CN117982558A - Salicornia bigelovii oral liquid capable of dispelling effects of alcohol and protecting liver and preparation method thereof - Google Patents
Salicornia bigelovii oral liquid capable of dispelling effects of alcohol and protecting liver and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver and a preparation method thereof, wherein the oral liquid comprises the following components: 40-50% of salicornia extract, 5-10% of silybum marianum extract, 5-10% of mulberry extract, 4-8% of fructo-oligosaccharide and 30-40% of purified water. Extracting flavonoid components in the Salicornia bigelovii by using an ultrasonic cell crushing auxiliary ethanol reflux method, extracting active components in the Silybum marianum and the mulberry by using a hot water leaching method, compounding according to a proportion, and filtering by using a fine filtering device to prepare the Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting the liver. The oral liquid has the effects of dispelling the effects of alcohol, activating spleen, protecting liver and nourishing liver, can improve the detoxification capacity of the liver, and is suitable for liver injury caused by drinking. Research results show that the oral liquid can obviously relieve liver damage after drinking, improve mental state and relieve harm to organisms caused by liver damage after drinking.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and in particular relates to a Salicornia anti-hangover and liver-protecting oral liquid and a preparation method thereof.
Background
Alcoholic liver disease (ALD LIVER DISEASE) is a toxic liver injury caused by long-term high-volume drinking, and is initially manifested as hepatic cell steatosis, and further progresses to alcoholic hepatitis, eventually leading to liver fibrosis and alcoholic cirrhosis, and can also induce extensive hepatic cell damage or even liver failure in the case of short-term severe alcoholism. Due to the ubiquitous phenomenon of excessive drinking, ALD diseased people are gradually increased, and harm is caused to human health. Currently, there are many methods for treating alcoholic liver disease, including drug therapy, nutritional support, liver transplantation, and the like. Although the traditional Chinese medicine composition can achieve a certain treatment effect on the alcoholic liver disease, no obvious specific medicine is available for treating the alcoholic liver disease so far.
The human body can generate a great deal of acetaldehyde through metabolism of liver after taking alcohol. Acetaldehyde is absorbed in the intestine and generates a large amount of free radicals. Free radicals are detrimental to biological membranes. A large amount of O 2-, active oxygen, etc. are generated and are normally purged by GSH. Increased consumption of antioxidants in humans can lead to reduced scavenging capacity, and prolonged drinking can lead to significant accumulation of malondialdehyde, and in severe cases, necrosis or fibrosis of the liver. The flavonoid substances can quickly decompose ethanol, reduce the concentration of ethanol in blood, remove a large amount of free radicals generated by lipid oxidation, and protect liver cells from being damaged by free radical electrophilic compounds. Flavonoid substances (Flavonoid) are compounds derived from fruits, vegetables, tea, seeds or plant roots, have wide pharmacological activity and lower toxicity, have different degrees of prevention and treatment effects on cardiovascular diseases, nerves, endocrine diseases, immune diseases, digestive respiratory diseases and other diseases, and have great value in clinical application. In recent years, flavonoids have been developed to prevent and treat alcoholic liver injury.
Salicornia is a plant of Salicornia of Chenopodiaceae (Chenopodiaceae) and grows on wet heavy saline soil of saline land, salt lake side and sea side, and river valley, and is a green plant with stems and leaves. Is mainly distributed in Jiangsu, north China, shandong, northwest, liaoning and other places. The Salicornia bigelovii is green healthy food beneficial to human bodies, has fresh and cool taste, is salty and crisp, is rich in nutrition and high in utilization value, contains various nutritional ingredients such as fatty acid, protein, mineral substances, vitamins and the like, and is a recognized green organic vegetable. Research shows that the active substance components separated from the Salicornia bigelovii comprises flavonoid, alkaloids, polysaccharide, saponin and the like. The active components have the functions of anti-inflammatory, antioxidant, anti-tumor, blood sugar and blood lipid reducing, etc. The total amount of the flavonoid substances in the Salicornia bigelovii extract is up to 60%, and the flavonoid substances have a certain effect on detoxification of alcoholism, so that the Salicornia bigelovii has higher nutritive value and good development prospect. In addition, the salicornia has strong salt tolerance, and can absorb the salt in the saline-alkali soil year by year, so that the salicornia can be planted to fully utilize the beach and coastal barren lands and improve the geology of the saline-alkali soil. Therefore, the development of the Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver has great application value and social benefit.
