CN107334913B - Black garlic extract and preparation method and application thereof - Google Patents
Black garlic extract and preparation method and application thereof Download PDFInfo
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- CN107334913B CN107334913B CN201611139223.1A CN201611139223A CN107334913B CN 107334913 B CN107334913 B CN 107334913B CN 201611139223 A CN201611139223 A CN 201611139223A CN 107334913 B CN107334913 B CN 107334913B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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Abstract
The invention discloses a black garlic extract, a preparation method and application thereof, wherein the black garlic extract provided by the invention is prepared by the following preparation method: the method comprises the steps of extracting black garlic powder with ethyl acetate to obtain a first crude extract insoluble in ethyl acetate, extracting the first crude extract with n-butyl alcohol to obtain a first black garlic extract soluble in n-butyl alcohol and a second crude extract insoluble in n-butyl alcohol, extracting the second crude extract insoluble in n-butyl alcohol with water to obtain a second black garlic extract soluble in water, wherein the first black garlic extract and the second black garlic extract have liver protection effect and can be applied to preparation of liver protection medicines and health care products.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the field of medicines and foods, in particular to a black garlic extract and a preparation method and application thereof.
[ background of the invention ]
Liver cancer is the 5 th most common cancer worldwide and is a common cause of cancer-related death. Hepatitis refers to the damage and inflammation of liver cells, and causes of hepatitis include viruses, alcohol, drugs, and the like. The liver inflammation can increase the incidence of liver cirrhosis and liver cancer. Because the Chinese people are always well-conditioned by the viral hepatitis and the daily work and rest and the change of the dietary habits, the probability of liver damage is increased, and the requirements of liver-protecting medicines and health-care products are wide.
The garlic is used as a medicine and food dual-purpose medicinal material besides being used as a daily food material or a spice. Record of traditional Chinese medical book: the garlic has pungent taste and warm nature, and has the effects of warming spleen and stomach, promoting digestion, regulating qi, detoxifying and killing insects. The research report also indicates that the garlic has good physiological activities of resisting oxidation, resisting bacteria, preventing cardiovascular diseases and the like and good anti-tumor effect.
The preparation process of the black garlic mainly comprises the step of controlling the screened fresh garlic to ferment and mature under the environment of high temperature and high humidity, which is a Meiner reaction. In the process of the Meina reaction, the peculiar allicin of garlic is converted into sulfide without garlic odor and with low irritation, gamma-glutamylcysteine (gamma-glutamylcysteine) is converted into S-allyl cysteine (SAC), protein is decomposed into amino acid, carbohydrate is decomposed into fructose, garlic cloves are also converted into black brown from original white, and finally, the garlic cloves are cooled and dried to obtain the garlic clove. The black garlic is soft and elastic, is soft and sweet after being eaten, has no spicy feeling and unpleasant smell of garlic, can be directly eaten, and is a popular health food in European and American countries, Japan, Korea and the like in recent years. The formation of the Maillard Reaction Products (MRP) in the food is believed to impart special color and flavor to the food, and research also indicates that the formation of the Maillard reaction products in the black garlic process causes the MRP of saccharides and amino acids to have stronger antioxidant activity than that of garlic. However, few further discuss other uses of black garlic extract.
[ summary of the invention ]
The invention aims to provide a black garlic water extract, a preparation method thereof and application of the black garlic water in liver-protecting medicines and liver-protecting health products.
Based on the above purpose, the invention comprises the following contents:
the black garlic extract is characterized by being obtained by the following preparation method:
extracting black garlic powder with ethyl acetate to obtain a first crude extract insoluble in ethyl acetate, and extracting the first crude extract with n-butanol to obtain a first black garlic extract soluble in n-butanol and a second crude extract insoluble in n-butanol, wherein the weight ratio of ethyl acetate to black garlic powder is 3-10:1, and the weight ratio of n-butanol to the first crude extract is 3-10: 1.
Further extracting the second crude extract insoluble in n-butanol with water to obtain a second black garlic extract dissolved in water, wherein the weight ratio of water to the second crude extract is 3-10: 1.
Further, the black garlic extracts extracted according to the previous steps, namely the first black garlic extract dissolved in the n-butanol layer and the second black garlic extract dissolved in the water layer, are applied to liver protection medicines or liver protection health food.
The invention uses solvents with different polarity substances for extraction, and is used for extracting the most main black garlic extract with liver protection function which can permeate through solvents with high polarity substances or water-soluble substances.
