KR102691530B1 - Method for increasing anti-obesity compounds of fruits of Cudrania tricuspidata and a composition for improving and preventing obesity comprising the increased anti-obesity compounds - Google Patents

Abstract

The present invention discloses a method for enhancing an anti-obesity component in the fruit of the mulberry tree and a composition for improving and preventing obesity comprising the active component.
According to the method according to the present invention, 6,8-diprenylgenistein, an anti-obesity active ingredient, can be increased by 50% or more in the mulberry fruit. The fermented mulberry fruit according to the present invention significantly increases 6,8-diprenylgenistein, an anti-obesity active ingredient, and is therefore useful as a functional food material for improving and preventing obesity. A pill containing the fermented mulberry fruit according to the present invention is not only useful for improving and preventing obesity, but also has high convenience of intake.

Description

Method for increasing anti-obesity compounds of fruits of Cudrania tricuspidata and a composition for improving and preventing obesity comprising the increased anti-obesity compounds

The present invention relates to a method for enhancing an anti-obesity component in mulberry fruit and a composition for improving and preventing obesity comprising the active component, and more specifically, to a method for enhancing 6,8-diprenylgenistein (DPG), an anti-obesity component, by fermenting mulberry fruit with a Lactobacillus plantarum strain, and a functional food composition for improving and preventing obesity comprising the enhanced anti-obesity component.

Recently, the obese population is increasing due to excessive nutrient intake and decreased physical activity due to the increase in processed foods and eating out. The World Health Organization (WHO) classifies obesity as a disease rather than a phenomenon or symptom, and reported that the number of obese people worldwide will increase to 1.5 billion in 2015, becoming a serious health problem. The cause of obesity is not clearly known, but it is not caused by a single condition, but rather by various causes such as inappropriate eating and living habits, genetic factors, and decreased physical activity (Korean J. Food Science Nutrition 22(1)110-12.2009).

Obesity is a condition in which excessive body fat accumulates due to an imbalance between energy intake and expenditure, which increases the number and size of fat cells. Energy in the body is stored in fat cells in the form of triglycerides, and when energy sources are depleted, the stored fat is broken down into free fatty acids and glycerol and used as an energy source. However, excessive energy intake promotes the differentiation of fat cells and increases the amount of fat stored in the body, which is a direct cause of obesity (Biochem J. 1;435(3):723-32 2011). Obesity is a risk factor that increases the incidence of various diseases as well as changes in body shape due to fat accumulation in the viscera and abdomen. When visceral fat accumulates excessively, problems with sugar metabolism in the body occur, and symptoms such as abnormal hormone secretion and abnormal cytokine secretion occur. Obesity causes an increase in neutral fat and LDL cholesterol and a decrease in HDL cholesterol, which leads to abnormal fat metabolism in the body, reduces insulin receptors in tissues, and also reduces insulin sensitivity, which inhibits the transport of glucose into cells, causing hyperglycemia and diabetes. In addition, obesity is known to be closely related to the occurrence of metabolic diseases such as hyperlipidemia, cardiovascular disease, cancer, respiratory disorders, stroke, and osteoarthritis.

Since conventional anti-obesity drugs such as orlistat and sibutramin are known to have serious side effects such as vomiting, constipation, gastrointestinal disorders, and cardiovascular diseases, efforts to develop effective and safe anti-obesity substances are ongoing. Retinol, vitamin E, vitamin U, and rhizome extract have been reported as substances that inhibit fat cell differentiation (Patent Publication No. 10-2021-0078832), and there is a need for more research and development to develop anti-obesity ingredients made of natural substances that are safe and can be consumed continuously and to enhance them.

Accordingly, the inventors of the present invention have conducted continuous research to meet the needs of the prior art and have confirmed that 6,8-diprenylgenistein derived from mulberry fruit has anti-obesity activity and that when mulberry fruit is fermented with Lactobacillus plantarum strain, the anti-obesity component is significantly enhanced and thus the invention is useful as a food composition for improving and preventing obesity.

Accordingly, the purpose of the present invention is to provide a method for increasing 6,8-diprenylgenistein by fermenting mulberry fruit with a Lactobacillus plantarum strain.

Another object of the present invention is to provide a fermented product of mulberry fruit containing 6,8-diprenylgenistein promoted by the above method.

