AU2020103370A4 - Preparation of non-allergic urushiol by a food chemical method and its products - Google Patents
Preparation of non-allergic urushiol by a food chemical method and its products Download PDFInfo
- Publication number
- AU2020103370A4 AU2020103370A4 AU2020103370A AU2020103370A AU2020103370A4 AU 2020103370 A4 AU2020103370 A4 AU 2020103370A4 AU 2020103370 A AU2020103370 A AU 2020103370A AU 2020103370 A AU2020103370 A AU 2020103370A AU 2020103370 A4 AU2020103370 A4 AU 2020103370A4
- Authority
- AU
- Australia
- Prior art keywords
- urushiol
- allergic
- stir
- amino acid
- solvent
- 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
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
- C07D209/16—Tryptamines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C221/00—Preparation of compounds containing amino groups and doubly-bound oxygen atoms bound to the same carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
- C07C225/02—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to acyclic carbon atoms of the carbon skeleton
- C07C225/14—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being unsaturated
- C07C225/16—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being unsaturated and containing six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
- C07C319/14—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides
- C07C319/20—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides by reactions not involving the formation of sulfide groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
- C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/29—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a method for preparing non-allergic urushiol by a food chemical
technology and the products thereof. The invention aims at the mechanism of urushiol
hypersensitivity, and takes the non-toxic and harmless amino acid as the auxiliary material, and
introduces an amino acid group on a phenol hydroxyl of urushiol to form urushiol amino acid
conjugates; thus, when the semi-antigen urushiol comes into contact with human or animal
skin, it prevents urushiol from being oxidized into electrophilic o-quinones compound by the
enzyme in the body, and further avoids organic allergic reaction caused by intact antigen as a
result of the reaction between o-quinones compound and the cytomembrane protein. The non
allergic urushiol-amino acid conjugate is prepared, and through a patch test on guinea pig's
skin and human body, the desensitization effect can reach more than 99.0%. The activity of
non-allergic urushiol was found to be unchanged and even enhanced by antivirus, anti
oxidation and anti-bacterial tests. The invented non-allergic urushiol products can be used in
food, health products and medicine.
Description
Preparation of non-allergic urushiol by a food chemical method and its products
[0001] The invention related to the fields of food, health products and medicine specifically refers to a food chemical method for preparing non-allergic urushiol and the products produced thereof.
[0002] Raw lacquer from the excretion of Rhus vernicifera, Rhus succedanea and Melanorrhoea usitata is mainly composed of urushiol (60-70%), water (20-30%), plant gum (4-10%), laccase (1.5 2%) and water insoluble glycoprotein (3-5%). The main structure of urushiol in lacquer is o benzene-2-phenol. The side chain of urushiol is the olefin containing 15 or 17 carbon atoms of 1, 2 or 3 unsaturated bonds. The position of unsaturated bonds is not fixed. Because of its excellent high gloss, high hardness, corrosion resistance, wear resistance, acid resistance, solvent resistance, heat resistance, water insulation, insulation and glossiness, it, is the top-grade paint for military, industry, agriculture and forestry, and furniture and is reputed as the "king of paint". At the same time, its main component urushiol has anti-oxidation, anti-bacterial, anti-tumor, anti-virus and anti fatty liver and other effects. There is great potential for use in food, health products and medicines. However, about 60% of the population is allergic to urushiol, with their skin appearing swelling, erythema, pruritus and other symptoms after contact with raw lacquer and some even suffering partial skin burn-like blisters, which makes people turn pale once hearing its name so as to limit its application.
[0003] Urushiol is a semi-antigen, but when the semi-antigen urushiol comes into contact with the skin, the catalytic reaction of the enzyme in vivo oxidizes urushiol to form an electrophilic o quinone compound, which reacts with the keratin or protein on the cell membrane at the positions of 4, 5 and 6 of the Catechol ring to form a complete antigen. After the protein-urushiol complex form complete antigens, Langerhans cells, which are captured and activated by Langerhans cells, aggregated in the lymph nodes and presented antigen information to the effector T cells which will release cytokines,. Cytokines will combine with different target cells: macrophages, lymphocytes and other types of cell tofinally show pathological reactions such as contact dermatitis, erythema, papules, vesicles and swelling.
[0004] At present, the main preparation method of non-allergic urushiol is to modify the structure of urushiol by using chemical reagents. The defects of the existing technology are that structural modification mainly with chemical reagents will generally apply or introduce harmful reagents or groups and the desensitization effect is not complete.
[0005] The purpose of the invention is to provide a method for preparing non-allergic urushiol using food chemistry technology and its products to solve the problems raised in the above technical background.
