KR20240025853A - Composition comprising extract of Solanum nigrum for immune-enhancement and anti-obesity - Google Patents

Abstract

The present invention relates to a composition for improving immunity and anti-obesity containing an extract of Camilla as an active ingredient. The extract of Camajung shows immune-promoting activity by inducing the production of TLR4/JNK-mediated immunomodulatory factors in macrophages, activating phagocytosis, and activating TLR4/JNK-mediated macrophage autophagy. In addition, the extract of Camilla inhibits lipogenesis by inhibiting the expression of PPARγ, CEBPα, FABP4, and Adiponectin in adipocytes, induces lipolysis by inhibiting Perilipin-1 and inducing phosphorylation of AMPK, and promotes adipocyte autophagy through LC3-II and SQSTM1/p62. It exhibits anti-obesity activity by suppressing excessive lipid accumulation in adipocytes by inducing browning of white fat through induction of predation and production of UCP-1, PGC-1α, and PRDM16. Accordingly, when applying the Camazhong extract of the present invention, it is possible to easily use a health functional food that shows comprehensive efficacy for immune enhancement and anti-obesity.

Description

Composition for immune enhancement and anti-obesity comprising extract of Solanum nigrum as an active ingredient {Composition comprising extract of Solanum nigrum for immune-enhancement and anti-obesity}

The present invention relates to a composition for improving immunity and anti-obesity containing Solanum nigrum extract as an active ingredient.

Humans are exposed to a variety of pathogenic microorganisms and viruses throughout their lives, but the host immune system protects the body from these attacks. The innate immune response is the first defense system that protects the human body from external harmful pathogens such as pathogenic microorganisms and viruses. Among immune cells responsible for the innate immune response, macrophages are representative immune cells that have phagocytosis and antigen presentation functions against pathogenic microorganisms or viruses. This action of macrophages is increased by pathogenic microorganisms or viruses and is increased by immune-enhancing factors such as interferon (IFN). Activated macrophages produce nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). It secretes immunostimulatory factors, which increase the phagocytosis of macrophages against infectious agents such as pathogenic microorganisms and viruses and cytotoxicity against cancer cells. Moreover, these immunostimulatory factors secreted by activated macrophages increase the activity of helper T cells and natural killer cells (NK cells), promote maturation of B cells and expansion of clones, and promote early immunity. It plays an important role in function. Therefore, it is known that inducing the activation of macrophages strengthens both the innate and adaptive immune systems and contributes to the improvement of the human body's immunity. Toll-like receptors (TLRs) of macrophages recognize foreign pathogens and induce activation of host defense mechanisms. Activation of these TLRs is known to activate both innate and adaptive immune responses. Among TLRs, TLR4 is known to be a major target that can improve immune function in patients with immune diseases, and drugs that stimulate TLR4 are actually used clinically.

Recent studies have reported that autophagy can increase T cell responses by regulating the functions of antigen presenting cells and T cells. In particular, activation of macrophage autophagy through TLR4 stimulation has been reported to enhance innate and adaptive immune responses by increasing macrophage antigen processing and presentation. In autophagy, microtubule-related protein 1A/1B-light chain 3 (LC3) has been used to investigate autophagosomes and autophagic activity and plays a role in recruiting cargo to autophagosomes. Because LC3-II formed from LC3-I is localized to autophagosomes, the amount of LC3-II is known to be directly proportional to the number of autophagosomes and autophagy-related structures. p62/sequestosome 1 (SQSTM1) is a selective autophagy receptor and is important for delivering diverse ubiquitinated cargo to the autophagic pathway. Therefore, increases in LC3-II and p62/SQSTM1 are known to be key indicators of autophagy.

Since diseases in the body often result in a state of reduced immune function, substances that can improve immune function in cancer patients, the elderly, etc. are required. To date, various treatments and adjuvants for the purpose of improving immunity have been developed and marketed, but the use of relatively safer adjuvants rather than treatments is suggested as a preferable direction due to side effects caused by long-term use. Substances that can enhance immune function can be obtained from chemical compounds or animal or plant sources. However, in the case of chemical compounds, the pharmacological mechanism may vary or toxic side effects may occur, and if pharmaceutical raw materials are obtained from animals, there is a risk of side effects such as the discovery of toxic substances that cause zoonotic viruses or rare incurable diseases. It is pointed out as In addition, since it is advantageous in terms of speed and cost to use plants rather than obtain substances using chemical compounds or animals, there is a demand for the development of natural immune-boosting substances derived from plants without side effects.