[ description of the drawings ]
FIG. 1 is a graph of serum alanine aminotransferase ALT effect of black garlic extract on mice treated with carbon tetrachloride.
FIG. 2 is a graph showing the effect of black garlic extract on the serum aspartate aminotransferase AST of mice treated with carbon tetrachloride.
FIG. 3 is a staining chart of liver injury tissues of mice, the liver tissues were photographed by H & E (400X) staining, and arrows indicate necrotic cells, wherein A is a control group; b is a sample treated with carbon tetrachloride; c is a sample of the pretreated silymarin group; d is a sample of the black garlic extract with the n-butanol layer of 200mg/kg which is pretreated; e is a sample of black garlic extract with a pre-treated n-butanol layer of 500 mg/kg; f is a sample of the water layer black garlic extract pretreated by 200 mg/kg; g is a sample of aqueous black garlic extract pretreated by 200 mg/kg.
Fig. 4, graph of the effect of black garlic extract on the hepatic malondialdehyde MDA content of mice treated with carbon tetrachloride.
Fig. 5, graph of the effect of black garlic extract on the liver glutathione GSH content of carbon tetrachloride-treated mice.
FIG. 6 is a graph showing the effect of black garlic extract on the CAT content in the liver of mice treated with carbon tetrachloride.
FIG. 7 is a graph showing the effect of black garlic extract on the liver SOD content of mice treated with carbon tetrachloride.
FIG. 8 is a graph showing the effect of black garlic extract on the GSH-Px content of the liver of mice treated with carbon tetrachloride.
FIG. 9 is a graph showing the effect of black garlic extract on the GSH-Rd content of the liver of mice treated with carbon tetrachloride.
[ detailed description ] embodiments
The invention provides a preparation method of black garlic extract, which comprises the steps of extracting black garlic powder with ethyl acetate to obtain a first crude extract insoluble in ethyl acetate, and extracting the first crude extract with n-butyl alcohol to obtain a first black garlic extract soluble in n-butyl alcohol and a second crude extract insoluble in n-butyl alcohol. The extracting step of the present invention may further comprise extracting the second crude extract with water to obtain a second black garlic extract dissolved in water. The black garlic extracts extracted according to the steps, namely the first black garlic extract dissolved in the n-butanol layer and the second black garlic extract dissolved in the water layer have the liver protection function, and can be applied to preparing liver protection medicines or preparing foods containing the black garlic extracts.
The weight ratio of the ethyl acetate to the black garlic powder is 3-10:1, the weight ratio of the n-butanol to the first crude extract is 3-10:1, and the weight ratio of the water to the second crude extract is 3-10: 1. The number of times of extraction by each solvent may be 1 to 5 times, and preferably 2 or 3 times.
Example (b):
preparation of extract of first, black garlic
Crushing black garlic, weighing, extracting with ethyl acetate 5 times of black garlic weight, filtering while hot, and continuously extracting for 3 times to obtain extract insoluble in ethyl acetate. Extracting the crude extract insoluble in ethyl acetate with 5 times of n-butanol, filtering, and continuously extracting for 3 times to obtain black Bulbus Allii extract soluble in n-butanol layer and crude extract insoluble in n-butanol layer. Extracting the crude extract insoluble in n-butanol with 5 times of water, filtering, and continuously extracting for 3 times to obtain black Bulbus Allii extract dissolved in water layer. The following experiment was performed after 3 black garlic extracts of different polarities were obtained from the ethyl acetate layer black garlic extract, the n-butanol layer black garlic extract, and the water layer black garlic extract.
Liver protection experiment of black garlic extract
Carbon tetrachloride induced liver injury
1. Inducing liver damage by carbon tetrachloride
In the experimental mode, a carbon tetrachloride-induced liver injury mode is adopted, and male mice of the C57BL/6 line are randomly grouped after being pre-cultured for 7 days, namely a control group, a carbon tetrachloride group, a silymarin group (200mg/kg), a black garlic ethyl acetate layer group (200mg/kg and 500mg/kg), a black garlic n-butyl alcohol layer group (200mg/kg and 500mg/kg) and a black garlic water layer group (200mg/kg and 500mg/kg), respectively. In the experiment, the control group and the carbon tetrachloride group orally administer water, and the silymarin group and the black garlic group orally administer silymarin and each black garlic layered extract respectively for 7 consecutive days. On day 7, animals of each group were fasted for 16 hours, and the animals were injected with olive oil in the abdominal cavity of the control group at an injection dose of 10ml/kg, and other groups were injected with carbon tetrachloride of 0.2% dissolved in olive oil in the abdominal cavity to induce liver damage at an injection dose of 10 ml/kg.
