolved 3. n-butanol is added ethyl alcohol, is formulated as the solution of 1mg/mL, passes through silicagel column (blade diameter length ratio 1:15, silica gel
Grain size is 200-300 μm), it is eluted with methanol dichloromethane solvent system, methanol dichloromethane volume ratio is 0:100、
20:80、40:60、60:40、80:20、100:0 is eluted successively, sample rate 3mL/min, elution rate be 15 drop/
Min, eluent determine the highest fraction of content through benzyl carbinol glycosides assay, after concentrate drying, obtain fraction 1；
4. fraction 1 plus water redissolve, it is formulated as the solution of 1mg/mL, passes through HPD100 resins (blade diameter length ratio 1:9) it, washes
De-, sample rate 3mL/min, elution rate is 15 drops/min, and eluent determines content highest through benzyl carbinol glycosides assay
Fraction, be freeze-dried after concentration up to callicarpa nudiflora total benzyl carbinol glycosides.
(5) preparation of callicarpa nudiflora polysaccharide
1. callicarpa nudiflora water extract, is added 5 times of 8% trichloroacetic acid (w/v), 8 DEG C of standing 30h are repeated 2 times, centrifuge
20min is concentrated under reduced pressure into the 1/10 of supernatant volume；
2. 100% ethyl alcohol, 8 DEG C of precipitation 48h is added in the concentrate into step (1), sediment uses absolute ethyl alcohol, third successively
Ketone, ether wash 3 times, are dried in vacuo to obtain callicarpa nudiflora Thick many candies；
After 3. callicarpa nudiflora Thick many candies add water to redissolve, it is formulated as the solution of 1mg/mL, (blade diameter length ratio is by HPD100 resins
1:9), water elution, sample rate 3mL/min, elution rate are 15 drops/min, are freeze-dried after eluent concentration to obtain the final product.
The beautyberry extract composition of embodiment 4-9 treatment children's herpanginas
The composite formula of embodiment 4-9 is referring to following table.
Above-mentioned composition can conventionally add corresponding auxiliary material, be prepared as oral solution, particle electuary, capsule, tablet
Or dispersant.
The beautyberry extract composition of comparative example 1-4 treatment children's herpanginas
The composite formula of comparative example 1-4 is referring to following table.
Above-mentioned composition can conventionally add corresponding auxiliary material, be prepared as oral solution, particle electuary, capsule, tablet
Or dispersant.
6 callicarpa nudiflora water extract of comparative example
It takes callicarpa nudiflora, adds water to cook twice, every time plus 10 times of amount water, 2 hours for the first time, the 2nd time 1 hour, merge and decoct
Liquid, filtration, filtrate concentration are dried to dry extract up to callicarpa nudiflora water extract.
Callicarpa nudiflora water extract can conventionally add auxiliary material, be prepared as oral solution, particle electuary, capsule, tablet
Or dispersant.
7 callicarpa nudiflora alcohol extract of comparative example
It takes callicarpa nudiflora, 50 DEG C of 95% ethyl alcohol reflux extraction 3h, filtration, filtrate concentration is added to be dried to dry extract up to naked
Flower Callicarpa bodinieri Levl. alcohol extract.
Callicarpa nudiflora alcohol extract can conventionally add auxiliary material, be prepared as oral solution, particle electuary, capsule, tablet
Or dispersant.
The extracorporeal antivirus effect of 10 beautyberry extract composition of embodiment is studied
The granular preparation and commercially available nakedflower that drug to be measured chooses embodiment 6-7, prepared by the composition of comparative example 1-7 are purple
Pearl particle (the general positive medicine company in Jiangxi) is studied.
Virus to be measured chooses CVB2 and CVA2.
The culture of 10.1 viruses
Hep-2 cell strains cryopreservation tube takes out from liquid nitrogen container, puts into 37 DEG C~42 DEG C warm water melt immediately, superclean bench
Interior to move into liquid in cryopreservation tube in centrifuge tube, 1000r/min centrifuges 5min, abandons frozen stock solution, is added 8ml's a concentration of 10%
Lower sediment cell is gently blown and beaten mixing by 1640 cell culture fluids, is transferred in culture bottle with pipette, in 37 DEG C, 5%CO2
It is cultivated in constant incubator.After cells grow up to the individual layer is passed on.
Virus is inoculated in oneself through growing up on the Hep-2 cells of single layer respectively, add RPMI-1640 maintaining liquids 8ml set 37 DEG C,
5%CO2It is cultivated in Virus culture case, and sets cell controls, observed day by day.When 80% or more lesion occurs in cell, repeatedly
Freeze thawing 3 times is gently blown and beaten with suction pipe, and 1000r/min centrifuges 5min, takes supernatant quantitative separating in small green bottle, adhesive plaster sealing,
Be put in -80 DEG C of refrigerators freeze it is spare.