Obesity is a condition in which body fat is accumulated excessively to the extent that it has a detrimental effect on health. When the calories consumed from food exceed the calories consumed through physical activity, it is accumulated as body fat. For men, when body fat is more than 25% of body weight, and for women, when it is more than 30% of body weight, it is called obesity. Obesity is a condition in which excessive fat is accumulated in the body and is a risk factor that not only causes cosmetic problems but also causes various adult diseases. Obesity is a cause of fatty liver disease, hyperlipidemia, osteoarthritis, gallstones, high blood pressure, diabetes, and cardiovascular disease. Additionally, it is known that prostate cancer, rectal cancer, and colon cancer are associated with obesity in men, and breast, ovarian, and uterine cancer are known to be associated with obesity in women. In particular, it has been reported that obesity is an important risk factor for heart disease and can have a significant impact on the structure and function of the heart. As it is known that the risk of heart failure increases rapidly as obesity increases in childhood, it is necessary to address obesity in health management. This is emerging as an important issue. The causes of obesity include diseases and genetic factors such as abnormalities in hypothalamic function and energy metabolism, but most obesity is caused by lifestyle habits caused by excessive nutritional intake and reduced physical activity. Recently, due to Western eating patterns and convenience of life, the consumption of ready-to-eat foods has increased and caused lack of exercise, leading to a continuous increase in the incidence of obesity, and this trend is expected to become more severe over time. Recently, as society recognizes obesity as a disease, various pharmaceutical companies are accelerating the development of drugs to treat obesity. Xenical (Xenical TM, Roche Pharmaceuticals, Switzerland), a fat absorption inhibitor, is one of the most widely used obesity treatments worldwide. Xenical's ingredient orlistat binds to digested fat and inhibits absorption in the intestines, thereby excreting some of the fat content during meals. Other drugs that have been developed include Reductil TM (Abbott, USA) and Exolise TM (Atopama, France), which promote satiety. However, the commercialized drugs have been reported to have side effects including liver damage, gastrointestinal bleeding, bronchitis, and kidney stones, and are at risk of developing heart disease, respiratory disease, and neurological disease. Therefore, when using currently commercialized obesity treatments, there is a need to develop materials that have excellent obesity improvement effects with fewer side effects due to problems such as stability as mentioned above.

Obesity is characterized by excessive accumulation of lipid droplets in fat cells and an increase in white adipose tissue (WAT). WAT is known to be caused by an increase in the size of adipocytes (adipocyte hypertrophy) and the number of adipocytes (adipocyte hyperplasia). The increase in the number of adipocytes in WAT is induced by a process in which preadipocytes differentiate into mature adipocytes called adipogenesis. Therefore, because lipogenesis can lead to obesity, inhibition of lipogenesis is considered a target for obesity treatment. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer binding protein α (CEBPα) are known to be key adipogenic transcription factors during adipocyte differentiation. It has been reported that inhibition of PPARγ and CEBPα expression in an obese mouse model suppresses weight gain by reducing adipocyte differentiation and adipogenesis. Fatty acid binding protein 4 (FABP4) also plays an important role in the differentiation of preadipocytes into mature adipocytes and the accumulation of lipid droplets. FABP4 is overexpressed during adipogenesis and autonomously accounts for 6% of total cytoplasmic proteins in mature adipocytes. do. Overexpression of FABP4 promotes the development of obesity and type 2 diabetes, hypertension, and cardiovascular disease caused by obesity. Additionally, adiponectin secreted from adipocytes promotes adipocyte differentiation and induces the accumulation of lipid droplets in adipocytes.

Lipolysis is a catabolic process of triacylglycerol (TAG) stored in lipid droplets in adipose tissue. During lipolysis, TAG is hydrolyzed into diacylglycerol (DAG), monoacylglycerol (MAG), fatty acids (FA), and glycerol. Since a decrease in lipolysis is frequently observed in obesity, reducing stored fat by promoting the hydrolysis of TAG through lipolysis is considered a major target for obesity treatment. Adipose triacylglycerol lipase (ATGL) is an enzyme that hydrolyzes TAG into DAG and FA, and ATGL deficiency has been reported to reduce lipolysis and cause lipid accumulation in adipose tissue. Hormone-sensitive lipase (HSL) is known to be an enzyme involved in the hydrolysis of TAG to DAG and DAG to MAG. In addition to ATGL and HSL, an important factor involved in lipolysis is Perilipin-1. Perilipin-1 surrounds lipid droplets present in adipocytes, and Perilipin-1 deficiency promotes lipolysis and reduces WAT. In line with this, research has shown that overexpression of Perilipin-11 inhibits TAG hydrolysis and increases TAG storage. Therefore, inhibition of perilipin-1 is being used as a new molecular target for the treatment of obesity.

Adipose tissue can traditionally be divided into white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is known to store excess energy in the form of TAG, while BAT is known to dissipate excess energy as heat. Therefore, increasing BAT is known to be effective in reducing body fat because BAT burns fat and releases energy as heat. AMP-activated protein kinase (AMPK), known as an important metabolic sensor and regulator of the body's energy balance, activates BAT by inducing WAT browning, inhibiting fatty acid synthesis, and promoting fatty acid oxidation to maintain mitochondrial homeostasis. do. Uncoupling protein 1 (UCP-1), which is mainly expressed in BAT, is located in the inner mitochondrial membrane and plays a role in releasing energy as heat by deactivating the respiratory chain in ATP synthesis (Cannon and Nedergaard, 2004). Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), which regulates mitochondrial biogenesis and respiration, is known to be involved in thermogenesis in BAT, and expression of PGC-1α in WAT induces BAT. PRDM16 is a key factor determining the development of brown fat and promotes the browning process of white adipose tissue. In fact, there are reports that an increase in energy expenditure was observed in animal models transgenic with PRDM16.

Autophagy is a catabolic process in which damaged cytoplasmic organelles are removed or reused through degradation. Damaged cytoplasmic organelles are isolated inside double-membrane vesicles, which are autophagosomes, and the autophagosomes fuse with lysosomes to decompose the damaged cytoplasmic organelles. This autophagy has been reported to degrade cytoplasmic lipid droplets in lipid metabolism and reduce triacylglycerol stored in lipid droplets. Additionally, continuous activation of autophagy inhibits the maturation of adipocytes through the lipolysis mechanism. These suggest that induction of adipocyte autophagy may reduce the accumulation of excessive lipid droplets in mature adipocytes.