Silybum marianum (L.) is an annual or biennial herb of the genus Silybum of the family Compositae. Bitter and cool, has the effects of clearing heat and promoting diuresis, soothing liver and promoting bile flow, is clinically used for protecting liver, reducing blood fat and resisting atherosclerosis plaque formation, has the effects of protecting cardiovascular system, and resisting cerebral ischemia, and has the effects of resisting platelet aggregation, and silybum marianum extract has been used as a liver-protecting medicament.
The mulberry is the mature fruit of the mulberry, is the ear of the mulberry of the Moraceae plant, has a lot of sweet juice and is one of fruits commonly eaten by people. Mulberry is sweet and cold in nature, enters heart, liver and kidney meridians, has the effects of nourishing yin and supplementing blood, can treat yin deficiency, less fluid and insomnia, and also has good effects of nourishing heart, liver and kidney, nourishing blood and dispelling wind.
However, no anti-hangover and liver-protecting medicine has been reported at present by using Salicornia as a main material and using raw materials such as silybum marianum and mulberry as auxiliary materials.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the Salicornia bigelovii oral liquid for dispelling the effects of alcohol and activating spleen, protecting liver and nourishing liver and the preparation method thereof, can improve the detoxification capacity of the liver, is suitable for liver injury caused by drinking, can obviously relieve the liver injury after drinking, improves the mental state and relieves the harm of the liver injury after drinking to organisms.
The invention is realized by the following technical scheme:
The Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver comprises the following components in percentage by mass: 40-50% of salicornia extract, 5-10% of silybum marianum extract, 5-10% of mulberry extract, 4-8% of fructo-oligosaccharide and 30-40% of purified water.
A preparation method of the Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver comprises the following steps:
Step 1) pretreatment of materials: selecting fresh tender stems of the Salicornia bigelovii, cleaning with clear water, removing sediment, draining water, drying, and grinding into powder for later use; drying herba Silybi Mariani and Mori fructus, and grinding into powder;
Step 2) extraction of active ingredients: taking Salicornia bigelovii powder, adding ethyl acetate, standing overnight, removing ester soluble components, filtering, and volatilizing residual ethyl acetate component; dissolving the processed Salicornia Herbacea powder in 60% ethanol water solution by ultrasonic assisted extraction method, and ultrasonic extracting; repeating the extraction for 2 times, mixing the filtrates, removing solvent, and freeze drying to obtain Salicornia Herbacea extract;
Respectively weighing silybum marianum and mulberry powder, and respectively obtaining silybum marianum extract and mulberry extract by adopting a heating reflux extraction method, wherein the heating reflux extraction method is as follows: the first extraction is carried out according to the feed liquid ratio of 1g: extracting for 1.5h by 12mL of heating reflux, and extracting for the second time according to the feed liquid ratio of 1g: extracting with 10mL of heat under reflux for 1.5h, mixing the filtrates, removing solvent, and freeze drying;
Step 3) preparation of oral liquid: compounding the Salicornia bigelovii extract, the Silybum marianum extract, the mulberry extract, the fructo-oligosaccharide and the purified water according to a certain proportion, filtering, fine filtering, sterilizing the filling bottles in advance, and bottling.
Preferably, the feed liquid ratio of the salicornia powder to ethyl acetate in the step 2) is 1g:4mL.
Preferably, the feed liquid ratio of the treated salicornia powder to the ethanol aqueous solution in step 2) is 1g:20mL.
Preferably, the power of the ultrasonic extraction in the step 2) is 150W, the time is 1h, and the extraction temperature is 50 DEG C
The beneficial effects of the invention are as follows:
According to the invention, the Salicornia bigelovii, silybum marianum and mulberry are taken as raw materials, flavonoid components in the Salicornia bigelovii are extracted by an ultrasonic cell crushing auxiliary ethanol reflux method, active components in the silybum marianum and mulberry are extracted by a hot water leaching method, the active components are compounded according to a proportion, and the filtration is carried out by a fine filtration device, so that the Salicornia bigelovii oral liquid for dispelling effects of alcohol and protecting liver is prepared. The flavonoid substances can quickly decompose the ethanol so as to reduce the concentration of the ethanol in blood, and can also remove a large amount of free radicals harmful to biological membranes generated by lipid oxidation, thereby achieving the effects of dispelling the effects of alcohol and protecting the liver. Research results show that the production process of the Salicornia anti-hangover and liver-protecting oral liquid is simple, the production period can be greatly shortened, continuous production can be realized, and a plurality of problems in intermittent production are avoided. The acquisition channels of the raw materials of the Salicornia bigelovii, the silybum marianum and the mulberries are simple; the preparation process of the product is simple and easy to implement, the production cost is general, and the quality is stable and controllable; and the mechanical assembly line operation is easy to realize, and the industrial production requirement can be met.
