CN103536747B - A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology - Google Patents
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology Download PDFInfo
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Abstract
The present invention relates to a kind of processing method of Bulbus Fritillariae Cirrhosae, be specifically related to a kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology.Comprise the following steps: (1) select materials (2) making beating: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 10 ~ 20wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.7 ~ 0.8 times is added; (3) pre-freeze: add the Tween 80 of serosity quality 10 ~ 15wt% before pre-freeze and fully stir; (4) lyophilization, requires that moisture control is at below 6wt%; (5) break micropowder and break into micropowder by vibration type micronizing grinding mixing roll.The Bulbus Fritillariae Cirrhosae superfine powder adopting the inventive method to prepare, farthest can retain the active component in Bulbus Fritillariae Cirrhosae.
Description
Technical field
The present invention relates to a kind of processing method of Bulbus Fritillariae Cirrhosae, be specifically related to a kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology.
Background technology
Prior art, when concocting this herb of Bulbus Fritillariae Cirrhosae, is normally dried section.Modern study shows, traditional concocting method is by active component many for loss.
Application number is the Chinese invention patent of 02154200.7; disclose a kind of preparation method of cow-bezoar snake bile Sichuan fritillary bulb dispersive; include the processing method of Bulbus Fritillariae Cirrhosae in the method, Bulbus Fritillariae Cirrhosae powder was broken into 80 ~ 160 object fine powders, be preferably ground into 200 ~ 300 object fine powders.In the method, directly Bulbus Fritillariae Cirrhosae powder is broken into micropowder, causes active component in Bulbus Fritillariae Cirrhosae greatly to lack.
Summary of the invention
The active component that the object of the invention is for solving Bulbus Fritillariae Cirrhosae superfine powder in prior art retains not high enough technical problem, provides a kind of preparation technology of Bulbus Fritillariae Cirrhosae superfine powder.The method farthest can retain the active component in Bulbus Fritillariae Cirrhosae, and obtained Bulbus Fritillariae Cirrhosae superfine powder rehydration is also greatly improved.
Above-mentioned technical purpose of the present invention is achieved by the following technical programs:
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 10 ~ 20wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.7 ~ 0.8 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 10 ~ 15wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll.
Preferred as technique scheme, step (5) is played micropowder specific requirement and is, makes grain size of micropowder more than 95% reach less than 10 μm.
Preferred as technique scheme, the moisture content of described Bulbus Fritillariae Cirrhosae superfine powder is lower than 5%.
Chinese medicine is in traditional Preparation process process, and the effective ingredient such as vegetable protein, polysaccharide can be damaged.The present invention is by pre-freeze after the making beating that adds water, and Bulbus Fritillariae Cirrhosae medical material is first through freezing before distillation dehydration, is formed stable " skeleton ", by low-temperature negative-pressure dehydrate, is prepared into Bulbus Fritillariae Cirrhosae superfine powder finally by vibration type micronizing grinding mixing roll.According to the feature of micronizing, when Chinese medicine carries out superfine grinding, affected many factors: first, the various and complicated component of herbal species, not all medicine is all applicable to superfine grinding, neither powder more thin better.Containing the medical material of armaticity, volatile ingredient, sporoderm-broken rate is high, is conducive to the abundant exposure emission and absorption of effective ingredient, but while pulverizing, the loss of volatile component can not be ignored, and therefore this kind of medical material needs to be crushed to what degree needs to study further; Secondly, be macromolecular medical material for some active components, being crushed to what degree could not saboteur's structure also need to investigate further, and some medical material containing toxic component also will using the index of the stripping of toxic component as an investigation in addition.Except kind is different, also has moisture, former particle size distribution, pulverize the factors such as environment.Therefore, up to the present, realize the superfine grinding of same fineness with same equipment, same disintegrating process, also acquire a certain degree of difficulty.According to concrete medicine, super-fine processing can only be carried out for specific requirement.Therefore, the adjustment carrying out a large amount of tests and disintegrating process and pulverizing parameter is needed.
