Donkey-hide gelatin traditional Chinese medicine composition for invigorating qi and blood, beautifying and removing freckles as well as preparation method and application thereof

Abstract

The invention discloses a donkey-hide gelatin traditional Chinese medicine composition capable of invigorating qi and blood and beautifying and removing freckles and a preparation method and application thereof, and relates to the field of traditional Chinese medicine compositions.The donkey-hide gelatin traditional Chinese medicine composition is prepared from, by weight, 3-10 parts of donkey-hide gelatin and 40-60 parts of Chinese herbal medicine active matter; a preparation method of the Chinese herbal medicine active matter comprises the following steps: crushing Chinese herbal medicines to obtain mixed powder, then embrittling the mixed powder, adding a composite plant acid solution after the embrittling treatment is finished, dipping, heating, carrying out reduced pressure distillation, carrying out alcohol precipitation, filtering, distilling filtrate to remove alcohol, concentrating and drying to obtain the Chinese herbal medicine active matter. In the formula of the Chinese herbal medicine active matters, donkey-hide gelatin can nourish yin, enrich blood and stop bleeding, and Chinese dates can nourish blood and tonify qi, so that the Chinese herbal medicine active matters have the effects of nourishing blood, tonify qi and stop bleeding and are used for treating bleeding syndromes caused by deficiency of qi and blood. The poria cocos is used for tonifying the spleen and harmonizing the stomach, the Chinese dates are used for tonifying the spleen and harmonizing the stomach, and the Chinese dates and the donkey-hide gelatin are used for nourishing blood and tonifying qi; the raw materials jointly play the effects of nourishing blood, tonifying qi, strengthening the spleen and stomach, and achieve the health-care effect of enhancing the immunity of the organism.

Description

Technical Field

[0001] The present invention relates to the field of Chinese medicine compositions, and in particular to a Chinese medicine composition of donkey-hide gelatin for replenishing qi and blood, beautifying and removing freckles, and a preparation method and application thereof. Background Art

[0002] Due to the accelerated pace of life, changes in dietary structure and a stressful lifestyle, people have been in a sub-healthy state for a long time. This state has not been improved for a long time, resulting in anemia, insomnia and low immunity. At present, there are many products on the market for replenishing qi and blood, beauty and freckle removal, among which the active ingredients of traditional Chinese medicine preparations are often less, which affects the effective use of important ingredients. Therefore, it is necessary to further improve it. Summary of the invention

[0003] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide a Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood, beautifying and removing freckles, and a preparation method and application thereof.

[0004] The technical solution of the present invention is as follows:

[0005] A Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood, beautifying and removing freckles, comprising the following raw materials in parts by weight:

[0006] 3-10 parts of donkey glue and 40-60 parts of Chinese herbal active ingredients;

[0007] The preparation method of the Chinese herbal medicine active ingredient is as follows:

[0008] The Chinese herbal medicine is crushed to obtain a mixed powder, and then the mixed powder is embrittled. After the embrittlement treatment, a composite plant acid solution is added for immersion, heating, vacuum distillation, alcohol precipitation, filtering, distilling the filtrate to remove alcohol, concentrating, and drying to obtain the product.

[0009] Preferably, the Chinese herbal medicine comprises wolfberry, polygonatum, poria and jujube in a mass ratio of 3-5:4-6:2-3:1-5; or;

[0010] The Chinese herbal medicines include American ginseng, longan pulp, wolfberry, angelica, red dates and iron preparations in a mass ratio of 3-7:4-8:2-5:1-5:3-5:0.1-0.5.

[0011] Preferably, the embrittlement treatment is:

[0012] Add the mixed powder into a mixed aqueous solution of sodium chloride, amino acid and chitosan, stir, spray polyvinyl alcohol solution, let stand, take out, and embrittle at -30 to -10°C under vacuum environment and pressure of 1-5kpa for 3-6h, and then embrittle at 60-80°C for 2-3h.

[0013] Preferably, the mass percentages of sodium chloride, amino acid and chitosan in water are 1-5%, 3-5% and 10-15% respectively.

[0014] Preferably, the spraying amount of the polyethylene glycol is 3-10% of the mass of the mixed powder.

[0015] Preferably, the amino acids include one or more of glycine, proline, alanine, phenylalanine, glutamic acid, and arginine.

[0016] Preferably, the composite plant acid solution comprises an aqueous solution of citric acid, malic acid, oxalic acid and sodium citrate in a molar ratio of 1:1-2:2-3:0.5-1.

[0017] Preferably, the concentration of the sodium citrate aqueous solution is 0.05-3 mol / L.

[0018] The invention also discloses a method for preparing a Chinese medicinal composition of donkey-hide gelatin for invigorating qi and blood and beautifying and removing freckles. The method comprises grinding donkey gelatin into powder to obtain donkey gelatin powder, and uniformly mixing the donkey gelatin powder with active Chinese herbal medicine to obtain the composition.

[0019] The present invention also discloses an application of any of the above-mentioned donkey-hide gelatin Chinese medicine compositions for replenishing qi and blood and beautifying and removing freckles, characterized in that the finished dosage form of the Chinese medicine composition is at least one of pills, tablets, capsules, granules, oral liquids, and decoctions.

[0020] Donkey glue, also known as donkey glue, is a glue block obtained by depilating the skin of the donkey, an equine animal. It tastes sweet and is neutral in nature. It enters the lung, liver, and kidney meridians. Functions: replenishing blood, stopping bleeding, nourishing yin, moistening the lungs, and calming the fetus.

[0021] Lycium barbarum is the dried mature fruit of Ningxia Lycium barbarum, a plant of the Solanaceae family. It is harvested in summer and autumn when it is orange-red, dried until the skin is wrinkled, and then exposed to the sun until the skin is dry and hard and the flesh is soft, and the fruit stem is removed; or it is dried at low temperature with hot air, and the fruit stem is removed. Lycium barbarum is sweet and neutral. It enters the liver and kidney meridians. It nourishes the liver and kidneys, improves the essence and improves the eyesight. It is used for asthenia, deficiency of essence, soreness of waist and knees, dizziness and tinnitus, internal heat and thirst, blood deficiency and sallow complexion, and blurred vision.

