CN110801013B - Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof - Google Patents

Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof Download PDF

Info

Publication number
CN110801013B
CN110801013B CN201911132220.9A CN201911132220A CN110801013B CN 110801013 B CN110801013 B CN 110801013B CN 201911132220 A CN201911132220 A CN 201911132220A CN 110801013 B CN110801013 B CN 110801013B
Authority
CN
China
Prior art keywords
urushiol
immunoglobulin
raw lacquer
peptide
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911132220.9A
Other languages
Chinese (zh)
Other versions
CN110801013A (en
Inventor
张存莉
范晨
王乾
苏嘉烨
聂俊莲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Northwest A&F University
Original Assignee
Northwest A&F University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Northwest A&F University filed Critical Northwest A&F University
Priority to CN201911132220.9A priority Critical patent/CN110801013B/en
Publication of CN110801013A publication Critical patent/CN110801013A/en
Application granted granted Critical
Publication of CN110801013B publication Critical patent/CN110801013B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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)
    • A61K36/22—Anacardiaceae (Sumac family), e.g. smoketree, sumac or poison oak
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39583—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials not provided for elsewhere, e.g. haptens, coenzymes
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The invention discloses a method for preparing non-allergic raw lacquer or urushiol by a biochemical method and a product thereof. The invention is based on the mechanism of urushiol allergy, the urushiol and one or more of protein, peptide or amino acid are combined in vitro to form a urushiol complex, the tolerance of an organism to anaphylactic reaction is enhanced, the urushiol loses the chance of being combined with direct skin protein, the anaphylactic reaction of the skin is avoided, and the non-allergic raw lacquer or urushiol is obtained. The protein, peptide or amino acid and urushiol are reversibly complexed without really damaging the structure of urushiol, so that the original biological activities of the urushiol such as oxidation resistance, tumor resistance, bacteria resistance and the like are not changed or lost, and the urushiol is easily digested and catabolized in vivo; can be used for developing raw lacquer or urushiol food, health product and medicine; the invention has the advantages of ingenious design, no addition and residue of toxic and harmful reagents in the preparation process and guarantee of the safety of the finished product.