2. Preparation of blood and liver homogenized liquid
And (3) collecting blood from carotid artery 24 hours after the mouse is anesthetized after carbon tetrachloride injection, centrifuging whole blood, separating serum, and taking supernatant for determining biochemical index of liver function. Dissecting and taking liver to prepare liver sample, and taking out liver large leaf part to fix with formalin for pathological section; diluting the rest liver samples with normal saline at a mass ratio of 1:1, homogenizing, centrifuging, collecting supernatant, and storing at-80 deg.C for analysis of liver tissue enzyme activity.
(II) biochemical index detection of serum liver function
A mouse blood sample is placed at room temperature for 1 hour to be coagulated, a centrifuge is used for centrifuging at 4 ℃ and 3000rpm per minute for 10 minutes, serum is separated, and supernate is taken to detect liver function biochemical indexes such as alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and the like by an automatic biochemical analyzer.
(III) pathological examination
Mice are sacrificed, liver tissues of 1 cm square are cut at the same position of the maximum right lobe and are placed into 10% neutral formalin for further H & E pathological staining, and the degree of damage of the liver tissues is observed.
The inflammation of liver tissue and the necrosis of liver cells are analyzed in a semi-quantitative way, the degree of liver injury is evaluated by the percentage of the injury area in a tissue section, and the degree is divided into four grades according to the scoring method published by Knodell et al, wherein the four grades are respectively as follows: 0: no lesions (none, 0%); 1: slight (slide, 1-25%); 2: mild (mil, 26-50%); 3: moderate (moderate, 51-75%); 4: severe (service, 76-100%).
(IV) discussion of Activity mechanism
1. Liver lipid peroxidation product (MDA) assay
Malonaldehyde MDA and thiometabinic acid TBA are subjected to a complete reaction at an acidic high temperature to form a red compound TBARS. The complex has a maximum absorbance at 532nm and the MDA content is determined by plotting a standard curve against 1,1,3,3-tetraethoxypropane (1,1,3,3-tetraethoxypropane, TEP).
2. Determination of Glutathione (GSH) content in liver
Taking 50 mu l of liver homogenized liquid, adding 50 mu l of 10% trichloroacetic acid (TCA), centrifuging after ice bath for 5 minutes, taking out 25 mu l of supernatant, adding 600 mu l of normal saline, mixing uniformly, adding 125 mu l of 5,5-Dithio-bis- (2-nitrobenzoic acid) (5,5-Dithio-bis- (2-nitrobenzoic acid), DTNB) solution, reacting for 5 minutes, measuring the absorbance value under the wavelength of 412nm, taking reduced GSH as a reference standard substance as a standard curve to calculate the GSH content in the sample, and expressing the result in mu mol/mg protein.
3. Liver tissue antioxidant enzyme activity determination
The activity of antioxidant enzymes including Catalase (CAT), Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px), Glutathione reductase (GSH-Rd) and the like is measured by using a commercially available reagent kit and an automatic biochemical analyzer, wherein the result of CAT is expressed in mu mol/mg protein, and the result of SOD, GSH-Px and GSH-Rd is expressed in U/mg protein.
(V) statistical analysis
The data of the experiment result is analyzed by single factor to determine the significance of the difference by Scheffe's multiple range test (One-way ANOVA), and the p value is less than 0.05, which is considered to have statistical significance. The pathological tissue analysis is characterized in that nonparametric Kruskal-Wallis analysis is adopted, and a Mann-Whitney U-test (Mann-Whitney U-test) is adopted to detect the significance of the difference between non-parametric Kruskal-Wallis analysis and non-parametric Kruskal-Wallis analysis, and when the p value is less than 0.05, the statistical significance is considered. The values in the figures are expressed as mean ± standard deviation (n ═ 10). # is the result of comparison with the control group, and # means p <0.05, # # means p <0.01, # # means p < 0.001. As compared to the group of carbon tetrachloride, p <0.05, p <0.01, p < 0.001. L is the low dose (200mg/kg) and H is the high dose (500 mg/kg).
(VI) results of the experiment
1. Determination of serum biochemical values
Determination of alanine aminotransferase ALT and aspartate aminotransferase AST in serum
Referring to fig. 1 and 2, after carbon tetrachloride was administered, ALT and AST in serum were significantly higher in the group of carbon tetrachloride than in the control group, indicating severe liver injury, while ALT and AST in serum were significantly decreased in the group of n-butanol and water layers when they were administered orally, whereas there was no significant difference in the group of ethyl acetate.