Solanum nigrum is an annual herbaceous plant from the Solanaceae family that grows throughout Korea and is widely used in oriental medicine for treating colds, bronchitis, nephritis, high blood pressure, jaundice, and boils. The whole plant contains various ingredients such as solanine, phenol, flavonoids, tannin, rutin, and vitamins A and C, and is known to have anti-inflammatory, blood sugar-lowering, and anti-cancer effects. It is also known to have anti-inflammatory, anti-hypertensive, and anti-cancer effects. It has been reported to have an antibacterial effect that inhibits the growth of.

Accordingly, while researching the various physiological activities of the Blackthorn extract, the present inventors confirmed that the Blackberry extract had immune-boosting and anti-obesity effects and completed the present invention.

Republic of Korea Patent Publication No. 10-2015-0071282 (Title of the invention: Pharmaceutical composition or food composition with TGR5 agonist activity, Applicant: Korea Food Research Institute, Publication date: June 26, 2015) Republic of Korea Patent Publication No. 10-2005-0107257 (Title of the invention: Processed food with added kimchi, Applicant: Lee Il-soo, Publication date: November 11, 2005)

The purpose of the present invention is to provide a composition for improving immunity and anti-obesity containing Solanum nigrum extract as an active ingredient.

The present invention relates to a composition for improving immunity and anti-obesity containing Solanum nigrum extract as an active ingredient.

The extract of Camilla may be an extract of a mixture of leaves and stems of Camilla. The leaves and stems may be mixed at a weight ratio of 1:0.5 to 1:2. Additionally, the Camajung extract may be a water extract. Preferably, it may be a filtrate obtained by adding black pepper to water, stirring at 4 to 80° C. for 1 to 24 hours, and then filtering it.

The extract of Camajung may have minimal cytotoxicity.

When preparing the extract of the present invention, the filtrate can be dried and powdered through conventional drying methods such as freeze-drying, hot air drying, and spray drying.

The extract of Camajung is characterized by increasing the production of immunomodulatory factors or activating phagocytosis in macrophages.

In order to be used as an immune-boosting and anti-obesity composition, the extract of Camilla is preferably treated at an effective concentration of 12.5 to 100 μg/ml. The extract of Camazhong induces autophagy in macrophages.

In addition, the Camajung extract can induce inhibition of lipid formation in adipocytes, activation of lipolysis, activation of adipocyte autophagy, or activation of browning of white fat. Accordingly, the extract can induce browning activation of white fat and improve energy consumption and energy metabolism.

Accordingly, the extract of the present invention can be applied to immune-boosting and anti-obesity food compositions, health functional foods, pet feed compositions, anti-obesity pharmaceutical compositions, etc.

The extract of the present invention is also prepared by dissolving it in water and using at least one solvent selected from the group consisting of n-hexane, methylene chloride, acetone, chloroform, ethyl acetate, and n-butanol, as a common method in the art. Thus, it can be additionally fractionated and prepared as a fraction.

As a device for extracting the extract or fraction of the above-mentioned Blackberry extract, a conventional extraction device, an ultrasonic grinding extractor, or a fractionator can be used. The solvent can be removed from the Camajung extract prepared in this way by hot air drying, reduced pressure drying, or freeze drying. In addition, the extract or fraction of the cauliflower can be purified and used using column chromatography.

The extract of the soybean root is obtained by using known methods used for separation and extraction of plant components, such as extraction with organic solvents (alcohol, ether, acetone, etc.), distribution of hexane and water, and column chromatography, according to commercial methods. It can be used after fractionation or purification using a combination of methods.

The chromatography includes silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, and medium pressure liquid chromatography. chromatography), thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

In addition, the present invention provides a pharmaceutical composition for improving immunity and anti-obesity containing an extract of Camo japonicum. The extract of Camilla may be added in an amount of 0.001 to 100% by weight to the pharmaceutical composition of the present invention.

The pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by adding at least one excipient, for example, starch, calcium carbonate, sucrose, or lactose to the Camilla extract of the present invention. It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection. The extract of the present invention has almost no toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

In addition, the present invention provides a health functional food for immunity promotion and anti-obesity containing a Camazhong extract and a foodologically acceptable food supplement. The extract of Camazoni can be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes the form of tablets, capsules, pills, or liquid, and foods to which the extract of the present invention can be added include, for example, various drinks, meat, sausages, bread, candy, These include snacks, noodles, ice cream, dairy products, soups, electrolyte beverages, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

The present invention relates to a composition for improving immunity and anti-obesity containing an extract of Camilla as an active ingredient. The extract of Camajung shows immune-promoting activity by inducing the production of TLR4/JNK-mediated immunomodulatory factors in macrophages, activating phagocytosis, and activating TLR4/JNK-mediated macrophage autophagy. In addition, the extract of Camilla inhibits lipogenesis by inhibiting the expression of PPARγ, CEBPα, FABP4, and Adiponectin in adipocytes, induces lipolysis by inhibiting Perilipin-1 and inducing phosphorylation of AMPK, and promotes adipocyte autophagy through LC3-II and SQSTM1/p62. It exhibits anti-obesity activity by suppressing excessive lipid accumulation in adipocytes by inducing browning of white fat through induction of predation and production of UCP-1, PGC-1α, and PRDM16. Accordingly, when applying the Camazhong extract of the present invention, it is possible to easily use a health functional food that shows comprehensive efficacy for immune enhancement and anti-obesity.