[0022] Polygonatum sibiricum is the rhizome of Polygonatum sibiricum, Polygonatum cyrtonema, Polygonatum jejuni, Polygonatum yunnanensis, Polygonatum cyrtonema, etc. It tastes sweet. It is neutral in nature. It enters the spleen, lung, and kidney meridians. Functions: tonifying the middle and replenishing qi, moistening the heart and lungs, tonifying the kidney and replenishing essence, and strengthening tendons and bones. Indications: deficiency and cold and heat, hemoptysis due to tuberculosis, physical weakness and loss of appetite after illness, weak tibia, rheumatic pain, madness and ringworm.

[0023] Poria cocos is the dried sclerotium of the plant Poria cocos of the Polyporaceae family. It tastes sweet and light, and is neutral in nature. It enters the heart, spleen, and lung meridians. Functions: dispelling dampness and promoting diuresis, benefiting the spleen and stomach, and calming the mind. Indications: difficulty urinating, edema, phlegm and cough, nausea and vomiting, diarrhea, spermatorrhea, stranguria, palpitations, and forgetfulness.

[0024] Jujube is the mature fruit of the plant jujube of the Rhamnaceae family. It is also known as red dates (Medical Introduction), thousand dates (Famous Doctors' Records), etc. It tastes sweet and is warm in nature. It enters the spleen and stomach meridians. Functions: tonifying the spleen and stomach, replenishing qi and producing body fluid, regulating the body's defense system, and detoxifying drugs. Indications: poor appetite due to weak stomach, loose stools due to weak spleen, insufficient qi, blood and body fluids, disharmony between the body's defense system and the body's defense system, palpitations, and irritability in women.

[0025] American ginseng is cold in nature and bitter in taste. It enters the heart, lungs, and kidneys. It has the functions of reducing fever and clearing fire, reducing deficiency heat, replenishing yin, promoting body fluid and lowering blood pressure.

[0026] Angelica sinensis has the effects of nourishing blood and blood, regulating menstruation and relieving pain, moisturizing and lubricating the intestines, anti-cancer, anti-aging, and immunity.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention first embrittles the raw material to make it have a loose porous structure, and then immerses it in a composite plant acid solution so that the active substance is fully dissolved, thereby increasing the content of the active substance.

[0029] (2) The embrittlement treatment adopted by the present invention is pre-immersed in a mixed aqueous solution containing sodium chloride, amino acids and chitosan. On the one hand, the mixed powder can fully absorb water. On the other hand, the amino acids inhibit the diffusion of active substances in the mixed powder into the matrix, that is, the mixed powder fully absorbs water and reduces the loss of active substances. In addition, polyvinyl alcohol is sprayed to form a film on the surface of the gel, which further improves the moisturizing effect, allowing the mixed powder to fully absorb water, thereby improving the subsequent frost heave effect of the mixed powder and further improving the dissolution of active substances.

[0030] (3) The composite plant acid solution used in the present invention is combined with the sodium citrate solution to maintain the pH value within a stable range, and the active polysaccharide is separated from the raw material by using organic acid molecules, and the glycosidic bonds of the plant high-molecular active polysaccharide are appropriately cut to achieve partial degradation, thereby greatly improving the acid leaching effect.

[0031] (4) The donkey glue in the prescription of the Chinese herbal active ingredients of the present invention nourishes yin, replenishes blood and stops bleeding, and the jujube nourishes blood and replenishes qi. The combination of the two has the effects of nourishing blood, replenishing qi and stopping bleeding, and is used to treat bleeding syndrome caused by insufficient qi and blood. Polygonatum sibiricum replenishes qi, replenishes the kidney and replenishes essence, and wolfberry is good at replenishing essence and qi. Poria replenishes the spleen and harmonizes the stomach, and jujube replenishes the spleen and harmonizes the stomach. Jujube and donkey glue nourish blood and replenish qi; together they play the effects of nourishing blood and replenishing qi, strengthening the spleen and harmonizing the stomach, and achieving the health care effect of enhancing the body's immunity. DETAILED DESCRIPTION

[0032] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0033] Example 1

[0034] The donkey glue is ground into powder to obtain donkey glue powder, and the donkey glue powder is evenly mixed with active Chinese herbal medicines to prepare a Chinese herbal medicine composition of donkey-hide glue for replenishing qi and blood and beautifying and removing spots.

[0035] A Chinese medicine composition comprises the following raw materials in parts by weight: 8 parts of donkey glue and 50 parts of Chinese herbal medicine active ingredients;

[0036] The preparation method of the Chinese herbal medicine active ingredient is as follows:

[0037] The Chinese herbal medicine is crushed to obtain a mixed powder, and then the mixed powder is embrittled. After the embrittlement treatment, a composite plant acid solution is added for immersion, heating, vacuum distillation, alcohol precipitation, filtering, distilling the filtrate to remove alcohol, concentrating, and drying to obtain the product.

[0038] The Chinese herbal medicine comprises wolfberry, polygonatum, tuckahoe and jujube in a mass ratio of 5:4:2:3;

[0039] The embrittlement treatment is:

[0040] The mixed powder was added into a mixed aqueous solution of sodium chloride, amino acid and chitosan, stirred, sprayed with polyvinyl alcohol solution, allowed to stand, taken out, cooled to -30°C under vacuum and pressure of 3 kPa for 4 h, and then embrittled at 70°C for 2 h.

[0041] The mass percentages of the sodium chloride, amino acid and chitosan in water are 3%, 4% and 15% respectively.

[0042] The spraying amount of the polyethylene glycol is 7% of the mass of the mixed powder.

[0043] The amino acids include glycine.

[0044] The composite plant acid solution comprises an aqueous solution of citric acid, malic acid, oxalic acid and sodium citrate in a molar ratio of 1:2:2:0.5. The amount of the composite plant acid solution added is twice the mass of the mixed powder.