Drawings
FIG. 1 is a diagram of a pathological section of liver tissue of a mouse;
in fig. 1: a is a blank control group; b is a model group; c is positive control group; d is a Salicornia oral liquid group;
FIG. 2 is a graph showing the results of fluorescent quantitative PCR for different inflammatory factors;
in fig. 2: a is the relative expression amount of IL-1 beta mRNA; b is the relative expression amount of IL-6 mRNA; c is the relative expression level of TNF-alpha mRNA; d is the relative expression amount of CYP2E1 mRNA.
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific embodiments.
Example 1
The Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver is prepared by compounding the following raw materials in percentage by mass: 40-50% of salicornia extract, 5-10% of silybum marianum extract, 5-10% of mulberry extract, 4-8% of fructo-oligosaccharide and 30-40% of purified water.
A preparation method of the Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver comprises the following specific steps:
(1) Pretreatment of materials:
Selecting fresh tender stems of Salicornia bigelovii, cleaning with clear water, removing sediment, draining water, drying, and grinding into powder for later use. And (3) drying the silybum marianum and the mulberries, and grinding the dried silybum marianum and the mulberries into powder for standby.
(2) Obtaining active ingredients:
100g of Salicornia bigelovii powder was taken, 400mL of ethyl acetate was added and allowed to stand overnight, most of the ester-soluble components were removed, and the residual ethyl acetate component was filtered off and volatilized. The extract is dissolved in 60% ethanol water solution with a feed liquid ratio of 1:20 (g/mL) by using an ultrasonic auxiliary extraction method, and is subjected to ultrasonic extraction for 1h at 150W, wherein the extraction temperature is 50 ℃. Repeating the extraction for 2 times, mixing the filtrates, removing solvent by rotary evaporator, and freeze drying to obtain Salicornia Bigella Maxim active ingredient (Salicornia Bigella Maxim extract).
Respectively weighing a proper amount of silybum marianum and mulberry powder, and respectively obtaining silybum marianum active ingredients (silybum marianum extract) and mulberry active ingredients (mulberry extract) by adopting a heating reflux extraction method for standby. The heating reflux extraction method comprises the following steps: the first extraction is carried out for 1.5 hours by heating and refluxing according to the feed liquid ratio of 1:12 (g/mL), the second extraction is carried out for 1.5 hours by heating and refluxing according to the feed liquid ratio of 1:10 (g/mL), the two filtrates are combined, the solvent is removed, and the freeze-drying is carried out, thus obtaining the product.
(3) Preparation of oral liquid:
The preparation method comprises the steps of compounding 40-50% of the Salicornia bigelovii extract, 5-10% of the Silybum marianum extract, 5-10% of the mulberry extract, 4-8% of fructo-oligosaccharide and 30-40% of purified water according to the mass ratio, filtering by a fine filtering device, pre-sterilizing and sterilizing a filling bottle, and bottling to obtain the Salicornia bigelovii oral liquid product (hereinafter referred to as Salicornia bigelovii oral liquid for short).
The results show that the preparation of the Salicornia Bigella anti-hangover and liver-protecting oral liquid product produced by the technology and the equipment has no sediment after several months. The method can remove thallus, impurities and clarify medicinal liquid, and the medicinal liquid can not precipitate after long-term storage.
Example 2
The liver protection effect of the Salicornia oral liquid prepared in example 1 was studied:
32 healthy male mice were randomly divided into a blank group, a model group, a positive control group (silymarin 200 mg/kg), and a Salicornia oral liquid dosage group (100 mg/kg), 8 each, and administered by intragastric administration. The blank group and the model group are respectively filled with 5mL/kg of physiological saline, the positive control group is filled with 200mg/kg of silymarin as a liver protecting drug for stomach every day, the administration time is 28 days, and the other groups except the blank group are filled with 6g/kg of gastric alcohol to make a mouse acute liver injury model, and the operation is carried out once every 12 hours for 3 times.
1. Influence of Salicornia bigelovii anti-alcoholic liver protection oral liquid on liver coefficient of alcoholic liver injury mice
Immediately dissecting the mice after euthanasia, taking out liver tissues, rapidly putting into ice-cold physiological saline, washing off surface blood stains, absorbing surface moisture by using water absorbing paper, precisely weighing the weight of the livers after removing fat tissues and fascia on the livers, and calculating the visceral organ index of the livers of the mice by combining the body weights of the mice.