Figure 1 shows the results of confirming the effect of the mixed extract (SNAP) of the leaves and stems of Capsicum japonicus (SNAP) under each extraction condition (temperature, time, and solvent) on the immune-enhancing factors NO, iNOS, IL-β, and TNF-α in macrophages. indicates.
Figure 2 shows the results of confirming NO production, gene expression of iNOS, COX-2, IL-β, IL-6, and TNF-α by treating macrophages with mixed extracts (SNAP) of the leaves and stems of Camilla at different concentrations.
Figure 3 shows the results of inducing activation of phagocytosis by treating macrophages with mixed extracts of leaves and stems (SNAP) of Capsicum ginseng at different concentrations.
Figure 4 shows that in macrophages, the mixed extract (SNAP) of the leaves and stems of Blackberry is treated with a TLR2/4 inhibitor, and the extract induces the expression of TLR4 to produce NO, iNOS, COX-2, IL-β, and IL-6. and the results of confirming gene expression of TNF-α are shown.
Figure 5 shows the results of confirming the gene expression of NO production, iNOS, COX-2, IL-β, IL-6, and TNF-α after treating macrophages with a MAPK inhibitor to inhibit various signaling pathways, showing that JNK is It was confirmed that it is a major upstream kinase involved in the production of immune-enhancing factors by mixed extracts of leaves and stems (SNAP).
Figure 6 shows the results of confirming through protein expression that the mixed extract (SNAP) of the leaves and stems of Capsicum ginseng phosphorylates JNK in macrophages and induces JNK activation through TLR4.
Figure 7 shows that the mixed extract of Camazum leaves and stems (SNAP) activates autophagy in macrophages through activation of TLR4 and JNK.
Figure 8 shows the results showing that lipid accumulation was suppressed by treating adipocytes with a mixed extract (SNAP) of the leaves and stems of Blackberry under different extraction conditions.
Figure 9 is a result showing that the mixed extract of the leaves and stems of Camilla (SNAP) inhibits lipid accumulation in adipocytes in a concentration-dependent manner.
Figure 10 confirms the efficacy of the mixed extract of blackcurrant leaves and stems (SNAP) to inhibit adipogenesis, induce lipolysis, induce autophagy of adipocytes, and induce browning of adipocytes. Shows the results.
- In each of the drawings of FIGS. 1 to 10, the gene/protein expression image results were quantified and presented in graphs.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the content introduced here is thorough and complete, and to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1: Preparation of Capsicum ginseng extract (stem and leaves)>

In order to prepare the Camazhong extract of the present invention, the Camazhong extract was prepared using distilled water as an extraction solvent. As the extraction raw material, a mixture of the stems and leaves of Capsicum ginseng was used.

Specifically, the stems and leaves of Capsicum ginseng were washed 2-3 times with distilled water, dried in a hot air dryer, mixed with equal amounts of stems and leaves, pulverized in a grinder, and stored at -20°C. Water equivalent to 20 times the volume was added to 10 g of the mixture of the stems and leaves of the sesame seeds, and extraction was performed at a temperature range of 4°C to 80°C and a time period of 1 hour to 24 hours to obtain each extraction liquid. Afterwards, the obtained liquid was filtered and freeze-dried to obtain the final mixed extract (SNAP) of the leaves and stems of Camazhong.

<Comparative Example 1. Preparation of comparative extract>

An extract derived from Camilla was prepared according to the method of Example 1, but the raw material sample, solvent, extraction temperature, and extraction time were used as shown in Table 1 below.

raw material sample Extraction solvent Extraction time (hour) Extraction temperature (℃) Capsicum leaves and fruit (1:1-w:w) water 6 80 blackcurrant fruit water 6 80 blackcurrant leaves water 6 80 Stem of black crayfish water 6 80 Blackcurrant leaves and stems Ethanol 50% (v/v) aqueous solution 6 30 Blackcurrant leaves and stems Ethanol 30% (v/v) aqueous solution 6 30 Blackcurrant leaves and stems Ethanol (99%) 6 30

<Experimental Example 1: Cell culture and confirmation of cytotoxicity of Camazhong extract>

Mouse macrophages, RAW264.7, and preadipocytes, 3T3-L1, were purchased from American Type Culture Collection (Manassas, VA, USA). RAW264.7 and 3T3-L1 cells were grown in Dulbecco's Modified Eagle Medium (DMEM)/F-12 1:1 Modified Medium (Lonza, Walkersville, MD, USA) with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA). ), 100U/ml penicillin and 100㎍/ml streptomycin (Gibco BRL, Grand Island, NY, USA) were used to culture at 37°C and 5% CO ₂ .

Before the experiment, all extracts from the cayenne pepper were dissolved in sterilized water and used.

Meanwhile, the MTT assay was used to evaluate cytotoxicity. This assay uses yellow water-soluble tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide] ( It uses the principle of reducing MTT) into water-insoluble dark purple MTT formazan crystals, and is a method of evaluating cytotoxicity by measuring the absorbance of the crystals at an appropriate wavelength (mainly 500-600 nm).