[0045] The concentration of the sodium citrate aqueous solution is 0.05 mol / L.

[0046] Example 2

[0047] The donkey glue is ground into powder to obtain donkey glue powder, and the donkey glue powder is evenly mixed with active Chinese herbal medicines to prepare a Chinese herbal medicine composition of donkey-hide glue for replenishing qi and blood and beautifying and removing spots.

[0048] A Chinese medicine composition comprises the following raw materials in parts by weight: 8 parts of donkey glue and 50 parts of Chinese herbal medicine active ingredients;

[0049] The preparation method of the Chinese herbal medicine active ingredient is as follows:

[0050] The Chinese herbal medicine is crushed to obtain a mixed powder, and then the mixed powder is embrittled. After the embrittlement treatment, a composite plant acid solution is added for immersion, heating, vacuum distillation, alcohol precipitation, filtering, distilling the filtrate to remove alcohol, concentrating, and drying to obtain the product.

[0051] The Chinese herbal medicines include American ginseng, longan pulp, wolfberry, angelica, red dates and ferrous lactate in a mass ratio of 6:5:3:2:4:0.3.

[0052] The embrittlement treatment is:

[0053] The mixed powder was added into a mixed aqueous solution of sodium chloride, amino acid and chitosan, stirred, sprayed with polyvinyl alcohol solution, allowed to stand, taken out, cooled to -30°C under vacuum and pressure of 3 kPa for 4 h, and then embrittled at 70°C for 2 h.

[0054] The mass percentages of the sodium chloride, amino acid and chitosan in water are 3%, 4% and 15% respectively.

[0055] The spraying amount of the polyethylene glycol is 7% of the mass of the mixed powder.

[0056] The amino acids include glycine.

[0057] The composite plant acid solution comprises an aqueous solution of citric acid, malic acid, oxalic acid and sodium citrate in a molar ratio of 1:2:2:0.5. The amount of the composite plant acid solution added is twice the mass of the mixed powder.

[0058] The concentration of the sodium citrate aqueous solution is 0.05 mol / L.

[0059] Example 3

[0060] The donkey glue is ground into powder to obtain donkey glue powder, and the donkey glue powder is evenly mixed with active Chinese herbal medicines to prepare a Chinese herbal medicine composition of donkey-hide glue for replenishing qi and blood and beautifying and removing spots.

[0061] A Chinese medicine composition comprises the following raw materials in parts by weight: 8 parts of donkey glue and 60 parts of Chinese herbal medicine active ingredients;

[0062] The preparation method of the Chinese herbal medicine active ingredient is as follows:

[0063] The Chinese herbal medicine is crushed to obtain a mixed powder, and then the mixed powder is embrittled. After the embrittlement treatment, a composite plant acid solution is added for immersion, heating, vacuum distillation, alcohol precipitation, filtering, distilling the filtrate to remove alcohol, concentrating, and drying to obtain the product.

[0064] The Chinese herbal medicine comprises wolfberry, polygonatum, tuckahoe and jujube in a mass ratio of 5:4:2:1;

[0065] The embrittlement treatment is:

[0066] The mixed powder was added into a mixed aqueous solution of sodium chloride, amino acid and chitosan, stirred, sprayed with polyvinyl alcohol solution, allowed to stand, taken out, cooled to -30°C under vacuum and pressure of 3 kPa for 4 h, and then embrittled at 70°C for 2 h.

[0067] The mass percentages of the sodium chloride, amino acid and chitosan in water are 3%, 4% and 12% respectively.

[0068] The spraying amount of the polyethylene glycol is 8% of the mass of the mixed powder.

[0069] The amino acids include arginine.

[0070] The composite plant acid solution comprises an aqueous solution of citric acid, malic acid, oxalic acid and sodium citrate in a molar ratio of 1:2:2:0.5. The amount of the composite plant acid solution added is twice the mass of the mixed powder.

[0071] The concentration of the sodium citrate aqueous solution is 1 mol / L.

[0072] Example 4

[0073] The difference from Example 1 is that the spraying amount of the polyethylene glycol is 10% of the mass of the mixed powder.

[0074] Example 5

[0075] Different from Example 1, the composite plant acid solution includes an aqueous solution of citric acid, malic acid, oxalic acid and sodium citrate in a molar ratio of 1:1:3:0.7, and the concentration of the sodium citrate aqueous solution is 0.05 mol / L.

[0076] Comparative Example 1

[0077] The difference from Example 1 is that no embrittlement treatment is performed, that is, the preparation method of the Chinese herbal active ingredient is as follows:

[0078] The Chinese herbal medicine is crushed to obtain a mixed powder, and then the mixed powder is added into a composite plant acid solution for immersion, heated, vacuum distilled, alcohol precipitated, filtered, the filtrate is distilled to remove alcohol, concentrated, and dried to obtain the product.

[0079] Comparative Example 2

[0080] The difference from Example 1 is that there is no immersion treatment in the composite plant acid solution, that is, the Chinese herbal medicine is crushed to obtain a mixed powder, which is then embrittled. After the embrittlement treatment, it is immersed in water, heated for extraction, distilled under reduced pressure, precipitated with alcohol, filtered, the filtrate is distilled to remove alcohol, concentrated, and dried to obtain the product.

[0081] Comparative Example 3

[0082] Different from Example 1, the composite plant acid solution comprises citric acid, malic acid and oxalic acid solutions in a molar ratio of 1:2:2. The amount of the composite plant acid solution added is twice the mass of the mixed powder.

[0083] The above embodiments and comparative examples were subjected to performance tests, and the test results are shown in Table 1.

[0084] Test 1:

[0085] Take wolfberry, polygonatum, poria, and jujube, add 10 times the amount of water and boil twice, each time for 2 hours, combine the decoctions, filter, and use the crude polysaccharide in the filtrate as the control sample to calculate the growth rate of the crude polysaccharide content of the embodiment and the comparative example compared with the control sample.

