KR102183670B1 - Process for preparing alcoholic fatty liver relaxation composition using Moringa oleifera and composition thereof - Google Patents

Abstract

The present invention, the first step of drying Moringa leaves as a main material; A second step of pulverizing Moringa leaves to generate powder and drying it again; A third step of first extraction by reacting the Moringa leaf powder in a state immersed in a mixed solution of water and ethanol on an ultrasonic extractor; A fourth step of secondary extraction by reacting the primary extract while being immersed in water on an ultrasonic extractor; A fifth step of filtering the secondary extract to remove impurities, and then concentrating by removing 90-99wt% of the solvent with a rotary evaporator; And a sixth step of freeze-drying the concentrate at a temperature of -40 to 70°C to obtain a composition from which the solvent is completely removed. In addition, according to another aspect of the present invention, in the relief composition of alcoholic fatty liver using Moringa leaf extract: (A) powder using Moringa leaf, (B) fractional extraction of a mixed solution of water and ethanol To do.
In the present invention, after fractionally extracting Moringa leaves, the relief composition of alcoholic fatty liver can be most effectively and completely obtained, and then used as various raw materials, such as pharmaceutical raw materials, and it is possible to easily change the formulation, and it is easy to absorb the human body and at the lowest cost. The effect of suppressing inflammation and oxidative stress caused by the liver while performing the function of preventing and mitigating alcoholic liver damage in terms of functional and morphological aspects through an alcoholic fatty liver relief composition that can mass-produce high-additive substances in a mass-processed form. Is expected.

Description

TECHNICAL FIELD [Process for preparing alcoholic fatty liver relaxation composition using Moringa oleifera and composition thereof]

The present invention relates to a relief composition for alcoholic fatty liver using moringa, and more specifically, after obtaining the most effective and intact relief composition for alcoholic fatty liver obtained by fractional extraction of Moringa leaves, and functional food ingredients including pharmaceutical raw materials and It relates to a method for preparing an alcoholic fatty liver relief composition using moringa that can be utilized organically and functionally in drinking materials and the like, and a composition thereof.

In recent years, due to changes in eating and drinking habits in Korea, liver diseases caused by alcohol abuse are increasing more and more. And according to the announcement of the National Statistical Office, alcohol is known to be the cause of about 60% of deaths from liver disease. As such, it is the cause of the problem that modern people are easily exposed to excessive alcohol intake, and this is being greatly illuminated as one of the social problems that cannot be ignored. These alcoholic liver diseases are caused by long-term and continuous excessive intake of alcohol, and liver damage is caused by metabolites such as acetaldehydes, hydroxyl radicals and hydrogen peroxide, which are metabolites generated during the metabolic process. It starts in the form of alcoholic fatty liver diseases, and if appropriate treatment and management are performed at this stage, it can prevent deterioration of steatohepatitis, cirrhosis, and hepatocellular carcinoma. However, even after repeated intake of alcohol, irreversible liver damage occurs, and in severe cases, most of the liver function is lost, leading to death. For this reason, in order to suppress irreversible liver damage, it is urgent to develop a preventive treatment or food at the stage of fatty liver.

On the other hand, before the development of the currently widely used treatments for liver function, they mainly used plant resources and natural products to take the place of liver treatment. Accordingly, plant-based natural products generally contain a variety of useful substances, and it can be said that safety compared to chemical synthetic substances has been sufficiently secured through the accumulation of human experience that has been used for a long time, so interest in natural products as pharmaceutical materials. Although this is greatly increasing, the ideal therapeutic drug using natural products with low side effects and high therapeutic efficiency is not being developed properly.

Moringa oleifera viewed from this point of view is a plant that lives widely in subtropical regions such as India and has high nutritional value, and has been widely used as feed for livestock as well as humans. It is also a medicinal plant that has been used for one purpose. As a traditional therapy, most parts such as roots, seeds, leaves, and fruits have been used to treat diseases such as paralytic seizures, tumors, scurvy, and skin infections. The pharmacological research on Moringa includes verification of antibacterial and antioxidant efficacy by variety, relieving kidney fibrosis, inhibiting breast cancer cell proliferation, antihistamine effect, alleviating skin aging and inflammation, inhibiting liver fibrosis and antibacterial, antitumor, and anticancer The effect is known.

In addition, Moringa leaves are found to contain a lot of nutrients that are easy to digest, such as proteins, vitamins, and minerals, and are in the spotlight as a supplementary source of dietary protein. They are excellent in preventing heart disease and liver damage and inflammation, and removing cholesterol. It is also known to be effective. Despite the weakness of these various Moringa leaves, Moringa against alcohol-induced liver damage Since research on the efficacy of leaves has not been carried out much until now, there is an urgent need for utilization plans and various treatments.

And as the prior art related to the above, “a beverage composition containing vitamins and moringa leaf extract and a method for manufacturing the same” of Korean Patent Publication No. 10-1408837, and “Moringa fermented beverage and its manufacturing method” of Korean Patent Publication No. 10-1748948 Its manufacturing method”, “A composition for treating or preventing cancer containing a water-soluble extract of Moringa leaves as an active ingredient, and a method for preparing extracts of Moringa leaves” of Korean Patent Publication No. 10-1803852, Korean Patent Publication No. 10 -1827601 "Cosmetic composition containing mixed extract of Moringa leaf and upper leaf having antioxidant and anti-inflammatory activity", etc. are known.

However, the above-described prior art documents do not clearly present a technical analysis and related technical content of an alcoholic fatty liver relief composition obtained by fractional extraction of Moringa leaves. Accordingly, as a result of in-depth research efforts of the applicants on Moringa to develop a natural product for the treatment of alcoholic fatty liver, the present invention was developed after confirming that it exhibits therapeutic and preventive efficacy for inhibiting inflammation and oxidative stress induced in the liver. Was done. In other words, Moringa leaf extract obtained by using water as an extraction solvent of Moringa oleifera leaves prevents and relieves alcoholic liver damage in terms of functional and morphology, while preventing inflammation and oxidative stress caused by the liver. The present invention was completed after it was confirmed that it exhibited an inhibitory therapeutic effect.

