CN106580871B - Vaginal gel and preparation method thereof - Google Patents
Vaginal gel and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
Abstract
The invention relates to the technical field of preparation of vaginitis medicaments, in particular to a vaginal gel and a preparation method thereof, which solve the problem that the prior vaginal gel product has unsatisfactory antibacterial, lubricating and moisturizing effects and mainly comprises the following components: sodium hyaluronate, vitamin E succinate modified hyaluronic acid, EGF (epidermal growth factor), vitamin C, calcium chloride, pentanediol, glycerol, xanthan gum and a buffer system taking disodium hydrogen phosphate-citric acid as gel. The vagina mucosa gel has anti-inflammatory activity, can improve the vagina wettability, is used for treating vagina inflammation, relieving vagina dryness symptoms or used as a human body lubricant, has the effect of promoting the repair of the vagina mucosa squamous epithelial cell layer, can improve the thinning of the vagina mucosa and repair small injury in the vagina, enables the vagina mucosa to recover elasticity and softness, and is suitable for people suffering from vaginitis and vagina dryness.
Description
Technical Field
The invention relates to the technical field of preparation of vaginitis medicaments, in particular to a vaginal gel and a preparation method thereof.
Background
Vaginitis is a common and frequently encountered disease in women and includes bacterial vaginitis, mycotic vaginitis and trichomonas vaginitis. The clinical symptoms of vaginitis are mainly manifested by increased vaginal secretion, peculiar smell and pruritus vulvae, and cause infection of cervix and uterus and pelvic inflammatory diseases. The main population of colpoxerosis or atrophic vaginitis is postmenopausal women, and about half of postmenopausal women have the problem of vaginal dryness according to survey and statistics. The vaginal mucosa consists of non-keratinized stratified squamous epithelium rich in glycogen and basement membrane rich in fiber. The stratified squamous epithelium is rich in glycogen during the growth period, and the vaginal mucosa is moist and elastic. After menopause, due to estrogen depletion, epithelial atrophy, loss of the squamous cell layer, only the basal and accessory basal layer cells which are malnutritional and in which villi easily fall are retained, in which case the vaginal mucosa is vulnerable to bacterial infections and penetration of toxic metabolites and is prone to infection by the formation of minor lacerations. Under normal conditions, the vaginal epithelium has a balance mechanism which can lead to sufficient hydration and lubrication in the vagina and ensure that the vaginal tissue is soft and elastic. After menopause, the female has symptoms of vaginal dryness and the like caused by the fact that vaginal secretion is reduced, vaginal lubrication is insufficient, a physiological protective layer cannot be formed, and epithelial cells are easy to damage or die.
The treatment products for vaginitis or colpoxerosis are divided into three categories: 1. western medicines, such as metronidazole, chlorhexidine, miconazole and ketoconazole, achieve sterilization and antibiosis, and the western medicines have quick effect but are easy to generate drug resistance and have side effects; 2. traditional Chinese medicines, such as fructus Cnidii, radix Sophorae Flavescentis, cortex Phellodendri, Alumen, Glycyrrhrizae radix, Borneolum Syntheticum and Kochiae fructus, etc., have heat-clearing and detoxicating, and parasite killing and itching relieving effects, and can not relieve symptoms in time with slow effect when used alone; 3. the instruments are divided into two types, one is treatment by using ozone or red light physiotherapy equipment, the cost is high when a patient needs to go to a hospital regularly for treatment or buy the treatment equipment by himself, the other is vagina bacteriostatic gel such as chitosan and silver ions, the biological bacteriostatic raw materials are adopted, the vaginitis is treated instantly, conveniently, quickly and economically, and the use and curative effect of the gel are widely accepted. The prior vaginal gel product has unsatisfactory effects of bacteriostasis, lubrication and moisture preservation.
Disclosure of Invention
The invention aims to solve the technical defects and provide a vaginal gel which can safely and effectively treat vaginitis and relieve female vaginal dryness and atrophy symptoms and a preparation method thereof.
In order to achieve the purpose, the invention designs a vaginal gel which comprises the following main components: sodium hyaluronate, vitamin E succinate modified hyaluronic acid, EGF (epidermal growth factor), vitamin C, calcium chloride, pentanediol, glycerol, xanthan gum and a buffer system taking disodium hydrogen phosphate-citric acid as gel.