[0086] Table 1 Crude polysaccharide test results of Examples and Comparative Examples

[0087]

[0088]

[0089] As can be seen from Table 1, the dissolution rate of the embodiment is higher than that of the comparative example, and the main reasons may be as follows: From the analysis of comparative examples 1-3, it can be seen that the embodiment first embrittles the raw material to make it have a loose porous structure, and then immerses it in a composite plant acid solution, so that its active substance is fully dissolved, thereby increasing the content of the active substance. Furthermore, the mixed aqueous solution containing sodium chloride, amino acids and chitosan is used in advance to immerse, on the one hand, so that the mixed powder fully absorbs water, on the other hand, the amino acid inhibits the diffusion of the active substance of the mixed powder into the matrix, that is, the mixed powder fully absorbs water and reduces the loss of active substances, and further sprays polyvinyl alcohol to form a film on the surface of the gel, and the moistening effect is further improved, so that the mixed powder fully absorbs water, so that the frost heave effect of the subsequent mixed powder is improved, and the dissolution of the active substance is further improved. In addition, the composite plant acid solution used in the embodiment is combined with a sodium citrate solution to maintain the pH value within a stable range, and the active polysaccharide is separated from the raw material by organic acid molecules, and the glycosidic bonds of the plant macromolecular active polysaccharide are appropriately cut off to achieve its partial degradation, which greatly improves the acid leaching effect.

[0090] Test 2: Evaluation of immunity enhancement function

[0091] The sample of Example 1 was made into pellets and the following tests were carried out.

[0092] Experimental animals and grouping: 80 clean-grade Kunming male mice and 40 female mice weighing 18g to 23g were provided by the Experimental Animal Farm of the College of Animal Science and Technology of Hunan Agricultural University (the experimental animal production license number is SCXK (Xiang) 2003-0003). Each group of 40 mice formed a large group, with a total of three groups. Immunization group I (male) was used for carbon clearance experiment; Immunization group II (male) was used for ConA-induced mouse lymphocyte transformation experiment and NK cell activity determination; Immunization group II (female) was used for organ-body ratio determination, mouse peritoneal macrophage phagocytosis of chicken red blood cells experiment, delayed-type hypersensitivity experiment, and half hemolysis value (HC 50 ) and the number of antibody-producing cells. Each large group of 40 mice was randomly divided into 4 groups according to body weight, namely the control group and the low-, medium- and high-dose groups.

[0093] Experimental environmental conditions: temperature 21°C-24°C, humidity 56%-58%, experimental animal use license number is SYXK (Xiang) 2003-0002.

[0094] Dose selection and sample processing: According to the recommended oral dosage for humans, the low, medium and high doses were set to 1.667 g / kg·bw, 3.333 g / kg·bw and 10.000 g / kg·bw (equivalent to 5, 10 and 30 times the recommended dosage for humans, respectively). During the test, 16.67 g, 33.33 g and 100.00 g of the granules of Example 1 were added with distilled water to 200 mL to prepare the test solution of the corresponding concentration. The mice were gavaged at a volume of 0.2 ml / 10 g-bw, once a day, for 30 consecutive days. The control group was gavaged with an equal volume of distilled water.

[0095] Main instruments and reagents: animal scale, analytical balance, clean bench, carbon dioxide incubator, centrifuge, 722 spectrophotometer, constant temperature water bath, liquid scintillator, microplate reader, microscope, etc. Sterile surgical instruments, vernier caliper, microsyringe, cell counter, 24-well and 96-well flat-bottom cell culture plates, 96-well U-shaped cell culture plates, glass dishes, gauze, test tubes, slide racks, 200-mesh nets, timers, hemoglobin pipettes, slides, etc.

[0096] Sheep red blood cells (SRBC), physiological saline, Hank's solution (pH7.2-7.4), RPMI1640 culture medium, calf serum, penicillin-streptomycin, ConA, 1% acetic acid, 1mol HCL solution, acidic isopropanol (96mL isopropanol plus 1mol HCL solution 4m), MTT, PBS buffer (Ph7.2-7.4), complement (guinea pig serum), SA buffer, agarose, Dulbecco's reagent, YAC-1 cells, sodium lactate, nitro tetrazolium chloride, phenazine dimethyl sulfate, oxidized coenzyme I, 0.2molL Tris-HCL buffer, 1% NP40, India ink, 0.1% sodium carbonate, chicken red blood cells, methanol, Giemsa stain, etc.

[0097] Experimental methods:

[0098] Determination of organ / body weight ratio: The mice were sacrificed after weighing, and the spleen and thymus were removed and weighed / calculated on an electronic analytical balance.

[0099] Delayed-type hypersensitivity (DTH) (plantar thickening method) Mice were sensitized by intraperitoneal injection of 2% (v / v) SRBC (0.2 ml / mouse) 4 days after being sensitized, and then 20% (v / v) SRBC (20ul / mouse) was subcutaneously injected at the measurement site. The thickness of the left hind paw was measured 24h after injection. The same site was measured three times and the average value was taken. The difference in paw thickness before and after the attack (plantar swelling) was used to express the degree of DTH.

[0100] ConA-induced mouse lymphocyte transformation test (MTT method) The spleen was sterilely removed and placed in a small dish containing an appropriate amount of sterile Hanks solution to prepare a cell suspension, which was filtered through a 200-mesh screen. The cells were washed three times with Hanks solution and centrifuged for 10 minutes each time (1000 rpm). The cells were then suspended in 2 ml complete culture medium, the number of live cells was counted, and the cell concentration was adjusted to 5×10 with RPMI1640 culture medium. 6 / mL. Then add the cell suspension into two wells of a 24-well culture plate, 1mL per well, add 50uLConA solution (equivalent to 5μg / mL) to one well, and the other well as a control, set to 5% carbon dioxide, and culture at 37℃ for 72h. 4h before the end of the culture, gently aspirate 0.7m of supernatant from each well, add RPMI1640 culture medium without calf serum, and add 50uL / well of MTT (5mg / mL), and continue to culture for 4h. After the culture is completed, add 1mL of acidic isopropanol to each well, blow and mix well to completely dissolve the purple crystals. Then measure the liquid with an enzyme marker at a wavelength of 570nm. The proliferation capacity of lymphocytes is expressed by the optical density value of the well with ConA minus the optical density value of the well without ConA.