[0006] The invention aims at the mechanism of urushiol hypersensitivity and is realized by the following methods:, it takes the non-toxic and harmless amino acid as the auxiliary material, and introduces an amino acid group on a phenol hydroxyl of urushiol to form urushiol amino acid conjugates; thus, when the semi-antigen urushiol comes into contact with human or animal skin, it prevents urushiol from being oxidized into electrophilic o-quinones compound by the enzyme in the body, and further avoids organic allergic reaction caused by intact antigen as a result of the reaction between o-quinones compound and the cytomembrane protein. The non-allergic urushiol-amino acid conjugate is prepared, and through a patch test on guinea pig's skin and human body, the desensitization effect can reach more than 99.0%. The activity of urushiol is found to be unchanged. The biological activity will not decrease thanks to the non-allergic urushiol which can be broadly applied into urushiol allergic elimination, urushiol eating, healthy products and medicines.
[0007] In order to achieve the above purpose, the invention provides the following technical scheme: aiming at the mechanism of urushiol hypersensitivity, taking the non-toxic and harmless amino acid as auxiliary material, introducing an aminoacyl amino acid group on a phenol hydroxyl group of urushiol to form urushiol amino acid conjugates by means of food chemistry, so that when the semi antigen urushiol comes into contact with the skin, it avoids the oxidation of urushiol into electrophilic o-quinone compounds by the enzyme in vivo, prevents the reaction of o-quinone compounds with cell membrane proteins to form intact antigens, and generates the non-allergic urushiol of urushiol- amino acid conjugate. For the preparation flow chart and the structure of the produced non-allergic urushiol, refer to the figure attached in the instruction.
[0008] It specifically includes the following steps:
[0009] S1, source of urushiol:
[0010] Urushiol can come from natural raw paint or refined paint produced by Rhus vernicifera, Rhus succedanea and Melanorrhoea usitata of different places of origin, different varieties and different seasons.
[0011] Its molecular formula is:
[0012] The R structure thereof can be
R=C17H93
Or
Any one of them.
[0013] S2, phenol hydroxyl of urushiol introduces benzyl protective group:
[0014] Amid the reactor, the solvent is added according to the ratio of ethanol: purified water-(1.5-2.5) L: IL; then little by little pour (1.5-2.0)mol/L sodium hydroxide solution into the solvent: according to the volume ration of solvent: sodium hydroxide solution=(12-18) L: IL, stir the mixed liquid evenly and heat to 60-85°C5o lye ratio is added slowly (1.5-2.0) mol/L
urushiol=(1-3): 1 mole ratio, add urushiol and stir for 10-30 minutes. In 30 min, according to the molar ratio of urushiol: benzyl chloride=10:(10-12), add benzyl chloride, maintain the temperature at 60-85 5 and keep reaction for 1 h-3 h which will stop when the oil droplet floating urushiol turns into the solution state.
[0015] Transfer out the reaction liquid from the reactor, remove the solvent through reduced pressure distillation, cool to normal temperature; add in HClat the density of (4.5-6.5)mol/L in the volume of about 20%-30% of the reaction liquid to carry out acidification, stir slowly, start to filter when the amount of solid precipitate no longer changes, transfer the filter cake into (1.5-2.5) mol/L NaOH solution and stir slowly, stop adding alkali and filtering when the cake is fully dissolved, drop concentrated hydrochloric acid into the obtained liquid for acidification, stop reacting and filtering when the precipitate no longer increases, wash the solid filter cake with distilled water to normal dry and recrystallize to produce product 1 whose main component is o benzyl urushiol.
[0016] S3 urushiol hydroxyl introduced amino acids:
[0017] Mix product 1 (o-benzoxy urushiol), amino acid and 4-2 methylaminopyridine (DMAP) according to the molar ration of 1:(1.2-1.5): (2-2.5), add ethanol into the mixture until complete dissolution and then, cool the reaction solution to 2-0°Creaction solution to equal volumed 1-ethyl
(3-2 methylaminopropyl) carbinyl 2-imide hydrochloride (EDCI) at the concentration of 2.mol/LL, stir for 5-10 min after the dropping at the temperature of 0--2C and then stir another 3-5h at room
temperature, remove the solvent through reduced pressure distillation after the reaction stops, extract with CH 2 C1 2 /H 2 0, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, filter to remove the solvent leaving brown powder and recrystal to result in product 2 whose main component is o-benzyl aminoacyl urushiol.
[0018] The amino acids can be any or any combination of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamine, lysine, arginine, histidine, aspartic acid, and glutamic acid etc.
[0019] S4, elimination of urushiol hydroxybenzyl protective group:
[0020] Dissolve generated product 2 in anhydrous ethanol, filter to remove the insoluble substance, add 10% palladium carbon (Pd/C) catalyst under stirring, stir in H2 atmosphere for 4-7h, filter to remove the insoluble substance, recrystallize the filtrate with anhydrous ethanol under vacuum distillation to gain product 3 which replaces the urushiol with aminoacyl group.