The present invention, in pulping process, with the addition of microcrystalline Cellulose.Microcrystalline Cellulose is a kind of conventional medical auxiliary materials, and it is present in reasonability in medicine and repeatedly proves with scientific being through, and therefore it can be used as adjuvant, is at least out of question at medical angle.In the present invention, the effect of microcrystalline Cellulose is not obviously traditional value as medical accessory, and it can help the effective ingredient of the making beating of raw material and protection raw material not to be destroyed in pulping process.
Because the extra interpolation of microcrystalline Cellulose it is also proposed new problem for present invention process, that is, during last micronizing, the resistance from material increases, and makes vibration type micronizing grind mixing roll usually overheated, and polishing effect is also undesirable.The present inventor, after employing Tween 80, solves this problem.Tween 80 is also a kind of conventional pharmaceutical auxiliaries, the same with microcrystalline Cellulose, is safe and reliable equally.The detailed directions of Tween 80 is: before pre-freeze, add the Tween 80 of serosity quality 10 ~ 15wt% and fully stir, then pre-freeze.
The Bulbus Fritillariae Cirrhosae superfine powder steady quality prepared by the present invention, can preserve for a long time at normal temperatures, its rehydration is fabulous, and can absorb water rapidly recovery, and its color and luster, quality and fresh goods are substantially identical.In addition, tested by In Vitro Dissolution, the medical material under known unit mass, Bulbus Fritillariae Cirrhosae superfine powder dry Bulbus Fritillariae Cirrhosae rear than the process of preparing Chinese medicine prepared by the inventive method, containing more effective ingredient.Also find in experimentation, Bulbus Fritillariae Cirrhosae superfine powder prepared by the inventive method, after inhaling the recovery of a small amount of water, in thick, and the Bulbus Fritillariae Cirrhosae superfine powder adopting traditional method to concoct, after restoring with waiting water gaging, sliminess is obviously not enough.Known by method of the present invention, the active component of Bulbus Fritillariae Cirrhosae medical material can be remained to a greater degree.Found by pharmacodynamic study, Bulbus Fritillariae Cirrhosae superfine powder prepared by the inventive method, the drug effect of its smaller dose can be equal to the drug effect of dry Bulbus Fritillariae Cirrhosae extractum clinical medicine dose.
The present invention has following technique effect:
(1) the Bulbus Fritillariae Cirrhosae superfine powder adopting the inventive method to prepare, farthest can retain the active component in Bulbus Fritillariae Cirrhosae;
(2) the Bulbus Fritillariae Cirrhosae superfine powder adopting the inventive method to prepare, the drug effect of its smaller dose can be equal to the drug effect of dry Bulbus Fritillariae Cirrhosae extractum clinical medicine dose;
(3) the Bulbus Fritillariae Cirrhosae superfine powder adopting the inventive method to prepare, steady quality, can preserve at normal temperatures for a long time;
(4) the fresh Bulbus Fritillariae Cirrhosae superfine powder adopting the inventive method to prepare, its rehydration is fabulous, and can absorb water rapidly recovery, and its color and luster, quality and fresh goods are substantially identical.
Accompanying drawing explanation
Fig. 1 is imperialine reference substance canonical plotting;
Fig. 2 is the imperialine canonical plotting of making beating micropowder;
Fig. 3 is Bulbus Fritillariae Cirrhosae micropowder, coarse powder and extractum In Vitro Dissolution compare.
Detailed description of the invention
This detailed description of the invention is only explanation of the invention; it is not limitation of the present invention; as long as any change that those skilled in the art can be done after having read description of the present invention, within claim limited range of the present invention, all will be subject to the protection of Patent Law.
embodiment 1
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 10wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.70 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 10wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll, and make grain size of micropowder more than 95% reach less than 10 μm, moisture content is lower than 5%.
embodiment 2
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 12wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.72 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 11wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll, and make grain size of micropowder more than 95% reach less than 10 μm, moisture content is lower than 5%.
embodiment 3
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 14wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.74 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 12wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll, and make grain size of micropowder more than 95% reach less than 10 μm, moisture content is lower than 5%.
embodiment 4
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 16wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.76 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 13wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll, and make grain size of micropowder more than 95% reach less than 10 μm, moisture content is lower than 5%.
embodiment 5
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 18wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.78 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 14wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll, and make grain size of micropowder more than 95% reach less than 10 μm, moisture content is lower than 5%.
embodiment 6
A kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 20wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.80 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 15wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll, and make grain size of micropowder more than 95% reach less than 10 μm, moisture content is lower than 5%.
ultraviolet content method and interpretation of result
The preparation of reference substance solution
Get imperialine reference substance 0.00301g, accurately weighed, add chloroform 15ml and make the solution of every 1ml containing 0.2mg, to obtain final product.