Description

Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof
Technical Field
The invention relates to the fields of food, health care products and medicines, in particular to a method for preparing non-allergic raw lacquer or urushiol by a biochemical method and a product thereof.
Background
Raw lacquer is the secretion of the phloem of Rhus vernicifera, rhus succinanea and Melanorrhoea usitata of the family Anacardiaceae, and is mainly applied to the paint industry in the past to make lacquerware, lacquer paintings and the like; the dried lacquer also has the effects of removing blood stasis, stimulating the menstrual flow, removing food retention, killing parasites, protecting the cardiovascular system and the like, and is widely used for pharmacy.
The main components of the raw lacquer comprise urushiol (60-70%), water (20-30%), vegetable gum (4-10%), laccase (1.5-2%), water-insoluble glycoprotein (3-5%) and the like. Urushiol is a catechol of an olefin having 15 or 17 carbon atoms in a side chain and 1, 2 or 3 unsaturated bonds in the chain.
Urushiol is a hapten, and when the hapten urushiol is contacted with skin, the catalytic reaction of in vivo enzyme oxidizes urushiol to form an electrophilic o-quinone compound, and the urushiol reacts with keratin on a cell membrane at the 4,5 and 6 positions of a catechol ring to form a complete antigen; after the protein-urushiol complex forms complete antigen, the protein-urushiol complex is captured by Langerhans cells (Langerhans cells), activated Langerhans cells gather in lymph nodes, antigen information is presented to effector T cells, the effector cells release cytokines, the cytokines are combined with macrophages, lymphocytes and other different types of target cells, finally pathological reactions such as contact dermatitis, erythema, papules, vesicles, swelling and the like are shown, mild patients have systemic itching and skin ulceration, severe patients can cause blindness, finger dry gangrene, arrhythmia, serous cavity effusion to cause death and other diseases, and the utilization and development of raw lacquer or urushiol are severely restricted.
Modern pharmacological research finds that urushiol has various good effects of resisting oxidation, bacteria, tumors and viruses, treating fatty liver and the like, so that raw lacquer or urushiol which eliminates the urushiol allergy obstacle has great application in the fields of food, health care products and medicines.
At present, the main methods for desensitization of raw lacquer are structural modification of urushiol and degradation of allergen urushiol by using bacterial strains.
The prior technical method for carrying out structural modification on urushiol has the defects that the structure of a urushiol functional group in raw lacquer is changed, and when carrying out structural modification on urushiol, a toxic and harmful reagent or group is generally used or introduced, so that the edible, health-care and medicinal values of raw lacquer are threatened, and the desensitization effect is not thorough; patent CN106075795B discloses that degrading urushiol by using Streptomyces griseus var. Ferrugineus results in loss of urushiol value and resource waste.
At present, no method and product for eliminating the allergy of raw lacquer or urushiol without destroying the basic structure of urushiol and changing the bioactivity of urushiol exist.
Disclosure of Invention
The invention aims to provide a method for preparing non-allergic raw lacquer or urushiol by a biochemical method and a product thereof, so as to solve the problems in the background technology.
The invention aims at the mechanism of raw lacquer allergy and is realized by the following method: under the specific condition, according to a certain process, adding immunoglobulin into raw lacquer or urushiol, adding one or more of protein, peptide and amino acid, and making urushiol and one or more of protein, peptide and amino acid form urushiol complex in vitro. On one hand, the immunoglobulin is an antibody and has an immune reaction to generate an antigen-antibody compound, so that urushiol loses the sensitization effect; in another aspect, the proteins, peptides and amino acids are amphoteric compounds containing a basic group-NH 2 The urushiol contains acidic phenolic hydroxyl groups, and the acid-base group combination enables the urushiol to form a complex with protein, peptide and amino acid in vitro, so that the urushiol loses oxidation reaction when contacting with skin keratin or cell membrane protein, and the formation of urushiol-protein antigen in vivo is avoided. Even if urushiol is in contact with the skin and oxidized into electrophilic o-phenylenediquinones, acidic groups of proteins, peptides and amino acids which are complexed with urushiol immediately compete to combine with 4,5,6 positions of o-phenylenediphenol ring to form in-vitro protein-urushiol complex, so that the binding of urushiol with skin keratin and cell membrane proteins is avoided to form antigen.
The urushiol complex formed by the non-allergic raw lacquer or the urushiol and one or more of the proteins, the peptides or the amino acids, wherein the proteins, the peptides or the amino acids and the urushiol are in reversible complexation, the structure of the urushiol is not really destroyed, the original biological activities of the urushiol such as oxidation resistance, tumor resistance, bacteria resistance and the like are not changed or lost, and the urushiol is easily digested and decomposed in vivo, so that the urushiol complex can be used for developing raw lacquer or urushiol foods, health care products and medicines.
Specifically, the method for preparing the non-allergic raw lacquer or urushiol by a biochemical method comprises the following steps:
s1, preparing an immunoglobulin activating solution:
dissolving trisodium citrate, citric acid, glucose, 0.9% physiological saline water = (22.0-25.0) kg (8.0-10.0) kg (24.5-27.5) kg and 1000L in a sterile environment to prepare an immunoglobulin activation matrix, placing the immunoglobulin activation matrix in a constant-temperature incubator at 28-32 ℃ for activation for 2-4h, and adding immunoglobulin to prepare an immunoglobulin activation solution; the immunoglobulin activation solution activates a matrix: the volume mass ratio of the immunoglobulin is (100-150) L: (5-8) kg.
The raw lacquer is natural lacquer and refined lacquer from Rhus vernicifera, rhus succinanea and Melanorrhoea uselta, etc.
Immunoglobulins and edible proteins, peptides and amino acids are either commercially available or self-made.
S2, preparing non-allergic raw lacquer or urushiol:
under the condition of room temperature, taking the immune globulin activation liquid, slowly adding the immune globulin activation liquid into raw lacquer or urushiol according to the volume ratio of the raw lacquer or urushiol to the immune globulin activation liquid =100L (10-17) L, stirring uniformly, then respectively adding the edible protein, the peptide, the amino acid or one or more mixtures of the edible protein, the peptide and the amino acid according to the volume mass ratio of the raw lacquer or urushiol to the edible protein/peptide/amino acid/peptide + amino acid =100L (20-30) kg/(5-10) kg/(10-20) kg/(5-30) kg), and stirring until the solution is in a uniform gel state.