First, RAW 264.7 cells were distributed into 96 wells at a concentration of 1 × 10 ⁵ cells/well and cultured for 24 hours. Then, the extracts for each condition were treated at concentrations of 0, 50, 100, and 200 ㎍/mL, respectively, and cultured for 24 hours. After adding MTT at a concentration of 1 mg/mL and reacting in an incubator at 37°C for 2 hours, dimethyl sulfoxide (DMSO) was added and the absorbance was measured at 570 nm using a microplate reader.

As a result, it was found that in all extracts (temperature, time) of the mixture of stems and leaves of Capsicum ginseng prepared in Example 1, more than 95% of the cells survived at all concentrations and did not exhibit cytotoxicity.

In comparison, among the extracts of Comparative Example 1, the single extract of the fruit of the Red Capsicum or the mixed extract of the leaves and fruits had a cell viability of less than 60% at a concentration of 50 μg/mL or more, which was confirmed to be cytotoxic. This means that the fruit of the Blackberry is known to be toxic and inedible, so it can be seen that toxic substances contained in the fruit have been introduced into the extract.

In addition to this, no toxicity was found in the water extract of the leaves of Camilla, the water extract of the stems of Camilla, the 50% (v/v) ethanol aqueous solution extract of the leaves and stems, and the ethanol extract of the leaves and stems, like each extract in Example 1.

<Experimental Example 2: Measurement of immunomodulatory agent production-inducing activity of mixed extracts of leaves and stems of Camilla>

The mixed extract (SNAP) of the leaves and stems of Blackcurrant root contains the immune regulators NO (nitric oxide), iNOS (inducible nitric oxide synthase), COX-2 (Cyclooxygenase-2), IL-1β (Interluekin 1 beta), IL- To determine whether the production of Interleukin 6 (Interleukin 6) and TNF-α (Tumor necrosis factor alpha) was increased, it was measured by Griess analysis and RT-PCR.

For Griess analysis to measure NO production, first, RAW264.7 cells (2 × 10 ⁵ cells/well) were cultured in a 12-well plate for 24 hours, and then treated with mixed extract (SNAP) of Solanum leaves and stems. and cultured for an additional 24 hours. Afterwards, 100 μl of cell culture medium was taken and mixed with an equal amount of Griess reagent (Sigma Aldrich) and reacted at room temperature for 15 minutes. After reaction, the absorbance was measured at 540 nm using a UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to evaluate the effect of the mixed extract of Capsicum ginseng leaves and stems (SNAP) on the expression of iNOS, COX-2, IL-1β, IL-6, and TNF-α. carried out. For this purpose, RAW264.7 cells (2 × 10 ⁵ cells/well) were cultured in a 12-well plate for 24 hours, then treated with mixed extract of blackthorn leaves and stems (SNAP) and cultured for an additional 24 hours. After all treatments were completed, total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA was analyzed quantitatively. Then, cDNA was synthesized from 1 μg of total RNA using the Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA). Afterwards, PCR was performed using the PCR Master Mix Kit (Promega, Madison, WI, USA) and the primers shown in Table 2. PCR results were visualized using agarose gel electrophoresis. The density of mRNA bands was calculated using the software UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA).

primer name base sequence iNOS F 5'-ttgtgcatcgacctaggctggaa-3' iNOS R 5'-gacctttcgcattagcatggaagc-3' COX-2F 5'-gtactggctcatgctggacga-3' COX-2R 5'-caccatacactgccaggtcagcaa-3' IL-1βF 5'-ggcaggcagtatcactcatt-3' IL-1βR 5'-cccaaggccacaggtattt-3' IL-6F 5'-gaggatacactcccaacagacc-3' IL-6 R 5'-aagtgcatcatcgttgttcataca-3' TNF-α F 5'-tggaactggcagaagaggca-3' TNF-α R 5'-tgctcctccacttggtggtt-3' GAPDH F 5'-ggactgtggtcatgagcccttcca-3' GAPDH R 5'-actcacggcaaattcaacggcac-3'

As a result, as shown in Figures 1a and 1b, the mixed extract of leaves and stems (SNAP) of Capsicum japonicus significantly increased the immune regulators NO, iNOS, IL-1β, and TNF-α in the extracts at each temperature and time. increased the production of

However, as shown in Figure 1c, unlike when extracted with 100% distilled water, the mixed extract (SNAP) of the leaves and stems of Camo japonicum contains ethanol, NO, iNOS, The activities of IL-1β, IL-6, and TNF-α were very minimal.

Based on these results, additional research was conducted using a mixed extract (SNAP) of the leaves and stems of Capsicum ginseng extracted for 6 hours at 80°C, which has the best immune-boosting ability using water as a solvent. The same treatment experiment was performed, and as shown in Figures 2a and 2b, the mixed extract (SNAP) of the leaves and stems of Blackberry extracted at 80°C for 6 hours concentration-dependently increased the levels of immune regulators NO, iNOS, COX-2, It can be confirmed that the production of IL-1β, IL-6, and TNF-α is increased.

<Experimental Example 3: Measurement of the effect of mixed extracts of leaves and stems of Capsicum ginseng on macrophage phagocytosis>

Immune modulators secreted by macrophages are known to increase phagocytosis of macrophages, and phagocytosis of macrophages is used as an indicator of macrophage activation. Therefore, Neutral red uptake assay was performed to evaluate the effect of mixed extract of Camajung leaves and stems (SNAP) on phagocytosis of RAW264.7 cells.