[0021] S5, Allergic detection of non-allergic urushiol
[0022] Choose SD guinea pigs whose weight are (300 20) g and depilatory area (30 mm2, choose unhairing qualified guinea pigs after 24 hours upon the hair removal, divide each half of the male and female randomly into white vaseline control group (matrix control group) and urushiol control group, apply the modified urushiol to the animals of 10 per group with a dosage of high, medium and low degree, respectively 0.2g/kg, 0.1g/kg, 0.05g/kg (equivalent to 50 times 25times and 12.5 times the clinic dosage). Upon continuous observation of 12-15d after 24h of the medicine application, it is found that the guinea pigs have normal hair and the ones in high, medium and low dosage groups appear smooth, tender and rosy skin without itching, swelling or fester etc.
[0023] 210 volunteers took part in the human body patch test for urushiol desensitization. It is verified that the desensitization effect of urushiol is excellent and the desensitization rate reaches over 99.0%.
[0024] S6, Bioactivity detection of non-allergic urushiol
[0025] Antiviral activity detection of non-allergic urushiol: dilute the solvents before and after urushiol desensitization with 5% of the maintenance media to 8 concentration gradients at the proportion of 2:1, seperately inoculate them in the cell well-grown 96 pore plate attached with 4 reperforations, cell control holes and blank control holes, cultivate in 5% C02 virus incubator at °C, stop the cultivation when 90% cells are diseased under the microscope, and then measure OD
value according to the operation of CCK-8 reagent kit (add 10 p LCCK-8 for dyeing, cultivate in 5%
C02 virus incubator at 35°C5rus 5h and measure OD value with 520nm wave length). Use Reed
Muench formula to work out half medicine poisoning concentration TC50 and confirm the highest non-poisonous concentration TCO. It is verified that non-allergic urushiol has much higher cytotoxicity to Hep-2 and RD than ribavirin, but 7% lower than urushiol basic solution.
[0026] Non-allergic urushiol scavenging ability test: in the range of certain concentration (0.31-10 mmol/L), the scavenging rate of superoxide anion radical is linear with VC concentration, the linear regression equation is y=8.975 x+5.827(the determinant coefficient R2 is 0.99573, the corrected determinant coefficient R2 Adj is 0.9962). By putting the scavenging rate of superoxide anion free radical into the aforesaid linear equation can figure out the scavenging capacity of the superoxide anion radical of the non-allergic urushiol is (18.74 2.12) molVC/g which is relatively higher than that of the basic urushiol solution. The concentration of Trolox is in the range of 1(480 mol/L), which is linearly related to the scavenging capacity of DPPH radical (the determinant coefficient R2 is 0.9892, the corrected determinant coefficient R2Adj is 0.9817), and the regression equation was: y=0.979 x+5.836. According to this formula, the scavenging capacity of DPPH radical of the sample non-allergic urushiol is (0.73+0.08)mol Trolox/g, proving that the free radical scavenging rate of the amino acid desensitized urushiol does not fall.
[0027]Non-allergic urushiol antibacterial, anti-tumor and other biological activities are not reduced too.
[0028] Compared with the prior art, the beneficial effect of the invention is: in the structure of urushiol, the innocuous amino acid group is introduced, so as to avoid being oxidized into o-benzoquinone in contact with the skin to form a complete antigen with the protein of the cell membrane in the body; the introduced amino acid group is easily interpreted by the enzyme drop in the body to release the urushiol hydroxyl group, which basically retains the bioactivity of urushiol, and can be used in the development of raw lacquer food, health products, drugs and non-allergic coatings; the urushiol structure does not contain any poisonous and harmful group so as to ensure the safety and liability offinished products; both the guinea pig test and human body patch test prove that the desensitization rate is above 99.4%.
[0029] Figure 1 is a flowchart for preparing non-allergic urushiol.
[0030] Following is a step-by-step description of the technical scheme of the patent in the light of specific implementation methods.
[0031] Embodiment I, tryptophan-urushiol desensitization
[0032] S1, add ethanol (C2H50H, 3L) and water (H20, 1.5L) to the 10L reactor, slowly add sodium hydroxide (NaOH 320mL, 1.5mol/L), stir until dissolved and heat to 70COo il urushiol (average
molecular weight 314) 143g, stir for 15 min, drop benzyl chloride (molecular weight 126.59) 63g within 30 min, keep the temperature at 85C after dropping, continue the reaction for 3 hours. The
reaction stops when the oil-dropurushiol turns into a solution state;
[0033] S2, transfer the reaction solution to a single-mouth bottle, evaporate the solvent by decompression, cool to room temperature, then add 1,000mL 6 mol/LHCl to acidify, filter when there is no solid precipitation, then transfer the filter cake into 550mL, 1.5mol/L NaOH solution, stir for min when the cake is completely dissolved, then filter again, transfer thefiltrate to 2,OOOmL flask, drop concentrated hydrochloric acid to the filtrate to acidify, stop the reaction when the precipitate no longer increases, extract and filter the separated powder, then wash the cake with distilled water to normal state and dry it resulting black brown to shallow taupe brown powder. The main component of product 1 is o-benzyl urushiol (average molecular weight 405).