[0101] Antibody-producing cell detection (Jerne modified slide method) is as follows:

[0102] Sheep blood was collected and washed with saline for 3 times, centrifuged (2000r / min) for 10min each time, and the packed SRBC was prepared into a 2% (v / v) cell suspension with saline, and 0.2ml was injected into the peritoneal cavity of each mouse. The mice were killed 4 days after immunization, and the spleen was taken out, gently torn, and the cell suspension was prepared with Hanks solution, filtered with a 200-mesh screen, washed, centrifuged twice, and finally the cells were suspended in 5ml Hanks solution. After the surface culture medium is heated and dissolved, it is mixed with an equal amount of Hanks solution of pH 7.4 and 2 times concentration, and then divided into small test tubes, 0.5 ml per tube. Then, 10% SRBC 50ul (v / v) and 20ul spleen cell suspension prepared with SA solution are added into the tube. After rapid mixing, pour it onto a glass slide that has been brushed with a thin layer of agarose. After the agarose solidifies, place the glass slide horizontally on a glass slide rack and incubate it in a carbon dioxide incubator for 1.5 hours. Then, complement diluted with SA solution (1:10) is added into the groove of the glass slide. After continuing to incubate for 1.5 hours, the number of hemolytic plaques is counted.

[0103] Half hemolysis value (HC 50 ) is determined as follows:

[0104] Take sheep blood, wash it with saline for 3 times, and inject 2% (v / v, prepared with saline) packed RBC 0.2mL into each mouse for immunization. 5 days later, remove the eyeball and take blood into a centrifuge tube, place it for about 1 hour, peel off the coagulated blood from the tube wall, let the serum fully precipitate, centrifuge at 2000npm for 10min, and collect the serum. Dilute the serum 200 times with SA buffer, take 1ml and put it in a test tube, add 10% (v / v, prepared with SA buffer) packed SRBC 0.5ml, complement 1mL (diluted with SA buffer at 1:10) in turn. Set up a control tube without serum (replaced with SA buffer). Keep it in a 37℃ constant temperature water bath for 30min, and terminate the reaction in an ice bath. Centrifuge at 2000rpm for 10min, take 1ml of the supernatant, and add 3ml of Du's reagent. At the same time, take 0.25 mL of 10% (v / v, prepared with SA buffer) compressed SRBC, add Dulbecco's reagent to 4 mL in another test tube, mix thoroughly, and place for 10 minutes. Then, use the control tube as a blank and measure the optical density of each tube at 540 nm. The amount of hemolysin is measured by the half hemolysis value (HC 50 ) is expressed as follows:

[0105] Half hemolysis value (HC 50 ) = optical density of sample / optical density of SRBC at half hemolysis × dilution factor

[0106] The method of the mouse carbon clearance experiment is as follows:

[0107] The mice were injected with Indian ink diluted 4 times with physiological saline through the tail vein, 0.1 mL per 10 g body weight. The time was immediately started after the ink was injected. At 2 and 10 minutes after the ink was injected, 20 ul of blood was collected from the medial canthal vein plexus, added to 2 mL of 0.1% NaCO3 solution, and shaken well. 0.1% Na2CO3 solution was used as a blank control, and the optical density (OD) was measured colorimetrically at a wavelength of 600 nm using a 722 spectrophotometer. The mice were killed, and the liver and spleen were taken out, weighed, and the phagocytic index a was calculated.

[0108] Experiment on phagocytosis of chicken red blood cells by mouse peritoneal macrophages (semi-in vivo method) Mice were intraperitoneally injected with 1 mL of 20% (v / v, prepared with saline) packed chicken red blood cell (2000rpm, 10min) suspension, and then killed by cervical dislocation at intervals of 30min. 1 mL of peritoneal macrophage washing fluid was taken and dropped on a glass slide, placed in an enamel box padded with wet gauze, and incubated in a 37°C incubator for 30min. After incubation, rinse in saline to remove unattached cells. Dry, fix with methanol:acetone (1:1), stain with 4% (v / v) Giemsa-phosphate buffer, rinse with distilled water and dry. Count under an oil immersion lens, count 100 macrophages per slide, and calculate the phagocytosis rate and phagocytosis index according to the following formula:

[0109] Phagocytic rate % = number of macrophages phagocytosing chicken red blood cells / number of macrophages counted × 100

[0110] Phagocytic index = total number of phagocytic chicken red blood cells / number of macrophages counted

[0111] Determination of NK cell activity (isotope 3 H-TdR assay), the method is as follows:

[0112] YAC-1 cells that grew well 24 hours after subculturing (survival rate>95%) were collected and plated at 1×10 6 / mLYAC-1 cell suspension plus suspension plus 3 The cells were labeled with 10 μCi of H-TdR and cultured in a 37°C, 5% carbon dioxide incubator for 2 h, with shaking once every 30 min. The labeled cells were washed three times with culture medium and resuspended in culture medium to a cell concentration of 1×10 5 The mice were killed by cervical dislocation, and the spleen was removed aseptically to prepare a spleen cell suspension. The cells were washed three times with Hank's solution, centrifuged at 1000 rpm for 10 min each time, and then suspended with 2 mL of RPMI1640 complete culture medium containing 10% calf serum. The cells were stained with trypan blue and counted (the number of viable cells should be above 95%). The cell concentration was adjusted to 1×10 7 / mL. Add 100uL of labeled target cells to each well of a 96-well plate, 100mL of effector cells to the test wells, 100uI of culture medium to the blank control wells, and 100uL of TritonX-100 to the maximum release wells. Set up 3 replicate wells for each sample and culture at 37°C with 5% carbon dioxide for 4 hours. Collect the cells on glass fiber filter paper using a multi-head cell collector and measure the number of pulses per minute (cpm) using a liquid scintillation meter.