Further, the immunoglobulin in step S1 may be one or a combination of immunoglobulin G (IgG), immunoglobulin a (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE).
Further, the protein in step S1 may be one or a combination of several of egg white, milk, beans, grains, animal protein, and the like.
Furthermore, the peptide compound can be one or a combination of several of milk peptide, bean peptide, cereal peptide, egg white peptide, livestock peptide, aquatic peptide, silk protein peptide, composite peptide, glutathione and the like.
Further, the amino acid may be one or a combination of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (gin), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), and the like.
The product prepared by the method for preparing the non-allergic raw lacquer or the urushiol.
The product prepared by the invention also passes guinea pig desensitization test:
selecting SD guinea pig with weight (300 + -20) g and depilatory area 30mm 2 After 24h of depilation, 50 guinea pigs which are qualified for depilation are selected, the males and females are respectively divided into a white vaseline control group (a substrate control group) and a raw lacquer stock solution control group at random, the administration and smearing dosage of the raw lacquer or urushiol high, medium and low dosage groups in the third step are 0.2g/kg, 0.1g/kg and 0.05g/kg (which are respectively equal to 50 times, 25 times and 12.5 times of the clinical dosage), and 10 animals in each group are selected; after 24h of administration, the tested substances are removed, and the hair of the guinea pig is normal, and the skin of the guinea pig in the high, medium and low dose groups is smooth and ruddy, has no pruritus, red swelling, ulceration and the like and is continuously observed for 12-15 days.
The product prepared by the invention passes the human body desensitization test:
selecting a qualified spot tester 210 as spot test volunteers, applying 2.5-4.0g of the third step of modified raw lacquer or urushiol in the spot tester by a closed spot test method, and applying an external adhesive tape on the back of the testee (female is attached to the inner side of the forearm); removing the test substance for 12-24 h; the first observation is made at an interval of 30 minutes, and the second and subsequent observations are made at 24, 48, 72, 96, 120, 144, 168 hours respectively; note that the tape reacted only; the test proves that the positive rate of the volunteers is 0.5 percent and the desensitization rate reaches 99.5 percent within 0.5-168h of removing the test substance.
Compared with the prior art, the invention has the beneficial effects that:
one of the technical advantages of the invention is that when the raw lacquer or the urushiol is desensitized, the basic structure of the urushiol is kept, the biological activity of the urushiol is not changed or lost, and the non-allergic raw lacquer still has the original biological activity of the urushiol such as oxidation resistance, tumor resistance, bacteria resistance and the like, and can be used for developing raw lacquer food, health care products and medicines;
the non-allergic raw lacquer has the second technical advantage that no toxic and harmful reagent is added or left in the preparation process, so that the safety of a finished product is ensured;
the third technical advantage of the non-allergic raw lacquer is that through double verification of a guinea pig test and a human body patch test, the desensitization rate is ensured to be more than 99.5%, the injury of the raw lacquer to a human body can be eliminated, and the physical and psychological trauma of raw lacquer practitioners is reduced.
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Example 1 preparation of immunoglobulin G + threonine non-allergenic Lacquer
In a sterile environment, 100mL of immunoglobulin activation matrix is configured according to the mass volume ratio of trisodium citrate, citric acid, glucose and 0.9% physiological saline water = (22.0-25.0) kg, (8.0-10.0) kg, (24.5-27.5) kg and 1000L, the immunoglobulin activation matrix is activated in a constant-temperature incubator at 28-32 ℃ for 2-4h, and 8G of immunoglobulin G is added to prepare the immunoglobulin G activation solution.
Under the environment with the temperature of 22-35 ℃, according to the proportion of raw lacquer, immunoglobulin activating solution, threonine =100L (10-17) L (10-20) kg, 530mL of immunoglobulin activating solution is taken, 3600mL of raw lacquer is gradually added while stirring, 450g of threonine is added after stirring uniformly, and the solution is stirred until the solution is in a uniform gel state.
Selecting SD guinea pig with weight (300 + -20) g and depilatory area 30mm 2 After 24 hours of depilation, 50 guinea pigs with qualified depilation are selected, the male and female are divided into a white vaseline control group (matrix control group) and a raw lacquer stock solution control group at random, the administration and coating doses of the modified raw lacquer high, medium and low dose groups are 0.2g/kg, 0.1g/kg and 0.05g/kg (respectively equivalent to 50 times, 25 times and 12.5 times of clinical dose), and 10 animals in each group are selected. After 24h of administration, the tested substances are removed, and the hair of the guinea pig is normal for 12-15d, and the skin of the guinea pig in the high, medium and low dose groups is smooth, ruddy, free of pruritus, red swelling and ulceration and the like.
A qualified spot tester, 210 name spot test volunteers, is selected, 2.5-4.0g of modified raw lacquer is coated in the spot tester by a closed spot test method, and an external adhesive tape is pasted on the back of a test subject (female is pasted on the inner side of the forearm). Removing the test substance for 12-24 h. The first observation was made at 30 minute intervals and the 2 nd and subsequent daily observations were made at 24, 48, 72, 96, 120, 144, 168 hours, respectively. Note that the tape reacted negligible. The test proves that the positive rate of the volunteers is 0.48% and the desensitization rate reaches 99.52% within 0.5-168h of removing the test substance.
