Application of melanin of Taihe silky fowl

Abstract

The invention discloses application of melanin of Taihe silky fowl. The invention provides application of melanin in Taihe silky fowl bones to preparation of a blood replenishing medicine. The Taihe black-bone chicken melanin is extracted from Taihe black-bone chicken bones and / or meat. A mouse blood deficiency model is established, Taihe silky fowl melanin is given to the mouse through intragastric administration, and it is found that the Taihe silky fowl melanin can significantly improve the ischemic state of the model mouse.

Description

Technical Field

[0001] The invention belongs to the technical field of biological natural product extraction and relates to the application of Taihe black-bone chicken melanin. Background Art

[0002] Taihe Black Chicken ( Gallus gallus domesticus Brisson is a traditional animal medicine in my country. It has the effects of "tonifying deficiency, strengthening yang, and tonifying yin and kidney". Modern pharmacological research has found that Taihe black chicken has the effects of anti-oxidation, anti-aging, anti-fatigue, anti-mutagenicity, blood tonic, cardiovascular system protection, inhibition of non-enzymatic glycosylation, prevention and treatment of osteoporosis, treatment of Parkinson's disease, and treatment of diabetes. Melanin is an important medicinal ingredient of Taihe black chicken, which has multiple functions such as eliminating free radicals, anti-oxidation, improving body immunity, and delaying aging. The bone tissue of Taihe black chicken accounts for one-third of the total mass of Taihe black chicken, and the study found that the deposition of melanin in the bone of Taihe black chicken is obvious. However, whether it is used as medicine or daily consumption, bone melanin is not fully utilized. Most of the existing basic research on Taihe black chicken melanin is aimed at skin and meat tissue melanin, which undoubtedly causes waste and does not give full play to the maximum therapeutic effect of Taihe black chicken. Therefore, in order to realize the utilization of Taihe black chicken bone tissue waste and further improve the use value of Taihe black chicken, it is urgent to develop and study the extraction method and pharmacological effect of Taihe black chicken bone melanin. Summary of the invention

[0003] The invention aims to provide application of Taihe black-bone chicken melanin.

[0004] The invention provides application of melanin in Taihe black-bone chicken bones in preparing blood-replenishing medicine.

[0005] In the above application, the Taihe black-bone chicken melanin is extracted from Taihe black-bone chicken bones and / or meat.

[0006] In the above application, the Taihe black-bone chicken melanin is extracted from Taihe black-bone chicken bones, comprising the following steps: 1) The bone tissue of Taihe black-bone chicken is dried and crushed to obtain bone tissue powder; 2) adding anhydrous ethanol to the bone tissue powder, mixing, refluxing in a water bath, filtering while hot after the refluxing is completed, collecting the filter residue and drying it to obtain defatted bone tissue powder; 3) mixing the defatted bone tissue powder with water, pre-cooking and cooling, adding composite protease and stirring, inactivating the enzyme and filtering while hot, collecting the filter residue and drying it to obtain a deproteinized bone tissue sample; 4) soaking the deproteinized bone tissue sample in lactic acid, extracting it at high temperature, centrifuging and discarding the supernatant, adding lactic acid to the precipitate and extracting it at high temperature, centrifuging and discarding the supernatant, washing and drying the residue, and obtaining the melanin in Taihe black chicken bone.

[0007] In the above application, in step 1), the drying temperature may be 50-70°C.

[0008] In the above application, in step 2), the mass ratio of the bone tissue powder to the volume of the anhydrous ethanol is 1 g: 2 ml; The temperature of the water bath is 90°C, the number of reflux is 4 times, and the time of each reflux is 1 hour; The drying temperature may be 50-70°C, specifically 60°C.

[0009] In the above application, in step 3), the volume ratio of the defatted bone tissue powder and water can be 1:6; The precooking temperature may be 85-100°C (specifically 90°C) and the precooking time may be 25-35 min (specifically 30 min); The composite protease includes protein endo-nuclease, exo-nuclease and flavor enzyme, which are purchased from Shanghai Yuanye Biotechnology Co., Ltd., with the product catalog number of S10155; The temperature of the heat preservation and stirring can be 55°C, and the pH value can be 7.5; The mass ratio of the defatted bone tissue powder to the composite protease may be 100:4.