[0113] NK cell activity = (1-experimental well cpm / (blank control well cpm-maximum release well cpm)) × 100%

[0114] Experimental data statistics: Excel and Spss software were used for statistical analysis. When using Spss software for analysis, the data were first tested for homogeneity of variance. If the variances were homogeneous, one-way analysis of variance was used for overall comparison. If differences were found, the Dunnett method was used for pairwise comparisons between the means of multiple dose groups and a control group. If the variances were not homogeneous, the original data were appropriately transformed. After the test for homogeneity of variance was met, the transformed data were used for statistics. If the purpose of homogeneity of variance was still not achieved after the variable transformation, the rank sum test was used for statistics. If the overall comparison was found to be different, the Tamhmane's T2 test, which did not require homogeneity of variance, was used for pairwise comparisons.

[0115] The effects of the sample in Example 1 on the body weight of mice are as follows:

[0116] Table 2 Initial body weight of mice in each group

[0117]

[0118]

[0119] Table 3 Mid-term body weight of mice in each group

[0120]

[0121] Table 4 Final body weight of mice in each group

[0122]

[0123] Table 5 Weight gain of mice in each group (x±s)

[0124]

[0125]

[0126] The results are shown in Tables 2-5. After the variance homogeneity test, the variances of the body weights of mice in each group at the beginning, middle and end of the experiment and the weight gain of mice during the experiment were the same. The results of one-way analysis of variance showed that there were no significant differences in the above indicators among the groups (P>0.05).

[0127] The effects on the ratio of immune organs to body weight of mice are shown in Table 6. After the variance homogeneity test, the variance of thymus / body weight and spleen / body weight of mice in each group was homogeneous. The results of one-way analysis of variance showed that there was no significant difference in the two indicators between the groups (P>0.05).

[0128] Table 6 Effects on the ratio of immune organs to body weight of mice

[0129]

[0130] The results of the effects on the cellular immune function of mice are as follows:

[0131] The effect on delayed-type hypersensitivity (DTH) in mice is shown in Table 7. After the homogeneity test, the variance of the swelling degree of the paw of each group of mice was homogeneous, and the one-way analysis of variance showed that there was a significant difference between the groups (P=0.006). Further pairwise comparisons between the means of multiple dose groups and a control group were performed using the Dunnett method, and the results showed that the swelling degree of the paw of mice in the medium and high dose groups was significantly higher than that in the control group (P<0.01). This suggests that the sample can improve the ability of delayed-type hypersensitivity in mice.

[0132] Table 7 Effects on delayed-type hypersensitivity (DTH) in mice

[0133]

[0134]

[0135] The effect of mouse ConA-induced mouse lymphocyte transformation ability experiment is shown in Table 8. The variance homogeneity test showed that the variance of lymphocyte proliferation ability of each group of mice was homogeneous, and the one-way analysis of variance showed that there was no significant difference among the groups (P>0.05).

[0136] Table 8 Effect on mouse lymphocyte transformation ability experiment

[0137]

[0138] The effects on humoral immunity are as follows: The effects on the number of antibody-producing cells in mice are shown in Table 9. The variance homogeneity test showed that the variance of the hemolytic plaques in each group of mice was homogeneous. The one-way analysis of variance showed that there were significant differences among the groups (P=0.006). The Dunnett method was further used to compare the means of multiple dose groups with a control group. The results showed that the number of hemolytic plaques in the middle and high dose groups was significantly higher than that in the control group (P<0.05 or P<0.01). This indicates that the sample can significantly improve the antibody production ability of mice.

[0139] Table 9 Effect on the number of mouse antibody-producing cells

[0140] Dosage Group Dosage (g / kg.bw) Number of animals <![CDATA[Number of hemolytic plaques (×10 3 / total spleen)]]> P-value Control group 0.000 10 19.6±6.3 - Low dose 1.667 10 24.8±8.8 0.364 Medium dose 3.333 10 30.3±8.5 0.016 High dose 10.000 10 32.2±8.9 0.004

[0141] Mouse half serum (HC 50 ) is shown in Table 10. After the variance homogeneity test, the variance of the half hemolysis value of mice in each group was homogeneous. The one-way analysis of variance showed that there was no significant difference among the groups in the overall comparison (P>0.05, see Table 10).

[0142] Table 10: Mouse half-lysed serum (HC 50 )

[0143]

[0144] The effects on mouse monocyte-macrophage function are as follows:

[0145] The effects on carbon clearance of mouse monocytes and macrophages are shown in Table 11. After the variance homogeneity test, the variance of the phagocytic index of each group of mice was homogeneous, and the univariate analysis showed that there was no significant difference among the groups (P>0.05), indicating that the sample had no significant effect on the carbon clearance ability of mice.

[0146] Table 11 Effects on carbon clearance of mouse monocytes and macrophages

[0147]

[0148] The effects on the ability of mouse macrophages to phagocytose chicken red blood cells are shown in Tables 12 and 13. After the variance homogeneity test, the square root arcsine transformation value of the phagocytosis rate and the phagocytosis index of each group of mice were homogeneous. The one-way ANOVA showed that there was no significant difference in the above two indicators between the groups (P>0.05). This indicates that the sample has no significant effect on the ability of mouse macrophages to phagocytose chicken red blood cells.

[0149] Table 12 Effects on the phagocytic rate of chicken red blood cells by mouse macrophages

[0150]

[0151]

[0152] Table 13 Effect of phagocytic index of chicken red blood cells on mouse macrophages

[0153]

[0154] The effects on the activity of mouse NK cells are shown in Table 14. After the variance homogeneity test, the variance of the square root arcsine transformed values ​​of the NK cell activity of mice in each group was homogeneous. The one-way analysis of variance showed that there was no significant difference among the groups in the overall comparison (P>0.05).