And (3) comparing the desensitized raw lacquer with the stock solution, pouring a certain amount of bacterial liquid into a plate culture medium which is cooled to about 50-60 ℃ by adopting a pre-bacterial liquid pouring plate method, uniformly mixing, pouring a plate (about 20-25 mL/plate), horizontally standing and solidifying to prepare a bacterial-containing plate. Dividing the plate into 3 regions by using a color pen, drilling a circular hole with the diameter of 5.5mm on the test plate by using a sterilized steel pipe in each region, carefully picking out a small culture medium block to make a circular hole, injecting 120-200 mu L of raw lacquer stock solution into one hole, injecting 120-200 mu L of non-allergic raw lacquer into the other hole, using the last hole as a control, pre-diffusing at 4-6 ℃ for 1-2h, culturing at 35-37 ℃ for 12-24h, and culturing mould in an incubator at 25-28 ℃ for 24-48h. Taking out the cultured test plate, measuring the diameter of the inhibition zone by using a vernier caliper by adopting a cross method, expressing the size of the inhibition zone by using the diameter, making 3 parallels for each bacterium, selecting the plate with an obvious inhibition zone to measure the diameter of the inhibition zone, and taking the average value of 3 repeated experiments as a result. The maximum difference of the sizes of the inhibition zones of the non-allergic raw lacquer and the raw lacquer stock solution is not more than 1.0-1.5 percent through verification, which proves that the inhibition activity of the raw lacquer is maintained while the allergy of the raw lacquer is eliminated.
Example 2 preparation of non-allergenic Lacquer with immunoglobulin A + Casein
300mL of immunoglobulin activation matrix is prepared according to the mass-volume ratio of trisodium citrate, citric acid, glucose and 0.9% physiological saline water = (22.0-25.0) kg (8.0-10.0) kg (24.5-27.5) kg and 1000L in a sterile environment, the immunoglobulin activation matrix is activated for 2-4h in a constant-temperature incubator at the temperature of 28-32 ℃, and 24g of immunoglobulin A is added to prepare the immunoglobulin activation solution.
Under the environment with the temperature of 22-35 ℃, according to the proportion of raw lacquer, immunoglobulin activating solution and edible protein =100L (10-17) L (20-30) kg, taking 725mL of immunoglobulin A activating solution, gradually adding 4500mL of raw lacquer while stirring, after stirring uniformly, adding 1050g of casein, and stirring until the solution is in a uniform gel state.
Selecting SD guinea pig with weight of 300 + -20 g and depilatory area of 30mm 2 After 24h of depilation, 50 guinea pigs which are qualified for depilation are selected, the male and female guinea pigs are respectively divided into a white vaseline control group (substrate control group) and a raw lacquer stock solution control group at random, the administration and smearing dosages of the high, medium and low dose groups of the modified raw lacquer are 0.2g/kg, 0.1g/kg and 0.05g/kg (respectively equivalent to 50 times, 25 times and 12.5 times of the clinical dosage), and 10 animals in each group are selected. After 24h of administration, the tested substances are removed, and the hair of the guinea pig is normal, and the skin of the guinea pig in the high, medium and low dose groups is smooth and ruddy, has no pruritus, red swelling, ulceration and the like and is continuously observed for 12-15 days.
A qualified spot tester, 210 name spot test volunteers, is selected, 1.5-2.0g of modified raw lacquer is coated in the spot tester by a closed spot test method, and an external adhesive tape is pasted on the back of a test subject (female is pasted on the inner side of the forearm). Removing the test substance for 12-24 h. The first observation was made at 30 minute intervals and the 2 nd and subsequent daily observations were made at 24, 48, 72, 96, 120, 144, 168 hours, respectively. Note that the tape reacted only insignificantly. The test proves that the positive rate of the volunteers is 0.4 within 0.5-168h after the test object is removed, and the desensitization rate reaches 99.52%.
Preparing 6mL of solution from 4mL of non-allergic raw lacquer by using 2% DMSO solution to serve as a test solution A; preparing 6mL solution from 4mL raw lacquer stock solution and 2% DMSO solution as test solution B; 50mg of ribavirin is prepared into a solution of 50mg/mL by using a 2% DMSO solution to serve as a positive control drug test solution.
Diluting the sample solution with 4% maintenance solution at a ratio of two times to 10 concentration gradients, sequentially inoculating into 96-well plate with grown cells, setting 4 multiple wells, cell control well and blank control well, setting at 32 deg.C and 5% CO 2 Culturing in a virus incubator, observing under an inverted microscope, terminating the culture when 90% of cells have lesions, and determining the CO content at 35 deg.C and 5% according to the procedure of CCK-8 kit (staining with 10 μ LCCK-8 per well, and incubating at 35 deg.C and 5% 2 Culturing in a virus incubator for 3h, measuring absorbance OD value at 520nm wavelength by using a microplate reader), and measuring the absorbance OD value. Calculating half toxic concentration TC of medicine by applying Reed-Muench formula 50 And determining the maximum nontoxic concentration TC 0 . TC of Hep-2 (human laryngeal carcinoma cell) by verification of non-allergic raw lacquer 50 Is 1.687,TC 0 Was 0.896. TC of non-allergic raw lacquer against RD (human rhabdomyosarcoma cells) 50 Is 1.789,TC 0 It was 1.023. The cytotoxicity is far greater than ribavirin, but 0.1 percent greater than the toxicity of raw lacquer stock solution to cells. Proves that the antiviral activity of the raw lacquer is retained while the allergy of the raw lacquer is eliminated.
Example 3 preparation of non-allergic raw Lacquer by immunoglobulin M + Casein phosphopeptide + phenylalanine
500mL of immunoglobulin activation matrix is prepared according to the mass-volume ratio of trisodium citrate, citric acid, glucose and 0.9% physiological saline water = (22.0-25.0) kg (8.0-10.0) kg (24.5-27.5) kg and 1000L in a sterile environment, the immunoglobulin activation matrix is activated for 2-4h in a constant-temperature incubator at the temperature of 28-32 ℃, and 48g of immunoglobulin M is added to prepare the immunoglobulin activation solution.
Taking 555mL of immunoglobulin M activation solution at the temperature of 22-35 ℃, gradually adding 3450mL of raw lacquer, 290g of casein phosphopeptide and 220g of phenylalanine while stirring according to the proportion of 5-30 kg of raw lacquer, immunoglobulin activation solution, edible protein, peptide and amino acid =100L and (10-17) L, and stirring until the solution is in a uniform gel state after stirring uniformly.
Selecting SD guinea pig with weight of 300 + -20 g and depilatory area of 30mm 2 After 24h of depilation, 50 guinea pigs which are qualified for depilation are selected, the male and female guinea pigs are respectively divided into a white vaseline control group (substrate control group) and a raw lacquer stock solution control group at random, the administration and smearing dosages of the high, medium and low dose groups of the modified raw lacquer are 0.2g/kg, 0.1g/kg and 0.05g/kg (respectively equivalent to 50 times, 25 times and 12.5 times of the clinical dosage), and 10 animals in each group are selected. After 24h of administration, the tested substances are removed, and the hair of the guinea pig is normal, and the skin of the guinea pig in the high, medium and low dose groups is smooth and ruddy, has no pruritus, red swelling, ulceration and the like and is continuously observed for 12-15 days.