The preparation of standard curve
Precision measures reference substance solution 0.1ml, 0.2ml, 0.4ml, 0.6ml, 1.0ml, put in 25ml tool plug test tube, add chloroform respectively to 10.0ml, add water 5ml, again precision of precision adds 0.05% bromocresol green buffer and (gets bromocresol green 0.05g, dissolving is made with 0.2mol/L sodium hydroxide solution 6ml, add potassium dihydrogen phosphate 1g, add water and make dissolving and be diluted to 100ml, to obtain final product) 2ml, close plug, violent jolting, is transferred in separatory funnel, places 30 minutes.Get chloroform liquid, filter, get subsequent filtrate with dry filter paper, with corresponding reagent for blank, according to ultraviolet visible spectrophotometry (annex V A), measure absorbance at the wavelength place of 415nm, take absorbance as vertical coordinate, concentration is abscissa, drawing standard curve.
Algoscopy
Sample thief is about 2g, accurately weighed, put in tool plug conical flask, add strong ammonia solution 3ml, infiltrate 1 hour, add chloroform-methanol (4:1) mixed solution 40m1, put 80 DEG C of heating in water bath to reflux 2 hours, let cool, filter, filtrate is put in 50ml volumetric flask, medicinal residues are washed 2-3 time with appropriate chloroform-methanol (4:1) mixed solution, washing liquid is incorporated in same volumetric flask, add chloroform-methanol (4:1) mixed solution and be settled to scale, shake up precision and measure 2-5ml, put in 25ml tool plug test tube, evaporate to dryness in water-bath, precision adds chloroform 10ml makes dissolving, method under the preparation of sighting target directrix curve, from " precision add water 5ml ", measure absorbance in accordance with the law, the weight (mg) of imperialine in need testing solution is read from standard curve, calculate, obtain.
methodological study
Precision test
Precision measures reference substance solution 0.6ml, puts in 25ml tool plug test tube, the method under the preparation of sighting target directrix curve, from " adding chloroform to 10ml ", measure absorbance in accordance with the law, calculate relative standard deviation (RSD), to investigate the precision of instrument.
Result shows that imperialine reference substance is 0.45% in the relative standard deviation (RSD) of 415nm place absorbance, shows that the method precision is good.
stability test
Bulbus Fritillariae Cirrhosae making beating micropowder is about 2g, accurately weighed, according to the preparation method of test sample under 2.2.1.3 algoscopy, measures absorbance at 0min, 5min, 10min, 15min, 20min, 25min in accordance with the law, calculates relative standard deviation.
Result show sample stability relative standard deviation (RSD) is 1.53%, shows that this sample is good at 25min internal stability.
repeatability is tested
Get the preparation method of 6 batch sample according to test sample under algoscopy, measure absorbance in accordance with the law, and calculate its relative standard deviation.
It is 2.58% that result shows 6 batch sample repeatability relative standard deviations (RSD), and between each batch, diversity is less.
average recovery
Get the portion in repeatability sample, absorbance is surveyed, and content is known.Then the standardize solution before from this part of test sample processing procedure is to that step in 50ml measuring bottle, precision pipettes six parts, every part 5ml respectively to six test tube, add appropriate imperialine reference substance, preparation method under 2.2.1.3 algoscopy item, from " in water-bath evaporate to dryness ", measure absorbance in accordance with the law, calculate the response rate.
Result show sample average recovery is all between 95%-105%, and meansigma methods is 102.0%, and relative standard deviation is 2.37%, process stabilizing.
the assay of imperialine
According to the preparation method drawing standard curve of standard curve.Get the sample micropowder under different disposal method respectively, by preparation method under algoscopy, measure absorbance in accordance with the law, calculate the content of imperialine by the dry product of Bulbus Fritillariae Cirrhosae micropowder.