[0034] S3, dissolve the sample out of the obtained black brown product 1 powder (500g), 326g tryptophan (relative molecular weight 204.2) and 330g DMAP (relative molecular weight 122.17) with absolute ethyl alcohol, cool the reaction to 1 to drop same volume 2.mol/L EDCI (relative molecular weight 191.7) to the reaction step by step, stir at 0 at r d8 min after dropping, then stir at room temperature for 4.5h, remove the solvent after the reaction by vacuum distillation, extract with CH2C12/H20, dry with anhydrous magnesium sulfate, filter to remove the solvent to get the black brown powder.
[0035] S4, dissolve the obtained black-brown powder (350g) in anhydrous ethanol (900 mL), add 10% Pd/C (15g) under stirring, stir in the atmosphere of hydrogen for 5h, filtering out the insoluble substance, distill the filtrate under the condition of decompression, recrystallize the anhydrous alcohol to get product 3 what is called trytophanyl urushiol.
[0036] S5, Choose SD guinea pigs whose weight are (300 20) g and depilatory area 30 mm2, choose unhairing qualified guinea pigs after 24 hours upon the hair removal, divide each half of the male and female randomly into white vaseline control group (matrix control group) and urushiol control group, apply the modified urushiol to the animals of 10 per group with a dosage of high, medium and low degree, respectively 0.2g/kg, 0.lg/kg, 0.05g/kg (equivalent to 50 times 25times and12.5 times the clinic dosage). Upon continuous observation of 12-15d after 24h of the medicine application, it is found that the guinea pigs have normal hair and the ones in high, medium and low dosage groups appear smooth, tender and rosy skin without itching, swelling or fester etc.
[0037] Select qualified spot tester and 210 attendants as the patch test volunteers, and adopt closed patch test method. Apply modified urushiol 2.5-4.g produced on step three to the spot apparatus which is pasted with the external adhesive tape to the back of the attendants (or the inner side of the forearm for the female). Remove the tested substance after 12-24h. The first observation is made after an interval of 30 minutes, and the second and subsequent daily observations are made at 24, 48, 72, 96,120,144,168 hours. Note that the tape reaction is not considered. The test proves that the positive rate of the volunteers is 0.58% and the desensitization rate reaches 99.42% during 0.5-168h after the tested substance has been removed.
[0038] S6, take the method of bacteria solution pre-adding and injecting plate to compare the non allergic urushiol and the original solution. Inject certain amount of the bacteria solution into the plate culture medium which have cooled to about 50-60°C, mix them evenly and pour into the plate (about
-25 mL/plate), bacteria containing plate is prepared upon horizontal standing and solidification.
Divide the plate into three regions on average, and make a circular hole with a diameter of 5.5 mm on the test plate with sterilized steel tube in each area, carefully remove small medium blocks to make a round hole, and inject 120-200 L raw lacquer solution into one hole, and 150-230 P L non-allergic
urushiol into the second hole and leave the third hole as a control, pre-diffuse the mold at 4-6°Cwas iI
2 hand cultivate it at 35-37°C7nd 12-24 h, cultivate the mold in the incubator at 25-28 8 the 24-48h.
Measure the diameter of the bacteriostatic circle with vernier caliper by cross method to express the size of the bacteriostatic circle. Each bacterium is in 3 parallels. Choose the plate with obvious bacteriostatic circle to measure the diameter of the bacteriostatic circle. The average value of the three repeated experiments is deemed as the result. The maximum difference between non-allergic urushiol and urushiol solution is not more than 0.3-0.5%, proving that the bacteriostatic activity of raw lacquer is preserved while the allergy to raw lacquer is eliminated.
[0039] Implementation case 2, methionine-urushiol desensitization
[0040] Si, add ethanol (C2H50H, 5L) and water (H20, 2.5L) to the 10L reactor, slowly add sodium hydroxide (NaOH 600mL, 1.5mol/L), stir until dissolved and heat to 80°C stir urushiol (average
molecular weight 314) 251g, stir for 10 min, drop benzyl chloride (molecular weight 126.59) 125g within 25 min, keep the temperature at 65Cmin, keep the temperature at t to ide cquer2.5 hours. The
reaction stops when the oil-dropurushiol turns into a solution state.
[0041] S2, transfer the reaction solution to a single-mouth bottle, evaporate the solvent by decompression, cool to room temperature, then add 1,600 mL 5 mol/L HCl to acidify, filter when there is no solid precipitation, then transfer the filter cake into 500 mL, 1.5 mol/L NaOH solution, stir for 50 min when the cake is completely dissolved, then filter again, transfer thefiltrate to 2,OOOmL flask, drop concentrated hydrochloric acid to the filtrate to acidify, stop the reaction when the precipitate no longer increases, extract and filter the separated powder, then wash the cake with distilled water to normal state and dry it resulting black brown to shallow taupe brown powder. The main component of product 1 is o-benzyl urushiol (average molecular weight 405).