[0010] In the above application, in step 3), the temperature after cooling may be 50-60°C (specifically 55°C), the temperature of the heat preservation and stirring may be 55°C, and the time may be 2-2.5 h (specifically 2 h); The enzyme inactivation temperature may be 90°C and the time may be 30 min; The temperature for drying the filter residue may be 50-70°C (specifically 60°C).

[0011] In the above application, in step 4), the mass percentage concentration of the lactic acid aqueous solution may be 50%; The material-liquid ratio of the deproteinized bone tissue sample to the lactic acid is 1 g:6 ml; The centrifugation rate may be 10000 rpm / min, and the time may be 10-20 min; since the melanin particles suspended in the lactic acid solution are relatively small, high-speed and long-term centrifugation is used to ensure that the melanin particles are completely precipitated; The solid-liquid ratio of the precipitate to the lactic acid aqueous solution may be 1 g:6 ml.

[0012] In the above application, in step 4), the temperature of the high temperature extraction process can be 115°C and the time can be 1 hour. If the extraction time is too short, the bone digestion may be incomplete, and if the extraction time is too long, the autoclave may be damaged or even dangerous. The centrifugal speed after the high temperature extraction can be 10000 rpm / min, and the time can be 10-20 min; the number of high temperature extraction and centrifugal treatment after the high temperature extraction can be 5 times; The residue is washed with hot water, the temperature of the hot water may be 60-70°C, the volume ratio of the residue to the hot water may be 1:5-8; the number of washings may be 3-6 times; The drying temperature of the filter residue may be 50-70°C, specifically 60°C.

[0013] The present invention has the following beneficial effects: The present invention can effectively remove fat, protein and a large amount of insoluble calcium salts in bone tissue through three steps of anhydrous ethanol reflux defatting-protease hydrolysis-lactic acid hydrolysis. Lactic acid is more suitable for later melanin product development and industrial production than hydrochloric acid, and the method is simple and the extraction efficiency is high. The present invention establishes a blood deficiency model in mice, and orally administers Taihe black-bone chicken melanin to mice, and finds that Taihe black-bone chicken melanin can significantly improve the ischemic state of the model mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the transmission electron microscope image of the mentioned melanin; Figure 2 This is the ultraviolet absorption spectrum of Taihe black-bone chicken melanin, Figure 2 (a) is the ultraviolet absorption spectrum of Taihe black-bone chicken melanin, (b) is the ultraviolet absorption spectrum of Taihe black-bone chicken melanin; Figure 3 is the effect of lactic acid concentration on melanin extraction; Figure 3 (a) shows the melanin yield under the lactic acid concentration factor, and (b) shows the melanin relative fluorescence intensity under the lactic acid concentration factor. Figure 4 is the effect of the solid-liquid ratio on melanin extraction; Figure 4 (a) is the material-liquid ratio factor to examine the melanin yield, (b) is the material-liquid ratio factor to examine the melanin relative fluorescence intensity; Figure 5 The effect of extraction temperature on melanin extraction; Figure 5 (a) shows the extraction temperature factor to examine the melanin yield, and (b) shows the extraction temperature factor to examine the melanin relative fluorescence intensity. DETAILED DESCRIPTION

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0016] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0017] Example A method for extracting melanin from Taihe black-bone chicken bones, the specific steps are as follows: 1. Take Taihe black-bone chicken, peel off the bone tissue, dry at 60℃, and crush the bone tissue using a powder grinder; 2. Degreasing by reflux with anhydrous ethanol: Take bone tissue powder, add anhydrous ethanol according to the ratio of solid to liquid, and reflux in a 90 ℃ water bath for 4 times, each time for 1 hour. After the reflux, filter while hot, collect the filter residue and dry it in a 60 ℃ oven to obtain the defatted bone tissue powder; 3. Protease hydrolysis: Take the defatted bone tissue powder, add 6 times the volume of water, pre-cook at 90 ℃ for 30 min, cool to 55 ℃, add 4% of the weight of the bone tissue composite protease (specific composition: protein endo-nuclease, exo-nuclease and flavor enzyme; purchased from Shanghai Yuanye Biotechnology Co., Ltd., product catalog number S10155), keep warm and stir at 55 ℃ for 2 h, inactivate the enzyme at 90 ℃ for 30 min, filter while hot, and dry the filter residue in an oven at 60 ℃ to obtain the bone tissue sample after protein removal.