“Beverage composition containing vitamins and Moringa leaf extract and method for manufacturing the same” in Korean Patent Publication No. 10-1408837 (Registration Date: 2014. 06. 11.) “Moringa fermented beverage and its manufacturing method” in Korean Patent Publication No. 10-1748948 (Registration Date: 2017. 06. 13.) Korean Patent Publication No. 10-1803852, “A composition for treating or preventing cancer containing a water-soluble extract of Moringa leaves as an active ingredient and a method for preparing extracts of Moringa leaves” (Registration date: 2017. 11. 27.) “Cosmetic composition containing mixed extract of Moringa leaf and upper leaf having antioxidant and anti-inflammatory activity” in Korean Patent Publication No. 10-1827601 (Registration Date: 02. 02. 2018)

(Non-Patent Document 1) Sreelatha S, Jeyachitra A, Padma PR. 2011. Antiproliferation and induction of apoptosis by Moringa oleifera leaf extract on human cancer cells. Food Chem Toxicol. 49: 1270-1275. (Non-Patent Document 2) Ndhlala AR, Mulaudzi R, Ncube B, Abdelgadir HA, du Plooy CP, Van Staden J. 2014. Antioxidant, antimicrobial and phytochemical variations in thirteen Moringa oleifera Lam. cultivars. Molecules. 19: 10480-10494. (Non-Patent Document 3) Park SH, Chang YC. 2012. Anti-fibrotic effects by moringa root extract in rat kidney fibroblast. J Life Sci. 22: 1371-1377. (Non-Patent Document 4) Cho HJ, Chang YC. 2014. Extract of moringa root inhibits PMA-induced invasion of breast chancer cells. J Life Sci. 24: 8-13. (Non-Patent Document 5) Thakur S, Verma A. 2013. Antihistaminic effect of Moringa oleifera seed extract. Int J Pharm Res Allied Sci. 2: 56-59. (Non-Patent Document 6) Abdull RAF, Ibrahim MD, Kntayya SB. 2014. Health benefits of Moringa oleifera. Asian Pac J Cancer Prev. 15: 8571-8576. (Non-Patent Document 7) Ruckmani K, Davimani S, Jayakar B, Anandan R. 1998. Anti-ulcer activity of the alkali preparation of the root and fresh leaf juice of Moringa oleifera Lam. Ancient Sci Life. 17: 220-223. (Non-Patent Document 8) Van Wyk B, Van Whk P. 1997. Field Guide to Trees of Southern Africa. Random House Struik. 2: 316-317. (Non-Patent Document 9) Lee HJ, Chang YC. 2012. Suppression of TNF-α-induced inflammation by extract from different parts of Moringa oleifera in HaCaT cells. J Life Sci. 22: 1254-1260. (Non-Patent Document 10) Ghasi S, Nwobodo E, Ofili JO. 2000. Hypocholesterolemic effects of crude extract of leaf of Moringa oleifera Lam in high-fat diet fed wistar rats. J Ethnopharmacol. 69: 21-25. (Non-Patent Document 11) Okuda T, Baes AU, Nishijima W, Okada M. 2001. Coagulation mechanism of salt solution-extracted active component in Moringa oleifera seeds. Water Res. 35: 830-834.

It is an object of the present invention to fundamentally improve the conventional problems as described above, after obtaining the most effective and intact relief composition of alcoholic fatty liver obtained by fractional extraction of Moringa leaves, and then functional food ingredients and drinking ingredients, etc. Alcoholic liver in terms of functional and morphology through an alleviating composition of alcoholic fatty liver that can be easily absorbed by the human body and mass-produced high-additive substances at the lowest cost while being used as various raw materials of It is intended to provide a method for preparing an alcoholic fatty liver relief composition and a composition thereof using moringa capable of suppressing inflammation and oxidative stress caused by the liver while performing damage prevention and relief functions.

One aspect of the present invention for achieving the above object, the first step of drying Moringa leaves as a main material; A second step of generating the Moringa leaves passed through the first step into a powder of 100-3,000 mesh with a pulverizer, and re-drying them at room temperature for 7 to 10 days; After making the temperature of the ultrasonic extractor in the range of 10 to 70°C for the second step, 1.8 kg of Moringa leaf powder was immersed in 20 L of a mixed solution of 30 wt% water and 70 wt% ethanol for 12 to 24 hours. A third step of reacting three times in a state to obtain a primary extract; After the first extract passed through the third step in a temperature range of 10 to 70°C in an ultrasonic extractor, 1.8 kg of the first extract was immersed in 20 ℓ of a mixed solution of 30 wt% water and 70 wt% ethanol for 12 to 24 hours. A fourth step of reacting three times to obtain a secondary extract; A fifth step of filtering the secondary extract passed through the fourth step to remove impurities, and then concentrating by removing 90-99wt% of the solvent with a rotary evaporator; And a sixth step of freeze-drying the concentrate passed through the fifth step at a temperature of -40 to 70°C to obtain a composition from which the solvent is completely removed.

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In addition, according to another aspect of the present invention, in forming the relief composition of alcoholic fatty liver using Moringa leaf extract: (A) 35-50 wt% of powder using Moringa leaf, and (B) 30 wt% of water and 70 wt of ethanol It is characterized by consisting of fractional extraction of 50 ~ 65wt% of the mixed solution mixed with %.

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On the other hand, prior to this, terms or words used in the present specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventors have developed the concept of terms in order to explain their own invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that it can be properly defined. Therefore, the embodiments described in the present specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all the technical spirit of the present invention, and thus various alternatives that can be substituted for them at the time of application It should be understood that there may be equivalents and variations.

According to the present invention, after fractional extraction of Moringa leaves, an alleviating composition of alcoholic fatty liver is most effectively obtained and used as a variety of raw materials such as medical raw materials, functional food raw materials and drinking raw materials, and free formulation change can be easily performed. In addition, it provides an effect that can be easily absorbed by the human body and mass-produced high-additive materials in a mass-processed form at the lowest cost.

In addition, the relieving composition for alcoholic fatty liver is expected to be effective in suppressing inflammation and oxidative stress caused by the liver while performing the function of preventing and alleviating alcoholic liver damage in terms of functional and morphological aspects.