The measurement of 10.2 virus virulences
Virus is done into 10 multiple proportions with RPMI-1640 maintaining liquids and is serially diluted various concentration, it is longitudinal to repeat 4 holes, it is inoculated with successively
In 96 orifice plates that oneself grows up to the Hep-2 cells of single layer, while setting cell controls.37 DEG C, 5%CO2It is cultivated in Virus culture case,
It is observed day by day under inverted microscope, 5d is observed continuously, liquid suction in plate hole is abandoned, 1% dimethyl diaminophenazine chloride 50 μ L, 37 DEG C, 5%CO are added2
1 hour in incubator, dye liquor is abandoned, excess dyestuff is fully washed to paint with washing lotion, 100 μ L of destainer, room temperature decoloration l0min is added to use
Microplate reader measures OD values in 540mn wavelength.The half infection concentration TCID of virus liquid is calculated according to Reed-Muench formula50。
Cell survival rate=each group OD values/normal cell OD value × 100%
Cytopathy variability=1- cell survival rates
Cell ratio away from=(be higher than 50% lesion rate -50%)/(being less than 50% lesion rate higher than 50% lesion rate -) ×
100%
TCID50=Antilog (Iog is higher than 50%CPE percentage viral dilution+ratio away from × dilution gfactor logarithm)
According to TCID50As a result, virus stock solution used is diluted to 10-6-10-7It uses.
10.3 drugs are to cytotoxicity assay
Drug to be measured RPMI-1640 maintaining liquids are pressed into two doubling dilutions, 7 concentration gradients, are inoculated in oneself successively through growing up to
In 96 orifice plates of the Hep-2 cells of single layer, and set 4 multiple holes and cell control well；37 DEG C, 5%CO2It is cultivated in incubator, by
Day observation cytopathy, is observed continuously 3d, calculates the maximal non-toxic concentration of drug.There is lesion person and is judged to drug toxicity in cell,
Neutral red staining, OD values are measured with microplate reader in 540nm wavelength, and it is dense to calculate the poisoning of drug half using Reed-Muench formula
Spend (TC50), and determine minimum non-toxic concn (TC0)。
TC50=[Antilog (log higher than 50%CPE percentage viral dilution+ratio away from)] × C
10.4 Antiviral breedings
Drug to be measured reinstates bis- multiple proportions of RPMI-1640 from non-toxic concn and is serially diluted 7 concentration, is inoculated in per 50 μ L of hole
Grow up in 96 plate holes of single layer Hep-2 cells, adds 50 μ L virus liquids per hole, while setting Ribavirin positive controls, disease
Malicious control group and blanc cell control group, every group of 4 multiple holes；The blank culture of equivalent is added in virus control group and blanc cell group
Base.5%CO2It is cultivated in incubator, observes cytopathy, when there is 90% or more lesion in virus control, 1% dimethyl diaminophenazine chloride dye
Color is used in combination microplate reader to measure OD values in 540nm wavelength.Drug medium effective concentration (EC is calculated using Reed-Muench methods50)
With therapeutic index (TI).
EC50=[Antilog (and log higher than 50%CPE percentage viral dilutions value-ratio away from)] × C
TI=half toxic concentrations (TC50)/medium effective concentration (EC50)
10.5 experimental results
Therapeutic index of the different callicarpa nudiflora drugs of table 1 to CVB2 and CVA2 viruses
| TI | CVB2 | CVA2 |
| Embodiment 6 | 70.2a | 84.1a |
| Embodiment 7 | 73.6a | 85.4a |
| Comparative example 1 | 48.6b | 55.9b |
| Comparative example 2 | 54.5b | 61.3b |
| Comparative example 3 | 50.1b | 58.7b |
| Comparative example 4 | 57.9c | 65.7c |
| Comparative example 5 | 64.7c | 70.2c |
| Comparative example 6 | 59.1c | 66.5c |
| Comparative example 7 | 61.4c | 67.9c |
| Commercially available callicarpa nudiflora particle | 40.7d | 56.9b |
| Ribavirin positive controls | 67.4a | 74.4d |
Note：Being represented between different letters in same row has significant difference.
As shown in Table 1, beautyberry extract composition provided by the invention effect in terms of anti-CVA2 and CVB2 viruses
Significantly.More than treatment in 2 kinds of children's herpangina virus, the component of callicarpa nudiflora composition provided by the invention lacks one
Can not, lacked respectively in comparative example 1-4 callicarpa nudiflora general flavone, callicarpa nudiflora total phenolics, callicarpa nudiflora total benzyl carbinol glycosides and
Callicarpa nudiflora polysaccharide, antiviral effect are remarkably decreased, and illustrate there is the effect mutually cooperateed between four kinds of components.In addition,
The content of what object of beautyberry extract group provided by the invention is obtained by strictly screening and matching, and is adjusted in comparative example 5
Whole four proportioning, antiviral significant effect decline.