[0018] 4. Lactic acid hydrolysis: Take the defatted and deproteinized bone tissue sample, add lactic acid aqueous solution to soak overnight, place it in a high-pressure sterilizer for high-temperature extraction for 1 hour, then centrifuge at 10,000 rpm / min for 20 min, discard the supernatant, repeat the above high-temperature extraction steps 5 times, and wash the residue with 6 times the volume of 60-70°C hot water 3 times. After each washing, centrifuge at 10,000 rpm / min for 20 min, discard the supernatant, and finally place the residue in a 60°C oven to dry to obtain Taihe black chicken bone melanin.

[0019] The structure was confirmed as follows: pass Figure 1 It can be seen from the transmission electron microscope image that the black particles are the melanin mentioned in the present invention; like Figure 2 As shown, by comparing with the ultraviolet absorption peak of melanin extracted from Taihe black chicken, the Taihe black chicken bone melanin of the present invention has the same ultraviolet absorption peak.

[0020] According to the above method, the influencing factors in the extraction method were investigated: 1. Effect of lactic acid concentration on the extraction of melanin from Taihe black-bone chicken The solid-liquid ratio was fixed at 1:6 (m:V, g:ml), the extraction temperature was 125 ℃, the number of extractions was 5, the extraction time was 1 h each time, and the lactic acid concentrations of 30%, 40%, 50%, 60%, and 70% were used as the investigation objects. The melanin yield and the relative fluorescence intensity of melanin were used as evaluation indicators to investigate the effect of lactic acid concentration on the extraction of melanin from Taihe black chicken bones. The results are as follows Figure 3 Considering the melanin production and relative fluorescence intensity, 50% lactic acid was selected for subsequent single factor experiments.

[0021] 2. Effect of solid-liquid ratio on the extraction of melanin from Taihe black-bone chicken bones The lactic acid concentration was fixed at 50%, the extraction temperature was 125 ℃, the number of extractions was 5, the extraction time was 1 h each time, the solid-liquid ratio (m:V, g:ml) of 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 was used as the investigation objects, and the melanin yield and melanin relative fluorescence intensity were used as evaluation indicators to investigate the effect of solid-liquid ratio (m:V) on the extraction of melanin from Taihe black chicken bones. The results are shown in Figure 4 Considering the melanin yield and relative fluorescence intensity, a 1:7 solid-liquid ratio was selected for subsequent single factor experiments.

[0022] 3. Effect of extraction temperature on the extraction of melanin from Taihe black-bone chicken The lactic acid concentration was fixed at 50%, the solid-liquid ratio (m:V, g:ml) was 1:7, the number of extractions was 5, the extraction time was 1h each, the extraction temperatures were 110 ℃, 115 ℃, 120 ℃, 125 ℃, and 130 ℃, and the melanin yield and relative fluorescence intensity of melanin were used as evaluation indicators to investigate the effect of extraction temperature on the extraction of melanin from Taihe black chicken bones. The results are shown in Figure 2. Figure 5 Considering the melanin yield and relative fluorescence intensity, the extraction temperature of 120 ℃ was selected for subsequent experiments.

[0023] 4. Orthogonal test of Taihe black chicken bone melanin extraction Based on the single factor experiment, the three-factor design L9 (3 3 ) Orthogonal experiment, taking melanin yield and melanin relative fluorescence intensity as evaluation indicators, the lactic acid extraction process of Taihe black chicken bone melanin was further optimized. The results are shown in Table 1-2.