[0155] Table 14 Effects on mouse NK cell activity

[0156]

[0157] In summary, the samples of 1.667g / kg·bw, 3.333g / kg·bw, and 10.000g / kg·bw were orally administered to mice for 30 days. The doses of 3.333g / kg·bw and 10.000g / kg·bw could significantly increase the delayed-type hypersensitivity and antibody-producing cell count of mice, and each dose had no significant effect on the weight gain of mice, the thymus / body weight ratio, the spleen / body weight ratio, the NK cell activity, the half hemolysis value, the carbon clearance capacity of mice, the ability of macrophages to phagocytose chicken red blood cells, and the ConA-induced transformation capacity of mouse lymphocytes. According to the judgment criteria in the Technical Specifications for Inspection and Evaluation of Health Foods (2003 Edition), the samples are considered to have the effect of enhancing immunity.

[0158] In the formula of the Chinese herbal active ingredients in the embodiment, donkey glue nourishes yin, replenishes blood and stops bleeding, and jujube nourishes blood and replenishes qi. The combination of the two has the effects of nourishing blood, replenishing qi and stopping bleeding, and is used to treat bleeding syndrome caused by insufficient qi and blood. Polygonatum sibiricum replenishes qi, nourishes the kidney and replenishes essence, and wolfberry is good at replenishing essence and qi. Poria cocos replenishes the spleen and stomach, and jujube replenishes the spleen and stomach. Jujube and donkey glue nourish blood and replenish qi; together they play the effects of nourishing blood and replenishing qi, strengthening the spleen and stomach, and achieving the health care effect of enhancing the body's immunity. After using donkey glue to replenish qi and blood, the face becomes ruddy and the chloasma disappears, which has a certain freckle removal effect.

[0159] Example 2 was made into an oral liquid and the following tests were performed:

[0160] The effects of the samples in Example 2 on the body weight of mice are as follows:

[0161] The serum hemolysin and delayed-type hypersensitivity reaction test groups are shown in Table 15. After variance analysis, there were no significant differences in initial body weight (F=0.8266, P=0.4871), mid-term body weight (F=0.3683, P=0.7762), final body weight (F=0.3203, P=0.8106), and weight gain (F=0.0657, P=0.9778) between each dose group and the control group (P>0.05).

[0162] Table 15 Initial body weight of mice in each group (serum hemolysin and delayed-type hypersensitivity reaction measurement group)

[0163]

[0164] The phagocytic chicken red blood cells and lymphocyte transfer test groups are shown in Table 16. By variance analysis, compared with the control group, the initial body weight of each dose group was F=0.8994, P=0.4500; the mid-term body weight was F=0.3169, P=0.8130; the final body weight was F=0.6144, P=0.6097: the body weight gain value was F=0.0272, P=0.9938, and there was no significant difference (P>0.05).

[0165] Table 16 Effect on mouse body weight (phagocytosis of chicken red blood cells and lymphocyte transfer assay group)

[0166]

[0167] The NK cell activity determination group is shown in Table 17. After variance analysis, compared with the control group, the initial body weight of each dose group was F=0.1286, P=0.9425; the mid-term body weight was F=0.1825, P=0.9076; the final body weight was F=0.0876, P=0.9664; the body weight gain value was F=0.2538, P=0.8581, and there was no significant difference (P>0.05).

[0168] Table 17 Effect on mouse body weight (NK cell activity assay group)

[0169]

[0170] The carbon clearance test group is shown in Table 18. After variance analysis, the initial body weight F = 0.0565, P = 0.9820; mid-term body weight F = 0.3929, P = 0.7588; final body weight F = 0.0966, P -0.9614; body weight gain F = 0.1442, P = 0.9327 of each dose group were compared with the control group, and there was no significant difference (P>0.05).

[0171] Table 18 Effect on mouse body weight (Carbon clearance test group)

[0172]

[0173]

[0174] The effects on the organ / body weight ratio of mice are as follows:

[0175] The spleen / body weight ratio was analyzed by variance analysis, F=0.4177, P=0.7413. There was no significant difference in spleen / body weight ratio between mice in each dose group and mice in the control group (P>0.05), as shown in Table 19.

[0176] The thymus / body weight ratio was analyzed by variance analysis, F=0.1820, P=0.9080. There was no significant difference in the thymus / body weight ratio between the mice in each dose group and the control group (P>0.05), see Table 19.

[0177] Table 19 Effects on the ratio of mouse organs to body weight

[0178]

[0179] The effect on delayed hypersensitivity (DH) in mice is shown in Table 20. After analysis of variance, F = 3.0474, P = 0.0396. 24 hours after antigen attack, there was no significant difference in the degree of paw swelling between the 3.3ml / kg.bw and 6.7ml / kg.bw dose groups and the control group (P>0.05), and there was a significant difference in the degree of paw swelling between the 10.0ml / kg.bw dose group and the control group (P<0.05).

[0180] Table 20 Effect on delayed-type hypersensitivity (DTH) in mice

[0181] Dosage group (ml / kg.bw) Number of animals Toe swelling (mm) Control group 11 0.25±0.15 3.3 11 0.28±0.19 6.7 11 0.27±0.12 10.0 11 0.43±0.16*

[0182] *P<0.05 compared with the control group

[0183] ConA-induced mouse spleen lymphocyte transformation experiment (MTT method):

[0184] After variance analysis, F=5.7916, P=0.0022. There was no significant difference in optical density between the 3.3ml / kg.bw and 6.7ml / kg.bw dose groups and the control group mice (P>0.05). There was a significant difference in optical density between the 10.0ml / kg.bw dose group and the control group mice (P<0.01), see Table 21.

[0185] Table 21 Effects on mouse lymphocyte transformation

[0186] Dosage group (ml / kg.bw) Number of animals Optical density difference Control group 11 0.342±0.058 3.3 11 0.349±0.048 6.7 11 0.368±0.047 10.0 11 0.428±0.061**

[0187] **P<0.01 compared with the control group

[0188] The effects on the production of serum hemolysin in mice are as follows: After analysis of variance, F=3.4985, P=0.0241. There was no significant difference in the anti-volume counts between the 3.3ml / kg.bw and 6.7ml / kg.bw dose groups and the control group mice (P>0.05), and there was a significant difference in the anti-volume counts between the 10.0ml / kg.bw dose group and the control group mice (P<0.05), see Table 22.