The callicarpa nudiflora extract obtained by conventional water extraction and alcohol extracting method and commercially available callicarpa nudiflora particle,
Complicated component, content is indefinite, can not play maximum effect, therefore its antiviral effect is also not so good as provided by the invention group
Close object.
11 beautyberry extract composition of embodiment treats the mouse of CVB2 and CVA2 infection
BALB/C mice is taken, it is random to be grouped, it is divided into Normal group, model group and the administration group (composition of embodiment 6-7
The granular preparation of preparation, commercially available callicarpa nudiflora particle group), with 4 times of TCID50Rat is injected intraperitoneally in virus quantity, normal group
Normal saline is given, gastric infusion immediately after 1 hour, model group and normal group give normal saline；Successive administration
Survival mice is put to death after 14 days, 14 days, eye socket takes hematometry LDH, SOD and MDA.
Influence of 2 beautyberry extract of table to CVB2 virus infected mice Serum Indexes
| LDH(U/L) | SOD(U/L) | MDA(umol/L) | |
| Blank group | 690.3±111.2a | 300.1±57.1a | 11.8±1.6a |
| Viral group | 1371.8±416.1b | 104.6±56.1b | 28.7±4.1b |
| Embodiment 6 | 764.4±271.1a | 269.7±37.6a | 13.4±2.7a |
| Embodiment 7 | 731.8±242.6a | 278.3±34.9a | 12.7±2.8a |
| Callicarpa nudiflora particle | 938.9±286.1c | 210.7±56.8c | 18.1±4.9c |
Note：Being represented between different letters in same row has significant difference.
Influence of 3 beautyberry extract of table to CVA2 virus infected mice Serum Indexes
| LDH(U/L) | SOD(U/L) | MDA(umol/L) | |
| Blank group | 620.4±245.9a | 274.1±50.3a | 10.4±4.3a |
| Viral group | 1482.4±450.2b | 139.4±50.4b | 36.9±5.2b |
| Embodiment 6 | 678.4±156.6a | 259.1±60.7a | 15.1±5.7a |
| Embodiment 7 | 632.1±319.5a | 265.7±43.1a | 13.9±3.8a |
| Callicarpa nudiflora particle | 890.4±334.8c | 210.1±53.1c | 23.1±4.2c |
Note：Being represented between different letters in same row has significant difference.
By table 2-3 it is found that the significant effect in terms of the mouse for the treatment of CVA2 and CVB2 viruses infection of embodiment 7, and commercially available
It is callicarpa nudiflora to compare, it is commercially available it is callicarpa nudiflora it is with obvious effects be not so good as the embodiment of the present invention 7, absolutely prove provided by the invention naked
Flower Japanese beauty-berry composition, by carrying out rational content proportioning after purification, mutually collaboration plays a role, and substantially increases
It inhibits the effect of CVA2 and CVB2 viruses.
12 beautyberry extract composition clinical treatment curative effect of embodiment
Select the herpangina patient 500 made a definite diagnosis, wherein male 552, women 448,5 years old left side of average age
The right side, patient are randomly divided into treatment group's (callicarpa nudiflora particle and commercially available callicarpa nudiflora particle prepared by embodiment 7, comparative example 1-7
Group) and control group, 1 bag every time for the treatment of group；3 times a day, control group takes Cefaclor, by weight 20~40mg/kg on the one, divides
It gives for 3 times, but a daily amount is no more than 1g.30 days as a treatment course simultaneously observe patient symptom improvement situation.
The course of disease for the treatment of group and control group case, degrees of symptoms are almost the same, and no significant difference is comparable.
Curative effect judges：
It is effective：Above-mentioned symptom is relieved, and body is clearly better.Effectively：Above-mentioned symptom is alleviated, and body has inclination
Turn.In vain：It without alleviation or is aggravated by above-mentioned symptom after treatment.