[0042] S3, dissolve the sample out of the obtained black brown product 1 powder (200g), 102g methionine (relative molecular weight 149.2) and 119g DMAP (relative molecular weight 122.17) with absolute ethyl alcohol, cool the reaction to 2 with absolute ethyl 2.mol/L EDCI (relative molecular weight 191.7) to the reaction step by step, stir at0°Cto th8 min after dropping, then stir at room
temperature for 3h, remove the solvent after the reaction by vacuum distillation, extract with CH2C12/H20, dry with anhydrous magnesium sulfate, filter to remove the solvent to get the black brown powder.
[0043] S4, dissolve the obtained black-brown powder (100g) in anhydrous ethanol (300 mL), add 10% Pd/C (20g) under stirring, stir in the atmosphere of hydrogen for 5h, filtering out the insoluble substance, distill the filtrate under the condition of decompression, recrystallize the anhydrous alcohol to get product 3 what is called methionine urushiol.
[0044] Choose SD guinea pigs whose weight are (300 20) g and depilatory area 30 mm2 , choose unhairing qualified guinea pigs after 24 hours upon the hair removal, divide each half of the male and female randomly into white vaseline control group (matrix control group) and urushiol control group, apply the modified urushiol to the animals of 10 per group with a dosage of high, medium and low degree, respectively 0.2g/kg, 0.lg/kg, 0.05g/kg (equivalent to 50 times 25times and 12.5 times the clinic dosage). Upon continuous observation of 12-15d after 24h of the medicine application, it is found that the guinea pigs have normal hair and the ones in high, medium and low dosage groups appear smooth, tender and rosy skin without itching, swelling or fester etc..
[0045] Select qualified spot tester and 210 attendants as the patch test volunteers, and adopt closed patch test method. Apply modified urushiol 2.5-4.Og produced on step three to the spot apparatus which is pasted with the external adhesive tape to the back of the attendants (or the inner side of the forearm for the female). Remove the tested substance after 12-24h. The first observation is made after an interval of 30 minutes, and the second and subsequent daily observations are made at 24, 48, 72, 96,120,144,168 hours. Note that the tape reaction is not considered. The test proves that the positive rate of the volunteers is 0.06% and the desensitization rate reaches 99.04% during 0.5 168h after the tested substance has been removed.
[0046] S6, Non-allergic urushiol scavenging ability test: in the range of certain concentration (0.31-10 mmol/L), the scavenging rate of superoxide anion radical is linear with VC concentration, the linear regression equation is y=8.975 x+5.827(the determinant coefficient R2 is 0.99573, the corrected determinant coefficient R2 Adj is 0.9962). By putting the scavenging rate of the mangnolia officinalis polysaccharide superoxide anion free radical into the aforesaid linear equation can figure out the scavenging capacity of the superoxide anion radical of the non-allergic urushiol is (18.74 2.12) pmolVC/g.
[0047] The concentration of Trolox is in the range of 1(480[amol/L), which is linearly related to the scavenging capacity of DPPH radical (the determinant coefficient R2 is 0.9892, the corrected determinant coefficient R2Adj is 0.9817), and the regression equation was: y=0.979 x+5.836. According to this formula, the scavenging capacity of DPPH radical of the sample non-allergic urushiol is (0.73008)pmol Trolox/g, proving that the free radical scavenging rate of the methionine desensitized urushiol does not fall.
[0048] Implementation case 3, serine-urushiol desensitization
[0049] Si, add ethanol (C2H50H, 5.5L) and water (H20, 2.5L) to the 10L reactor, slowly add 500ml sodium hydroxide (NaOH, 1.8mol/L), stir until dissolved and heat to 75C5stir urushiol
(average molecular weight 314) 90g, stir for 20 min, drop benzyl chloride (molecular weight 126.59) 41g within 30 min, keep the temperature at 75Cmin, keep the temperature at t to the
scave2.5 hours. The reaction stops when the oil-drop urushiol turns into a solution state.
[0050] S2, transfer the reaction solution to a single-mouth bottle, evaporate the solvent by decompression, cool to room temperature, then add 2,150 mL 6 mol/L HCl to acidify, filter when there is no solid precipitation, then transfer the filter cake into 700 mL, 1.5 mol/L NaOH solution, stir for 50 min when the cake is completely dissolved, then filter again, transfer the filtrate to 2,OOOmL flask, drop concentrated hydrochloric acid (200mL) to the filtrate to acidify, stop the reaction when the precipitate no longer increases, extract and filter the separated powder, then wash the cake with distilled water to normal state and dry it resulting black brown to shallow taupe brown powder. The main component of product 1 is o-benzyl urushiol (average molecular weight 405).
[0051] S3, dissolve the sample out of the obtained black brown product 1 powder (200g), 1.22g tryptophan (relative molecular weight 204.2) and 130g DMAP (relative molecular weight 122.17) with absolute ethyl alcohol, cool the reaction to 2Cwith absolute ethy 2.mol/L EDCI (relative
molecular weight 191.7) to the reaction step by step, stir at -1°Ctoth7 min after dropping, then
stir at room temperature for 4h, remove the solvent after the reaction by vacuum distillation, extract with CH2C12/H20, dry with anhydrous magnesium sulfate, filter to remove the solvent to get the black brown powder.