The mass concentration of the main components is as follows: sodium hyaluronate: 0.2-2%; vitamin E succinate modified hyaluronic acid: 0.1 to 1 percent; EGF: 0.001-0.005%; vitamin C: 0.003-0.006%; calcium chloride: 0.01 to 0.02 percent; and (3) pentanediol: 2 to 10 percent; glycerol: 5 to 10 percent; transparent xanthan gum: 1 to 2 percent.
The molecular formula of the vitamin E succinate modified hyaluronic acid is shown as
Wherein R is
or-H.
Preferably, the method comprises the following steps: the molecular weight of the sodium hyaluronate is 40-100 ten thousand daltons.
The preparation method of the vaginal gel comprises the steps of adding sodium hyaluronate, hyaluronic acid modified by vitamin E succinate, EGF, vitamin C and calcium chloride into deionized water under stirring, and mixing until the sodium hyaluronate, the hyaluronic acid modified by vitamin E succinate, the EGF, the vitamin C and the calcium chloride are dissolved; adding pentanediol, glycerol and transparent xanthan gum under stirring, and mixing until dissolving to obtain a uniform system; adding ionized water, stirring, and adjusting pH of the mixture gel to 4.5 + -0.5 with disodium hydrogen phosphate-citric acid buffer system to obtain the final product.
The vitamin E succinate modified hyaluronic acid is prepared by the following method:
ves activation: dissolving Vitamin E Succinate (VES) in dimethyl sulfoxide (DMSO)
Adding dicarbonyl imidazole (CDI for short) with the same molar ratio with VES carboxyl, activating the VES carboxyl by CDI, and generating VES-CDI intermediate product;
VES-HA production: dissolving micromolecular sodium hyaluronate with the molecular weight range of 5000Da-20kDa in anhydrous formamide, adding 4-dimethylaminopyridine (DMAP for short) as a catalyst, stirring and dissolving at normal temperature, slowly dropwise adding VES-CDI obtained in the step (a), stirring at normal temperature for 20h-32h, and carrying out esterification reaction on hydroxyl of the sodium hyaluronate and VES carboxyl to obtain a vitamin E succinate modified hyaluronic acid (VES-HA for short) polymer;
c. and (3) dialysis and freeze-drying: adding acetone with the volume 2-3 times of that of the reaction solution for precipitation, performing suction filtration to obtain a precipitate, adding water for redissolving the precipitate, dialyzing the precipitate for 2d-3d with pure water, and performing freeze drying on the dialyzate to obtain vitamin E succinate modified hyaluronic acid (VES-HA) freeze-dried powder.
Hyaluronic acid (abbreviated as H)A) HA is a linear glycosaminoglycan composed of repeating disaccharide units glucuronic acid and N-acetylglucosamine, which are connected intramolecularly by 1-4 glycosidic bonds and β (1-3), under normal physiological conditions, the repeating disaccharide units in HA are 25000, and have a molecular weight of about 106-107Da, the HA HAs large molecular weight, and a large number of random coil structures are arranged in the molecule, so that the HA molecule HAs excellent viscoelastic property. HA HAs anti-infective and anti-inflammatory effects, and the specific mode of action is as follows: HA HAs high viscoelasticity, and high permeability of cell abnormality is reduced through regulation, so that invasion of pathogenic microorganisms such as bacteria and the like is prevented; HA induces fibroblasts and keratinocytes to secrete a series of cytokines that act as pro-inflammatory factors in the immune response; HA influences the functions of neutrophils and macrophages and HAs the regulation and control effects on migration, adhesion, phagocytosis and invasion pathogen killing of the neutrophils and the macrophages; HA is useful as a scavenger of inflammatory-related molecules such as metalloproteases, thereby inhibiting the development of an inflammatory response. Due to the fact that HA is large in molecular weight and good in viscoelastic performance, lubricity of tissues can be supplemented or increased after application, in addition, HA molecular surface groups are very hydrophilic, water can be absorbed and retained, and a moisturizing effect can be achieved after application. HA molecules have desirable physiological and biological properties, such as good biocompatibility, good biodegradability, and non-immune prototypes. Two different molecular weights of HA were added to the vaginal gel: macromolecular HA of 40kDa-100kDa plays a role in moisturizing and lubricating; the 5000Da-20kDa micromolecule HA HAs the effects of repairing small injury and recovering a physiological protective layer of a vagina, and the micromolecule HA HAs different anti-inflammatory and bacteriostatic effects. HA metabolizes very fast in vertebrates, about 30% of HA in human body is metabolized and replaced every day, of course, HA half-life difference is large at different parts in human body, and human skinThe half-life of HA in the joint is about 1 day, and the half-life of HA in the joint can reach 20 days. At present, hyaluronic acid is mostly produced by adopting a microbial fermentation method, and sodium salt is used for increasing the ionic strength or adjusting the pH value in the production process, so most of HA finished products exist in the form of sodium salt, namely sodium hyaluronate, and macromolecular HA is added in the form of sodium hyaluronate.