2. Pathological tissue section examination
The evaluation of liver injury lesions was performed by dividing the degree of vacuolization (vacuolization), inflammation (inflammation) and necrosis (coaggulant necrosis) of hepatocytes into four grades, and referring to fig. 3 and table 1, when carbon tetrachloride was administered for induction, liver tissues were significantly affected compared to the control group, while mice orally administered with n-butanol layer group and water layer group were significantly improved in the degree of liver injury (fig. 3, D-G), and the degree of hepatocyte necrosis was significantly reduced compared to the carbon tetrachloride group. Therefore, it is known from the results of pathological tissue section that the n-butanol layer group and the water layer group extract have a protective effect on carbon tetrachloride-induced liver injury. Since no significant effect is imparted to the ethyl acetate layer group, the ethyl acetate layer group data is not shown in the following chart.
TABLE 1
3. Biochemical value determination of liver
1) Liver lipid peroxidation product MDA content determination
Referring to FIG. 4, after induction by carbon tetrachloride, the lipid peroxidation product MDA was produced in large amounts, indicating that the liver was under free radical attack, whereas after oral administration of n-butanol and water layers of 200mg/kg, 500mg/kg and silymarin of 200mg/kg, it was found that the MDA content was effectively reduced.
2) Liver GSH content determination
GSH is an important non-enzymatic antioxidant substance and can participate in different enzymatic reactions, and the reduction of GSH content in tissues caused by carbon tetrachloride is an important index of toxicity caused by carbon tetrachloride. As shown in FIG. 5, the GSH content was significantly lower than that in the control group under the induction of carbon tetrachloride, and the administration of 200mg/kg, 500mg/kg and 200mg/kg of silymarin to the n-butanol and water-phase groups significantly increased the GSH content in the liver.
3) Determination of the activity of liver antioxidant enzymes
As shown in FIGS. 6 to 9, after carbon tetrachloride administration, the activities of CAT, SOD, GSH-Px and GSH-Rd in liver were significantly decreased in the carbon tetrachloride group, while the activities of CAT, SOD, GSH-Px and GSH-Rd in liver were significantly increased in the n-butanol group and the water group.
The invention discloses that the black garlic extract obtained by extracting with n-butanol and water has the effect of protecting liver, so that the black garlic extract can be applied to preparing a liver-protecting medicine or preparing food containing the black garlic extract. Experiments prove that the black garlic extract can be orally taken at ordinary times to reduce the degree of acute liver injury.
The foregoing is by way of example only, and not limiting. It is intended that all equivalent modifications or variations without departing from the spirit and scope of the present invention shall be included in the appended claims.
Claims (3)
1. The black garlic extract with the liver protection function is characterized by being obtained by the following preparation method:
extracting the black garlic powder with ethyl acetate to obtain a first crude extract insoluble in ethyl acetate, and extracting the first crude extract with n-butanol to obtain a first black garlic extract soluble in n-butanol and a second crude extract insoluble in n-butanol, wherein the weight ratio of ethyl acetate to black garlic powder is 5:1, and the weight ratio of n-butanol to the first crude extract is 5: 1.
2. The black garlic extract having liver protecting function according to claim 1, wherein the second crude extract insoluble in n-butanol is extracted with water to obtain a second black garlic extract dissolved in water, wherein the weight ratio of water to the second crude extract is 5: 1.
3. Use of the black garlic extract with liver protecting function of claim 1 or 2 in preparing liver protecting medicine or health food.
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| CN106174260A (en) * | 2016-07-11 | 2016-12-07 | 来鑫（厦门）生物科技有限公司 | A kind of black Bulbus Allii water extract purposes in promoting intestinal emptying and containing the food of black Bulbus Allii water extract and medicine |
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Non-Patent Citations (3)
| Title |
|---|
| Hepatoprotective Effect of Aged Black Garlic Extract in Rodents;Jung Hyu Shin,et.al;《Toxicological Research》;20140301;第30卷(第1期);全文 * |
| 黑蒜多酚粗提物体内抗氧化功能研究;杨贺晴;《食品科学技术学报》;20161125;第34卷(第6期);第41页摘要倒数第2-3行 * |
| 黑蒜药理作用的研究进展;刘晓旭;《中国保健营养（中旬刊）》;20130430(第4期);全文 * |
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