Another object of the present invention is to provide a functional food composition for improving and preventing obesity, which comprises a fermented product of the mulberry fruit.

In order to achieve the above object of the present invention, the present invention provides a method for increasing 6,8-diprenylgenistein by fermenting mulberry fruit with a Lactobacillus plantarum strain.

Cudrania tricuspidata B. is a deciduous small tree or shrub in the mulberry family that is native to Korea, China, and Japan. Cudrania tricuspidata leaves are used in oriental medicine to treat eczema, mumps, diabetes, chronic lumbago, and bruises, and the stems are mainly used to treat women's edema and blood clots, and the fruit is known to be used to clear heat and promote blood circulation. In addition, the fruit has been used in the private sector as a tonic, stroke, diuretic, and anthelmintic. However, it is not known that 6,8-diprenylgenistein, which has anti-obesity activity derived from the fruit of the Cudrania tricuspidata, is significantly enhanced when the fruit of the Cudrania tricuspidata is fermented with Lactobacillus plantarum strain.

In the present invention, 6,8-diprenylgenistein, a compound having anti-obesity activity derived from the fruit of the mulberry tree, has a structure represented by the following chemical formula 1:

[Chemical Formula 1]

In the present invention, fermentation is performed by fermenting the fruit of the mulberry tree with a Lactobacillus plantarum strain.

The fruit of the mulberry tree can be used preferably in powdered form, but is not limited thereto.

Preferably, the fermentation can be carried out by adding 3 to 7 times (v/w) of water to the mulberry fruit powder, inoculating the Lactobacillus plantarum culture solution at a concentration of 1 to 3% (v/w) based on the mulberry fruit powder, and fermenting at 25 to 40°C for 2 to 5 days.

If the inoculum amount of Lactobacillus plantarum strain is less than 1% (v/w), the fermentation rate may be delayed, if the fermentation temperature is less than 25℃, the fermentation period may be long, which may lead to contamination by bacteria, and if it exceeds 40℃, the growth of the strain may stop, and if the fermentation period is less than 2 days, the fermentation period may not be sufficient, which may result in low production of physiologically active substances, and if it exceeds 5 days, over-fermentation may occur, causing the produced physiologically active substances to be decomposed.

According to the present invention, when the fruit of the mulberry tree is fermented with the Lactobacillus plantarum strain, the content of 6,8-diprenylgenistein is increased by 50% or more (Test Example 1, Table 4, and Fig. 2).

According to the present invention, 6,8-diprenylgenistein derived from the fermented product of the mulberry fruit has excellent lipid accumulation inhibition activity without cytotoxicity (Test Examples 2 and 3, Figs. 3 to 5).

According to another object of the present invention, a fermented product of mulberry fruit containing 6,8-diprenylgenistein promoted by the above method is provided.

The fermented mulberry fruit according to the present invention contains 6,8-diprenylgenistein in a content increased by 50% or more compared to the mulberry fruit before fermentation.

According to another object of the present invention, a functional food composition for improving and preventing obesity is provided, which comprises a fermented product of the mulberry fruit.

The above functional food refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body according to the Health Functional Food Act, and the functionality may mean consumption for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.

In the present invention, the functional food may include conventional food additives, and the suitability of the food additives may be judged based on the specifications and standards for the relevant items in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

The functional food of the present invention can be used in various ways in foods and beverages for preventing or improving obesity, and for example, can be used in various foods, beverages, gum, tea, vitamin complexes, health functional food supplements, food additives, etc.

In the present invention, the functional food can be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powder, capsules, liquid solutions, and pills, and is preferably processed into a pill.

The above-mentioned ring-shaped functional food can be prepared by molding a mixture of the above-mentioned compound, excipient, binder, disintegrant, etc., by an appropriate method, and, if necessary, can be coated with white sugar or another appropriate coating agent, or can be coated with starch, talc, or an appropriate substance.

The ring according to the present invention may additionally contain auxiliary materials in addition to the fermented mulberry fruit according to the present invention.

In the present invention, auxiliary materials that can be used include psyllium husk, eucommia ulmoides, dried yul, aloe powder, bok berry, magnolia berry, gilt root, garlic, Ophiopogon japonicus, mugwort, almond seed, and peony root.