Result shows, and Bulbus Fritillariae Cirrhosae micropowder imperialine content is higher than coarse powder, and making beating powder content is again higher than oven dry and cold dry micropowder.
bulbus Fritillariae Cirrhosae micro powder granule, common flour, extractum dissolution in vitro are tested
Get Bulbus Fritillariae Cirrhosae making beating micropowder 10g respectively, drop in 500ml dissolution medium; Bulbus Fritillariae Cirrhosae coarse powder 10g drops in 500ml dissolution medium; Bulbus Fritillariae Cirrhosae decoction pieces 10g, adding distil water 400ml, reflux two hours at 150 DEG C, centrifugal filtration, filtrate decoction is settled to 100ml, drops in 400ml dissolution medium.According to Chinese Pharmacopoeia (version annex in 2010) dissolution method slurry processes, rotating speed 100r/min, temperature is (37 scholar 0.5) DEG C, respectively at 5,15,30,45,60,90min samples 5mL, supplements the water of identical temperature and volume in stripping rotor simultaneously.The centrifugal rear film excessively of the sample taken out, the filtrate precision of each sample pipettes 1ml, add chloroform 10ml, distilled water 5ml, the bromocresol green solution 2ml of 0.05%, violent jolting, be transferred in paging funnel and leave standstill 15 minutes, get chloroform layer, then cross leaching subsequent filtrate, measure absorbance at 415nm wavelength place.Sample size, in the average content of Bulbus Fritillariae Cirrhosae micropowder quality standard, is 0.0541%, calculates this In Vitro Dissolution of various kinds percentage rate.
Dissolution in vitro experimental result shows, and in 5min, Bulbus Fritillariae Cirrhosae micropowder dissolution rate in vitro is faster than common flour and extractum, and in 90min, accumulation stripping quantity is also higher, meets the rapid dissolution characteristic of effective ingredient after micro mist cell breaking cellular wall.
Claims (3)
1. a Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology, comprises the following steps:
(1) select materials: get Bulbus Fritillariae Cirrhosae and wash, remove impurity, drain;
(2) pull an oar: raw material Bulbus Fritillariae Cirrhosae adds the microcrystalline Cellulose powder of raw materials quality 10 ~ 20wt% after tentatively pulverizing, then by the rotation of mixer batch can, described raw material is fully mixed with described microcrystalline Cellulose, obtain batch mixing; Then the water making beating of batch mixing quality 0.7 ~ 0.8 times is added;
(3) pre-freeze: pre-freeze after making beating, pre-freeze to be frozen into solid for standard, and maintains the solid state that is frozen into 5 ~ 10 hours, pre-freezing temperature-8 ~-18 DEG C; Described pre-freeze, also will add the Tween 80 of serosity quality 10 ~ 15wt% and fully stir before pre-freeze;
(4) lyophilization: pre-freeze postlyophilization, cryodesiccated controlled condition is at-45 ~-50 DEG C, 24 ~ 120 hours time, requires that moisture control is at below 6wt% simultaneously;
(5) micropowder is broken: the powder agglomates obtained after lyophilization is broken into micropowder by vibration type micronizing grinding mixing roll.
2. a kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology according to claim 1, is characterized in that: step (5) is played micropowder specific requirement and is, makes grain size of micropowder more than 95% reach less than 10 μm.
3. a kind of Bulbus Fritillariae Cirrhosae ultra-fine powder preparation technology according to claim 1 and 2, is characterized in that: the moisture content of described Bulbus Fritillariae Cirrhosae superfine powder is lower than 5%.
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Address after: 313300 No.1 Road, health pharmaceutical garden, Anji Economic Development Zone, Huzhou, Zhejiang Patentee after: Zhejiang Zuo Libaicao prepared slices of Chinese crude drugs company limited Address before: 313300, Huzhou Anji Economic Development Zone, Zhejiang Province Health Medical Park No.1 road Zhejiang Chinese Herbal Medicine Co., Ltd. Patentee before: Zhejiang Baicao Traditional Chinese Medicine Pieces Co., Ltd. |