4 callicarpa nudiflora granule therapy children herpangina result of table
| Effective (example) | Effectively (example) | (example) in vain | Obvious effective rate (%) | |
| Control group | 40 | 6 | 4 | 80 |
| Embodiment 7 | 46 | 2 | 2 | 92 |
| Comparative example 1 | 22 | 13 | 15 | 44 |
| Comparative example 2 | 26 | 11 | 13 | 52 |
| Comparative example 3 | 24 | 13 | 13 | 48 |
| Comparative example 4 | 27 | 12 | 11 | 54 |
| Comparative example 5 | 33 | 15 | 2 | 66 |
| Comparative example 6 | 30 | 12 | 8 | 60 |
| Comparative example 7 | 31 | 13 | 6 | 62 |
| Callicarpa nudiflora particle | 20 | 8 | 22 | 40 |
As shown in Table 4, the composition of beautyberry extract provided by the invention, in treatment children herpangina side
The effect of face, is substantially better than commercially available callicarpa nudiflora particle, and for commercially available callicarpa nudiflora particle due to its complicated component, content is unknown, treatment
The effect of there is corresponding decline.
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, the embodiment not to
The scope of the claims of the present invention is limited, all equivalence enforcements or change without departing from carried out by the present invention are intended to be limited solely by the technology of the present invention
In the range of scheme.
Claims (10)
1. a kind of composition for treating children's herpangina, which is characterized in that the composition in parts by weight, by
40-50 parts of callicarpa nudiflora general flavones, 10-20 parts of callicarpa nudiflora total phenolics, the total benzyl carbinol glycosides of 20-30 parts of nakedflower purple strains and 2-6 parts
Callicarpa nudiflora polysaccharide composition.
2. composition according to claim 1, which is characterized in that component is carried by callicarpa nudiflora water in the composition
Object purifies to obtain.
3. composition according to claim 2, which is characterized in that the preparation method of the callicarpa nudiflora water extract is：It is naked
Flower Callicarpa bodinieri Levl. adds water to cook 1-3 times, and merging filtrate is concentrated into medicinal extract shape to get callicarpa nudiflora water extract.
4. composition according to claim 3, which is characterized in that the preparation method of the callicarpa nudiflora general flavone include with
Lower step：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and obtains callicarpa nudiflora ethanol solution；
(2) for callicarpa nudiflora ethanol solution by AB-8 resins, ethyl alcohol is eluent, collects eluent, is dried in vacuo up to nakedflower
Purple beautyberry total flavone.
5. composition according to claim 3, which is characterized in that the preparation method of the callicarpa nudiflora total phenolics include with
Lower step：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and obtains callicarpa nudiflora ethanol solution；
(2) by callicarpa nudiflora ethanol solution, petroleum ether, dichloromethane, ethyl acetate extraction is sequentially added, after concentrate drying, is obtained
To ethyl acetate phase；
(3) it by D101 macroreticular resins after concentrating ethyl acetate phase, is eluted with ethanol solution, collects eluent, vacuum drying
Up to callicarpa nudiflora total phenolics.
6. composition according to claim 3, which is characterized in that the preparation method packet of callicarpa nudiflora total benzyl carbinol glycosides
Include following steps：
(1) callicarpa nudiflora water extract, adds ethyl alcohol to dissolve, and obtains callicarpa nudiflora ethanol solution；
(2) by callicarpa nudiflora ethanol solution, petroleum ether, ethyl acetate, extracting n-butyl alcohol are sequentially added, after concentrate drying, is obtained
N-butanol phase；
(3) after mutually redissolving n-butanol, silicagel column and HPD100 resin columns are passed sequentially through, eluent is freeze-dried after concentrating is
.
7. composition according to claim 3, which is characterized in that the preparation method of the callicarpa nudiflora polysaccharide includes following
Step：
(1) trichloroacetic acid, centrifugation, supernatant concentration is added in callicarpa nudiflora water extract；
(2) ethanol precipitation 24-48h is added, sediment is washed with absolute ethyl alcohol, acetone, ether successively, is dried in vacuo to obtain nakedflower purple
Pearl Thick many candies；
(4) after callicarpa nudiflora Thick many candies redissolve, by HPD100 resins, water elution is freeze-dried after eluent concentration to obtain the final product.
8. a kind of pharmaceutical preparation including claim 1-7 any one of them treatment children's herpangina, feature exist
In the preparation is made of claim 1-7 any one of them composition and pharmaceutically acceptable auxiliary material.
9. claim 1-7 any one of them composition is in preparing treatment and/or pre- child-resistant herpangina drug
Application.
10. application according to claim 9, which is characterized in that the composition is preparing treatment and/or preventing by CVA2
And/or the application in children's herpangina drug caused by CVB2.
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Address after: 343100 Innovation Avenue 278, Jinggangshan Economic and Technological Development Zone, Ji'an City, Jiangxi Province Applicant after: Jiangxi Puzheng Pharmaceutical Co., Ltd. Address before: 343100 No. 5 Ji'an Avenue, Jinggangshan Economic and Technological Development Zone, Ji'an City, Jiangxi Province Applicant before: Jiangxi Pozin Pharmaceutical Co., Ltd. |
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