A qualified spot tester, 210 name spot test volunteers, is selected, 2.5-4.0g of modified raw lacquer is coated in the spot tester by a closed spot test method, and an external adhesive tape is pasted on the back of a test subject (female is pasted on the inner side of the forearm). Removing the test substance for 12-24 h. The first observation was made at 30 minute intervals and the 2 nd and subsequent daily observations were made at 24, 48, 72, 96, 120, 144, 168 hours, respectively. Note that the tape reacted only insignificantly. The test proves that the positive rate of the volunteers is 0.4 within 0.5-168h after the test object is removed, and the desensitization rate reaches 99.52%.
FRAP is often used to detect the reducing power of a sample. Measuring FRAP values with concentrations of 15, 30, 60, 120, 240 and 480 mu mol/L by using Trolox as a standard substance, and calculating a linear regression equation to obtain an equation of y =0.0017x-0.001 (determination coefficient R) 2 0.9946, and the coefficient R is determined after correction 2 A dj 0.9937). The FRAP of the non-allergic raw lacquer is (2.22 +/-0.41) mu molTrolox/mg, and the difference with the FRAP of raw lacquer stock solution is within +/-0.03.
ABTS of Trolox in the range of 1.25-60. Mu. Mol/L + The clearance is linearly related to its concentration (coefficient of determination R) 2 0.9972, and the coefficient R determined after correction 2 A dj At 0.9952) and the standard curve is y =1.588x-0.558. ABTS of non-allergic raw lacquer is calculated by the calculation + ABTS of scavenging capacity (0.68. + -. 0.06) μmol Trolox/mg, with raw lacquer stock solution + Phase of scavenging abilityThe difference is within +/-0.01, and the anti-oxidation activity of the raw lacquer is maintained while the allergy elimination of the raw lacquer is realized.
Example 4 preparation of non-allergenic urushiol from immunoglobulin G + serine
200mL of immunoglobulin activation matrix is prepared according to the mass-volume ratio of trisodium citrate, citric acid, glucose and 0.9% physiological saline water = (22.0-25.0) kg (8.0-10.0) kg (24.5-27.5) kg and 1000L in a sterile environment, the immunoglobulin activation matrix is activated in a constant temperature incubator at the temperature of 28-32 ℃ for 2-4h, and 16G of immunoglobulin G is added to prepare the immunoglobulin activation solution.
Under the environment with the temperature of 22-35 ℃, according to urushiol and immunoglobulin activating solution, amino acid =100L (10-17) L (10-20) kg, 170mL of immunoglobulin G activating solution is taken, and then, 1330mL of urushiol is gradually added while stirring, after stirring uniformly, 150G of serine is added, and the solution is stirred until the solution is in a uniform gel state.
Selecting SD guinea pig with weight (300 + -20) g and depilatory area 30mm 2 After 24h of depilation, 50 guinea pigs which are qualified for depilation are selected, the male and female animals are respectively divided into a white vaseline control group (matrix control group) and a urushiol stock solution control group at random, the administration and smearing dosages of the modified urushiol high, medium and low dosage groups are 0.2g/kg, 0.1g/kg and 0.05g/kg (respectively equivalent to 50 times, 25 times and 12.5 times of the clinical dosage), and 10 animals in each group are selected. After 24h of administration, the tested substances are removed, and the hair of the guinea pig is normal, and the skin of the guinea pig in the high, medium and low dose groups is smooth and ruddy, has no pruritus, red swelling, ulceration and the like and is continuously observed for 12-15 days.
A qualified spot tester (210 name) is selected as a spot test volunteer, 2.5-4.0g of non-allergic urushiol is coated in the spot tester by a closed spot test method, and an external adhesive tape is pasted on the back of the testee (female is pasted on the inner side of the forearm). Removing the test substance for 12-24 h. The first observation was made at 30 minute intervals and the 2 nd and subsequent daily observations were made at 24, 48, 72, 96, 120, 144, 168 hours, respectively. Note that the tape reacted negligible. The test proves that the positive rate of the volunteers is 0.4 within 0.5-168h after the test object is removed, and the desensitization rate reaches 99.52%.
And (3) comparing the desensitized urushiol with the stock solution, pouring a certain amount of bacteria solution into a plate culture medium which is cooled to about 50-60 ℃ by adopting a pre-bacteria solution pouring plate method, uniformly mixing, pouring the plate (about 20-25 mL/plate), horizontally standing and solidifying to prepare a bacteria-containing plate. Dividing the plate into 3 regions by using a color pen, drilling a circular hole with the diameter of 5.5mm on the test plate by using a sterilized steel pipe in each region, carefully picking out a small piece of culture medium to prepare a circular hole, injecting 120-200 mu L of urushiol into one hole, injecting 120-200 mu L of non-allergic raw lacquer into the other hole, using the last hole as a control, pre-diffusing at 4-6 ℃ for 1-2h, culturing at 35-37 ℃ for 12-24h, and culturing mould in an incubator at 25-28 ℃ for 24-48h. Taking out the cultured test plate, measuring the diameter of the inhibition zone by using a vernier caliper by adopting a cross method, expressing the size of the inhibition zone by using the diameter, making 3 parallels for each bacterium, selecting the plate with an obvious inhibition zone to measure the diameter of the inhibition zone, and taking the average value of 3 repeated experiments as a result. The maximum difference of the sizes of the inhibition zones of the non-allergic urushiol and the urushiol stock solution is not more than 0.5-0.3 percent through verification, and the fact that the urushiol is allergic to the urushiol is proved, and the antibacterial activity of the urushiol is kept.
Example 5 preparation of non-allergenic urushiol from immunoglobulin D + valine
In a sterile environment, 900mL of immunoglobulin activation matrix is prepared according to the mass ratio of trisodium citrate, citric acid, glucose and 0.9% physiological saline water = (22.0-25.0) kg, (8.0-10.0) kg, (24.5-27.5) kg and 1000L, the immunoglobulin activation matrix is activated in a constant-temperature incubator at 28-32 ℃ for 2-4h, and 72g of immunoglobulin is added to prepare an immunoglobulin D activation solution.
Under the environment with the temperature of 22-35 ℃, 580mL of immunoglobulin activation solution is taken according to urushiol and immunoglobulin activation solution, amino acid =100L (10-17) L (10-20), urushiol 5300mL is gradually added while stirring, 630g of valine is added after stirring uniformly, and the solution is stirred until the solution is in a uniform gel state.