The Vitamin E Succinate (VES) is a derivative of the vitamin E, the VES HAs obviously improved chemical stability compared with the vitamin E, the application range is wider, the carboxyl of the VES can be activated by an activating agent, and then the carboxyl of the VES can be condensed with the hydroxyl of the HA molecule to carry out esterification reaction under the action of a catalyst to form the VES modified HA molecule, so that the stability of the small molecular HA is improved on one hand, and the anti-inflammatory property and the antioxidant property of the vaginal gel are also improved on the other hand.
VES modified HA (VES-HA) is an amphiphilic hyaluronic acid derivative, HAs hydrophilicity, and the existence of a hydrophobic end can solubilize hydrophobic substances in a system in a physical embedding manner, so that the use of an organic solvent is avoided. The VES-HA combines the dual advantages of VES and HA while improving the stability of the small molecular HA, HAs multiplied anti-inflammatory effect, and can effectively resist various different types of vaginal inflammation. The VES-HA molecular structure generated by the esterification reaction is simple, HAs excellent biocompatibility, stability and biodegradability, and the method for modifying HA by VES is effective, economical, simple and easy to industrialize.
The vaginal gel and the preparation method thereof have anti-inflammatory activity, can improve the vaginal wettability, are used for treating vaginal inflammation, relieving vaginal dryness symptoms or used as a human body lubricant, have the effect of promoting the repair of squamous epithelial cell layers of vaginal mucosa, can improve the thinning of the vaginal mucosa and repair small injuries in vagina, restore the elasticity and softness of the vaginal mucosa, and are suitable for people suffering from vaginitis and vaginal dryness.
Detailed Description
The invention is further described below by way of examples.
Example 1:
the vaginal gel and the preparation method thereof described in the embodiment comprise the following components in mass concentration:
1. macromolecular sodium hyaluronate: 0.2 percent of
2.VES-HA：0.5％
3.EGF：0.001％
4. Vitamin C: 0.003%
5. Calcium chloride: 0.01 percent
6. And (3) pentanediol: 2 percent of
7. Glycerol: 10 percent of
8. Transparent xanthan gum: 1 percent of
The molecular weight of the macromolecular sodium hyaluronate is between 40 and 100 ten thousand daltons.
The preparation method comprises adding sodium hyaluronate, hyaluronic acid modified by vitamin E succinate, EGF, vitamin C and calcium chloride into deionized water with total water amount of about 98% under stirring, and mixing to dissolve; adding pentanediol, glycerol and transparent xanthan gum under stirring, and mixing until dissolving to obtain a uniform system; adding ionized water, stirring, and adjusting pH of the mixture gel to 4.5 + -0.5 with disodium hydrogen phosphate-citric acid buffer system to obtain the final product.
The vitamin E succinate modified hyaluronic acid is prepared by the following method:
ves activation: dissolving Vitamin E Succinate (VES) in dimethyl sulfoxide (DMSO), adding dicarbonyl imidazole (CDI) with molar ratio equal to VES carboxyl, activating VES carboxyl by CDI, and generating VES-CDI intermediate product;
VES-HA production: dissolving small-molecule sodium hyaluronate (with a molecular weight range of 5000Da-20kDa) in anhydrous formamide, adding 4-Dimethylaminopyridine (DMAP) as a catalyst, stirring and dissolving at normal temperature, slowly dropwise adding VES-CDI obtained in the step (a), stirring at normal temperature for 24 hours, and carrying out esterification reaction on hydroxyl of the sodium hyaluronate and VES carboxyl to obtain a vitamin E succinate modified hyaluronic acid (VES-HA) polymer;
c. and (3) dialysis and freeze-drying: adding acetone with the volume 2-3 times of that of the reaction solution for precipitation, performing suction filtration to obtain a precipitate, adding water for redissolving the precipitate, dialyzing with pure water for 2 days, and performing freeze drying on the dialysate to obtain vitamin E succinate modified hyaluronic acid (VES-HA) freeze-dried powder.