1 is an overall flow chart for preparing an alcoholic fatty liver relief composition using Moringa leaves according to the present invention
Figure 2 is a flow chart showing the extract yield by another solvent using the Moringa leaf according to the present invention
3 is an H&E-stained liver tissue photograph for confirming the effect of the Moringa fraction extracts according to the present invention on the morphology of liver tissue
Figure 4 is a photograph of H&E-stained liver tissue to confirm the effect of Moringa water extracts according to the present invention on the morphology of liver tissue
Figure 5 is a photograph of the immunostained liver for the morphological distribution of TNF-α on the effect of Moringa water extracts according to the present invention on the morphology of liver tissue
6 is a photograph of the result of searching for progression of liver fibrosis, which is a representative finding of liver cirrhosis, by performing picro sirius red for the degree of damage in the form of liver tissue by Moringa water extracts according to the present invention.
Figure 7 is an image photograph showing the effect of the functional difference of the liver moringa water extract according to the present invention on SPECT / CT image
Figure 8 is a chart showing the analysis conditions of the alcoholic fatty liver relief composition using Moringa according to the present invention

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

One aspect of the present invention relates to a method for preparing an alcoholic fatty liver relief composition using Moringa leaves, and to a method of obtaining the alcoholic fatty liver relief composition obtained by fractional extraction of Moringa leaves. Briefly explaining such a manufacturing method is to prepare an alcoholic fatty liver relief composition through drying, grinding, re-drying, primary extraction, secondary extraction, filtration, concentration, and freeze drying.

The first step of the present invention is dried using Moringa leaves as the main material. The first step is to select the main raw material and then dry it. The moringa leaves are used as the main raw material to be washed, and an automated device is introduced to stabilize the quality and improve productivity. Here, in the first step, the moringa leaves are washed and then naturally dried and provided.

In the second step of the present invention, the moringa leaves passed through the first step are pulverized to form powder and dried again. The second step is the step of pulverizing the main raw material that has gone through the drying step, i.e., moringa leaves, and then re-drying it.In particular, it is desirable to introduce a dedicated automation facility to improve productivity when the main raw material is pulverized and then produced as pulverized material. .

At this time, according to the detailed configuration of the present invention, in the second step, the Moringa leaves are produced in a powder state of 100-3,000 mesh with a grinder, and re-dried at room temperature for 7-10 days. It is produced in the form of a powder of 100-3,000 mesh using a dry main raw material, that is, Moringa leaves. Subsequently, the pulverized Moringa leaf powder is naturally dried again.

The third step of the present invention is extracted by reacting the Moringa leaf powder passed through the second step in a state immersed in a mixed solution of water and ethanol on an ultrasonic extractor. The third step is the primary extraction of powdered and re-dried Moringa leaf powder, and it is better to automate the process for quality stability and productivity improvement. This uses an ultrasonic extractor to immerse Moringa leaf powder in a mixed solution of water and ethanol, and then reacts it to obtain a primary extract.

At this time, according to the detailed configuration of the present invention, in the third step, after setting the temperature of the ultrasonic extractor in the range of 10 to 70°C, 1.8 kg of Moringa leaf powder was immersed in 20 ℓ of a mixed solution of water and ethanol for 12 to 24 hours. It reacts three times in a state to obtain a primary extract. This is done in an ultrasonic extractor (WUC-D22H, Wisd, Korea) in a state of immersing 1.8 kg of Moringa leaf powder in 20 ℓ of a mixed solution of water and ethanol for 12 to 24 hours under a temperature condition of 10 to 70°C. Repeated again 3 times to obtain the first extract.

The third step of the present invention is extracted by reacting the first extract through the second step in a state immersed in a mixed solution of 30 wt% water and 70 wt% ethanol on an ultrasonic extractor. The third step is a step of secondary extraction by secondary reaction of the first extract in the same manner as in the second step, and it is preferable to automate the process for quality stability and productivity improvement. This uses an ultrasonic extractor to immerse the primary extract in water and then obtains a secondary extract.

At this time, according to the detailed configuration of the present invention, in the fourth step, after setting the temperature of the ultrasonic extractor in the range of 10 to 70°C, 1.8 kg of the primary extract was added to 20 ℓ of a mixed solution of 30 wt% water and 70 wt% ethanol for 12 to 24 hours. It is characterized in that it reacts three times while immersed to obtain a secondary extract. This is done by immersing 1.8kg of the primary extract in 20ℓ of a mixed solution of 30wt% water and 70wt% ethanol under a temperature condition of 10~70℃ on an ultrasonic extractor (WUC-D22H, Wisd, Korea) for 12 to 24 hours. , This reaction is repeated three times to obtain a second extract.

The fifth step of the present invention is to remove impurities by filtering the secondary extract passed through the fourth step, and then concentrate by removing 90 to 99 wt% of the solvent with a rotary evaporator. The fifth step is the step of concentrating the secondary extract, and it is better to automate the process for quality stability and productivity improvement. This is the second extract obtained using the ultrasonic extractor Whatman No. 2 After filtration treatment with filter paper (Whatman International Ltd., Maidstone, UK) to remove impurities, use a rotary evaporator (EYELA N-1000, Rikakikai Co, Japan) to remove 90-99wt% of solvent. After removal, the ingredients are concentrated.

In the sixth step of the present invention, a composition obtained by completely removing the solvent is obtained by lyophilizing the concentrate through the fifth step at a temperature of -40 to 70°C. The sixth step is a step of completing the composition according to the present invention by lyophilizing the concentrate obtained by removing 90-99wt% of the solvent and concentrating the components, and it is preferable to introduce a dedicated automated facility to improve productivity. This completes a composition obtained by completely removing the solvent by freeze-drying the concentrate from 90 to 99 wt% of the solvent removed at a temperature of -40 to 70°C.

Another aspect of the present invention is proposed with respect to an alcoholic fatty liver relief composition using the Moringa leaf extract. In particular, CYP2E1 (generated and activated through various pathways including alcohol to produce toxic substances, typically through active ingredients of Moringa oleifera leaves, such as Acetaldehydes, Upperoxide anion radicals, Hydroxyl radicals, Hydrogen peroxide, etc. are known) In terms of functional and morphological aspects, alcoholic liver damage prevention and relief functions effectively reduce the expression level of alcoholic fatty liver, and the relief composition of alcoholic fatty liver relieves inflammation and oxidative stress caused by the liver. It was confirmed that it shows therapeutic and prophylactic efficacy to inhibit. As a result, it provides an alcoholic fatty liver relief composition containing the active ingredient of Moringa oleifera leaves.

Meanwhile, according to the present invention, in forming an alleviating composition for alcoholic fatty liver using Moringa leaf extract, (A) powder using Moringa leaf, (B) a mixed solution of 30 wt% of water and 70 wt% of ethanol is fractionated. It is characterized by being made by extraction.

As a detailed configuration of the present invention, the content ratio of the components is characterized by 50 to 65 wt% of a mixed solution of powder using Moringa leaves, water and ethane. As an example of the content ratio of such a composition, a mixture of 1.8 g of powder using Moringa leaves and 30 wt% of 70% mixed solution of water and 20 ℓ of ethanol are respectively used. Here, the alcoholic fatty liver relief composition according to the present invention is alcoholic through the above manufacturing method, that is, through drying of moringa leaves → crushing → re-drying → primary extraction → secondary extraction → filtration and concentration → freeze drying. Use a fatty liver relief composition.