[0052] S4, dissolve the obtained black-brown powder (175g) in anhydrous ethanol (750 mL), add 10% Pd/C (20g) under stirring, stir in the atmosphere of hydrogen for 5h, filtering out the insoluble substance, distill the filtrate under the condition of decompression, recrystallize the anhydrous alcohol to get product 3 what is called serine urushiol.
[0053] S5, Choose SD guinea pigs whose weight are (300 20) g and depilatory area 30 mm2, choose 50 unhairing qualified guinea pigs after 24 hours upon the hair removal, divide each half of the male and female randomly into white vaseline control group (matrix control group) and urushiol control group, apply the modified urushiol to the animals of 10 per group with a dosage of high, medium and low degree, respectively 0.2g/kg, O.lg/kg, 0.05g/kg (equivalent to 50times times and 12.5 times the clinic dosage). Upon continuous observation of 12-15d after 24h of the medicine application, it is found that the guinea pigs have normal hair and the ones in high, medium and low dosage groups appear smooth, tender and rosy skin without itching, swelling or fester etc..
[0054] S5, Select qualified spot tester and 210 attendants as the patch test volunteers, and adopt closed patch test method. Apply modified urushiol 2.5-4.0g produced on step three to the spot apparatus which is pasted with the external adhesive tape to the back of the attendants (or the inner side of the forearm for the female). Remove the tested substance after 12-24h. The first observation is made after an interval of 30 minutes, and the second and subsequent daily observations are made at 24, 48, 72, 96, 120, 144, 168 hours. Note that the tape reaction is not considered. The test proves that the positive rate of the volunteers is 1.53% and the desensitization rate reaches 98.47% during 0.5-168h after the tested substance has been removed.
[0055] S6, FRAP is usually taken as the reducing power of the tested sample. Choose Trolox as the standard item to measure its FRAP value when its concentration is respectively 19, 20, 40, 80, 160 and 320jmol/L, and then work out the linear regression equation as y=0.0017x-0.001
(determinate coefficient R2 is 0.9973 and corrected determinate coefficient R2Ady is 0.9954), while FRAP of non-allergic urushiol is (2.44 0.41) mol Trolox/g.
[0056] S7, in the range of 7-60 mol/L, the ABTS+- scavenging capacity of Trolox is linearly related to its concentration (the determinant coefficient R2 is 0.9982, the corrected determinant coefficient R2Adj is 0.9982) and the standard curve is y=1.742 x-0.618. The ABTS+- scavenging capacity of Trolox is (0.51 0.06) mol Trolox/g.
[0057] Implementation Case 4, Proline-urushiol desensitization.
[0058] SI, add ethanol (C2H50H, 7L) and water (H20, 3.5L) to the 10L reactor, slowly add sodium hydroxide (NaOH 700mL, 12mol/L), stir until dissolved and heat to 70°C stir urushiol
790g, stir for 30 min, drop benzyl chloride (molecular weight 126.59) 319g within 30 min, keep the temperature at 65C5in, keep the temperature at t to ide is 3 hours. The reaction stops
when the oil-drop urushiol turns into a solution state.
[0059] S2, transfer the reaction solution to a single-mouth bottle, evaporate the solvent by decompression, cool to room temperature, then add 250 mL 6mol/L HCl to acidify, filter when there is no solid precipitation, then transfer the filter cake into 400 mL, 1.5mol/L NaOH solution, stir for 60 min when the cake is completely dissolved, then filter again, transfer the filtrate to 2,000mL flask, drop concentrated hydrochloric acid to the filtrate to acidify, stop the reaction when the precipitate no longer increases, extract and filter the separated powder, then wash the cake with distilled water to normal state and dry it resulting black brown to shallow taupe brown powder. The main component of product 1 is o-benzyl urushiol (average molecular weight 405).
[0060] S3, dissolve the sample out of the obtained black brown product 1 powder (300g), 127g proline (relative molecular weight 115.1) and 180g DMAP (relative molecular weight 122.17) with absolute ethyl alcohol, cool the reaction to 2Cwith absolute ethyl2.mol/L EDCI (relative
molecular weight 191.7) to the reaction step by step, stir at -1°CtothlO min after dropping, then
stir at room temperature for 5h, remove the solvent after the reaction by vacuum distillation, extract with CH2C12/H20, dry with anhydrous magnesium sulfate, filter to remove the solvent to get the black brown powder.
[0061] S4, dissolve the obtained black-brown powder (220g) in anhydrous ethanol (950mL), add % Pd/C (5g) under stirring, stir in the atmosphere of hydrogen for 8h, filtering out the insoluble substance, distill the filtrate under the condition of decompression, recrystallize the anhydrous alcohol to get product 3 what is called proline urushiol.