[0024] Table 1 Orthogonal test level table L9 (3 3 )

[0025] Table 2 Orthogonal experimental design and results

[0026] Based on the melanin yield and fluorescence analysis results, experimental group 6, i.e., lactic acid concentration of 50%, solid-liquid ratio of 1:8, and extraction temperature of 115 ℃, was finally selected as the optimal extraction process, and a process stability test was carried out.

[0027] 5. Process stability test The RSD values ​​of melanin yield and melanin relative fluorescence intensity data in three process stability experiments were all less than 10%, indicating that the melanin extraction process had good stability, as shown in Table 3.

[0028] Table 3 Process stability test results

[0029] Second, Taihe black-bone chicken has a good blood-tonifying effect, but the effect is unclear. In order to reveal the anti-blood deficiency effect of Taihe black-bone chicken melanin, the present invention established a blood deficiency model in mice, and gavaged the mice with Taihe black-bone chicken melanin, and found that Taihe black-bone chicken melanin can significantly improve the ischemic state of the model mice.

[0030] The specific steps are as follows: 1. Use 90 clean mice, 6-8 weeks old, weighing 18-22 g. The experimental animals were kept at a temperature of 22-27°C, a relative humidity of 40-70%, and light and dark alternation for 12 hours; 2. The mice were fed adaptively for 5 days. During the adaptation period, they were allowed to drink water and eat freely, and were given maintenance feed; 3. After adaptive feeding, the subjects were randomly divided into groups according to body weight. The specific groups are shown in Table 4 below: Among them, the preparation method of Taihe black-bone chicken whole chicken water extract is as follows: cut the Taihe black-bone chicken into pieces, add water and boil for 1 hour, add paraffin while it is hot, stir to melt it, leave it overnight, discard the upper layer of paraffin and fat solids, beat into a homogenate, add papain and stir, adjust the pH to 6-7, hydrolyze at 50℃ for 5 hours, boil for 5 minutes, centrifuge, and reduce the pressure to concentrate the supernatant into a thick paste.

[0031] Table 4 Experimental groups of mice

[0032] 4. Starting from the first day of the experiment, the normal group and the model group were gavaged with 0.2 mL / 10g normal saline, and the other groups were gavaged with rhG-CSF positive drug, low, medium and high doses of melanin, and low, medium and high doses of Taihe black-bone chicken water extract for 30 consecutive days; 5. On the 25th to 27th day after the start of the experiment, mice in all groups except the normal group were intraperitoneally injected with cyclophosphamide (40 mg / kg) to establish the model; 6. During the administration period, observe the general condition of the mice every day, including whether they move quickly, whether their fur is shiny and dense, and whether the color of their auricles, nose lips, and tails is normal. Weigh their body weight every week and adjust the dosage according to their body weight; 7. One hour after the last administration, the eyeballs of mice in each group were removed to collect blood, and 0.3 mL of whole blood was collected in an EDTA-K2 anticoagulant tube; 8. Take 20 μL blood samples from each group and use a fully automatic blood analyzer to perform routine blood tests to detect the red blood cell count (RBC), white blood cell count (WBC), hemoglobin (HGB), and hematocrit (HCT) indicators in the anticoagulated whole blood of the mice.

[0033] 9. As shown in Table 5, the levels of WBC, RBC, HGB and HCT in the model group decreased significantly ( P <0.05), indicating that the model was successfully established. The WBC of the medium and high dose melanin groups and the low dose Taihe black-bone chicken water extract group were significantly higher than those of the model group, indicating that Taihe black-bone chicken melanin can improve the ischemic state and has the same effect as the whole chicken water extract.

[0034] Table 5 Blood test results of experimental mice

[0035] Note: Compared with the normal group, △ P <0.05; △△ P <0.01; compared with the model group, ** P <0.01.

Claims

1. Application of Taihe black-bone chicken melanin in the preparation of blood-replenishing medicine.

2. The use according to claim 1, characterized in that: The Taihe black-bone chicken melanin is extracted from Taihe black-bone chicken bones and / or meat.

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