As shown in table 1 below, the liver coefficients of the model group mice were significantly increased (P < 0.01) compared to the blank group, indicating that the model group was modeled successfully. Compared with the model group, the liver coefficient of the Salicornia oral liquid group is obviously reduced (P is less than 0.05), and compared with the positive control group, the viscera coefficient of the Salicornia oral liquid group is lower than that of the positive control group, which indicates that the Salicornia oral liquid can obviously inhibit liver cell edema.
Table 1 changes in liver coefficients of mice of each group
Table 1 notes: #P＜0.05、## P < 0.01 compared with the model group; *P＜0.05、** P < 0.01 compared to blank
2. Effects of Salicornia bigelovii extract on ALT and AST contents and SOD and GSH activities in serum of mice with alcoholic liver injury
After the dissection of the mice, blood in the heart is extracted by a disposable syringe, the hemolysis phenomenon is prevented after pollution is prevented in the process of absorption, the absorbed blood is placed into a centrifuge tube containing heparin, and the centrifuge tube is kept stand for 30min at room temperature and centrifuged at 3000rpm for 10min to prepare serum for standby. The ALT, AST, SOD and GSH in serum are all determined by using a kit purchased from Nanjing's institute of biological engineering, and all the operation steps are strictly performed according to the instructions in the kit.
As shown in Table 2 below, the ALT and AST levels in the serum of mice in the model group were significantly increased (P < 0.01) compared to the blank group, indicating successful modeling. Compared with the model group, the ALT and AST contents in the Salicornia oral liquid group are obviously reduced (P is less than 0.01) compared with the model group, and the difference has statistical significance. The reduced activity of SOD and GSH in the serum of mice in the model group (P < 0.05) indicates successful modeling. Compared with the model group, the activity of SOD and GSH in the high dose group (Salicornia oral liquid group) of the Salicornia extract is increased (P is less than 0.05), and the difference has statistical significance. Compared with the positive control group, the serum indexes of the Salicornia oral liquid group are better than those of the positive control group, which shows that the Salicornia oral liquid has stronger liver protection effect.
TABLE 2 Effect of Salicornia on alcoholic liver injury mice ALT, AST, SOD and GSH
Table 2 notes: *P<0.05、** P <0.01 compared to model group; #P<0.05、## P <0.01 compared to blank
3. Influence of Salicornia Herbacea extract on liver morphology of mice with alcoholic liver injury
Taking out the liver tissue of the mouse, weighing, then putting the liver tissue into 10% neutral formalin-containing fixed liquid for soaking for 72 hours, dehydrating and fixing, conventionally taking materials, embedding by paraffin, slicing by a slicing machine, wherein the thickness is 5 mu m, staining by H & E, and observing morphological changes of each liver tissue under a light microscope.
Fig. 1 is a pathological section of mouse liver tissue, fig. 1: a is a blank control group, B is a model group, C is a positive control group, D is a salicornia oral liquid group, and the observation is carried out under a 200-fold mirror by adopting an H & E staining method. As can be seen by the optical lens, the liver cells in the blank group shown in fig. 1A have clear structure, complete cell nucleus, plump cytoplasm, orderly arrangement and radial liver ropes; the mouse liver cells of the model group shown in fig. 1B show serious denaturation change, a large-scale edema appears around the cells, cell nucleus cavities, the liver cells show sheet necrosis, and liver sinuses disappear, which indicates that the modeling of the alcoholic liver injury of the mouse is successful; FIG. 1C shows that the peripheral edema of liver cells is obviously reduced by using silymarin as a positive control and compared with a model group, the liver cells are orderly arranged, the cell nucleus state is good, and the hepatic cable is radial; comparing the morphology of the mouse liver cells in the Salicornia oral liquid group of FIG. 1D with that of the mouse liver cells in the model group of FIG. 1B, the result shows that the liver tissue damage of the Salicornia oral liquid group is obviously reduced, the edema condition of the liver cells is gradually relieved, the liver ropes are gradually restored to the radial morphology, and the cell nuclei are gradually complete and clear. Compared with the silymarin positive control group in fig. 1C, the liver protection effect of the erigeron breviscapus is superior to that of silymarin.