The molecular formula of the obtained vitamin E succinate modified hyaluronic acid is shown as
Wherein R is
or-H.
Evaluation of anti-inflammatory action:
according to bacterial vaginitis, mycotic vaginitis and trichomonas vaginitis aimed by the vaginal gel disclosed by the invention, candida albicans and escherichia coli are selected as experimental strains.
Preparing a sample solution: the vaginal gel and water of the invention were diluted to 4 concentrations at 1:1, 1:2, 1:4 and 1:8 for use.
The method comprises the following steps:
two media were prepared: 25ml of each of the Sha's dextrose agar medium and the trypticase Soy peptone agar medium were poured into a petri dish having a diameter of 15cm under a laminar flow hood, respectively, and allowed to solidify.
0.4ml of candida albicans and escherichia coli suspension is respectively taken, the candida albicans is inoculated to a Shaw glucose agar culture medium, and the escherichia coli is inoculated to a tryptic soy peptone agar culture medium.
After the applied bacteria liquid is dried, the plate is divided into 4 parts with equal size by a sterilization blade under a laminar flow hood, and the plate is respectively loaded into different culture dishes by using sterile instruments.
Immersing a small piece of autoclaved filter paper into a sample to be tested, then respectively clamping the small piece of autoclaved filter paper by using sterilization forceps, putting the small piece of autoclaved filter paper into a separated plate, covering a plate cover, culturing for 40 hours in a BOD (biochemical oxygen demand) incubator at 32 ℃, and then respectively measuring the size of a bacteriostatic zone.
As a result: the bacteriostatic effect of the vaginal gel on candida albicans and escherichia coli is shown in Table 1
TABLE 1 bacteriostatic Effect of vaginal gels
| Bacterial strains | 1:1 dilution of sample liquid | 1:2 dilution of sample liquid | 1:4 dilution of sample liquid | 1:8 dilution of sample liquid |
| Candida albicans | 43.0mm | 40.5mm | 32.6mm | 25.3mm |
| Escherichia coli | 42.7mm | 41.8mm | 40.2mm | 30.8mm |
And (4) conclusion: as can be seen from Table 1, the vaginal gel of the present invention has significant bacteriostatic effects on Candida albicans and Escherichia coli. Evaluation of the long-lasting moisturizing action of the mixture gel:
three gel samples: the first group of normal vaginal gels, the second group of gels made in this example.
The method comprises the following steps: weighing 2 groups of samples respectively, recording the initial weight of the samples, placing the samples in a constant-temperature incubator with the humidity of 35% and the temperature of 28 ℃, weighing the water loss weight of the samples when the samples are placed for 24 hours, 48 hours and 72 hours respectively, and calculating the water loss rate according to the following formula:
water loss rate (initial weight of sample-weight of water lost from sample)/initial weight of sample 100%
As a result: the water loss rate of the samples was determined as shown in Table 2 below
TABLE 2 Water loss for different samples
And (4) conclusion: the vaginal gel of this example has high long-term lock-up capability.
The mass concentration of the main components of the invention can be in the following range: sodium hyaluronate: 0.2-2%; vitamin E succinate modified hyaluronic acid: 0.1 to 1 percent; EGF: 0.001-0.005%; vitamin C: 0.003-0.006%; calcium chloride: 0.01 to 0.02 percent; and (3) pentanediol: 2 to 10 percent; glycerol: 5 to 10 percent; transparent xanthan gum: 1 to 2 percent.
Claims (4)
1. A vaginal gel characterized by: the main components are as follows: sodium hyaluronate, hyaluronic acid modified by vitamin E succinate, EGF, vitamin C, calcium chloride, pentanediol, glycerol, transparent xanthan gum and a buffer system taking disodium hydrogen phosphate-citric acid as gel, wherein the mass concentration of the main components is as follows: sodium hyaluronate: 0.2-2%; vitamin E succinate modified hyaluronic acid: 0.1 to 1 percent; EGF: 0.001-0.005%; vitamin C: 0.003-0.006%; calcium chloride: 0.01 to 0.02 percent; and (3) pentanediol: 2 to 10 percent; glycerol: 5 to 10 percent; transparent xanthan gum: 1-2%, the molecular formula of the vitamin E succinate modified hyaluronic acid is
Wherein R is
or-H.