[0062] S5, Choose SD guinea pigs whose weight are (300 20) g and depilatory area 30 mm2, choose 50 unhairing qualified guinea pigs after 24 hours upon the hair removal, divide each half of the male and female randomly into white vaseline control group (matrix control group) and urushiol control group, apply the modified urushiol to the animals of 10 per group with a dosage of high, medium and low degree, respectively 0.2g/kg, 0.lg/kg, 0.05g/kg (equivalent to 50 times times and 12.5 times the clinic dosage). Upon continuous observation of 12-15d after 24h of the medicine application, it is found that the guinea pigs have normal hair and the ones in high, medium and low dosage groups appear smooth, tender and rosy skin without itching, swelling or fester etc.
[0063] Select qualified spot tester and 210 attendants as the patch test volunteers, and adopt closed patch test method. Apply modified urushiol 2.5-4.g produced on step three to the spot apparatus which is pasted with the external adhesive tape to the back of the attendants (or the inner side of the forearm for the female). Remove the tested substance after 12-24h. The first observation is made after an interval of 30 minutes, and the second and subsequent daily observations are made at 24, 48, 72, 96,120,144,168 hours. Note that the tape reaction is not considered. The test proves that the positive rate of the volunteers is 0.48% and the desensitization rate reaches 99.52% during 0.5-168h after the tested substance has been removed.
[0064] S6, match 8mL desensitized lacquer with 2% DMSO solution to get 6mL solution as test solution A; match 8mL raw lacquer with 2% DMSO solution to get 6mL solution as test solution B; match 1OOg ribavirin with 2% DMSO solution to get 1OOg/mL solution as positive control test solution.
[0065] Dilute the test solution with 4% of the maintenance media to 10 concentration gradients at the proportion of 2:1, separately inoculate them in the cell well-grown 96 pore plate attached with 4 reperforations, cell control holes and blank control holes, cultivate in 5% C02 virus incubator at 32Cvirus incubator at and blan90% cells are diseased under the microscope, and
then measure OD value according to the operation of CCK-8 reagent kit (add 10 p L CCK-8 for
dyeing, cultivate in 5% C02 virus incubator at 35C5rus 3h and measure OD value with 520nm
wave length). Use Reed-Muench formula to work out half medicine poisoning concentration TC50 and confirm the highest non-poisonous concentration TCO. It is proved that TC50 of desensitized lacquer to Hep-2 is 1.593 and TCO 0.798, while TC50 of desensitized lacquer to RD is 1.579 and TCO 1.125. The cytotoxicity is much higher than ribavirin, but 0.5% lower than basic urushiol solution, proving that the antiviral activity of urushiol is maintained during the process of desensitization.
[0066] The above is a detailed presentation of the best way to implement this patent, but this patent is not limited to the above-mentioned way of implementation. Within the knowledge of the general technical personnel in this field, any changes in line with the purpose of the patent is allowed.
Claims (5)
1. A food chemistry method for the preparation of non-allergic urushiol is characteristic of and specifically contains the following steps:
Si, phenol hydroxyl of urushiol introduces benzyl protective group:
the solvent is added according to the ratio of ethanol: purified water--(1.5-2.5) L: IL; then little by little pour (1.5-2.0) mol/L sodium hydroxide solution into the solvent: according to the volume ration of solvent: sodium hydroxide solution=(12-18) L: IL, stir the mixed liquid evenly and heat to -85°C5 stir the mixed liquid evenly and heat to t: urushiol=(1-3): 1 mole ratio, add urushiol and
stir for 10-30 minutes. In 30 min, according to the molar ratio of urushiol: benzyl chloride=10:(10 12), add benzyl chloride, maintain the temperature at 60-85°C5add benzyl chloride, ml h-3 h which
will stop when the oil droplet floating urushiol turns into the solution state; remove the solvent through reduced pressure distillation, cool to normal temperature; add in HCl at the density of (4.5 6.5)mol/L in the volume of about 20%-30% of the reaction liquid to carry out acidification, stir slowly, start to filter when the amount of solid precipitate no longer changes, transfer the filter cake into (1.5-2.5)mol/L NaOH solution and stir slowly, stop adding alkali and filtering when the cake is fully dissolved, drop concentrated hydrochloric acid into the obtained liquid for acidification, stop reacting and filtering when the precipitate no longer increases, wash the solid filter cake with distilled water to normal dry and recrystallize to produce product 1 whose main component is o benzyl urushiol.
S2, urushiol hydroxyl introduces aminoacyl amino acid group:
Mix product 1 (o-benzoxy urushiol), amino acid and 4-2 methylaminopyridine (DMAP) according to the molar ration of 1:(1.2-1.5): (2-2.5), add ethanol into the mixture until complete dissolution and then, cool the reaction solution to 2-C; then gradually add equal volumed 1-ethyl-(3-2
methylaminopropyl) carbinyl 2-imide hydrochloride (EDCI) at the concentration of 2.Omol/LL, stir for 5-10 min after the dropping at the temperature of 0--2C and then stir another 3-5h at room
temperature, remove the solvent through reduced pressure distillation after the reaction stops, extract with CH2 C1 2 /H 2 0, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, filter to remove the solvent leaving brown powder and recrystal to result in product 2 whose main component is o-benzyl aminoacyl urushiol.
S3, elimination of urushiol hydroxybenzyl protective group:
Dissolve generated product 2 in anhydrous ethanol, filter to remove the insoluble substance, add 10% palladium carbon (Pd/C) catalyst under stirring, stir in H2 atmosphere for 4-7h, filter to remove the insoluble substance, recrystallize the filtrate with anhydrous ethanol under vacuum distillation to gain product 3 which replaces the urushiol with aminoacyl group.
2. The method described in claim 1 is characterized in that the urushiol in step Sl is derived from natural raw paint or its modified paint.
3. The molecular formula of the non-allergic urushiol product by the method of food chemistry is:
OH
R1
R
4. The non-allergic urushiol product prepared by the food chemistry method according to claim 3 is characterized in that the R1 is an aminoacyl amino acid group.
5. The non-allergic urushiol product prepared by the food chemistry method according to claim 4, is characterized in that the aminoacyl amino acid group is one or any combination of several selected from alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid and glutamic acid, etc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020103370A AU2020103370A4 (en) | 2020-11-10 | 2020-11-10 | Preparation of non-allergic urushiol by a food chemical method and its products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2020103370A AU2020103370A4 (en) | 2020-11-10 | 2020-11-10 | Preparation of non-allergic urushiol by a food chemical method and its products |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020103370A4 true AU2020103370A4 (en) | 2021-01-28 |
Family
ID=74192160
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020103370A Active AU2020103370A4 (en) | 2020-11-10 | 2020-11-10 | Preparation of non-allergic urushiol by a food chemical method and its products |
Country Status (1)
| Country | Link |
|---|---|
| AU (1) | AU2020103370A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116675677A (en) * | 2023-08-02 | 2023-09-01 | 中国林业科学研究院林产化学工业研究所 | C8 urushiol derivative and preparation method and application thereof |
-
2020
- 2020-11-10 AU AU2020103370A patent/AU2020103370A4/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116675677A (en) * | 2023-08-02 | 2023-09-01 | 中国林业科学研究院林产化学工业研究所 | C8 urushiol derivative and preparation method and application thereof |
| CN116675677B (en) * | 2023-08-02 | 2023-09-26 | 中国林业科学研究院林产化学工业研究所 | C8 urushiol derivative and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020103370A4 (en) | Preparation of non-allergic urushiol by a food chemical method and its products | |
| CN106267243B (en) | A kind of molecular probe and its preparation method and application | |
| WO2012013136A1 (en) | Polypeptidic tyrosinase inhibitor | |
| HRP20030876A2 (en) | Photosensitiser and method for production thereof | |
| US20120245120A1 (en) | Zinc sucrose octasulfates, their preparation, and pharmaceutical and cosmetic uses thereof | |
| TW200831106A (en) | Antiinflammatory agent comprising 2-aminophenol or its derivatives as an effective ingredient(s) | |
| CN111000770A (en) | Whitening cream containing pearl peptide | |
| FR2936149A1 (en) | USE OF ALVERINE IN THE TREATMENT OF SKIN DISEASES | |
| US9597270B2 (en) | Hydrogel-forming material | |
| JPH05170636A (en) | Beautifying and whitening cosmetic | |
| JP2016222620A (en) | The active extract of phalaenopsis, preparation method and use thereof | |
| CN110746315B (en) | Method for preparing non-allergic urushiol by food chemical method and product thereof | |
| AU2020103121A4 (en) | Method for preparing non-allergic raw lacquer or urushiol by biochemical method and its products | |
| Zeng et al. | Coral-friendly and non-transdermal polymeric UV filter via the Biginelli reaction for in vivo UV protection | |
| BG98956A (en) | Compounds and products of the amadori, method for their preparation and use | |
| CN113786492B (en) | Polymer carrier for photodynamic therapy and preparation method and application thereof | |
| CA3064050A1 (en) | Extracts from arthrospira and uses thereof | |
| JPH04187618A (en) | Skin external preparation | |
| CN110801013A (en) | Method for preparing non-allergic raw lacquer or urushiol by biochemical method and product thereof | |
| CN112386520A (en) | Mesoderm injection composition for comprehensively improving skin color and preparation method thereof | |
| CN113368004B (en) | Polypeptide composition with anti-aging and repairing effects and preparation method and application thereof | |
| JPH06145040A (en) | Melanogenesis-suppressing agent | |
| JP2006131522A (en) | Cosmetic | |
| TWI500594B (en) | Crystal form a of 3(2-acetylamino-acetylamino)-propionic acid, its preparing method and application thereof | |
| Liu et al. | Strong infiltrative HHC36 antimicrobial peptide/silver nanoparticles-loaded carboxymethyl chitosan/sodium alginate hydrogel for acne vulgaris therapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) |