4. Fluorescent quantitative PCR detection of different inflammatory factors
Extracting total RNA of liver tissue by adopting a Trizol method, taking about 50mg of liver tissue, adding the liver tissue into a Trizol solution, dispersing by adopting a high-speed disperser, separating out the total RNA by precooled isopropanol, and washing redundant impurities by ethanol to obtain the total RNA. cDNA was obtained by reverse transcription using the K1622 reverse transcription kit from Thermo company. The determination is carried out by adopting a fluorescent quantitative PCR instrument, the reverse transcription product 3 mu L is added into the RT-PCR reaction system composition ：Water,PCR grade(Vial2,colorless cap)3μL,PCR Primer 10×conc 2μL,Master Mix 2×conc(Vial1,green cap)12μL,, the total reaction volume is 20 mu L, and the fluorescent quantification is carried out by taking beta-actin as an internal reference gene.
FIG. 2 shows the results of fluorescent quantitative PCR of different inflammatory factors, FIG. 2: a is the relative expression level of IL-1 beta mRNA, B is the relative expression level of IL-6mRNA, C is the relative expression level of TNF-alpha mRNA, and D is the relative expression level of CYP2E1 mRNA.
As shown in FIG. 2, the expression levels of IL-1β, IL-6, TNF- α and CYP2E1 were significantly increased in the model group compared to the blank group, indicating that the model group was successfully modeled. A decrease in the expression levels of all three inflammatory factors was observed in the positive control group compared to the model group. The inflammatory mediators IL-1 beta, IL-6 and TNF-alpha associated with the Salicornia oral liquid groups in FIGS. 2A, 2B and 2C showed a decreasing trend compared with the model group. The Salicornia bigelovii oral liquid has the effect of protecting liver by inhibiting the expression of relevant inflammatory factors IL-1 beta, IL-6 and TNF-alpha. Elevated levels of CYP2E1 expression lead to increased oxidative stress, depletion of the antioxidant system in the body, massive rupture of the mitochondrial membrane of hepatocytes leading to liver damage, and significant decreases in CYP2E1 expression in fig. 2D, indicating that Salicornia bigelovii could reduce alcohol damage to the liver by modulating CYP2E1 expression levels.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting thereof; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (5)
1. The Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver is characterized by comprising the following components in percentage by mass: 40-50% of salicornia extract, 5-10% of silybum marianum extract, 5-10% of mulberry extract, 4-8% of fructo-oligosaccharide and 30-40% of purified water.
2. The preparation method of the Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver as claimed in claim 1, which is characterized by comprising the following steps:
Step 1) pretreatment of materials: selecting fresh tender stems of the Salicornia bigelovii, cleaning with clear water, removing sediment, draining water, drying, and grinding into powder for later use; drying herba Silybi Mariani and Mori fructus, and grinding into powder;
Step 2) extraction of active ingredients: taking Salicornia bigelovii powder, adding ethyl acetate, standing overnight, removing ester soluble components, filtering, and volatilizing residual ethyl acetate component; dissolving the processed Salicornia Herbacea powder in 60% ethanol water solution by ultrasonic assisted extraction method, and ultrasonic extracting; repeating the extraction for 2 times, mixing the filtrates, removing solvent, and freeze drying to obtain Salicornia Herbacea extract;
Respectively weighing silybum marianum and mulberry powder, and respectively obtaining silybum marianum extract and mulberry extract by adopting a heating reflux extraction method, wherein the heating reflux extraction method is as follows: the first extraction is carried out according to the feed liquid ratio of 1g: extracting for 1.5h by 12mL of heating reflux, and extracting for the second time according to the feed liquid ratio of 1g: extracting with 10mL of heat under reflux for 1.5h, mixing the filtrates, removing solvent, and freeze drying;
Step 3) preparation of oral liquid: compounding the Salicornia bigelovii extract, the Silybum marianum extract, the mulberry extract, the fructo-oligosaccharide and the purified water according to a certain proportion, filtering, fine filtering, sterilizing the filling bottles in advance, and bottling.
3. The preparation method of the salicornia anti-hangover and liver-protecting oral liquid according to claim 2, wherein the feed liquid ratio of the salicornia powder to the ethyl acetate in the step 2) is 1g:4mL.
4. The preparation method of the salicornia anti-hangover and liver-protecting oral liquid according to claim 2, wherein the feed liquid ratio of the treated salicornia powder to the ethanol aqueous solution in the step 2) is 1g:20mL.
5. The preparation method of the Salicornia bigelovii oral liquid for dispelling the effects of alcohol and protecting liver according to claim 2, wherein the power of ultrasonic extraction in the step 2) is 150W, the time is 1h, and the extraction temperature is 50 ℃.
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