Selecting SD guinea pig with weight of 300 + -20 g and depilatory area of 30mm 2 After 24h of depilation, 50 guinea pigs with qualified depilation are selected, the male and female animals are respectively divided into white vaseline control group (matrix control group) and urushiol stock solution control group at random, and the modified urushiol high, medium and low dosage groups are administered with smearing dosage of 0.2g/kg, 0.1g/kg and 0.05g/kg (respectively equivalent to that of modified urushiol high, medium and low dosage groups50, 25 and 12.5 times the clinical dose), 10 animals per group. After 24h of administration, the tested substances are removed, and the hair of the guinea pig is normal, and the skin of the guinea pig in high, medium and low dose groups is smooth and ruddy, has no pruritus, red swelling and ulceration and the like after continuous observation for 12-15 d.
A qualified spot tester, 210 name spot test volunteers, is selected, 2.5-4.0g of modified urushiol is coated in the spot tester by a closed spot test method, and an external adhesive tape is pasted on the back of a test subject (female is pasted on the inner side of the forearm). Removing the test substance for 12-24 h. The first observation was made at 30 minute intervals and the 2 nd and subsequent daily observations were made at 24, 48, 72, 96, 120, 144, 168 hours, respectively. Note that the tape reacted only insignificantly. The test proves that the positive rate of the volunteers is 0% and the desensitization rate reaches 100% within 0.5-168h of removing the test substance.
Preparing 6mL of solution from 4mL of non-allergic urushiol and 2% DMSO solution to serve as a test solution A; preparing 6mL of urushiol stock solution B as a test solution B by using 4mL of urushiol stock solution and 2% DMSO solution; 50mg of ribavirin is prepared into a solution of 50mg/mL by using a 2% DMSO solution to serve as a positive control drug test solution.
Diluting the sample solution with 4% maintenance solution at a ratio of 10 concentration gradients twice, sequentially inoculating into 96-well grown cells, setting 4 multiple wells, cell control well and blank control well, placing at 32 deg.C and 5% CO 2 Culturing in a virus incubator, observing under an inverted microscope, terminating the culture when 90% of cells have lesions, and determining the CO content at 35 deg.C and 5% according to the procedure of CCK-8 kit (staining with 10 μ LCCK-8 per well, and incubating at 35 deg.C and 5% 2 Culturing in a virus incubator for 3h, measuring absorbance OD value at 600nm wavelength by using a microplate reader), and measuring the absorbance OD value. Calculating the median toxic concentration of the medicine by using Reed-Muench formula, and determining the maximum nontoxic concentration TC 0 . Proved that the non-allergic urushiol is used for treating Hep-2 (human laryngeal carcinoma cell) TC 50 Is 1.578,TC 0 Is 0.824. TC desensitization to RD (human rhabdomyosarcoma cells) 50 Has a value of 0.452,TC 0 Is 0.221. The cytotoxicity is far higher than that of ribavirin, even the toxicity of the urushiol stock solution to cells is 7 percent lower, and the fact that the antiviral activity of urushiol is kept while the allergy of raw lacquer is eliminated is proved.
Example 6 preparation of non-allergenic urushiol from immunoglobulin E + Casein phosphopeptide + methionine
In a sterile environment, 800mL of immunoglobulin activation matrix is prepared according to the mass-volume ratio of trisodium citrate, citric acid, glucose and 0.9% physiological saline water = (22.0-25.0) kg, (8.0-10.0) kg, (24.5-27.5) kg and 1000L, the immunoglobulin activation matrix is activated in a constant temperature incubator at 28-32 ℃ for 2-4h, and 64g of immunoglobulin E is added to prepare the immunoglobulin activation solution.
Under the environment of 22-35 ℃, according to urushiol, immunoglobulin activation solution and peptide + amino acid =100L, (10-17) L, (5-30) kg, 850mL of immunoglobulin activation solution is taken, 5500mL of urushiol is gradually added while stirring, 70g of casein, 190g of casein phosphopeptide and 100g of methionine are added after uniform stirring, and the mixture is stirred until the solution is in a uniform gel state.
Selecting SD guinea pig with weight (300 + -20) g and depilatory area 30mm 2 After 24 hours of depilation, 50 guinea pigs which are qualified for depilation are selected, male and female are divided into a white vaseline control group (matrix control group) and a urushiol stock solution control group semi-randomly, the administration and coating doses of the modified urushiol high, medium and low dose groups are 0.2g/kg, 0.1g/kg and 0.05g/kg (which are respectively equivalent to 50 times, 25 times and 12.5 times of clinical dose), and 10 animals in each group are selected. After 24h of administration, the tested substances are removed, and the hair of the guinea pig is normal, and the skin of the guinea pig in the high, medium and low dose groups is smooth and ruddy, has no pruritus, red swelling, ulceration and the like and is continuously observed for 12-15 days.
A qualified spot tester, 210 name spot test volunteers, is selected, 2.5-4.0g of modified urushiol is coated in the spot tester by a closed spot test method, and an external adhesive tape is pasted on the back of a test subject (female is pasted on the inner side of the forearm). Removing the test substance for 12-24 h. The first observation was made at 30 minute intervals and the 2 nd and subsequent daily observations were made at 24, 48, 72, 96, 120, 144, 168 hours, respectively. Note that the tape reacted negligible. The test proves that the positive rate of the volunteers is 0.4 within 0.5-168h after the test object is removed, and the desensitization rate reaches 99.52%.
Within a certain concentration range (0.31-10 mmol/L), the scavenging rate of superoxide anion free radicals and VC concentration are in a linear relation, and the linear regression equation is y =8.975x +5.677(determination of coefficient R 2 0.9943, and the coefficient R was determined after correction 2 Adj is 0.9948). Substituting the measured clearance rate of the non-allergic urushiol superoxide anion free radical into the linear equation to obtain that the clearance capacity of the non-allergic urushiol superoxide anion free radical is (18.92 +/-2.42) mu mol VC/mg, and is within +/-0.01 of the clearance capacity of the urushiol stock solution superoxide anion free radical, which proves that the antioxidation activity of the urushiol is maintained while the urushiol is eliminated.
The concentration of Trolox is in a certain range (1.5-80 mu mol/L) and is in linear relation with DPPH free radical clearance (determining coefficient R) 2 Is 0.9892, and the coefficient R is determined after correction 2 Adj is 0.9817), the regression equation is: y =0.978x +5.823. According to this equation, the DPPH radical scavenging ability of the non-allergenic urushiol sample was (0.69. + -. 0.08). Mu. Mol Trolox/mg, which was within. + -. 0.02 from the DPPH radical scavenging ability of the urushiol stock solution, demonstrating that the antioxidant activity of urushiol is retained while the allergenicity to urushiol is eliminated.
Although the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