Specifically, RAW264.7 cells (2 × 10 ⁵ cells/well) were cultured in a 12-well plate for 24 hours, then treated with mixed extracts of black and white leaves and stems (SNAP) depending on the concentration, and incubated for an additional 24 hours. cultured. After 24 hours, each cell was washed three times with 1xPBS, then 1 ml of 0.01% neutral red solution was added to each cell and cultured for 2 hours. Afterwards, each cell was washed three times with 1xPBS, and then 1 ml of cell lysis buffer (ethanol acid:acetic acid = 1:1) was added to elute the neutral red solution absorbed by the cells. Absorbance was measured at 540 nm using a UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

As a result, as shown in Figure 3, the mixed extract of Camajung leaves and stems (SNAP) was found to significantly increase macrophage phagocytosis.

<Experimental Example 4: Confirmation of receptors related to the production of immune modulators induced by Camazum alla extract>

Since it was confirmed that the mixed extract (SNAP) of the leaves and stems of Camilla of the present invention increases the production of immune modulators, experiments related to major receptors related to macrophage activation were conducted to trace the mechanism. To this end, since Toll-like receptor 2/4 (TLR2/4) is known to be a major receptor involved in macrophage activation, we investigated the role of TLR2/4 on the production of immunomodulators induced by mixed extract of blackthorn leaves and stems (SNAP). The effect was evaluated.

Specifically, in RAW264.7 cells, TLR2 or TLR4 was inhibited with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor), and then treated with a mixed extract (SNAP) of Solanum leaves and stems for 24 hours. NO levels were analyzed by Griess analysis, and NOS, COX-2, IL-1β, IL-6, and TNF-α were analyzed by RT-PCR.

As a result, as shown in Figures 4a and 4b, TLR2 inhibition by C29 inhibits NO, NOS, COX-2, IL-1β, IL-6, and TNF-α by the mixed extract of blackthorn leaves and stems (SNAP). Although there was no effect on the production of production was significantly reduced.

Therefore, it is proven that the mixed extract of Solanum leaves and stems (SNAP) increases the production of immune modulators by inducing the expression of Toll-like receptor 4 (TLR4) in macrophages.

<Experimental Example 5: Confirmation of upstream kinases involved in the production of immunomodulators induced by mixed extracts of leaves and stems of Capsicum ginseng>

Since it is known that the activation of TLR4 induced by the mixed extract (SNAP) of the leaves and stems of the present invention induces the production of immune modulators in macrophages by activating the MAPK (mitogen-activated protein kinases) signaling pathway, macrophages We analyzed whether MAPK signaling is involved in the production of immune modulators mediated by the mixed extract of leaves and stems of Blackthorn (SNAP).

Specifically, RAW264.7 cells were pretreated with PD98059 (40μM), an ERK1/2 inhibitor, SB203580 (40μM), a p38 inhibitor, and SP600125 (40μM), a JNK inhibitor, before treating RAW264.7 cells with the mixed extract of leaves and stems (SNAP). After inhibiting each signaling pathway, the mixture was treated with a mixed extract (SNAP) of Blackthorn leaf and stem for 24 hours. NO levels were analyzed by Griess analysis, and NOS, COX-2, IL-1β, IL-6, and TNF-α were analyzed by RT-PCR.

As a result, as shown in Figures 5a and 5b, regardless of whether the mixed extract of Capsicum ginseng leaves and stems (SNAP) inhibits ERK1/2 and p38 signals in macrophages, NO, NOS, COX-2, IL-1β, IL-6 and TNF-α production were induced.

However, when JNK signaling was inhibited, the production of NO, NOS, COX-2, IL-1β, IL-6, and TNF-α produced by the mixed extract of blackthorn leaves and stems (SNAP) was reduced.

Therefore, it can be confirmed that the JNK signal is the main upstream kinase involved in the production of immune modulators induced by the mixed extract of leaves and stems of Blackthorn (SNAP).

<Experimental Example 6: Confirmation of JNK activation through TLR4 mediated by mixed extracts of leaves and stems of Blackberry>

Since it was confirmed through gene expression that the production of immune modulators was reduced due to JNK inhibition in macrophages treated with the mixed extract of leaves and stems of Blackthorn (SNAP) of the present invention, it was confirmed that the mixed extract of leaves and stems of Blackthorn (SNAP) activated JNK. We decided to confirm whether it was induced normally through protein expression using Western blotting. For this purpose, in addition to the extract, TAK-242TLR4 inhibitor) was also treated with the experimental group cells.

For the experiment, cells treated with mixed extracts of Solanum leaves and stems (SNAP) were washed three times with cold 1xPBS and then incubated with protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich). ) and reacted at 4°C for 30 minutes. Cells reacted with RIPA buffer were centrifuged at 15,000 rpm for 10 minutes at 4°C, the supernatant was taken, and proteins were analyzed using a BCA (bicinchoninic acid; BCA) protein analysis kit (Thermo Fisher Scientific, Waltham, MA USA). Analyzed quantitatively.