After administration of the alcoholic fatty liver relief composition of the present invention (hereinafter referred to as “Moringa fraction extract”), the rate of decrease in the occurrence of liver damage was examined by biopsy, and liver damage levels in serum were measured and compared. . In addition, to verify the administration effect of alcoholic fatty liver relief composition by concentration, blood tests and the reduction rate of the liver damaged area were measured. In terms of morphology, the inflamed area was searched through immunostaining, and the presence or absence of fibrosis was confirmed by Pikro Sirius Red staining. , Cytochrome P450 2E1 (CYP2E1), a major culprit of oxidative stress and an indicator of liver damage by producing active oxygen, was measured and compared by Western blotting technique. In addition, the degree of functional activity of the liver and the area of the lesion were investigated through nuclear medical imaging (liver single photon emission computed tomography, SPECT) to determine how alcoholic fatty liver relief compositions reduce alcoholic liver damage in vivo. Tested.

Hereinafter, preferred embodiments and experimental examples are presented to aid in understanding the present invention. However, the following examples and experimental examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by these examples.

First, for the evaluation of the alleviation effect of the alcoholic liver damage of the Moringa fraction extract, a mouse C57BL/6 line (Orient Bio, Korea) was used, and the mouse was at room temperature 20±2 using a constant temperature/humidity device in the animal breeding room. It was reared in individual cages in an environment with a relative humidity of 50±5% and a light/dark cycle of about 12 hours. In order to test the induction of liver damage due to alcohol and the efficacy of the Moringa leaf fraction extract, mice were divided into six experimental groups as shown in Table 1 below, and the experiment was conducted for 12 weeks.

On the other hand, in a state where feed was infinitely supplied to all experimental groups, Experimental Group 1 supplied distilled water to a water bottle as a normal control, and infinitely supplied by the mouse free will, as well as oral administration of distilled water at 0.1 mL once a day. I did. Experimental group 2 was a normal control group. Instead of negative water, 25% alcohol was supplied to a water bottle and 25% alcohol was administered orally once a day at 0.1 mL. Experimental groups 3, 4, 5, and 6 were Moringa treatment groups, and 25% alcohol was supplied to a water bottle instead of drinking water, and each Moringa fraction extract (Hexane, MC) at a concentration of 200 mg/kg in 0.1 mL 25% alcohol. , MeOH, water) was dissolved and administered orally every day.

That is, when 1.8 kg of Moringa leaves dried at room temperature were fractionally extracted in the order of Hexane, MC, MeOH, and water solvent, finally 63.18g, 28.45g, with yields of 3.51%, 1.58%, 8.74% and 2.87%, respectively, Fractional extracts of 157.44g and 51.76g were obtained, respectively.

[Experimental Example 1] Histopathology evaluation analysis of liver tissue

For histopathological evaluation of alcoholic fatty liver model mice, the day before the end of the experiment 12 weeks, 12 hours after fasting, anesthetize with isoflurane (Troikaa, India), an inhalation anesthetic, and then extract the liver and immediately 4% It was fixed in formalin (paraformaldehyde). After fixing the extracted liver in formalin for more than 24 hours, use an automatic embedding machine (TP-1020, LEICA, Germany) for 12 hours of water, 70% alcohol, 80% alcohol, 95% alcohol, 100% alcohol. Each was increased sequentially for 1 hour to induce a dehydration reaction, and finally, it was treated with 100% xylene for 3 hours to facilitate the penetration of paraffin. A microtome (KD-3358, WanTong Precision, which can be cut to a certain thickness by fixing the block after making a block with paraffin in the cleared tissue by immersing the liver that has undergone this clearing process with paraffin and making it easy for cutting). Instruments, China) were cut to a thickness of 5 μm, where the cells of the tissue may not appear overlapping when observed under a microscope. The obtained tissue section was attached to a slide glass, subjected to deparaffinization, and stained with haematoxylin and eosin (H&E) for observation with an optical microscope (Eclipse L200D, Nicon, Japan) to confirm the induction and alleviation effect of alcoholic fatty liver. The histopathological effect test experiment of the liver was repeated three times.

[Result 1] Results of histopathology evaluation of liver tissue

The results of performing H&E staining to confirm the histopathological changes of the liver and the alleviating effect of fatty liver through feeding of Moringa fraction extract and alcohol intake are shown in FIG. 3. That is, in Figure 3 (A) control, (B) 25% alcohol, (C) 25% alcohol treated with 200mg/kg hexane layer, (D) 25% alcohol treated with 200mg/kg MC layer, (E) 25% alcohol treated with 200 mg/kg MeOH layer and 25% alcohol treated with (F) 200 mg/kg water layer, of which (F) shows a magnificent effect on liver tissue (which is magnified 400 X. Scale bar = 100) Μm.)

3(A) shows that in the normal control group consisting of mice fed only distilled water, no alcohol damage was observed, so normal hepatocytes could be observed.In these hepatocytes, lipid droplets, the most representative indicator of fatty liver, were not at all. It was not observed, and even lipid accumulation was not seen. In addition, the shape of the nucleus was clearly observed in cells of relatively small size.

3B shows damage in liver tissue in the normal control group who consumed alcohol for 12 weeks. Balloon deformation, malolisosomes, and destroyed nuclei were observed in hepatocytes due to fat deposition, and this morphology and destroyed nuclei are indicators for determining fatty liver.

In Figure 3 (F), unlike the extract-treated group of Hexane, MC, and MeOH, in the water extract-treated group, a similar liver morphology was observed with that of the normal control group. In other Moringa-treated groups, malolithic and white lipid accumulation was also observed around and between the nuclei of hepatocytes, but the water extract-treated group showed a shape of hepatocytes close to normal. In addition, in the water extract treated group, a large number of lipid accumulation or destroyed nuclei characteristic of the alcohol treatment group were not observed at all.

[Experimental Example 2] Biochemical Analysis of Blood

In order to compare the levels of enzymes that are indicators of liver damage in blood, blood analysis was performed using a blood analyzer (BS-390, Mindray, WA, USA). For this purpose, about 1 mL of blood was collected through cardiac blood collection on the last day of 12 weeks after the experiment was completed. The collected blood was reacted at room temperature for 30 minutes, and then centrifuged at 1,4000 rpm for 10 minutes using an ultra-high-speed centrifuge to separate and obtain only the supernatant serum. The obtained serum was compared and measured for the enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), enzymes that are indicators of liver damage, and the amount of triglyceride (TG) was measured using a blood analyzer. . Blood biochemical analysis indicators were measured repeatedly three times.

