Method for manufacturing a composition for improving lipid metabolism disorders associated with menopausal estrogen reduction, comprising enzyme-treated Artemisia, Hop, and Monk fruit complex extract powder

Abstract

The present invention relates to a composition for improving lipid metabolism abnormalities due to menopausal estrogen reduction, comprising enzyme-treated complex extract powder of mugwort, hops, and monk fruit, and a method for preparing the same. The composition of the present invention includes mugwort, hops, and monk fruit in an optimal weight ratio, and uses a complex extract as an active ingredient in which the content of the active ingredient is maximized through a stepwise temperature-raising extraction process. In particular, the present invention can induce conversion into active aglycones by adding a bioconversion process using complex enzymes of a specific composition ratio, thereby exhibiting excellent effects in weight loss and improvement of lipid metabolism indicators through AMPK activation in a state of metabolic decline during menopause. Accordingly, the present invention is safe even with long-term use by using components derived from natural products and can be usefully utilized to improve the quality of life of menopausal women.

Description

Technology Field

[0001] The present invention relates to a composition for improving lipid metabolism abnormalities caused by menopausal estrogen reduction, comprising an enzyme-treated complex extract powder of Artemisia annua, hops, and monk fruit, and a method for preparing the same. Specifically, the invention relates to an anti-obesity and lipid metabolism improvement composition comprising a complex extract of Artemisia annua, hops, and monk fruit as an active ingredient, and maximizing the content and bioavailability of the active ingredient through a specific step-by-step temperature-raising extraction process and bioconversion technology. Background Technology

[0003] With the dramatic extension of human life expectancy due to advancements in modern medicine, 'Active Aging'—which involves going beyond mere life extension to maintain quality of life after middle age and embracing a healthy old age—is emerging as a key topic in the healthcare industry. In particular, for women, menopause, characterized by rapid hormonal changes during the life cycle, serves not only as a physical and mental turning point but also as a critical crisis point for metabolic health.

[0004] Estrogen, a female hormone, is a key factor in maintaining homeostasis that goes beyond simply regulating reproductive functions to controlling systemic lipid metabolism, maintaining vascular elasticity, and the distribution of fat cells. However, as estrogen secretion becomes drastically depleted upon entering menopause, the body reorganizes its metabolic pathways to accumulate body fat primarily around the abdominal viscera rather than in the buttocks or thighs. This rapid increase in visceral fat induces insulin resistance and raises blood triglyceride (TG) and low-density lipoprotein (LDL) levels, leading to chronic metabolic diseases such as hyperlipidemia, hypercholesterolemia, and dyslipidemia; furthermore, this becomes a critical cause of non-alcoholic fatty liver disease and cardiovascular diseases.

[0005] Currently, the medical community employs hormone replacement therapy (HRT) to manage menopausal metabolic syndrome; however, as research results have reported that this may increase the risk of developing certain cancers, such as breast and endometrial cancer, patients' psychological resistance to long-term use and ongoing safety controversies persist. To address this, various natural health supplements for women have been released on the market, but existing technologies have the following fundamental limitations.

[0006] First, existing methods for manufacturing herbal extracts adopt a uniform high-temperature extraction approach while overlooking the thermodynamic properties of the active ingredients in the raw materials. Medicinal herbs such as mugwort and hops contain both highly volatile essential oils and heat-sensitive flavonoids; however, with conventional hot water extraction methods, these key indicator components are largely lost or denatured by heat, often resulting in actual pharmacological activity falling short of expectations.

[0007] Second, most active ingredients in natural products exist in the form of sugar-bound 'glycosides.' These large-molecule glycosides have large molecular weights and low water solubility, resulting in significantly reduced absorption rates in the intestines. This can lead to an imbalance in bioavailability, where the metabolic efficiency of active ingredients varies widely depending on the state of the individual gut microbiome. In particular, for menopausal women with reduced metabolic function, even if they consume these natural ingredients, the concentration is insufficient to trigger lipid metabolism regulatory mechanisms, such as AMPK activation, within actual target cells.

[0008] Third, a simple mixing of raw materials alone cannot guarantee synergistic effects among complex ingredients. For ingredients with different pharmacological mechanisms to exert synergistic effects within the body without antagonism, it is necessary to establish precise formulation ratios and support them with engineering designs that enable each component to biochemically complement one another.

[0009] Therefore, for the safe metabolic management of menopausal women, there is an urgent need to develop an innovative solution that goes beyond simple nutritional supply. This solution integrates optimized process technology to extract active ingredients from raw materials in their whole spectrum with bioconversion technology to convert them into an active form that is easily bioavailable.

[0010] Accordingly, the inventors of the present invention conducted research to fundamentally improve the metabolic health of menopausal women by securing the active ingredients of natural raw materials without loss and maximizing bioavailability. As a result, they completed the present invention, which organically combines a specific blending ratio of mugwort, hops, and monk fruit with a stepwise temperature-raising extraction process and complex enzyme treatment technology. The composition according to the present invention can be utilized in various ways, such as for the prevention or improvement of menopausal obesity and lipid metabolism disorders, as well as for functional foods and pharmaceutical compositions. The problem to be solved

[0012] The present invention aims to provide an optimal extraction process capable of simultaneously satisfying the thermodynamic stability and solubility characteristics of the active ingredients of Artemisia vulgaris, hops, and monk fruit. Furthermore, it aims to provide a safe and effective composition capable of fundamentally improving menopausal metabolic abnormalities by maximizing obesity-inhibiting synergy through the optimal blending ratio of each raw material and increasing the absorption rate of active ingredients in the body by applying bioconversion technology using complex enzyme treatment. means of solving the problem

[0014] One aspect is mugwort ( Artemisia argyi ), hop( Humulus lupulus ) and monk fruit ( Siraitia grosvenorii The present invention provides a composition for the prevention or improvement of obesity or lipid metabolism abnormalities, comprising a complex extract of ) as an active ingredient.

[0015] In this specification, the term "Artemisia annua" may refer to the dried leaves and young stems of Artemisia annua or a closely related plant belonging to the Asteraceae family, and may act to inhibit the initial differentiation mechanism of fat cells by supplying flavonoid components such as eupatilin.

[0016] In this specification, the term "hop" may refer to the dried female flower spikes of the hop belonging to the Moraceae family, which may include active ingredients such as humulene and xanthohumol to assist in improving lipid metabolism in the composition of the present invention, and at the same time serve to impart a scent to the composition through its unique essential oil components.

[0017] In this specification, the term "Luo Han Guo" may refer to the fruit of the Luo Han Guo belonging to the Cucurbitaceae family, and may serve as a key material that activates the AMPK pathway, an energy metabolism switch in the body, by supplying a high concentration of a poorly soluble triterpene glycoside such as Mogroside V.

[0018] The complex extract of the present invention may refer to an active ingredient obtained through a stepwise temperature-raising extraction process from raw materials in which the mugwort, hops, and monk fruit are mixed in a specific weight ratio.

[0019] Specifically, the term "extract" may be used to encompass not only the solution obtained by extracting active ingredients from the mugwort, hops, and monk fruit, but also fractions, concentrates, or dried products obtained by further purifying or processing the solution. As long as the objective of the present invention can be achieved, the extract may include not only the crude extract prepared through the stepwise temperature-raising extraction process, but also fractions obtained by systematically fractionating the extract using various solvents, concentrates obtained by concentrating only the solids, or dried products obtained by pulverizing the extract using methods such as freeze-drying or spray-drying.

[0020] The above complex extract may be extracted with water, C1 to C4 lower alcohols, or a mixture of these solvents, specifically with ethanol at a concentration of 60 to 80% (v / v), more specifically with ethanol at a concentration of 70% (v / v).

[0021] In one experimental example, as a result of comparing and analyzing the extraction efficiency of indicator components according to the type and concentration of the extraction solvent, it was confirmed that in the example using 70% (v / v) ethanol as a solvent, the total content of cineol, eupatilin, and mogroside V was significantly higher compared to the comparative example using 100% anhydrous ethanol or the comparative example using only purified water.

[0022] This is due to the solvent polarity of ethanol at a concentration of 70% (v / v), which is optimized for simultaneously eluting components with different physicochemical solubility characteristics, such as volatile essential oil components, hydrophilic flavonoids, and sparingly soluble triterpene glycosides. When an alcohol solvent within the above concentration range is used in combination with the step-by-step heating process of the present invention, high-efficiency extraction of all components, which is difficult to achieve with a single solvent, becomes possible, which can support the anti-obesity and lipid metabolism improvement synergy of the final composition.

[0023] In addition, the complex extract may be characterized by being prepared by a stepwise extraction process comprising: a first extraction step at 20 to 35°C using an ethanol solvent at a concentration of 60 to 80% (v / v); a second extraction step at 40 to 55°C; and a third extraction step at 60 to 80°C.

[0024] Specifically, if the first extraction temperature exceeds 35°C, volatilization loss of aromatic components such as cineol and humulen with low boiling points may occur, which may reduce the physiological activity of the composition, and if the second extraction temperature exceeds 55°C, thermal denaturation and a sharp decrease in content of eupatilin, a heat-sensitive hydrophilic flavonoid, may occur.

[0025] In addition, if the above third extraction temperature is below 60℃, the solubility of mogroside V, a triterpene glycoside with a large molecular weight, is not sufficiently secured, which may cause the extraction efficiency to decrease rapidly, and conversely, if excessive heat exceeding 80℃ is applied, the chemical stability of the flavonoid components already dissolved is destroyed, and there is a risk that the quality of the final extract will deteriorate.

[0026] In one experimental example, it was confirmed that in the case of an example in which the stepwise temperature-raising extraction process according to the present invention was applied and the third extraction temperature was set to 75°C, the total sum content of the indicator component was significantly higher compared to comparative examples in which the third extraction temperature was lower or higher than the range of the present invention, or comparative examples in which batch extraction was performed at a high temperature of 80°C from the beginning. In particular, the example that underwent the above stepwise process showed a pattern of a full-component extract in which all active components derived from raw materials were harmoniously contained without being destroyed or lost, and it was confirmed that this plays a decisive role in maximizing the mutually complementary pharmacological mechanisms of each component.

[0027] The above complex extract may be characterized by being extracted by mixing mugwort, hops, and monk fruit in a weight ratio of 2:1:1.

[0028] If the mixing ratio of Artemisia annua exceeds the above range and is included in excess, the sensory acceptability of the composition may be reduced due to the characteristic essential oil components and strong bitter taste of Artemisia annua, and the balance with other components may be disrupted, potentially leading to a counterproductive effect of reduced fat differentiation inhibition efficiency. Conversely, if the mixing ratio of hops becomes excessive, the overall activity spectrum of the composition may shift toward the characteristic sedative and stabilizing effects of hops rather than lipid metabolism improvement, which may result in reduced efficiency in inhibiting body fat accumulation and enhancing metabolic activity. Furthermore, if the mixing ratio of Monk Fruit is excessive, the hygroscopicity of the final extract powder may increase excessively due to the high concentration of carbohydrate components and glycosides contained in Monk Fruit, which may impair the physical stability of the formulation.

[0029] In one experimental example, it was confirmed that the example with the 2:1:1 weight ratio of the present invention showed significantly superior lipid accumulation inhibitory activity compared to the comparative example with a simple equal mixing ratio or other mixing ratios, and in particular, it was confirmed that an inhibition rate of about 46.24% was recorded even without treatment with the complex enzyme composition.

[0030] The above composition further comprises a complex enzyme composition, wherein the complex enzyme composition may be characterized by naringinase, hesperidinase, and β-primeverosidase (β-) mixed in a weight ratio of 4:4:2.

[0031] In this specification, the term "complex enzyme composition" refers to a functional catalyst mixture added to convert high molecular weight glycoside components present in the mugwort, hop, and monk fruit extracts into low molecular weight non-glycoside forms that are easily bioabsorbed. This not only maximizes biological activity by improving the low bioavailability of natural active ingredients but also improves sensory quality by controlling the characteristic bitterness and off-flavor of the extracts.

[0032] In this specification, the term "naringinase" refers to an enzyme that hydrolyzes the sugar chains of flavonoid glycosides in an extract, which can increase the bioavailability of active ingredients while simultaneously structurally breaking down components that cause a strong bitter taste.

[0033] In this specification, the term “hesperidinase” refers to an enzyme that acts complementarily with naringinase to convert polyphenol compounds such as hesperidin into non-glycosidic hesperetin, which substantially increases the ability of the composition of the present invention to inhibit adipocyte differentiation and, in particular, can enhance antioxidant and blood circulation improvement activities through interaction with Artemisia annua and hop extracts.

[0034] In this specification, the term “β-primeverosidase” refers to an enzyme that specifically degrades primeroside, a disaccharide conjugate contained in monk fruit and other raw materials. By efficiently degrading complex glycoside structures that are difficult to process with general sugar-degrading enzymes, it induces synergistic effects with other enzymes and can gently adjust the overall flavor of the composition.

[0035] The naringinase, hesperidinase, and β-primeverosidase included in the above complex enzyme composition may be characterized by being mixed in a weight ratio of 4:4:2.

[0036] In one experimental example, it was confirmed that the example applying the 4:4:2 weight ratio of the present invention increased p-AMPK activity by approximately 3.92 times and p-ACC activity by approximately 4.25 times compared to the control group, while significantly reducing the expression of SREBP-1c, a key transcription factor that induces lipid synthesis, to 24.8%. This was determined to be because the series of bioconversion processes, in which naringinase and hesperidinase primarily degrade the sugar chains of the substrate and β-primeverosidase completes the final conversion into active aglycones, was optimized at the 4:4:2 ratio.

[0037] In this specification, the term “obesity” refers to a state in which adipose tissue in the body accumulates excessively beyond a normal range, and may encompass a pathological condition in which the overall metabolic function of the body is impaired due to an imbalance in energy metabolism, going beyond a mere increase in body weight.

[0038] In this specification, the term “lipid metabolism disorder” may refer to a condition in which impaired processes of lipid synthesis, transport, and breakdown in the body result in abnormal levels of lipid concentration in the blood or excessive deposition of fat in organs such as the liver.

[0039] In particular, in the present invention, the obesity and lipid metabolism abnormalities may include symptoms that occur as the lipid metabolism regulatory mechanism weakens due to hormonal imbalance caused by menopausal estrogen deficiency. Specifically, the lipid metabolism abnormalities may be one or more selected from the group consisting of hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, dyslipidemia, and fatty liver, but are not particularly limited thereto.

[0040] In this specification, the term "prevention" may refer to any act of suppressing or delaying the onset of obesity or lipid metabolism disorders through the ingestion or administration of a composition according to the present invention, and "improvement" may refer to any act of improving or beneficially altering a condition caused by obesity or lipid metabolism disorders through a composition according to the present invention. Specifically, this may refer to comprehensive therapeutic support activities that include processes such as reducing body weight by promoting the breakdown of already accumulated body fat or restoring abnormal lipid concentrations, such as hyperlipidemia or hypercholesterolemia, to normal levels, and normalizing biological metabolic functions and lowering the risk of related complications by alleviating symptoms such as fatty liver.

[0041] A composition comprising a complex extract of Artemisia annua, hops, and monk fruit according to the present invention as an active ingredient can be implemented in the form of a pharmaceutical composition or a food composition depending on its purpose and use.

[0042] The above “food composition” encompasses all forms of food intended to manage menopausal metabolic health and improve obesity and lipid metabolism abnormalities through daily consumption, and specifically may include health functional foods, functional beverages, general processed foods, etc.

[0043] The food composition of the present invention can be prepared in various formulations such as powder, granules, tablets, capsules, pills, liquids, jellies, bars, or gums, and may include food-grade acceptable carriers, excipients, diluents, sweeteners, or flavorings without limitation. In particular, monk fruit, which is one of the active ingredients, contains a high concentration of mogroside V and can simultaneously provide a natural sweetening effect, thus having the advantage of securing excellent sensory acceptability while minimizing the addition of separate sugars.

[0044] In addition, the food composition of the present invention can be prepared by further combining vitamins, minerals, amino acids, and dietary fiber for nutritional supplementation of menopausal women, and since the absorption rate of active ingredients in the body is maximized through stepwise temperature extraction and bioconversion technology, excellent lipid metabolism improvement effects can be expected even with a small amount of intake.

[0045] The above “pharmaceutical composition” may be used for the purpose of treating or assisting in diseases such as obesity, hyperlipidemia, hypercholesterolemia, dyslipidemia, and fatty liver caused by estrogen deficiency.

[0046] The pharmaceutical composition of the present invention may be formulated by including a pharmaceutically acceptable carrier, said carrier being one commonly used in formulation and may include, but is not particularly limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0047] The pharmaceutical composition of the present invention may be administered orally or parenterally. Formulations for oral administration include tablets, capsules, powders, granules, and liquids, while formulations for parenteral administration include injections and suspensions. In particular, the composition of the present invention exhibits pharmacological activity in which glycoside components are optimized and converted into the active form, the aglycone, through complex enzyme treatment at a specific weight ratio. This results in improved cell membrane permeability, which potently activates the AMPK and ACC pathways and significantly reduces the expression of SREBP-1c upon actual in vivo administration. Furthermore, since complex components derived from natural products are used, it is relatively safe from the side effects associated with conventional hormone replacement therapy (HRT) and can provide a suitable therapeutic adjuvant for menopausal women requiring long-term metabolic management.

[0049] Another aspect provides a method for preparing a composition for the prevention or improvement of obesity or lipid metabolism disorders, comprising: a) a step of preparing a raw material mixture by drying and grinding mugwort leaves, hops, and monk fruit, and then mixing them; b) a step of adding water, a C-1 to C4 lower alcohol, or a mixed solvent thereof to the raw material mixture and performing a first extraction at 20 to 35°C to obtain a first extract containing volatile essential oil components and heat-denatured active ingredients; c) a step of obtaining a second extract containing hydrophilic flavonoids by heating the mixture containing the first extract to 40 to 55°C to perform a second extraction; d) a step of obtaining a third extract containing insoluble triterpene glycosides and high molecular weight polysaccharides by heating the mixture containing the second extract to 60 to 80°C to perform a third extraction; and e) a step of obtaining a complex extract by filtering and concentrating the extract under reduced pressure. The same parts as described above apply equally to the above-described method.

[0050] The manufacturing method of the present invention is designed considering the thermodynamic stability and solubility characteristics of the active ingredients of each raw material, and by blocking the volatilization of aromatic components such as cineole and humulene in the first extraction step and increasing the extraction efficiency of mogroside, a sparingly soluble triterpene glycoside derived from monk fruit, in the third extraction step, it is possible to maximize the synergy of improving lipid metabolism through AMPK activation.

[0051] The manufacturing method of the present invention may further include the step of adding a complex enzyme composition to the complex extract of step e) and carrying out a bioconversion reaction at 40 to 55°C for 4 to 12 hours.

[0052] When the temperature is below 40℃ during the above bioconversion reaction, the enzyme activity is low, resulting in minimal conversion efficiency of the glycoside into an aglycone; when the temperature exceeds 55℃, the protein structure of the enzyme may be denatured, causing it to lose its catalytic function. Additionally, if the reaction time is less than 4 hours, it is difficult to reach the target concentration of the active ingredient, and if it exceeds 12 hours, the reaction reaches a saturated state, which may lead to reduced process efficiency or chemical degradation of the extracted ingredient.

[0053] In one experimental example, the pH of the complex extract according to one aspect was adjusted to a range of 4.5 to 5.5, and then a complex enzyme composition prepared to have a concentration of 0.5% (w / w) relative to the weight of the solids in the extract was added, and then an enzyme reaction was performed for 8 hours at a temperature of 50°C to convert the glycoside components in the extract into an aglycone, which is the active form.

[0054] In step a) above, the raw material mixture may be characterized by containing mugwort, hops, and monk fruit in a weight ratio of 2:1:1.

[0055] In addition, in the step of the bioconversion reaction, the complex enzyme composition may be characterized by naringinase, hesperidinase, and β-primeverosidase (β-) being mixed in a weight ratio of 4:4:2. Effects of the invention

[0057] The composition according to the present invention has the advantage of simultaneously securing major components such as cineole, eupatilin, and mogroside V in a maximized amount without thermal denaturation or volatile loss through a three-stage elevated heat extraction process designed considering the thermodynamic stability and solubility characteristics of the active ingredients of each raw material.

[0058] In particular, by mixing mugwort, hops, and monk fruit in specific weight ratios, an optimal chemical balance is achieved without mutual antagonism between the components, thereby exhibiting a unique technical synergy that effectively regulates the differentiation mechanism of adipocytes from the early stages. Furthermore, the present invention dramatically improves the absorption rate and bioavailability in the body by applying bioconversion technology using complex enzymes mixed in specific compositional ratios to optimize and convert glycoside components within the extract into their active form, aglycone.

[0059] This combination of technologies strongly activates the AMPK and ACC pathways to inhibit fat synthesis even when metabolism is reduced due to menopausal estrogen deficiency, and exhibits remarkable effects in fundamentally improving menopausal obesity and lipid metabolism abnormalities, such as inhibiting weight gain in vivo and restoring blood triglyceride and LDL cholesterol levels to normal levels. Specific details for implementing the invention

[0061] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0063] Preparation Example 1.

[0064] First, dried mugwort, hops, and monk fruit were ground to a size of 80 to 100 mesh using a grinder, and then 200g of mugwort, 100g of hops, and 100g of monk fruit were mixed in a weight ratio of 2:1:1 to prepare a raw material mixture. Ten times the weight of 70% (v / v) ethanol solvent was added to the raw material mixture, and a first extraction was performed by stirring for 2 hours while maintaining the temperature inside the extractor at 30℃ to prevent loss of volatile essential oil components.

[0065] Next, the internal temperature of the extractor was raised to 50℃ and a second extraction was performed for 3 hours to induce the elution of hydrophilic flavonoid components, and the temperature was raised again to 75℃ and a final third extraction was performed for 3 hours to elute insoluble triterpene glycosides and high molecular weight polysaccharide components within the monk fruit tissue.

[0066] After extraction was completed, the extract was sequentially filtered through a 100 mesh filter and a 5.0 μm microfilter to remove solids, and the obtained filtrate was concentrated under reduced pressure at a temperature of 45°C or lower to obtain a complex extract with a solid content of 20 Brix or higher (Example 1).

[0068] Preparation Example 2.

[0069] A complex extract was prepared by undergoing a stepwise temperature-raising extraction process from a mixture of mugwort, hops, and monk fruit using the same method as in Preparation Example 1 above.

[0070] Separately, a complex enzyme composition of the present invention was prepared by mixing naringinase, hesperidinase, and β-primeverosidase (β) in a weight ratio of 4:4:2. After adjusting the pH of the complex extract obtained in Preparation Example 1 to a range of 4.5 to 5.5, the complex enzyme composition prepared to a concentration of 0.5% (w / w) relative to the weight of the solids in the extract was added. Subsequently, an enzymatic reaction was carried out for 8 hours at a temperature of 50°C to convert the glycoside components in the extract into an aglycone, which is the active form.

[0071] After the reaction was completed, the composition was heated at 90°C for 10 minutes to inactivate the residual enzyme, and the final reaction product was freeze-dried to finally obtain a complex enzyme-treated composition in powder form (Example 2).

[0073] Experimental Example 1. Confirmation of Indicator Component Content According to Stepwise Temperature Increase Extraction Conditions and Extraction Solvent

[0074] First, we intended to verify the effect of the stepwise heating extraction process and extraction solvent according to the present invention on the extraction efficiency of the main active ingredient.

[0075] Dried mugwort, hops, and monk fruit were ground to a size of 80 to 100 mesh using a pin mill, and a raw material mixture was prepared by mixing 200g of mugwort, 100g of hops, and 100g of monk fruit in a weight ratio of 2:1:1. For each experimental group, purified water, 70% (v / v) ethanol, or 100% anhydrous ethanol was added as a solvent, and a first (30℃ for 2 hours), second (50℃ for 3 hours), and third (50~90℃ for 3 hours) process was performed in an extractor equipped with a reflux condenser, wherein the third temperature conditions were set to below 50℃, 75℃ (titration), and above 90℃ (Table 1).

[0076] All extracts were filtered and concentrated at 45°C or below using a rotary vacuum concentrator to be used as analytical samples, and the content of cineol, eupatilin, and mogroside V was quantitatively analyzed using an HPLC system.

[0077] [Table 1]

[0078]

[0079] [Table 2]

[0080]

[0082] As a result, in the case of Examples 1 and 2, which applied a 70% ethanol solvent and a three-stage extraction temperature of 75°C, the content of all indicator components was maximized with an optimized balance. Specifically, the volatile essential oil component cineole was found to be about 5.25 mg / g, the hydrophilic flavonoid eupatilin about 4.12 mg / g, and the sparingly soluble triterpene glycoside mogroside V about 12.80 mg / g, confirming an unrivaled extraction efficiency with a total content of about 22.17 mg / g. This demonstrates that the three-stage temperature-raising extraction process of the present invention accurately reflects the extraction critical point according to the physicochemical characteristics of each raw material component. In particular, the first low-temperature process fundamentally prevented the volatile loss of aromatic components with low boiling points, and the final third temperature-raising process was determined to have maximized the extraction yield by dramatically increasing the solubility of mogroside V, which is difficult to extract at medium-low temperatures with low energy levels.

[0083] On the other hand, even when using the same 70% ethanol, Comparative Example 1-1, where the 3rd stage temperature was below the range of the present invention, showed an extraction efficiency of the poorly soluble mogroside V of only 3.12 mg / g, while Comparative Example 1-2, where the temperature exceeded the range, showed a sharp decrease in content to 1.85 mg / g due to thermal damage to eupatilin. In addition, in Comparative Example 1-3, where high-temperature extraction was performed from the beginning, the cineol component volatilized, resulting in a content of only 1.15 mg / g, and Comparative Examples 1-4 to 1-11, where the solvent was changed to 100% ethanol or purified water, showed significantly lower overall component content compared to the examples due to differences in solvent polarity.

[0084] From this, it was finally confirmed that when the 70% ethanol solvent of the present invention and the three-step heating process conditions specified at 60 to 80°C are satisfied, an optimized extraction can be achieved to induce maximum elution of insoluble glycosides while maintaining the thermal stability of flavonoids, thereby enabling the simultaneous securing of all active ingredients at a maximized content.

[0086] Experimental Example 2. Confirmation of lipid accumulation inhibitory effect based on raw material blending ratio and enzyme treatment

[0087] We intended to confirm the difference in the inhibitory effect on lipid accumulation according to the raw material mixing ratio and whether or not enzyme treatment was performed according to the present invention.

[0088] First, 3T3-L1 cells (ATCC, USA), which are preadipocytes, were cultured in DMEM medium supplemented with 10% FBS in a 37°C 5% CO2 incubator. After 2 days, when the cells were completely confluenced, the medium was replaced with a differentiation-inducing substance, an MDI cocktail (0.5 mM IBMX, 1 μM Dexamethasone, 10 μg / mL Insulin), to induce adipocyte differentiation. At this time, samples of Examples 1 and 2 and Comparative Examples 2-1 to 2-5 with various formulation ratios were each treated at a concentration of 100 μg / mL and cultured for 8 days.

[0089] Subsequently, differentiated cells were fixed in 10% formalin for 1 hour, washed with purified water, and stained for 30 minutes using a solution of Oil Red O dissolved in 60% isopropanol. After washing the stained cells, lipid particle formation was observed using an inverted microscope. Finally, 100% isopropanol was added to elute the stain adsorbed onto the lipid globules, and the absorbance was measured at 520 nm using a microplate reader. The lipid accumulation inhibition rate was calculated as a percentage based on the absorbance of the untreated control group.

[0090] [Table 3]

[0091]

[0093] As a result, in the case of Example 1, which applied a specific 2:1:1 raw material mixing ratio in the present invention, an excellent lipid accumulation inhibition rate of approximately 46.24% was observed even without enzyme treatment, confirming significantly higher activity compared to Comparative Example 2-1, which had a simple uniform mixture, or Comparative Examples 2-2 and 2-3, which had different mixing ratios. In particular, in Comparative Examples 2-4 and 2-5, where the proportion of Artemisia annua was increased beyond the range of the present invention, a tendency for the lipid accumulation inhibition effect to decrease was observed. This demonstrates that the mechanism of early differentiation inhibition by eupatilin derived from Artemisia annua and the mechanism of metabolic activation by components derived from monk fruit and hops achieve an optimal chemical balance without mutual antagonism only at a specific weight ratio of 2:1:1, thereby exhibiting critical synergy.

[0094] In addition, Example 2, which included complex enzyme treatment, showed a significantly increased lipid accumulation inhibition rate of up to 80.99% and demonstrated an activity enhancement of approximately 1.7 times or more compared to Example 1. This is because the high concentration of glycoside components obtained through the stepwise heating process were largely converted into the active form, the aglycone, through bioconversion, thereby maximizing cell membrane permeability and binding affinity with target receptors. From this, it was confirmed that the combination of the raw material formulation ratio and the enzyme treatment process of the present invention can strongly inhibit the adipocyte differentiation mechanism from the early stages.

[0096] Experimental Example 3. Confirmation of lipid metabolism regulation and improvement effects in menopausal obese model mice

[0097] To confirm the effects of lipid metabolism regulation and improvement in menopausal obesity model mice, molecular biological mechanisms and biomarkers were analyzed using menopausal obesity model mice.

[0098] Specifically, estrogen deficiency was induced in 8-week-old female C57BL / 6 mice by ovariectomy (OVX). Following a one-week recovery period, the mice were fed a high-fat diet for eight weeks while the sample was administered orally daily. For mechanistic analysis, proteins were extracted from liver tissue after the experiment, and Western blot analysis was performed to quantify changes in the phosphorylation of AMPK and ACC, as well as the expression of SREBP-1c. Simultaneously, body weight was measured weekly during the experiment to calculate the increase, and serum triglyceride (TG) and LDL cholesterol concentrations isolated after the final blood collection were measured using a dedicated analysis kit to comprehensively evaluate the efficacy of improving lipid metabolism in vivo. The control group refers to a negative control group that was fed a high-fat diet for eight weeks after ovariectomy and received an equal amount of distilled water orally instead of the sample.

[0099] [Table 4]

[0100]

[0102] As a result, it was confirmed that Example 2, which applied the 4:4:2 weight ratio of the present invention, exhibited excellent metabolic regulatory ability by increasing p-AMPK activity by approximately 3.92 times and p-ACC activity by approximately 4.25 times compared to the control group, while significantly reducing the expression of SREBP-1c, a key transcription factor that induces lipid synthesis, to the level of 24.8%. This was determined to be the result of the efficacy of strongly triggering the AMPK activation pathway being maximized as a series of bioconversion processes, in which naringinase and hesperidinase primarily degrade the sugar chains of the substrate and β-primeverosidase completes the final conversion into active aglycones, were optimized at the 4:4:2 ratio.

[0103] Meanwhile, in the case of Comparative Examples 3-1 to 3-4, which had different composition ratios despite treating the same extract with enzymes, the p-AMPK activity level remained at half the level of Example 2, proving that the specific enzyme mixing ratio of 4:4:2 defined by the present invention is a critical value for most efficiently hydrolyzing complex glycosides in the extract to activate metabolic pathways.

[0104] [Table 5]

[0105]

[0107] In addition, it was confirmed that the groups administered Examples 1 and 2 to menopausal obesity model mice showed a significant improvement in body weight gain and blood lipid indicators compared to the control group. In particular, Example 2 recorded a body weight of 5.3g, which suppressed the body weight gain to approximately 65% ​​of the control group (15.2g), and demonstrated excellent efficacy in significantly restoring blood triglyceride and LDL cholesterol levels. This was determined to be due to the organic combination of high-concentration active ingredients obtained through the optimized three-stage heating process of the present invention and the conversion of active aglycones by complex enzyme treatment at a 4:4:2 ratio, thereby maximizing the efficiency of lipid metabolism in vivo.

[0108] On the other hand, in the case of Comparative Examples 3-1 to 3-4, in which the composition ratio of the complex enzyme falls outside the scope of the present invention, it was confirmed that despite undergoing enzyme treatment in the same manner as Example 2, the conversion efficiency of the active ingredient was low or the improvement in body weight and lipid levels was significantly lower than that of Example 2 due to conflicting interactions between the ingredients.

[0109] From this, it was confirmed that the stepwise temperature-raising extraction process and complex enzyme treatment with a specific composition ratio of the present invention have a significant effect in improving menopausal obesity and lipid metabolism abnormalities.

[0111] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 a) a step of preparing a raw material mixture by drying and grinding mugwort, hops, and monk fruit, and then mixing them; b) a step of obtaining a first extract containing volatile essential oil components and heat-denatured active ingredients by adding 10 times the weight of a 70% (v / v) concentration ethanol solvent to the raw material mixture and stirring while maintaining the temperature at 30°C to perform a first extraction; c) a step of obtaining a second extract containing hydrophilic flavonoids by raising the temperature of the mixture containing the first extract to 50°C to perform a second extraction; d) a step of obtaining a third extract containing insoluble triterpene glycosides and high-molecular-weight polysaccharides by raising the temperature of the mixture containing the second extract to 75°C to perform a third extraction; A method for preparing a composition for the prevention or improvement of obesity or lipid metabolism disorders, comprising the step of: e) filtering the extract and concentrating under reduced pressure at a temperature of 45°C or lower to obtain a complex extract; wherein the method further comprises the step of adding a complex enzyme composition to the complex extract of step e) and performing a bioconversion reaction at 50°C for 8 hours, and, after the bioconversion reaction is completed, heating at 90°C for 10 minutes to inactivate residual enzymes; and, after the heating is completed, freeze-drying the final reaction product to obtain it in powder form.

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