Claims (6)

1. A method for preparing non-allergic raw lacquer or urushiol by a biochemical method is characterized by comprising the following steps:
s1, preparing an immunoglobulin activating solution:
dissolving trisodium citrate, citric acid, glucose and 0.9% physiological saline water in a mass-volume ratio of (22.0-25.0) = (8.0-10.0) kg, (24.5-27.5) kg to 1000L to prepare an immunoglobulin activation matrix in a sterile environment, placing the immunoglobulin activation matrix in a constant-temperature incubator at the temperature of 28-32 ℃ for activation for 2-4h, and adding immunoglobulin to prepare an immunoglobulin activation solution; activating the substrate in the immunoglobulin activation solution in the step S1: the volume mass ratio of the immunoglobulin is (100-150) L: (5-8) kg;
s2, preparing non-allergic raw lacquer or urushiol:
under the condition of room temperature, taking the immunoglobulin activation solution, slowly adding the immunoglobulin activation solution into raw lacquer or urushiol while stirring according to the volume ratio of raw lacquer or urushiol to the immunoglobulin activation solution =100L (10-17) L, and after uniformly stirring, adding the immunoglobulin activation solution into the raw lacquer or urushiol according to the volume ratio of the raw lacquer or urushiol: edible protein/peptide/amino acid/edible protein + peptide + amino acid =100L (20-30) kg/(5-10) kg/(10-20) kg/(5-30) kg volume mass ratio), respectively adding edible protein or peptide or amino acid, or mixture of edible protein, peptide and amino acid, stirring until the solution is in uniform gel state.
2. The method of claim 1, wherein the immunoglobulin of step S1 is one of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE).
3. The method of claim 1, wherein the immunoglobulins and edible proteins, peptides and amino acids of steps S1 and S2 are commercially or self-made.
4. The method for preparing non-allergic raw lacquer or urushiol according to claim 1, wherein the edible protein in step S2 is one or more of egg white, milk, grains, and livestock protein.
5. The method for preparing non-allergic raw lacquer or urushiol according to claim 1, wherein the peptide in step S2 is one or more of milk peptide, cereal peptide, egg white peptide, livestock peptide, aquatic peptide, silk peptide, complex peptide, and glutathione.
6. The method of claim 1, wherein the amino acid in step S2 is one or more of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp) and glutamic acid (Glu) in any combination.
CN201911132220.9A 2019-11-19 2019-11-19 Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof Active CN110801013B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911132220.9A CN110801013B (en) 2019-11-19 2019-11-19 Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911132220.9A CN110801013B (en) 2019-11-19 2019-11-19 Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof

Publications (2)

Publication Number Publication Date
CN110801013A CN110801013A (en) 2020-02-18
CN110801013B true CN110801013B (en) 2023-03-31

Family

ID=69490563

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911132220.9A Active CN110801013B (en) 2019-11-19 2019-11-19 Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof

Country Status (1)

Country Link
CN (1) CN110801013B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116832389A (en) * 2023-07-14 2023-10-03 宁波博通文化创意有限公司 Improved process for desensitizing lacquer urushiol

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5409908A (en) * 1993-10-20 1995-04-25 Vyrex Corporation Complexing urushiols
WO1995026980A2 (en) * 1994-04-01 1995-10-12 Immulogic Pharmaceutical Corporation Haptenated peptides and uses thereof
CN1148505A (en) * 1995-08-11 1997-04-30 日本脏器制药株式会社 Activated immune globulin
US5643572A (en) * 1990-07-06 1997-07-01 Allergene, Inc. Methods and compositions for the modulation of host immune response to an allergen
CN106075795A (en) * 2016-06-28 2016-11-09 中国林业科学研究院林产化学工业研究所 A kind of bioanalysis removes the hypersensitive method of Toxicodendron verniciflnum (Stokes) F. A. Barkley (Rhus verniciflua Stokes) laccol

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6733764B2 (en) * 2000-06-14 2004-05-11 Alain Martin Immunostimulator anti-cancer compounds and methods for their use in the treatment of cancer
WO2013166218A1 (en) * 2012-05-01 2013-11-07 The Regents Of The University Of California Deactivation of urushiol and method of treatment and prevention of contact dermatitis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5643572A (en) * 1990-07-06 1997-07-01 Allergene, Inc. Methods and compositions for the modulation of host immune response to an allergen
US5409908A (en) * 1993-10-20 1995-04-25 Vyrex Corporation Complexing urushiols
WO1995026980A2 (en) * 1994-04-01 1995-10-12 Immulogic Pharmaceutical Corporation Haptenated peptides and uses thereof
CN1148505A (en) * 1995-08-11 1997-04-30 日本脏器制药株式会社 Activated immune globulin
CN106075795A (en) * 2016-06-28 2016-11-09 中国林业科学研究院林产化学工业研究所 A kind of bioanalysis removes the hypersensitive method of Toxicodendron verniciflnum (Stokes) F. A. Barkley (Rhus verniciflua Stokes) laccol

Also Published As

Publication number Publication date
CN110801013A (en) 2020-02-18

Similar Documents

Publication Publication Date Title
CN100512808C (en) Externally applied compound amino acid prepn for promoting wound healing and its preparation process and application thereof
TW200938840A (en) A method for in vitro study of immune modulation using pig blood cell
CA2534102A1 (en) Use of spermine and/or spermidine against skin ageing in dietary, pharmaceutical or cosmetic compositions
CN110801013B (en) Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof
AU3261799A (en) Inorganic nitrite and organic acid in combination as topical antiviral composition
GB2053680A (en) Aconite root extract
KR19990044835A (en) Herbal Extract
AU2020103121A4 (en) Method for preparing non-allergic raw lacquer or urushiol by biochemical method and its products
KR101779480B1 (en) Composition for prevention or treatment of depilation or improvement of hair growth having fermented peptone, and formulation having the same
Jayanti et al. Egg white albumin form complex with aspirin and caffeine and its role as free radical scavenger
Gray et al. Bilirubin-serum protein complexes and the van den Bergh reaction
CN108402473B (en) Oral brain polypeptide composition with memory improving function
BG62172B1 (en) Compounds and production of the amadori reaction, method for their preparation and application
Kietzmann et al. The mouse epidermis as a model in skin pharmacology: influence of age and sex on epidermal metabolic reactions and their circadian rhythms
AU2020103370A4 (en) Preparation of non-allergic urushiol by a food chemical method and its products
Flesch Epidermal iron pigments in red species
EP3288575B1 (en) Bioassimilable protein-melanin complex, preparation and uses
CN112386520A (en) Mesoderm injection composition for comprehensively improving skin color and preparation method thereof
CN110746315B (en) Method for preparing non-allergic urushiol by food chemical method and product thereof
Sueki et al. Effect of non-enzymatic glycosylation and heating on browning of human stratum corneum and nail
RU2350342C1 (en) Method of producing gel based on water extraction from unossified antlers
RU2363481C1 (en) Way of pantohematogen manufacture
KR101672883B1 (en) Microneedle for prevention of depilation, of improvement of hair growth
Burda et al. The end effector of ischemic tolerance present in blood plasma from double conditioned donors ameliorates trimethyltin provoked damage in brain
Al-Hussaini Alkaline phosphatase in fish gut

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant