Applications of bitter melon extract and its active ingredients

Abstract

Application of bitter melon extract and its active ingredients 3β,7β,25-trihydroxycucurbitacin-5,23(E)-diene-19-aldehyde (TCD) and momordicin II (MC II) in liver protection, wherein the bitter melon extract is provided by extraction with an alcohol solvent from at least one of the following groups: bitter melon vine, mature bitter melon fruit, overripe bitter melon fruit, immature bitter melon seeds, mature bitter melon seeds and overripe bitter melon seeds.

Description

[Technical Field]

[0001] This invention relates to the application of bitter melon extract and its active ingredients in liver protection, particularly in reducing lipid accumulation in hepatocytes, lowering liver fat content, reducing liver inflammation index, and preventing or treating fatty liver. [Previous Technology]

[0002] In Taiwan, the prevalence of fatty liver disease is very high. According to a 2023 survey by the Liver Disease Prevention and Treatment Foundation, the detection rate of fatty liver disease through abdominal ultrasound is as high as 54%. In other words, on average, one in two people has fatty liver disease. Fatty liver disease refers to the accumulation of excessive lipids in liver cells. Although there are no obvious symptoms, the lipids accumulated in liver cells can cause chronic inflammation of the liver. If left untreated for a long time, it may develop into liver fibrosis, cirrhosis, or even liver cancer.

[0003] Generally speaking, fatty liver can be improved through a healthy diet with less oil and salt, regular exercise, and weight control. However, due to changes in lifestyle, modern people generally have problems such as eating out for a long time, irregular work and rest, and lack of sufficient exercise, which has led to an increase in the incidence of fatty liver.

[0004] Currently, there is a severe shortage of drugs or health foods that can effectively improve fatty liver. Although there are liver-protecting drugs or health foods on the market that claim to contain artichoke or milk thistle as active ingredients, studies have shown that artichoke and milk thistle are not effective in improving fatty liver. The U.S. Food and Drug Administration (FDA) did not approve Rezdiffra (resimetiro) as a treatment for non-alcoholic steatohepatitis (NASH) until 2024. Therefore, the industry is still committed to developing health foods or drugs that can effectively treat fatty liver. [Summary of the Invention]

[0005] The inventors of this case have discovered that alcohol solvent extracts of bitter melon vines, mature bitter melon fruits, overripe bitter melon fruits, immature bitter melon seeds, mature bitter melon seeds, and overripe bitter melon seeds can effectively reduce oleic acid-induced lipid accumulation in hepatocytes, thus providing liver-protective effects such as reducing liver fat content and lowering liver inflammation index. Further research has found that using 3β,7β,25-trihydroxycucurbitacin-5,23(E)-diene-19-aldehyde (TCD) and momordicin II (MC II) alone from bitter melon extract can also provide the aforementioned benefits.

[0006] Therefore, one object of the present invention is to provide the use of bitter melon extract in the preparation of a composition for liver protection, wherein the bitter melon extract is provided by extraction with an alcohol solvent from at least one of the following groups: bitter melon vines, mature bitter melon fruits, overripe bitter melon fruits, immature bitter melon seeds, mature bitter melon seeds, and overripe bitter melon seeds. Preferably, the bitter melon extract is provided by extraction with an alcohol solvent from at least one of the following groups: bitter melon vines, mature bitter melon fruits, overripe bitter melon fruits, and mature bitter melon seeds; more preferably, the bitter melon extract is provided by extraction with an alcohol solvent from bitter melon vines. Furthermore, the bitter melon extract preferably contains TCD and / or MC II. On the other hand, if the composition is used in humans to provide liver protection benefits, the dosage of the composition, based on the dry weight of the bitter melon extract, is preferably 0.05 g / kg body weight to 15 g / kg body weight per day.

[0007] Another object of the present invention is to provide a use of an active ingredient in the preparation of a composition for liver protection, wherein the active ingredient is 3β,7β,25-trihydroxycucurbitacin-5,23(E)-diene-19-aldehyde (TCD), momordicin II (MC II), or a combination thereof. When using this composition in humans to provide liver protection benefits, the preferred dosage of the composition, based on the dry weight of the active ingredient, is 0.5 mg / kg body weight to 20 mg / kg body weight daily.

[0008] In the above-described uses according to the present invention, the composition is preferably used for one or more of the following: reducing hepatocyte lipid deposition, reducing liver fat content, and reducing liver inflammation index; more preferably for the prevention or treatment of fatty liver, especially non-alcoholic fatty liver.

Implementation Method

[0011] The following will describe the detailed technical content and some specific embodiments of the present invention so that those skilled in the art can understand the features of the present invention; however, the present invention may be practiced in different forms without departing from the spirit of the present invention, and the scope of protection of the present invention should not be interpreted as limited to those specifically stated in the specification.

[0012] Unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification (especially in the claims described below) shall be understood to include both singular and plural forms; the range of values ​​used (e.g., 5 to 100) shall be understood to include all rational numbers in that range and any rational numbers in that range, thus including all possible combinations of values ​​between the lowest and highest values ​​listed; the units “grams per kilogram of body weight” and “milligrams per kilogram of body weight” refer to the dosage per kilogram of individual body weight; “individual” means mammal, including humans or non-human animals.

[0013] The maturity of bitter melon fruit is judged based on the plumpness of the surface particles and the color of the peel. Among them, the surface particles of "unripe" bitter melon fruit are smaller, while the surface particles of "ripe" bitter melon fruit are larger and more distinct, and the peel of "overripe" bitter melon fruit will be yellow. The maturity of bitter melon seeds is judged based on the maturity of the bitter melon fruit at the time of seed collection. For example, seeds extracted from ripe bitter melon fruit are ripe bitter melon seeds. As for "ripe" bitter melon vines, it refers to vines that have grown fruit.

[0014] The inventors of this case have discovered that extracts obtained by extracting bitter gourd vines, mature bitter gourd fruits, overripe bitter gourd fruits, immature bitter gourd seeds, mature bitter gourd seeds, and / or overripe bitter gourd seeds with alcohol solvents can effectively reduce lipid accumulation in hepatocytes. Therefore, they can be used to reduce liver fat content and liver inflammation index, among other liver-protective effects. Further research on the aforementioned bitter gourd extracts revealed that 3β,7β,25-trihydroxycucurbitacin-5,23(E)-diene-19-aldehyde (TCD) and momordicin II (MC II) themselves can provide the aforementioned benefits.

[0015] Therefore, the present invention relates to the use of bitter melon extract in the preparation of a composition for liver protection, wherein the bitter melon extract is provided by extraction with an alcohol solvent from at least one of the following groups: bitter melon vines, mature bitter melon fruits, overripe bitter melon fruits, immature bitter melon seeds, mature bitter melon seeds and overripe bitter melon seeds.

[0016] In the use of the present invention, the bitter melon extract is preferably provided by extraction with an alcohol solvent from at least one of the following groups: bitter melon vines, mature bitter melon fruits, overripe bitter melon fruits, and mature bitter melon seeds; more preferably, it is provided by extraction with bitter melon vines (especially mature bitter melon vines). Examples of the alcohol solvent in the use of the present invention include, but are not limited to, methanol, ethanol, propanol, isopropanol, and butanol. In some embodiments of the present invention, methanol is used as the extraction solvent. The bitter melon extract preferably contains TCD and / or MC II.

[0017] When preparing the bitter melon extract of the present invention, the raw material may be pretreated as needed to improve the extraction efficiency. In some embodiments of the present invention, the raw material is cut into thin slices with a thickness of less than 2 cm (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 cm) before extraction.

[0018] As needed, the extract provided by the extraction operation can be processed to improve the convenience of storage and transportation. Such processing includes, but is not limited to: vacuum concentration, drying (e.g., hot air drying, freeze drying), sterilization, dilution, and solid-liquid separation (e.g., filtration, centrifugation, decantation). In some embodiments of the present invention, the bitter melon extract is provided in the form of a dry powder. In some embodiments of the present invention, the bitter melon extract is provided in the form of a concentrated liquid.

[0019] This invention also relates to the use of an active ingredient in the preparation of a composition for liver protection, wherein the active ingredient is TCD, MC II, or a combination thereof. In the use according to the invention, TCD and MC II may be commercially available, prepared by synthetic methods known in the art, or isolated from natural extracts. For example, but not limited to, TCD and MC II may be isolated from bitter melon extract.

[0020] In the use according to the present invention, the composition is used for at least one of the following: reducing lipid accumulation in hepatocytes, reducing liver fat content, and reducing liver inflammation index. In the use according to the present invention, the composition is used for the prevention or treatment of fatty liver, preferably for the prevention or treatment of non-alcoholic fatty liver disease.

[0021] In the use of the present invention, the composition may be a pharmaceutical composition or a food composition.

[0022] The pharmaceutical composition provided by the present invention can be used for systemic or local drug delivery and can be delivered through various drug delivery systems (DDS). For example, but not limited thereto, the pharmaceutical composition provided by the present invention can be delivered through an oral drug delivery system or an injectable drug delivery system.

[0023] The pharmaceutical composition provided according to the present invention may be in any suitable form without particular limitation, depending on the intended use and the corresponding suitable dosage form; for example, but not limited thereto, the pharmaceutical composition may be administered to the individual in need by one or more of the following methods: oral, subcutaneous injection, intramuscular injection, intraperitoneal injection and intravenous injection (including infusion and rapid injection).

[0024] Depending on the form and purpose of use, any pharmaceutically acceptable excipient or carrier that will not adversely affect the desired benefits of the bitter melon extract, TCD, and / or MC II of the present invention may be used to provide the pharmaceutical composition; wherein, the pharmaceutical composition comprises at least one of the bitter melon extract, TCD, and MC II of the present invention, and a pharmaceutically acceptable excipient or carrier; or, the pharmaceutical composition comprises at least one of the bitter melon extract, TCD, and MC II of the present invention, and a pharmaceutically acceptable excipient or carrier. The pharmaceutically acceptable excipient or carrier is known in the art and includes, but is not limited to: buffers, diluents, stabilizers, preservatives, absorption delay agents, disintegrants, solubilizers, thickeners, emulsifiers, antioxidants, binders, binders, thickeners, dispersants, suspending agents, lubricants, etc.

[0025] Taking oral dosage forms as an example, the pharmaceutical composition can be provided in a dosage form suitable for oral administration by any suitable method. Liquid dosage forms suitable for oral administration include syrups, oral liquids, suspensions, elixirs, etc., while solid dosage forms suitable for oral administration include powders, granules, lozenges, sugar-coated lozenges, enteric-coated lozenges, chewable lozenges, effervescent lozenges, film-coated lozenges, capsules, long-acting sustained-release lozenges, etc. Examples, but not limited to, of pharmaceutically acceptable excipients or carriers that can be used to provide the aforementioned liquid dosage forms include: water, saline, glucose (dextrose), glycerol, ethanol or its analogues, vegetable oil, mineral oil, polyethylene glycol, and combinations thereof; examples of pharmaceutically acceptable excipients or carriers that can be used to provide the aforementioned solid dosage forms include: cellulose, starch, talc, kaolinite, bentonite, sodium citrate, gelatin, agar, carboxymethyl cellulose, gum arabic, alginate, glyceryl monostearate, glyceryl distearate, calcium stearate, magnesium stearate, calcium carbonate, magnesium carbonate, and combinations thereof.

[0026] For injectable solutions or infusions suitable for injection, the pharmaceutical composition provided according to the present invention may contain one or more components such as isotropic solutions, salt buffers (e.g., phosphate buffer or citrate buffer), solubilizers, emulsifiers, 5% sugar solutions, etc., and may be provided in dosage forms such as intravenous infusions, emulsion intravenous infusions, dry powder injections, suspension injections, or dry powder suspension injections. Alternatively, the pharmaceutical composition may be prepared as a pre-injection solid, and before being administered to the individual in need, the pre-injection solid may be dissolved in other solutions or suspensions or emulsified to provide the desired injectable.

[0027] As needed, the pharmaceutical composition provided according to the present invention may further contain appropriate amounts of additives, such as colorants, coloring agents, etc., which can improve the feel of the pharmaceutical composition when in use, and buffers, preservatives, antiseptics, antibacterial agents, antifungal agents, etc., which can improve the stability and storability of the composition.

[0028] The food composition provided according to the present invention can be a beverage, a solid food, or a semi-solid food, and can be provided in the form of health food, health care food, functional food, nutritional supplement, or special nutritional food. For example, but not limited to, the food composition can be dairy products, processed meat products, bread, pasta products, biscuits, ice cream, lozenges, capsules, fruit juices, teas, sparkling water, alcoholic beverages, sports drinks, nutritional drinks, etc. Preferably, the food composition is provided in the form of a health food.

[0029] Furthermore, depending on the form of use and requirements, any suitable food additives may be contained in the food composition provided according to the present invention. For example, the food additives that may be used include, but are not limited to: preservatives, antibacterial agents, antifungal agents, antioxidants, bleaching agents, color-retaining agents, leavening agents, nutritional additives, coloring agents, flavoring agents (e.g., sweeteners), thickeners, binding agents, chemicals for the food industry, emulsifiers, and agents for quality improvement, brewing, and food manufacturing.

[0030] The recommended dosage, usage standards and conditions for specific groups (e.g., pregnant women, children, the elderly, etc.), or suggestions for taking it together with other foods or medicines can be indicated on the outer packaging of the health food, health care food, functional food, nutritional supplement food or special nutritional food provided according to the present invention, so as to enable users to take it on their own without the guidance of a doctor, pharmacist or relevant personnel without safety concerns.

[0031] Where necessary, one or more other liver-protecting ingredients may be added to the composition provided by the present invention to further enhance the efficacy of the composition or increase the flexibility and compatibility of the formulation, as long as the other liver-protecting ingredients do not have an adverse effect on the efficacy of the bitter melon extract, TCD and / or MC II of the present invention.

[0032] The composition provided by the present invention can be applied at different frequencies, such as once a day, multiple times a day, or once every few days, depending on the individual's needs, age, weight, health condition, and purpose of application. The content of bitter melon extract, TCD, and MC II in the composition provided by the present invention can also be adjusted according to actual application needs, for example, to the recommended daily dosage. When the composition of the present invention is used in humans to provide liver protection benefits, the dosage of the composition, based on the dry weight of bitter melon fruit extract, bitter melon vine extract, and / or bitter melon seed extract, can be from 0.05 g / kg body weight to 15 g / kg body weight daily, for example: 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0. 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.15, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.50, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 grams per kilogram of body weight, and the dosage may be selected from any two of the aforementioned values.

[0033] For example, when a composition prepared from bitter melon fruit extract is taken orally in humans, the dosage of the composition, based on the dry weight of the bitter melon fruit extract, may be 0.08 to 1 g / kg body weight per day, preferably 0.15 to 0.8 g / kg body weight per day, and more preferably 0.3 to 0.5 g / kg body weight per day. In one specific embodiment, the oral dosage of the composition, based on the dry weight of the bitter melon fruit extract, is 0.33 g / kg body weight per day. Furthermore, when a composition prepared from bitter melon vine extract is taken orally in humans, the dosage of the composition, based on the dry weight of the bitter melon vine extract, may be 0.05 to 0.8 g / kg body weight per day, preferably 0.1 to 0.6 g / kg body weight per day, and more preferably 0.15 to 0.3 g / kg body weight per day. In one specific embodiment, the oral dose of the composition, based on the dry weight of the bitter melon vine extract, is 0.2 g / kg body weight daily. Furthermore, when the composition prepared from bitter melon seed extract is used orally in humans, the dosage, based on the dry weight of the bitter melon seed extract, can be 1 to 15 g / kg body weight daily, preferably 2 to 10 g / kg body weight daily, and more preferably 4 to 8 g / kg body weight daily. In one specific embodiment, the oral dose of the composition, based on the dry weight of the bitter melon seed extract, is 4.57 g / kg body weight daily.

[0034] When the composition according to the present invention is used in humans to provide hepatoprotective benefits, the dosage of the composition, based on the dry weight of TCD and / or MC II, is from 0.5 mg / kg body weight to 20 mg / kg body weight per day, for example: 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg body weight per day, and the dosage may be within the range of any two of the aforementioned values.

[0035] For example, when a composition prepared from TCD is administered orally to a human individual, the dosage of the composition, based on the dry weight of TCD, may be 0.5 to 10 mg / kg body weight daily, preferably 0.8 to 8 mg / kg body weight daily, and more preferably 1 to 4.5 mg / kg body weight daily. In one specific embodiment, the dosage of the composition, based on the dry weight of TCD, is 1.83 mg / kg body weight daily. Furthermore, when a composition prepared from MC II is administered orally to a human individual, the dosage of the composition, based on the dry weight of MC II, may be 2 to 20 mg / kg body weight daily, preferably 3 to 12 mg / kg body weight daily, and more preferably 4 to 8 mg / kg body weight daily. In one specific embodiment, the dosage of the composition, based on the dry weight of MC II, is 4.45 mg / kg body weight daily.

[0036] The present invention will be further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the appended claims.

[0037] Example

[0038] Example 1: Effect of bitter melon extract on inhibiting lipid accumulation in hepatocytes

[0039] 1-1. Preparation of bitter melon extract

[0040] Take mature bitter melon vines, unripe bitter melon fruits, mature bitter melon fruits, overripe bitter melon fruits, unripe bitter melon seeds, mature bitter melon seeds and overripe bitter melon seeds, cut them into thin slices with a thickness of about 0.6 cm, freeze-dry them and grind them into powder, and store them in a -30ºC refrigerator.

[0041] Take 0.5 g of each of the above seven powder groups and add them separately to 50 mL methanol microwave-safe bottles (MARS Xpress plus vessel). Place each microwave-safe bottle in a sealed microwave apparatus (purchased from CEM, USA; model: MARS 6), set the temperature to 80ºC and the microwave power to 600 watts (4 bottles at a time), and then perform extraction. Afterward, cool and make up to 50 mL of each crude extract with methanol. Then, centrifuge at 4,000 rpm for 10 minutes, collect the supernatant and concentrate under reduced pressure to provide seven groups of bitter melon extracts for subsequent experiments.

[0042] 1-2. Efficacy Test

[0043] HepG2 cells (a human liver cancer cell line; purchased from the Bioresource Preservation and Research Center of the Food Industry Development Institute) were seeded in a transparent, flat-bottomed black 96-well cell culture dish at a density of 1×10⁴ cells per well. The cells were cultured in HepG2 cell culture medium (i.e., DMEM culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution) at 37ºC and 5% CO₂ for 24 hours. The cells were then divided into nine groups and cultured using different extracts provided in Examples 1-1 as follows: (1) Blank group: cultured in HepG2 cell culture medium for 48 hours; (2) Control group: cultured in HepG2 cell culture medium for 24 hours, then oleic acid (final concentration 0.3 mM) was added to the culture medium, and cultured for another 24 hours; (3) Bitter gourd vine group: cultured in a medium containing 5000… (3) After culturing HepG2 cells in a medium containing nacles / mL (ng / mL) of mature bitter melon vine extract for 24 hours, oleic acid (final concentration 0.3 mM) was added to the medium, and the cells were cultured for another 24 hours; (4) Immature bitter melon fruit group: The same procedure as the "bitter melon vine group" was followed, but the bitter melon vine extract was replaced with the immature bitter melon fruit extract; (5) Mature bitter melon fruit group: The same procedure as the "bitter melon vine group" was followed, but the bitter melon vine extract was replaced with the mature bitter melon fruit extract; (6) Overripe bitter melon fruit group: The same procedure as the "bitter melon vine group" was followed, but the bitter melon vine extract was replaced with the overripe bitter melon fruit extract; (7) Immature bitter melon seed group: The same procedure as the "bitter melon vine group" was followed, but the bitter melon vine extract was replaced with the immature bitter melon seed extract; (8) Mature bitter gourd seed group: The same as the "bitter gourd vine group" was used, but the bitter gourd vine extract was replaced with mature bitter gourd seed extract; and (9) Overripe bitter gourd seed group: The same as the "bitter gourd vine group" was used, but the bitter gourd vine extract was replaced with overripe bitter gourd seed extract.

[0044] Next, the cell culture medium was removed, and the cells were rinsed twice with phosphate-buffered saline (PBS). Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes. The cells were then rinsed again with PBS, and Nile red and DAPI staining agents were added to stain the oil droplets and nuclei of the hepatocytes, respectively, for 15 minutes. After staining, the cells were rinsed twice with PBS, and the oil droplet content in each group of cells was analyzed using a fluorometer. Using the oil droplet content of the control group cells as 100%, the relative lipid accumulation percentage of the other groups of cells was calculated. The results are shown in Figure 1.

[0045] As shown in Figure 1, compared with the control group, the cell lipid accumulation in the bitter melon vine group, the mature and overripe bitter melon fruit group, and the bitter melon seed group at different maturity levels was significantly reduced. Among them, the cell lipid accumulation in the bitter melon vine group, the mature bitter melon group, the overripe bitter melon group, and the mature bitter melon seed group decreased by at least about 50%, and the decrease in cell lipid accumulation in the bitter melon vine group was as high as about 80%. The foregoing results indicate that the bitter melon extract of the present invention can effectively reduce lipid accumulation in hepatocytes, and has the effect of reducing liver fat content and liver inflammation index, and can be used to prevent or treat fatty liver.

[0046] Example 2: Analysis of active ingredients in bitter melon extract

[0047] 2-1. Bitter melon vine extract

[0048] After reconstituted 10 mg of mature bitter melon vine extract provided in Example 1-1 with 10 mL of methanol, the extract was filtered through a 0.22 μm nylon membrane and then analyzed for components and content using liquid chromatography-tandem mass spectrometry (LC / MS / MS; Acquity ultra-performance LC liquid chromatography system and Xevo™ TQ-S mass spectrometer, both purchased from Waters Corporation, USA) under the following conditions: 1. Liquid chromatography system conditions: An Acquity UPLC BEH C18 column (100 × 2.1 mm, purchased from Waters Corporation, USA) was used; the column temperature was 35ºC; the flow rate was 0.3 mL / min; the mobile phases A and B were 5% methanol solution (containing 0.1% formic acid) and 100% methanol solution (containing 0.1% formic acid), respectively; the gradients were 0-1 min / 72% B, 1-2 min / 74% B, and 2-6 min / 74% B. B. 6-7 minutes / 85% B. 7-8 minutes / 85% B. 8-9.5 minutes / 99% B. 9.5-10.5 minutes / 99% B. 10.5-11 minutes / 72% B. 11-15 minutes / 72% B. 2. Mass spectrometer conditions: Electrolytic ionization (ESI) was used as the mass spectrometer ionization source; capillary voltage was 3.0 kV; desolvation flow and cone gas flows were 900 L / h and 150 L / h, respectively; desolvation temperature was 450ºC; multiple reaction monitoring (MRM) analysis mode was used.

[0049] As shown in Table 1, the extract of mature bitter melon vines contains 21 cucurbitane-type triterpenoids, with the top three in highest content being momordicine I (MC I), TCD, and MC II, in that order. Efficacy tests will be conducted on these three components in subsequent studies. Table 1 Element Content (μg / g) Momordicoside A (MA) ND Momordicoside L (ML) 239.96±11.37 TCD 1054.82±35.95 Momordicoside K (MK) 135.25±13.36 MC I 5433.61±576.39 MC II 313.29±12.57 Momordicoside I aglycone (MIa) ND Momordicoside G (MG) 0.73±0.10 Momordicoside F1 (MF1) ND Momordicoside I (MI) ND Momordicoside F2 (MF2) 1.22±0.08 Momordicoside P (MP) ND Kuguacin R (KR) ND Kuguacin J (KJ) ND Karavilagenin A (KAA) ND Kuguaglycoside C (KCC) ND Charantadiol A (CA) ND Karavilagenin D (KAD) ND Kuguacin N (KN) ND 19 (R+S) ND "ND" means "no detect"; "19(R+S)" refers to 5,19-epoxy-19R,25-dimethoxycucurbit-6,23-dien-3-ol and 5,19-epoxy-19S,25-dimethoxycucurbit-6,23-dien-3-ol.

[0050] 2-2. Bitter melon fruit extract and bitter melon seed extract

[0051] The contents of MC I, TCD and MC II in bitter melon fruit and seed extracts were determined in accordance with the method of Example 2-1. The results showed that in the bitter melon fruit extract, the MC I content was 577.90 μg / g to 116.19 μg / g, the TCD content was 609.89 μg / g to 58.8 μg / g, and the MC II content was 28.8 μg / g to 13.02 μg / g; in the bitter melon seed extract, the MC I content was 57.60 μg / g to 10.32 μg / g, the TCD content was 8.13 μg / g to 6.96 μg / g, and the MC II content was 2.03 μg / g to 2.04 μg / g.

[0052] Example 3: Effect of bitter melon active ingredients on inhibiting lipid accumulation in hepatocytes

[0053] 3-1. Efficacy Test

[0054] HepG2 cells were seeded in a transparent, flat-bottomed black 96-well cell culture dish at a density of 1×10⁴ cells per well and cultured in HepG2 cell culture medium (i.e., DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin solution) at 37ºC and 5% CO₂ for 24 hours. The cells were then divided into five groups and cultured as follows: (1) Blank group: cultured in HepG2 cell culture medium for 48 hours; (2) Control group: cultured in HepG2 cell culture medium for 24 hours, then oleic acid (final concentration 0.3 mM) was added to the culture medium and cultured for another 24 hours; (3) MC I group: cultured in HepG2 cell culture medium containing 1 μg / mL, 5 μg / mL, 10 μg / mL or 50 μg / mL MC I for 24 hours, then oleic acid (final concentration 0.3 mM) was added to the culture medium. (4) MC II group: Follow the same procedure as "MC I group", but replace the added MC I with MC II; (5) TCD group: Follow the same procedure as "MC I group", but replace the added MC I with TCD.

[0055] Next, the cells in each group were fixed, stained, and analyzed for oil droplet content in accordance with the methods of Examples 1-2. The oil droplet content of the control group cells was taken as 100%, and the relative lipid accumulation percentage of the cells in other groups was calculated. The results are shown in Figure 2.

[0056] As shown in Figure 2, compared with the control group, the percentage of lipid accumulation in cells of both the MC II group and the TCD group was significantly reduced at a dose of 50 μg / mL. Furthermore, the TCD group showed a significant reduction in lipid accumulation in hepatocytes even at a dose of 10 μg / mL. Unlike the TCD and MC II groups, the lipid accumulation in the MC I group did not differ significantly from the control group. These results indicate that both TCD and MC II can effectively reduce lipid accumulation in hepatocytes, thus reducing liver fat content and liver inflammation index, and can be used for the prevention or treatment of fatty liver.

[0057] 3-2. Half-maximal effective concentration (EC50) for inhibiting lipid accumulation in hepatocytes

[0058] The EC50 of MC II and TCD were calculated using the experimental results from 3-1, and the results are shown in Table 2. Table 2 Element EC50 (μg / mL) MC II 118.9 TCD 48.8

[0059] Next, the daily oral doses of MC II and TCD were calculated based on the following formulas. For an adult weighing 60 kg, the dosage of MC II is approximately 4.45 mg (dry weight) / kg body weight per day, and the dosage of TCD is approximately 1.83 mg (dry weight) / kg body weight per day. Furthermore, based on the content of MC II and TCD in the extracts, the oral dose of bitter melon fruit extract is approximately 0.33 g (dry weight) / kg body weight per day, the oral dose of bitter melon vine extract is approximately 0.2 g (dry weight) / kg body weight per day, and the oral dose of bitter melon seed extract is approximately 4.57 g (dry weight) / kg body weight per day. [Simplified Explanation of the Diagram]

[0009] Figure 1 is a bar chart showing the percentage of lipid accumulation in HepG2 cells treated with bitter melon extracts of different parts / maturities compared to the control group. In the figure, "#" indicates a p-value < 0.05 compared to the control group, "*" indicates a p-value < 0.05 compared to the control group, "**" indicates a p-value < 0.01 compared to the control group, and "***" indicates a p-value < 0.001 compared to the control group. Furthermore, the HepG2 cell line in the control group was not treated with oleic acid, while the HepG2 cells in the other groups were treated with oleic acid to induce lipid accumulation.

[0010] Figure 2 shows a bar chart of lipid accumulation (%) in HepG2 cells treated with different concentrations of bitter melon active ingredients compared to the control group. In the figure, "###" indicates a p-value < 0.001 compared to the blank group, "**" indicates a p-value < 0.01 compared to the control group, and "***" indicates a p-value < 0.001 compared to the control group. Furthermore, the HepG2 cell line in the blank group was not treated with oleic acid, while the HepG2 cells in the other groups were all treated with oleic acid to induce lipid accumulation.

Claims

1. Use of bitter melon extract in the preparation of a composition for non-alcoholic fatty liver disease, wherein the bitter melon extract is provided by extraction with an alcohol solvent from at least one of the following groups: mature bitter melon vines, mature bitter melon fruits, overripe bitter melon fruits and mature bitter melon seeds, wherein the bitter melon extract contains 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (TCD) and momordicine II (MC II).

2. As claimed in claim 1, wherein the composition is used for one or more of the following: reducing hepatocyte lipid deposition, reducing liver fat content, and reducing liver inflammation index.

3. For the purposes described in claim 1 or 2, the dosage of the component, based on the dry weight of the bitter melon extract, is from 0.05 g / kg body weight to 15 g / kg body weight per day.

4. Use of a separated active ingredient in the preparation of a composition for non-alcoholic fatty liver disease, wherein the active ingredient is 3β,7β,25-trihydroxycucurbitacin-5,23(E)-diene-19-aldehyde and momordicin II.

5. As claimed in paragraph 1 or 4, wherein the dosage of the composition, based on the dry weight of the active ingredient, is from 0.5 mg / kg body weight to 20 mg / kg body weight per day.

6. As claimed in claim 5, wherein the composition is used for one or more of the following: reducing hepatocyte lipid deposition, reducing liver fat content, and reducing liver inflammation index.

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