Afterwards, proteins were separated on SDS-PGAE and transferred to PVDF membrane. Membranes were reacted in blocking buffer (0.05% Tween 20 (TBS-T) containing 5% skim milk) for 1 hour at room temperature. Next, the PDVF membrane was washed with TBS-T buffer and then treated with specific primary antibodies in 5% BSA in 0.05% TBS-T and placed at 4°C for 16 hours. After primary antibody treatment, the PVDF membrane was washed with TBS-T buffer, and the secondary antibody was subsequently treated with blocking buffer and stirring at room temperature. After washing the PVDF membrane with TBS-T buffer, chemiluminescence was performed using ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and LI-COR C-DiGit Blot Scanner (Li-COR Biosciences, Lincoln, USA). NE, USA) was used to visualize it. The density of the bands in the Western blot was calculated using UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA).

As a result, as shown in Figures 6a and 6b, it was confirmed that the mixed extract of Camazum leaves and stems (SNAP) phosphorylates JNK starting 15 minutes after treatment in macrophages.

In addition, inhibition of TLR4 by TAK-242 significantly attenuated the mixed extract of blackthorn leaves and stems (SNAP)-induced JNK phosphorylation.

Accordingly, it can also be proven through protein expression that the mixed extract of the leaves and stems of Blackberry (SNAP) induces JNK activation through TLR4.

<Experimental Example 7: Confirmation of macrophage autophagy induction by mixed extract of leaves and stems of Capsicum ginseng>

TLR-mediated autophagy has been reported to promote immune responses through increased antigen processing and presentation in antigen-presenting cells. Additionally, it has recently been reported that stimulation of autophagy increases T cell responses by regulating the functions of both antigen presenting cells and T cells. Therefore, autophagy promoters are considered potential adjuvant candidates to enhance immune responses.

Accordingly, in order to confirm whether the mixed extract (SNAP) of the leaves and stems of Camilla of the present invention has an autophagy activation effect, the expression levels of LC3 and p62/SQSTM1 were determined through Western blotting. The conversion from LC3-I to LC3-II is used as a major indicator of autophagy activation. In addition, p62/SQSTM1 is known to promote the formation of autophagosomes by interacting with LC3.

As a result of the experiment, as shown in Figures 7a and 7b, the production of LC3-II increased from 15 minutes after treatment of macrophages with the mixed extract of leaves and stems of Blackthorn (SNAP), and p62/SQSTM1 (Sequestosome1) increased from 3 hours. The expression also began to increase. In addition, the expression of LC3-II and p62/SQSTM1 was increased in a concentration-dependent manner with respect to the extract. These results confirm that the mixed extract of leaves and stems (SNAP) of Camilla leaves induces autophagy in macrophages.

<Experimental Example 8: Confirmation of lipid accumulation inhibitory activity of mixed extracts of leaves and stems of Camilla>

3T3-L1 cells, which are preadipocytes, were distributed in a 6-well plate at 1 × 10 ⁵ per well, cultured for 6 days, and then differentiated in DMI medium (Adipogenic medium). MDI medium was used as 10% FBS/DMEM treated with 0.5 mM IBMX, 1 μM dexamethasone, and 1 μg/ml insulin. This point was considered differentiation day 0. After 2 days of differentiation, the medium was replaced with 1㎍/mL insulin medium, and after 2 days of culture, the medium was maintained with 10% FBS medium for up to 8 days. A mixed extract (SNAP) of leaves and stems of Camilla was added on day 0 of differentiation and treated for 8 days. After 8 days of differentiation, the degree of adipocyte differentiation and the degree of inhibition of differentiation caused by the mixed extract of black and white leaves and stems (SNAP) were analyzed through Oil Red O staining. After inducing adipocyte differentiation, the cells that had been treated with the mixed extract of the leaves and stems of Blackberry (SNAP) were washed with 1× phosphate buffered saline (PBS), then added with 10% (w/v) formalin and fixed at room temperature for 1 hour. Formalin was removed, each well with cells cultured was washed with 60% (v/v) isopropanol, and the isopropanol was completely blown off in the hood. Oil Red O solution was added to the dried cell culture well, left for 10 minutes, washed with distilled water, and adipocyte differentiation was confirmed by microscopic imaging. The stained cell culture wells were dissolved in 100% isopropanol and the absorbance was measured at 500 nm.

As a result, as shown in Figure 8a, the lipid formation inhibitory activity of the mixed extract (SNAP) of the leaves and stems of Camilla increased as the extraction temperature increased. At this time, it was confirmed that in the case of extracts extracted under conditions of 60-80°C, lipid accumulation was suppressed by up to 60-70% compared to the control group. In the same context, as shown in Figure 8b, when the mixed extract (SNAP) of Camazum leaves and stems was extracted at 80°C, lipid formation was suppressed by 60-70%, regardless of the extraction time.

Meanwhile, the same experiment was conducted on the water extract of the leaves of Camilla, the water extract of the stems of Camilla, the 50% (v/v) ethanol aqueous solution extract of the leaves and stems, and the ethanol extract of the leaves and stems, and the same concentration of 100㎍/ The efficacy of inhibiting lipid accumulation was found to be only 15 to 20% at ㎖ concentration, which was very minimal.

Based on these results, an additional experiment was performed as shown in Figure 9 on the mixed extract (SNAP) of Camazhia leaves and stems extracted using water as a solvent at 80°C for 6 hours at 80°C used in the immunity enhancement experiment. The efficacy of suppressing lipid accumulation was excellent.