[Result 2] Evaluation result on the biochemical components of blood

The results of a blood test to confirm the effect on the biochemical components in blood in the animal model administered with Moringa fraction extract and alcohol are shown in Table 2 below. As liver damage progresses, significant hematologic changes can be observed. Changes in serum levels of enzymes and triglycerides related to liver damage in the blood of mice treated with alcohol and Moringa fraction extracts at the same time were observed. Could.

Meanwhile, since AST and ALT in blood are generally known as enzymes released into the blood when liver tissue is damaged, they are widely used as indicators of liver function impairment. The experiment of the present invention also showed relatively elevated blood ALT and AST activities in the alcohol-treated group than in the normal control group ( p <0.05). However, significantly lower enzyme activity was observed in the MC of the alcohol-treated group and the Moringa-treated group administered with the Moringa fraction extract, and the treatment group when compared with the treatment group of the water extract ( p < 0.05), it could be observed that there was no significant difference in enzyme activity from the normal control group (Table 2 A, B). In addition, when comparing the triglyceride levels in the serum, it was observed that the triglyceride levels were significantly lower when comparing the Hexane of the Moringa treatment group and the water extract treatment group with the normal control group ( p <0.05) (Table 2A). .

In Experimental Examples 1 and 2 above, a biopsy was performed by administering 25% alcohol orally to C57BL/6 mice for 12 weeks. As a result, recovery was possible only by management. Was not observed, but nuclear damage and balloon deformation due to cell damage, which are frequently observed in alcoholic liver damage, were observed. However, in the Moringa water treatment group, the same amount of alcohol as the alcohol treatment group was ingested and administered orally, but the damage in liver tissues was significantly reduced compared to the other treatment groups (Fig. 3).

In addition, when liver damage occurs, ALT and AST of mitochondria are released into the blood, and their levels in the serum tend to increase.Table 2 (A) and (B) show that liver damage is caused by alcohol. The increased activity of ALT and AST was observed in the mice, and the decreased activity was observed in the treatment group of the Moringa water extract when compared with other solvent extracts. In addition, in (C) of [Table 2], the triglyceride (TG) in the form of VLDL, which was freed from the blood, was also observed at a high concentration in the alcohol-treated group, and a low concentration in the extract-treated group of Moringa water was shown. From this, it can be expected that among the Moringa fraction extracts, the extract using water as a solvent is effective against alcoholic liver disease.

On the other hand, as shown in Experimental Examples 1 and 2 and Results 1 and 2, in order to confirm the correlation with the concentration of the water extract-treated group, which showed the best efficacy in the evaluation of the alcoholic fatty liver relaxation effect of the Moringa fraction extract, Oral administration experiments by concentration were conducted.

A mouse (C57BL/6 line, Orient Bio, Korea), which is the same experimental animal as the previous experiment, was used, and the mouse was kept at a room temperature of 20±2℃ and a relative humidity of 50±5% using a constant temperature and humidity device in the animal breeding room. It was maintained and reared in a cage for each individual by a free diet method in an environment with a light and dark cycle of about 12 hours. In order to evaluate the induction of fatty liver due to alcohol and the efficacy of the water extract of Moringa leaves, mice were divided into 5 experimental groups as shown in Table 3 below, and the experiment was conducted for 12 weeks.

On the other hand, in a state where feed was infinitely supplied to all experimental groups, Experimental Group 1 was supplied with distilled water as a normal control in a water bottle and infinitely supplied by the mouse's free will, and 0.1 mL of distilled water was administered orally once a day. Proceeded. Experimental group 2 was an alcohol treatment group, 30% alcohol was supplied to a water bottle instead of negative water, and 0.1 mL of 30% alcohol was orally administered once a day. Experimental groups 3, 4, and 5 were extract-treated groups of Moringa water, and 30% alcohol was supplied to a water bottle instead of drinking water, and each of the concentrations of 100 mg/kg, 200 mg/kg, and 400 mg/kg in 0.1 mL 30% alcohol. Moringa water extract was dissolved and administered orally every day. In the previous experiment conducted with an alcohol treatment concentration of 25%, a clear difference was shown in the blood test part.In the tissue staining, also indicators of liver damage such as balloon deformity and Mallory corpuscle and lipid accumulation were observed. The concentration of alcohol treatment was increased to 30% in order to induce stronger liver damage because it tended to be somewhat insufficient in the observation of lipid droplets.

[Experimental Example 3] Analysis of histopathology evaluation of liver tissue

For histopathological evaluation of alcoholic liver injury model mice, after fasting 12 hours after fasting the day before the end of the experiment, the liver was anesthetized using Isoflurane (Troikaa, India), and then the liver was excised and decay progressed. It was fixed in 4% formalin (paraformaldehyde) to facilitate biopsy without failing. After fixing the extracted liver in formalin for more than 24 hours, use an automatic embedding machine (TP-1020, LEICA, Germany) for 12 hours of water, 70% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol. The concentration of was increased sequentially and treated for 1 hour each to induce a dehydration reaction, and finally, treated with 100% xylene for 3 hours to facilitate the penetration of paraffin to proceed with a clearing process. A microtome (KD-3358, WanTong Precision Instruments, China) that can be cut to a certain thickness by fixing the block after making a block so that the liver that has undergone this clearing process is immersed in a paraffin paste to fill the cleared tissue with paraffin and to facilitate cutting. ), the cells of the tissue were cut to a thickness of 5 μm that may not appear to overlap when observed under a microscope. After attaching the obtained tissue section to a slide glass to undergo deparaffinization and to meet the purpose, haematoxylin and eosin (H&E) staining, immunohistochemistry (TNF-α), and Picro sirius red staining were performed, and then an optical microscope (Eclipse L200D, Nicon, Japan) histologically confirmed the induction and alleviation effect of alcoholic fatty liver. The experiment to confirm the histopathological effect of these liver tissues was repeated three times.

[Result 3] Results of histopathology evaluation of liver tissue

The results of performing H&E staining, immunostaining, and picrosirius red to confirm the histopathological changes of the liver and the alleviating effect of fatty liver through administration of Moringa fraction extract and alcohol administration are shown in [Figs. 4] to [Fig. 6].