Preferably, the pill according to the present invention can be manufactured by mixing 5 wt% of fermented mulberry fruit, 15 wt% of psyllium husk, 15 wt% of Eucommia ulmoides, 15 wt% of dried yam, 15 wt% of aloe powder, 5 wt% of Nabokja berry, 5 wt% of Schisandra chinensis, 5 wt% of Platycodon grandiflorum, 5 wt% of garlic, 3.75 wt% of Ophiopogon japonicus, 3.75 wt% of Artemisia capillaris, 3.75 wt% of Aralia elata, and 3.75 wt% of Paeonia japonica.

According to the method of the present invention, 6,8-diprenylgenistein, an anti-obesity active ingredient in mulberry fruit, can be increased by 50% or more.

The fermented mulberry fruit according to the present invention significantly increases 6,8-diprenylgenistein, an anti-obesity active ingredient, and is therefore useful as a functional food material for improving and preventing obesity.

A pill containing a fermented product of mulberry fruit according to the present invention is not only useful for improving and preventing obesity, but is also highly convenient to consume.

Figure 1 is a chromatogram of a fermented product of mulberry fruit according to the present invention.
Figure 2 is a graph showing the results of analyzing the content of 6,8-diprenylgenistein (area of peak) in the composition of a fermented mulberry fruit prepared by a method according to the present invention. Control group (pre-fermented mulberry fruit), Example 1 (fermented mulberry fruit according to the present invention), Comparative Examples 1 and 2 (fermented mulberry fruit fermented with different strains).
Figure 3 is a graph showing the results of a cytotoxicity test of 6,8-diprenylgenistein, an anti-obesity active ingredient according to the present invention.
Figure 4 is a photograph showing the staining results of 3T3-L1 adipocytes treated with 6,8-diprenylgenistein, an anti-obesity active ingredient according to the present invention.
Figure 5 is a graph showing the results of analyzing the fat accumulation inhibition activity in 3T3-L1 adipocytes treated with 6,8-diprenylgenistein, an anti-obesity active ingredient according to the present invention.

Hereinafter, in order to help understand the present invention, the configuration and effects of the present invention will be described in more detail through specific examples. However, the following examples are merely provided to help understand the present invention more clearly, and the scope of the rights of the present invention is not limited by the following examples.

Manufacturing Example 1: Manufacturing of fermented mulberry fruit

Harvest and dry the fruits of the mulberry tree grown in Sancheong and Jinju. It was prepared by powdering (70-100 mesh).

The three lactic acid bacteria listed in Table 1 were obtained from the Korea Research Institute of Bioscience and Biotechnology (KCTC) and used for fermentation. The freeze-dried strains were inoculated onto MRS plate medium and cultured at the optimal growth temperature to obtain a single colony. The strains were stored by inoculating a single strain into MRS broth and culturing for 24 hours, then preparing a stock using glycerol and storing it in an ultra-low temperature freezer at 70℃. The strains were thawed and used whenever necessary. The thawed strains were subcultured once more and then used for inoculation.

Strain name Optimal growing temperature (℃) Source KCTC No. Comparative Example 1 Lactobacillus brevis 30 human feces 3498 Comparative Example 2 Lactobacillus casei 37 Cheese 3110 Comparative Example 3 Lactobacillus plantarum 30 silage 3103

After sterilizing the mulberry fruit powder at 80℃ for 20 minutes, the mixture was evenly mixed with sterilized distilled water equivalent to 3 times (v/w). Each seed cultured at 3× ¹⁰⁹ cfu/ml was inoculated at 1% (v/w) of the powder sample, and fermented for 72 hours at a low speed of 6-70 rpm at the optimal growth temperature in Table 1. After fermentation, the fermented product was completely dried below 60℃, powdered, and then analyzed as follows.

Test Example 1: Analysis of anti-obesity active ingredients

In Manufacturing Example 1, each fermented sample dried and powdered and the pre-fermented mulberry fruit powder (control example) were added 70% ethanol equivalent to 10 times (v/w), sonicated, filtered through a 0.45 ㎛ membrane filter, and then filtered through a 0.2 ㎛ filter immediately before device injection to prepare the analysis sample.

The 6,8-diprenylgenistein standard was precisely weighed, completely dissolved in methanol, filtered through a 0.2 ㎛ filter to prepare a standard stock solution, and diluted stepwise to prepare a standard calibration curve.

The high-performance liquid chromatography equipment used for component analysis of the analysis samples and standard materials was an Acquity UPLC H-class System (Waters, USA) equipped with a photodiode array (PDA) detector, and the analysis conditions were as shown in Table 2:

Parameter Condition Instrument Acquity UPLC H-class System Detector Acquity UPLC PDA detector Column BEH C18 (2.1 × 100 ㎜, 1.7 ㎛) Column Temp. 40℃ Absorbance 260 nm Injection Volume 2 uL Flow Rate 0.35 mL/min Mobile Phase A: 0.1% formic acid in water
B: acetonitrile Gradient Time(min) 0 19 20 25 A 95 20 95 95 B 5 80 5 5

The content of 6,8-diprenylgenistein, an anti-obesity active ingredient in the analysis sample, was calculated by substituting the peak area value on the chromatogram into the standard calibration curve to obtain the detection concentration, and then calculating the value using the formula in Table 3.

Indicator substance content (μg/g) = C × (a × b) / S C: Concentration of individual indicator substances in the test solution (μg/mL)
a: Total volume of test solution (mL)
b: Dilution factor
S: Sample weight (g)

The chromatogram for Example 1 (fermented mulberry fruit fermented with Lactobacillus plantarum) is shown in Fig. 1.

The 6,8-diprenylgenistein (DPG) content of the fermented products of Example 1, Comparative Example 1, and Comparative Example 2 compared to the control example (pre-fermented mulberry fruit powder) is shown in Table 4 and Figure 2:

DPG increase rate (%) compared to control Contrast example 100 Comparative Example 1 136 Comparative Example 2 125 Example 1 151

According to Table 4 and Figure 2, in the case of Example 1, it can be confirmed that the content of 6,8-diprenylgenistein increases by more than 50% compared to the unfermented mulberry fruit (control example).

Test Example 2: Adipocyte cytotoxicity analysis

Example 1 To confirm the effect of 6,8-diprenylgenistein, an anti-obesity active ingredient derived from fermented products, on preadipocytes themselves, cell viability was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colormetric assay, which measures the amount of living cells.

Specifically, the preadipocytes of Reference Example 1 were dispensed at a concentration of 1.0×10 ⁴ cells/well in a 96-well plate and cultured in a CO ₂ incubator (NU-4750G, NUAIRE) for 24 hours. Then, the samples were diluted to different concentrations (15.625, 31.25, 62.5, 125, 250 μM) of 6,8-diprenylgenistein extracted from the fermented product of Example 1 and cultured again for 24 hours. After culture, MTT (Promega, USA) reagent was added to each well, and after 4 hours of culture, the absorbance was measured at 490 nm using a spectrophotometer (SpectraMax M5, Molecular Devices, USA). The results are shown in Fig. 3.

As shown in Figure 3, 6,8-diprenylgenistein, an anti-obesity active ingredient of the present invention, was confirmed to have no cytotoxicity when treated at 15 to 250 μM.

Test Example 3: Analysis of adipocyte differentiation and fat accumulation inhibition ability

3T3-L1 adipocytes are cells derived from mouse embryonic fibroblast preadipocytes and have the property of differentiating into adipocytes when a differentiation-inducing substance such as insulin is added. Therefore, they are widely used in in vitro studies to study the effects of promoting adipocyte decomposition and inhibiting adipogenesis. In the present invention, in order to confirm the effect of the anti-obesity active ingredient derived from the fermentation product of Example 1 on inhibiting the differentiation of 3T3-L1 adipocytes, differentiation of preadipocytes was induced.

3T3-L1 (KCLB 10092.1) cells were used as a gift from the Korean Cell Line Bank. To differentiate 3T3-L1 preadipocytes into adipocytes, cells were cultured until confluence in a 24-well plate for cell culture. Differentiation was induced using differentiation induction media (MDI media) containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 ㎍/mL insulin, and 1 µM dexamethasone in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 units/mL penicillin-streptomycin (P/S, Gibco, USA). The cells were incubated for approximately 48 h at 37°C in a humidified CO2 atmosphere. After culturing in an incubator (5% CO2/95% air), the medium was replaced with insulin medium containing only insulin. At this time, samples diluted to different concentrations (15.625, 31.25, 62.5, 125, 250 μM) of 6,8-diprenylgenistein extracted from the fermented product of Example 1 were treated, and after culturing in an incubator under the same conditions for 48 hours again, the medium was replaced with DMEM medium supplemented with 10% FBS and 100 units/mL P/S every two days, and after confirming the formation of lipid droplets through an optical microscope, adipogenic differentiation was terminated.

Oil red O stains neutral fat, and because the lipid droplets that accumulate in the cytoplasm in a spherical shape during the process of adipocyte differentiation are neutral lipids, they can be stained, and thus the degree of lipolysis can be determined.

When differentiation into adipocytes was judged to be complete, the plate was washed once with cold phosphate buffered saline (PBS, Gibco, USA), fixed with 10% formalin at room temperature for more than 1 hour, and then washed twice with PBS to completely remove the formalin. After staining with Oil Red O dye (ORO stock solution: DW = 6:4) for 30 minutes, washed once with PBS, and then PBS was dispensed into each well. Intracellular lipid accumulation was observed under an optical microscope, and the resulting photograph is shown in Fig. 4.

After removing the PBS, the mixture was completely dried, the pigment was extracted with 100% isopropanol, transferred to a 96-well plate, and the absorbance was measured at 490 nm. The lipid accumulation inhibition ability was converted to % based on the control example (P) and shown in Figure 5.

As shown in Fig. 4, the lipid accumulation photographs in the Oil Red O staining solution confirm that lipid accumulation is significantly reduced as the concentration of 6,8-diprenylgenistein increases. Compared to the control example (P; cells differentiated into fat by treating with insulin without treating with the sample), lipid accumulation was inhibited in a concentration-dependent manner in differentiated 3T3-L1 adipocytes, and at concentrations of 125 and 250 μM, 6,8-diprenylgenistein inhibited lipid accumulation in 3T3-L1 cells almost similarly to the positive control example (N; normal cells not treated with insulin).

As shown in Figure 5, it can be confirmed that 6,8-diprenylgenistein at 125 and 250 μM reduces fat globule formation by more than 50% compared to the control example (P), and it can be confirmed that it is excellent in inhibiting fat differentiation as it appears at a similar level to the positive control example (N).

Manufacturing Example 2: Manufacturing of anti-obesity pills

After mixing 5 wt% of fermented mulberry fruit powder, 15 wt% of psyllium husk powder, 15 wt% of Eucommia ulmoides powder, 15 wt% of dried yul powder, 15 wt% of aloe powder, 5 wt% of Nabokja powder, 5 wt% of Schisandra chinensis powder, 5 wt% of Platycodon grandiflorum powder, 5 wt% of garlic powder, 3.75 wt% of Ophiopogon japonicus powder, 3.75 wt% of Artemisia capillaris powder, 3.75 wt% of Aquilegia chinensis powder, and 3.75 wt% of Peony root powder in a weight ratio, the mixture was made into pills by molding according to a conventional pill manufacturing method, dried at 65°C for 12 hours, and then packaged.

Claims (7)

A method for increasing 6,8-diprenylgenistein by fermenting mulberry fruit with Lactobacillus plantarum strain.
The above fermentation is performed by adding 3 to 7 times (v/w) of water to the powder of Kujibong fruit, inoculating the Lactobacillus plantarum culture solution at a concentration of 1 to 3% (v/w), and fermenting at 25 to 40°C for 2 to 5 days.
A method wherein the content of 6,8-diprenylgenistein in mulberry fruit is increased by more than 50% compared to before fermentation.
delete delete A fermented product of mulberry fruit containing 6,8-diprenylgenistein promoted by the method according to claim 1.
Functional food for improving and preventing obesity, containing fermented mulberry fruit according to Article 4.
delete A functional food for improving and preventing obesity, made into a pill form by mixing 5 wt% of fermented mulberry fruit according to Article 4, 15 wt% of psyllium husk, 15 wt% of Eucommia ulmoides, 15 wt% of dried yam, 15 wt% of aloe powder, 5 wt% of Nabokja berry, 5 wt% of Schisandra chinensis, 5 wt% of Platycodon grandiflorum, 5 wt% of garlic, 3.75 wt% of Ophiopogon japonicus, 3.75 wt% of Artemisia capillaris, 3.75 wt% of Peony root, and 3.75 wt% of Peony root.