2. The vaginal gel of claim 1, wherein: the molecular weight of the sodium hyaluronate is 40-100 ten thousand daltons.
3. The method of preparing a vaginal gel as claimed in claim 1 or claim 2 wherein: adding sodium hyaluronate, hyaluronic acid modified by vitamin E succinate, EGF, vitamin C and calcium chloride into deionized water under stirring, and mixing until the sodium hyaluronate, the EGF, the vitamin C and the calcium chloride are dissolved; adding pentanediol, glycerol and transparent xanthan gum under stirring, and mixing until dissolving to obtain a uniform system; adding ionized water, stirring, and adjusting pH of the mixture gel to 4.5 + -0.5 with disodium hydrogen phosphate-citric acid buffer system to obtain the final product.
4. A method of preparing a vaginal gel as claimed in claim 3 wherein: the vitamin E succinate modified hyaluronic acid is prepared by the following method:
ves activation: dissolving vitamin E succinate (VES for short) in dimethyl sulfoxide (DMSO for short), adding dicarbonyl imidazole (CDI for short) with the same molar ratio as VES carboxyl, activating the VES carboxyl by CDI, and generating VES-CDI intermediate product;
VES-HA production: dissolving micromolecular sodium hyaluronate with the molecular weight range of 5000Da-20kDa in anhydrous formamide, adding 4-dimethylaminopyridine (DMAP for short) as a catalyst, stirring and dissolving at normal temperature, slowly dropwise adding VES-CDI obtained in the step (a), stirring at normal temperature for 20h-32h, and carrying out esterification reaction on hydroxyl of the sodium hyaluronate and VES carboxyl to obtain a vitamin E succinate modified hyaluronic acid (VES-HA for short) polymer;
c. and (3) dialysis and freeze-drying: adding acetone with the volume 2-3 times of that of the reaction solution for precipitation, performing suction filtration to obtain a precipitate, adding water for redissolving the precipitate, dialyzing the precipitate for 2d-3d with pure water, and performing freeze drying on the dialyzate to obtain vitamin E succinate modified hyaluronic acid (VES-HA) freeze-dried powder.
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| CN108992662A (en) * | 2018-08-23 | 2018-12-14 | 杭州元研细胞生物科技有限公司 | A kind of vagina of the factor containing mescenchymal stem cell is spraying and preparation method thereof |
| CN109589404A (en) * | 2018-11-27 | 2019-04-09 | 杭州元研细胞生物科技有限公司 | One kind is for anti-ageing reparation product of female reproductive system and preparation method thereof |
| CN109464655B (en) * | 2019-01-08 | 2021-08-13 | 福建龙生生物科技有限公司 | External capsule preparation for preventing and treating vaginitis |
| CN110151981A (en) * | 2019-05-31 | 2019-08-23 | 杨平芝 | A kind of composition and preparation method thereof repaired vagina and treat vaginitis |
| CN110917132A (en) * | 2019-12-19 | 2020-03-27 | 浙江因诺泰医疗器械有限公司 | Vaginal squamous epithelium repair aqua and preparation method thereof |
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| CN104945538A (en) * | 2015-06-25 | 2015-09-30 | 浙江大学 | Hyaluronic acid vitamin E derivative and preparation and application |
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| CN103182070A (en) * | 2013-04-12 | 2013-07-03 | 江苏迪沃生物制品有限公司 | External composition, and preparation and application thereof |
| CN103191408A (en) * | 2013-04-19 | 2013-07-10 | 江苏迪沃生物制品有限公司 | Medicine composition and gel for treating colpitis and applications thereof |
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Effective date of registration: 20190227 Address after: 310000 Yuhang District, Hangzhou City, Zhejiang Province, No. 2959 Yuhangtang Road, Cangqian Street, 4 buildings and 1 storey Applicant after: Zhejiang Inotai Medical Devices Co., Ltd. Address before: Room 458, Building 3, No. 1, Luting Road, Cangqian Street, Hangzhou City, Zhejiang 310000 Applicant before: HANGZHOU YOUNG CELL BIOTECHNOLOGY CO., LTD. |
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