That is, in [Fig. 4] (A) control, (B) 30% alcohol, (C) 30% alcohol treated with 100 mg/kg water layer, (D) 30% alcohol treated with 200 mg/kg water layer, (E) The slides of (D) and (E) in the 30% alcohol treated with 400 mg/kg water layer and Moringa treatment group showed excellent improvement in alcoholic fatty liver (this is magnified 400 X. Scale bar = 100 μm.)

In (A) of [Fig. 4], in the normal control group consisting of mice fed only distilled water, no alcohol-induced damage was observed, so normal hepatocytes could be observed, and in these hepatocytes, lipid droplets, the most representative indicator of fatty liver Was not observed at all, and even lipid accumulation was not observed. In addition, unlike (A) of [Fig. 3], the shape of the nucleus was clearly observed in cells of a relatively small size.

In addition, as shown in (B) of [Fig. 4], in the alcohol-treated group to which alcohol was administered for 12 weeks, damage to the liver tissue could be observed. Fat deposition in hepatocytes was more damaged, and balloon deformation, lipid droplets, and destroyed nuclei were observed, and these forms and destroyed nuclei are representative criteria for determining fatty liver.

As in Experimental Examples 1 and 2, a liver morphology of a different aspect from the alcohol-treated group was observed in the moringa water extract-treated group (FIG. 4). The hepatocytes in the alcohol-treated group showed white lipid accumulation and lipid droplets in the periphery and between the nucleus, but in the water extract-treated group, the shape of hepatocytes was close to normal. In addition, in the water extract treatment group, except for the 100 mg/kg concentration group, a large number of lipid accumulation or destroyed nuclei characteristic of the alcohol treatment group were not found at all.

In addition, when the immunostaining was performed using TNF-α, a pro-inflammatory enzyme, in order to know the effect of Moringa water extract on liver damage caused by alcohol, it is as shown in [Fig. 5]. That is, in [Fig. 5], (A) normal control, (B) 30% alcohol, (C) 30% alcohol treated with 100 mg/kg water layer, (D) 30% alcohol treated with 200 mg/kg water layer, (E ) The slides of 30% alcohol and (D) and (E) treated with 400mg/kg water layer had low brown color fascinating and inflammatory factors (TNF-α). (This is magnified 100 times. Scale bar = 400 μm.)

In (D) and (E) of [Fig. 5], no TNF-α positive reaction was observed in the control group and in the 200 mg/kg and 400 mg/kg treatment groups of Moringa water. On the other hand, in the alcohol-treated group as in (B) of [Fig. 5], a strong positive reaction was observed around blood vessels, and as in (C) of [Fig. 5], the extract of Moringa water 100 mg/kg was also treated. A relatively weak TNF-α positive reaction could be observed.

As described above, the object of the present invention is the prevention of fatty liver, and therefore, liver fibrosis, which is an indicator of liver cirrhosis in which irreversible damage occurs, should not be observed. Accordingly, as a result of searching for progression of liver fibrosis, which is a representative finding of liver cirrhosis, histopathologically by proceeding with picro sirius red that develops red in collagen fibers, that is, in (A) normal control, (B) 30% alcohol, (C) 30% alcohol treated with 100 mg/kg water layer, (D) 30% alcohol treated with 200 mg/kg water layer, slides in this group showed no signs of fibrosis, which is a factor in cirrhosis of the liver. Magnification 200 times. Scale bar = 200 μm.)

As shown in Fig. 6, in all of the normal control group, the alcohol-treated group, and the Moringa water-treated group, it was observed that no color development was observed in the liver tissue except for the collagen layer forming the blood vessel wall.

[Experimental Example 4] Biochemical Analysis of Blood

In order to compare the levels of enzymes that are indicators of liver damage in blood, blood analysis was performed using a blood analyzer (BS-390, Mindray, WA, USA). For this purpose, about 1 mL of blood was collected through cardiac blood collection on the last day of 12 weeks after the experiment was completed. The collected blood was reacted at room temperature for 30 minutes, and then centrifuged at 1,4000 rpm for 10 minutes using an ultra-high-speed centrifuge to separate and obtain only the supernatant serum. The obtained serum was compared and measured for the enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), enzymes that are indicators of liver damage, and the amount of triglyceride (TG) was measured. Blood biochemical analysis indicators were measured repeatedly three times.

[Result 4] Evaluation result on the biochemical components of blood

The results of a blood test to determine the effect on the biochemical components in the blood in the animal model administered with Moringa fraction extract and alcohol are shown in Table 4 below. As liver damage progresses, significant changes in hematology can be observed, so albumin, a protein related to liver function in serum, and AST, a representative enzyme related to liver damage, in the blood of mice administered with alcohol and Moringa fraction extract at the same time. And it was possible to observe the change in the level of triglycerides (TG).

Meanwhile, in general, blood AST is widely used as an indicator of liver function impairment because it is known as an enzyme released into blood when liver tissue is damaged. The experiment of the present invention also showed relatively elevated blood AST activity in the alcohol-treated group than in the normal control group ( p <0.05). In addition, when compared with the alcohol-treated group and the water extract-treated group of Moringa, the concentration-dependent lowering enzyme activity was observed. Also, as in (B) of [Table 4], the 400 mg/kg concentration group was compared with the normal control group. Was observed that there was no significant difference in enzyme activity ( p <0.05). On the other hand, in the case of ALB, a protein specifically synthesized only in normal liver, as shown in (A) of [Table 5], the alcohol-treated group showed a low level, and the control group and the Moringa-treated group showed a relatively high level ( p. <0.05). In addition, when comparing the serum triglyceride levels as shown in (C) of [Table 5], it was observed that the triglyceride levels were significantly lower when comparing the water extract treatment group and the alcohol treatment group of the Moringa treatment group ( p. <0.05).

[Experimental Example 5] CYP2E1 expression analysis

For the purpose of comparing the expression level of CYP2E1 in alcoholic liver injury, 1 mL Tissue extracting buffer (containing 1 x protease inhibitor cocktail set I) was added to the liver tissue (50 mg), and the tissue was crushed using a homogenizer. The hepatocytes were homogenized through shock from repeated sonication. The resulting liver tissue lysate was centrifuged at 4° C. for 10 minutes to 10,000 g to obtain a supernatant. The prepared liver lysate supernatant was mixed with a final concentration of 5 x SDS-PAGE loading buffer (Biosesang, Korea), and then reacted at 100° C. for 1 hour, and then Western blot was performed. The Western blot process used SDS-Page gel electrophoresis, and in the development process, proteins with Precision plus protein dual color standards markers (BIO-RAD, CA, USA) were used to facilitate the location search according to the molecular weight of the target protein. After unfolding them, they were transferred from Gel to PVDF membrane. In addition, in order to prevent the untransferred protein from being contaminated with other proteins, the membrane was immersed in 10% Bovine Serum Albimin and blocked at room temperature for 1 hour, and then the primary antibodies (abcam, UK) of CYP2E1 were 4 at a ratio of 1:1000. It was reacted at ℃ for 12 hours. For signal detection, a secondary antibody diluted with Goat Anti-Rabbit lgG HRP (abcam, UK) at a ratio of 1:2000 was used, and anti-β actin antibodies HRP (abcam, UK) were 1:1000 for this antibody. It was diluted with to induce a reaction. The expression level of CYP2E1 was analyzed using a protein expression analyzer (XD-79, UVITEC, UK), and an image of the protein expression level band was analyzed using NIH-Image software. Protein analysis using Western blotting was repeated three or more times.

[Result 5] CYP2E1 expression analysis result

The increase of free radicals caused by alcohol is the cause of oxidative stress, and has a key role in liver damage caused by alcohol. In response to this, the living body expresses CYP2E1 high to relieve oxidative stress, which is the cause of liver damage. It is an important indicator to measure the degree of. For this reason, Western blotting was also performed in the experiment of the present invention to measure the expression level of CYP2E1 in the liver. The results are shown in [Table 5] below.

That is, (A) control group, (B) 30% alcohol, (C) 30% alcohol treated with 100 mg/kg water layer, (D) 30% alcohol treated with 200 mg/kg water layer and (E) 30% 400 mg/kg The expression of CYP2E1 in the water layer was in proportion to the concentration.

As shown in [Table 5], the normal control group administered with distilled water showed little expression of the protein level of CYP2E1, and the expression level of CYP2E1 was approximately 3 times higher in the alcohol-treated group than in the normal control group, and statistically significant. . In addition, as shown in (C, D, E) of [Table 5], in the extract-treated group of Moringa fed Moringa water, the expression level of CYP2E1 was higher than that of the normal control group, but showed a concentration-dependent decrease. From this, it is observed that CYP2E1 increases the protein expression level when alcohol is administered, and the protein expression level is suppressed in a concentration-dependent manner by the extract of Moringa water.

[Experimental Example 6] Analysis of liver lesion confirmation through nuclear medicine imaging method

Tin chloride and Tc ^99m have properties that are not adsorbed to damaged liver tissue but only to normal liver tissue. Using this property, the functional difference between the liver was more visible through SPECT imaging. Tc ^99m is extracted through 1ml of physiological saline solution to facilitate vascular injection in the Generator (Drytec, GE healthcare, NY, USA), and then tin chloride is added. Tc ^99m of 7 ⁺ has 0~6 ⁺ ions due to reduction. the ^99m Tc is the generation, stands in the form of double-Tc ^99m TcO _2¯ is adsorbed specifically on the liver (肝).

Tin chloride, acting on this principle, was inserted into the mouse body at a concentration of about 1 mCi for each mouse through venous blood vessels in the tail, and these isotopes were absorbed for about 1 hour so that they could be adsorbed to the liver through blood vessels throughout the body. After 1 hour, the mouse's anesthesia state was continuously maintained for easy shooting using the inhalation anesthetic agent Isofloran solution, and micro PET/SPECT (INVEON PET/SPECT/CT System, SIMENS, Germany) for 1 hour A scanned image was obtained.

[Result 6] Analysis of liver lesion identification through nuclear medical imaging

Nuclear medicine imaging is an imaging diagnostic method that evaluates biochemical changes or functional problems in a living body by imaging their distribution by administering radioactive substances adsorbed to a specific site so that various biochemical and functional activities can be visually seen. In the experiment of the present invention, SPECT was taken to confirm the functional activity of normal cells of the liver. In particular, SPECT is a method that can accurately locate a lesion by obtaining a cross-sectional image. When the liver is damaged or fatty liver occurs, destruction of hepatocytes or balloon deformation occurs, resulting in a loss of liver function because the density of hepatocytes decreases, and low levels of radioactive isotopes are observed during SPECT imaging.

In [Fig. 7] (A) normal control, (B) 30% alcohol, (C) 30% alcohol treated with 100 mg / kg aqueous layer, (D) 30% alcohol treated with 200 mg / kg aqueous layer and (E) 30 % 400mg/kg water layer.

In the present invention, as a result of comparing and analyzing the amount of Tc ^99m uptake between the normal control group and the alcohol-treated group, as shown in (A, B) of [Fig. 7], the alcohol-treated group showed significantly reduced Tc ^99m activity compared to the normal control group. I could confirm it. On the other hand, it was observed that Tc ^99m was absorbed in a concentration-dependent manner in the Moringa water treatment group as shown in (C to E) of FIG. 7.

As shown in Experimental Examples 1 to 6 and Results 1 to 6, as a result of conducting a biopsy by oral administration of 30% alcohol to C57BL/6 mice for 12 weeks, symptoms of liver cirrhosis or hepatitis, which are irreversible damages, were observed. It was not possible to do, but it showed the findings of the malory corpuscle and fatty liver, which are often observed in alcoholic liver injury. However, as shown in [Fig. 4] and [Fig. 5], in the Moringa water treatment group, the same amount of alcohol as the alcohol treatment group was ingested and administered orally, however, fat accumulation, inflammatory reaction and damage in the liver tissue tended to decrease in a concentration-dependent manner. .

And, as shown in (B) of [Table 4], AST, an index of liver damage, in the blood test showed relatively elevated blood activity in the alcohol-treated group than in the normal control group. In addition, when comparing the alcohol-treated group and the Moringa water extract-treated group, the concentration-dependent lowering enzyme activity was observed, and even in the 400 mg/kg concentration group, there was no significant difference in enzyme activity from the normal control group. Could. On the other hand, in the case of ALB, a protein specifically synthesized only in the normal liver as shown in (A) of [Table 4], the alcohol-treated group showed a low level, and the control group and the Moringa-treated group showed a relatively high level. In addition, when comparing the level of triglycerides in the serum as shown in (C) of [Table 4], it was observed that the level of triglycerides was significantly lower when comparing the water extract treatment group and the alcohol treatment group of the Moringa treatment group.