<Experimental Example 9: Regulating activity of lipid formation-related protein expression of mixed extracts of leaves and stems of Capsicum ginseng>

3T3-L1 cells, which are preadipocytes, were distributed in a 6-well plate at 1 × 10 ⁵ per well, cultured for 6 days, and then differentiated in DMI medium (Adipogenic medium). This point was considered differentiation day 0. After 2 days of differentiation, the medium was replaced with 1 ㎍/ml insulin medium, and after 2 days of culture, the medium was maintained with 10% FBS medium until day 8. Mixed extract (SNAP) of leaves and stems of Camilla was added on day 0 of differentiation and treated for 8 days. After 8 days of differentiation, cells were collected and the expression of proteins related to lipid formation was examined through Western blot analysis.

As shown in Figure 10a, the mixed extract (SNAP) of the leaves and stems of Chamomile leaves contains PPARγ (Peroxisome proliferator-activated receptor γ), C/EBPα (CCAAT/enhancer binding protein α), and FABP4 (Fatty acid-), which induce lipid formation. binding protein 4), suppressed the expression of Adiponectin.

In addition, as shown in Figure 10b, the mixed extract (SNAP) of the leaves and stems of Camilla inhibited the expression of Perilipin-1, which inhibits lipid decomposition, and the expression of AMPK (AMP-activated protein kinase), which promotes lipid decomposition. Increased phosphorylation.

Referring to the results in Figure 10c, it can be seen that the mixed extract of leaves and stems of Camilla (SNAP) increases the production of LC3-II and p62/SQSTM1, which are autophagy indicators that inhibit lipid accumulation.

Meanwhile, among adipocytes, the cells that suppress lipid accumulation are brown fat, and the factors that induce browning of white fat were also confirmed. As shown in Figure 10d, UCP-1 induces browning of white fat that suppresses lipid accumulation. , increased the production of PGC-1α and PRDM16. Therefore, the mixed extract of the leaves and stems of Solanum ginseng (SNAP) inhibits lipid formation in adipocytes, induces lipolysis, induces autophagy, and induces browning of white fat. In particular, it promotes energy consumption and energy metabolism through browning of white fat. It is believed to inhibit excessive lipid accumulation in fat cells.

<Formulation example 1. Pharmaceutical formulation>

200 g of the water extract (extraction conditions 80°C, 1 day) of the mixture of leaves and stems of Camilla of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. A 10% gelatin solution was added to this mixture, then pulverized and passed through a 14 mesh sieve. This was dried and 160g of potato starch, 50g of talc, and 5g of magnesium stearate were added to make the resulting mixture into tablets.

<Formulation example 2. Food production>

Formulation Example 2-1. Manufacturing of cooking seasoning

A cooking seasoning for health promotion was prepared by adding 1% by weight of the water extract (extraction conditions 80°C, 1 day) of the mixture of leaves and stems of the present invention to the cooking seasoning.

Formulation Example 2-2. Manufacturing of flour foods

The water extract (extraction conditions 80°C, 1 day) of the leaf and stem mixture of the present invention is added to flour at 0.1% by weight, and this mixture is used to prepare bread, cakes, cookies, crackers, and noodles for health promotion. Food was manufactured.

Formulation Example 2-3. Preparation of soups and gravies

The water extract (extraction conditions 80°C, 1 day) of the mixture of leaves and stems of the present invention was added to the soup and gravy at 0.1% by weight to prepare soup and gravy for health promotion.

Formulation Example 2-4. Manufacturing of dairy products

The water extract (extraction conditions 80°C, 1 day) of the leaves and stem mixture of the present invention was added to milk at 0.1% by weight, and the milk was used to prepare various dairy products such as butter and ice cream.

Formulation Example 2-5. Vegetable juice manufacturing

Vegetable juice for health promotion was prepared by adding 0.5 g of the water extract (extraction conditions 80°C, 1 day) of the mixture of leaves and stems of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Fruit juice manufacturing

A fruit juice for health promotion was prepared by adding 0.1 g of the water extract (extraction conditions 80°C, 1 day) of the mixture of leaves and stems of the present invention to 1,000 ml of apple juice or grape juice.

Claims (10)

A composition for improving immunity and anti-obesity, characterized in that it contains leaf and stem extracts of Solanum nigrum as an active ingredient. According to paragraph 1,
The extract is a composition for improving immunity and anti-obesity, characterized in that it has minimal cytotoxicity. According to paragraph 1,
The extract is a composition for promoting immunity and anti-obesity, characterized in that it induces increased production of immunomodulatory factors or activation of phagocytosis in macrophages. According to paragraph 1,
The extract is an immune-promoting and anti-obesity composition characterized by induction of autophagy in macrophages. According to paragraph 1,
The extract is an anti-obesity composition characterized in that it induces inhibition of lipid formation in adipocytes, activation of lipolysis, activation of adipocyte autophagy, or activation of browning of white fat. According to clause 5,
The extract is an anti-obesity composition characterized in that it has the effect of promoting energy consumption and energy metabolism by inducing browning activation of white fat. A food composition for immune enhancement and anti-obesity containing the composition of claim 1. A health functional food for immunity enhancement and anti-obesity containing the composition of claim 1. A feed composition for improving immunity and anti-obesity in companion animals containing the composition of claim 1. A pharmaceutical composition for anti-obesity with an immune-enhancing function containing the composition of claim 1.