[0189] Table 22 Effect of anti-hemolysin volume on mouse serum

[0190] Dosage group (ml / kg.bw) Number of animals Anti-volume number Control group 11 170.36±7.75 3.3 11 171.36±13.31 6.7 11 174.64±9.59 10.0 11 183.18±9.87*

[0191] *P<0.05 compared with the control group

[0192] Antibody-producing cell detection (Jerne modified slide method): After analysis of variance, F=8.6680, P=0.0001. There was no significant difference in the number of antibody-producing cells / 10° (splenocytes) between the 3.3ml / kg.bw dose group and the control group mice (P>0.05), there was a significant difference in the number of antibody-producing cells / 10" (splenocytes) between the 6.7ml / kg.bw dose group and the control group mice (P<0.05), and there was a significant difference in the number of antibody-producing cells / 10" (splenocytes) between the 10.0ml / kg.bw dose group and the control group mice (P<0.01), see Table 23.

[0193] Table 23 Effects on mouse antibody-producing cells

[0194] Dosage group (ml / kg.bw) Number of animals <![CDATA[Number of antibody-producing cells / 10 6 (splenocytes)]]> Control group 11 148.18±100.38 3.3 11 127.27±96.63 6.7 11 245.91±102.15* 10.0 11 312.27±89.79**

[0195] The percentage of phagocytosis was analyzed by variance analysis, F = 3.1610, P = 0.0349. There was no significant difference in the percentage of phagocytosis of chicken red blood cells by peritoneal macrophages in each dose group compared with the control group (P>0.05), see Table 24.

[0196] The phagocytic index was analyzed by variance analysis, F = 1.3207, P = 0.2811. There was no significant difference in the phagocytic index of chicken red blood cells by peritoneal macrophages in each dose group compared with the control group (P>0.05), as shown in Table 24.

[0197] Table 24 Effects on the ability of mouse peritoneal macrophages to phagocytose chicken red blood cells

[0198]

[0199] The carbon clearance experiment in mice is as follows: After analysis of variance, F=3.7513, P=0.0192. There was no significant difference in the phagocytic index a between the 3.3ml / kg.bw and 6.7ml / kg.bw dose groups and the control group mice (P>0.05), and there was a significant difference in the phagocytic index a between the 10.0ml / kg.bw dose group and the control group mice (P<0.05), see Table 25.

[0200] Table 25 Effects on carbon clearance in mice

[0201] Dosage group (ml / kg.bw) Number of animals Phagocytic index a Control group 10 6.20±0.77 3.3 10 6.11±0.64 6.7 10 6.50±0.56 10.0 10 6.91±0.28*

[0202] *P<0.05 compared with the control group

[0203] In summary, the products of the samples of Examples 1 and 2 have the effect of enhancing immunity, and the donkey glue and the active ingredients of Chinese herbal medicine have the effects of replenishing qi and blood, beautifying and removing spots.

[0204] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A Chinese medicinal composition of donkey-hide gelatin for nourishing qi and blood, beautifying and removing freckles, characterized in that: The invention comprises the following raw materials in parts by weight: 3-10 parts of donkey glue and 40-60 parts of Chinese herbal active ingredients; The preparation method of the Chinese herbal medicine active ingredient is as follows: The Chinese herbal medicine is crushed to obtain a mixed powder, and then the mixed powder is embrittled. After the embrittlement treatment, a composite plant acid solution is added for immersion, heating, vacuum distillation, alcohol precipitation, filtering, distilling the filtrate to remove alcohol, concentrating, and drying to obtain the product.

2. The Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood and beautifying and removing freckles according to claim 1, characterized in that: The Chinese herbal medicine comprises wolfberry, polygonatum, poria and jujube in a mass ratio of 3-5:4-6:2-3:1-5; or; The Chinese herbal medicines include American ginseng, longan pulp, wolfberry, angelica, red dates and iron preparations in a mass ratio of 3-7:4-8:2-5:1-5:3-5:0.1-0.

5.

3. The Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood and beautifying and removing freckles according to claim 1, characterized in that: The embrittlement treatment is: Add the mixed powder into a mixed aqueous solution of sodium chloride, amino acid and chitosan, stir, spray polyvinyl alcohol solution, let stand, take out, and embrittle at -30 to -10°C under vacuum environment and pressure of 1-5kpa for 3-6h, and then embrittle at 60-80°C for 2-3h.

4. The Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood and beautifying and removing freckles according to claim 3, characterized in that: The mass percentages of the sodium chloride, amino acid and chitosan in water are 1-5%, 3-5% and 10-15% respectively.

5. The Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood and beautifying and removing freckles according to claim 3, characterized in that: The spraying amount of the polyethylene glycol is 3-10% of the mass of the mixed powder.

6. The Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood and beautifying and removing freckles according to claim 3, characterized in that: The amino acids include one or more of glycine, proline, alanine, phenylalanine, glutamic acid, and arginine.

7. The Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood and beautifying and removing freckles according to claim 1, characterized in that: The composite plant acid solution comprises an aqueous solution of citric acid, malic acid, oxalic acid and sodium citrate in a molar concentration ratio of 1:1-2:2-3:0.5-1.

8. The Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood and beautifying and removing freckles according to claim 7, characterized in that: The concentration of the sodium citrate aqueous solution is 0.05-3 mol / L.

9. A method for preparing a Chinese medicinal composition of donkey-hide gelatin for replenishing qi and blood, beautifying and removing freckles according to any one of claims 1 to 8, characterized in that: The donkey glue is ground into powder to obtain donkey glue powder, and the donkey glue powder is evenly mixed with active ingredients of Chinese herbal medicine to obtain the product.

10. An application of the donkey-hide gelatin Chinese medicine composition for replenishing qi and blood, beautifying and removing freckles as claimed in any one of claims 1 to 8, characterized in that: The finished dosage form of the traditional Chinese medicine composition is at least one of pills, tablets, capsules, granules, oral liquids, and decoctions.

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