In addition, as shown in [Table 5], the normal control group administered with distilled water showed little expression of the protein level of CYP2E1, and the alcohol-treated group showed higher expression of CYP2E1 compared to the normal control group. And, as shown in (C, D, E) of FIG. 6, in the extract-treated group of Moringa-fed Moringa water, the expression level of CYP2E1 was higher than that of the normal control group, but showed a concentration-dependent decrease.

In addition, as a result of comparing and analyzing the amount of Tc ^99m absorbed between the normal control group and the alcohol-treated group as shown in (A, B) of [Fig. 7], the alcohol-treated group showed significantly reduced Tc ^99m activity compared to the normal control group. I could confirm. On the other hand, it was observed that Tc ^99m was uptaken in a concentration-dependent manner in the treatment group of Moringa water as in (C to E) of [Fig. 7].

From the above results, it is judged that the extract of Moringa water showed the alleviating effect of alcoholic liver damage in an animal model ingested with 30% alcohol.

In other words, according to the above experimental examples and results, once again,

first; As shown in Fig. 3, as a result of a biopsy by oral administration of alcohol to C57BL/6 mice for 12 weeks, symptoms of irreversible liver cirrhosis or hepatitis could not be observed, but Mallory corpuscles, which are frequently observed in alcoholic liver damage. And fatty liver were observed. However, in the Moringa water-treated group, the same amount of alcohol as the alcohol-treated group was ingested and administered orally, but fat accumulation, inflammatory reactions, and damage in the liver tissue were significantly reduced.

second; The increase of free radicals caused by alcohol is a cause of oxidative stress, and in response to this, in order to relieve oxidative stress, the living body highly expresses CYP2E1, which is considered an important index for measuring the degree of liver damage. CYP2E1 is produced and activated through various pathways including alcohol to produce toxic substances. Representative examples include acetaldehydes·uperoxide anion radical·hydroxyl radical·hydrogen peroxide, and the like. For this reason, CYP2E1 is considered to be an important indicator to measure the degree of liver damage, so the expression of CYP2E1 increases in proportion to the absolute amount and frequency of alcohol, and the high expression of CYP2E1 occupies a high proportion as the cause of the development of alcoholic fatty liver. have. Although CYP2E1, an enzyme responsible for alcohol oxidative stress, was observed in the alcohol-treated group to which only alcohol was administered orally (Fig. 6A, B), the extract-treated group of Moringa water was orally administered with the extract of Moringa water. It could be observed that the expression level of CYP2E1 decreased to a significant level (C to E in Fig. 6). From these results, it was shown that when the extract of Moringa water was administered at an appropriate concentration, the expression of CYP2E1 increased by alcohol in liver tissue was significantly reduced. It is believed that the oxidative stress that causes CYP2E1 also shows a high reduction rate due to the Moringa water extract.

third; Nuclear medicine imaging is an imaging diagnostic method in which a radioactive isotope adsorbed to a specific site is administered to a living body and imaged, and then the biochemical and functional activity of the target site is visually displayed to compare and evaluate. SPECT used in the experiment of the present invention is to acquire a tomography image using a workstation using a two-dimensional projection data obtained by rotating a gamma-ray camera around an experimental animal, and the location and damage of the lesion by simultaneously acquiring a planar image and a tomography image. It is a diagnostic method that is easy to accurately detect the degree. As a result of the experiment, similar results were observed in the physiological and histological forms of the normal control group and the high concentration of Moringa water extract-treated group in the SPECT (Fig. 7A, E). In the alcohol-treated group, it was found that the amount of accumulation of radioactive isotopes of low activity was visually indicated (FIG. 7B), and it was judged that the administration of the extract of Moringa water reduced alcoholic liver damage and fatty liver accumulation.

fourth; When liver damage occurs, mitochondrial ALT and AST are released into the blood, and their levels in serum tend to increase.In the experiment of the present invention, the activity of ALT and AST in mice caused liver damage by alcohol This increasing tendency was observed, and in the extract-treated group of Moringa water, the level was observed to decrease in proportion to the concentration (B in Table 5). In addition, a high concentration of triglyceride (TG) in the form of VLDL, which was freed from the blood, was also observed in the alcohol-treated group, and decreased proportionally to the concentration in the extract-treated group of Moringa water (C in Table 5). Albumin (ALB) produced by the normal liver showed a completely opposite trend (Table 5A).

According to the present invention, in summary, these results are obtained by fractionally extracting Moringa leaves, and after obtaining the most effective and intact relief composition of alcoholic fatty liver, it is used as a variety of raw materials such as pharmaceutical raw materials, functional food raw materials and drinking raw materials, Alcoholic liver damage prevention and mitigation function in functional and morphological aspects is performed through an alcoholic fatty liver relief composition that can easily change the formulation and is easily absorbed by the human body and can mass-produce high-additive substances at the lowest cost. Yet, it is expected to be effective in suppressing inflammation and oxidative stress caused by the liver.

The present invention is not limited to the disclosed embodiments, and it is obvious to those of ordinary skill in the art that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, such variations or modifications will have to belong to the claims of the present invention.

Claims (6)

A first step of drying moringa leaves as a raw material;
A second step of generating the Moringa leaf powder having passed through the first step into a powder of 100 to 3,000 mesh with a pulverizer, and re-drying it at room temperature for 7 to 10 days to obtain Moringa leaf powder;
The moringa leaf powder after the second step was immersed in 20ℓ of a mixed solution of 30wt% of water and 70wt% of ethanol for 12 to 24 hours at a temperature of 10 to 70℃ of an ultrasonic extractor for 12 to 24 hours. A third step of reacting to obtain a primary extract;
The first extract after the third step was reacted three times while immersing 1.8 kg of the first extract in 20 ℓ of a mixed solution of 30 wt% water and 70 wt% ethanol for 12 to 24 hours at a temperature of 10 to 70°C of an ultrasonic extractor. A fourth step of obtaining a secondary extract;
A fifth step of filtering the secondary extract after the fourth step to remove impurities, and then concentrating by removing 90-99wt% of the solvent with a rotary evaporation concentrator; And
A sixth step of obtaining a composition from which the solvent is completely removed by freeze-drying the concentrate passed through the fifth step at a temperature of -40 to 70°C; an alcoholic fatty liver relief composition using Moringa, comprising: Manufacturing method. delete delete delete In forming an alcoholic fatty liver relief composition using Moringa leaf extract through the manufacturing method of claim 1:
(A) 35-50wt% of powder using Moringa leaves, and
(B) An alleviation composition of alcoholic fatty liver using Moringa, characterized in that 50-65 wt% of a mixed solution of 30 wt% of water and 70 wt% of ethanol is fractionally extracted. delete

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