KR100535266B1 - Scrophularia buergeriana extract with anti-aging activity and a composition containing the extract - Google Patents

Abstract

The present invention relates to an extract of Hyunsam ginseng which has excellent activity of inhibiting cell aging and inhibiting aging in experimental animals, and a composition comprising the same. do.

Description

Extract of ginseng with excellent anti-aging activity and composition comprising same {SCROPHULARIA BUERGERIANA EXTRACT WITH ANTI-AGING ACTIVITY AND A COMPOSITION CONTAINING THE EXTRACT}

[Industrial use]

The present invention relates to a ginseng extract having excellent anti-aging activity and a composition comprising the same, and more particularly, to a composition containing the same, which has excellent anti-aging effect. Anti-aging substances can be used as raw materials for health food and beverages, cosmetics and medicines for adults and the elderly.

[Private Technology]

Delaying aging, degenerative diseases do not develop, and live a healthy, disease-free long-term, everyone's wish to find a substance that suppresses aging efforts from the past to the present continues. As a cause of aging, free radicals generated by metabolites of active oxygen species such as superoxide, hydrogen peroxide, hydroxyl group and singlet oxygen act on proteins, membranes and DNA in vivo to prevent oxidative damage. In order to suppress these actions, many researches on antioxidant components have been made, and researches to search for new materials having antioxidant activity have been actively conducted at home and abroad. However, aging is not only caused by oxidation but is influenced by numerous factors such as telomeres, cell replication capacity, gene damage and recovery ability. Therefore, in order to evaluate the aging inhibitory ability, not the antioxidant effect of natural products and drugs, There is a need for methods of using aging in aging or laboratory animals.

Most of the substances studied under the name of anti-aging agents are antioxidants. Vitamins C and E, thiol group N-acetyl-L-cysteine, S-allyl-L-cysteine, oltipraz, and other natural antioxidants β-carotene, curcumin, ellagic acid and furmaric acid have been studied. Recently, carnosine, an antioxidant present in muscle, has been reported to maintain cell morphology during aging, and PBN (α-phenyl-Nt-butyl nitrone) has long-life effects in cells and experimental animals. Not much has been reported but reported as anti-aging. Cells harvested from tissues of normal animals, including humans, do not continue to grow in cell cultures, stop after a limited number of divisions, and show the cells of aging animals. The number of cells that can divide is different according to the animals taken, and the number of divisions is known to be proportional to the maximum lifespan of the animal, which means that the life or aging of the animal is determined at the cellular level. . Fibroblasts have been mainly used for the study of cell aging, and changes in cell morphology and gene expression according to aging were investigated. Similar results have been reported for cells other than fibroblasts, and the phenomenon of cellular aging is accepted as a common phenomenon occurring in all cells. Human fibroblasts continue to divide about 50-70 times, gradually dividing, resulting in cessation of growth and the appearance of senescent cells without dying for a while. As aging begins, fibroblasts become elongated.

The best search markers of aging that show differences between young fibroblasts and aging fibroblasts are (1) cell division, enhanced expression during cell aging, (2) acidic β-galactosidase, (3) p21 and (4). ) p16, (5) c-fos, (6) hsp70, (7) microscopic cell morphology with reduced expression, and these indicate precisely whether the cells have aged, so developing an appropriate combination of anti-aging effects Can be used as a way to evaluate.

In experimental animals, blood composition, blood glucose, serum lipid, and organ weights change with age, and are used as an indirect factor in determining the degree of aging. In addition, aging mice lack muscle contraction, relaxation, and coordination, resulting in unnatural behavior and decreased activity. Long-term administration of natural products to naturally aged ICR mice can be used to evaluate the efficacy of anti-aging materials by measuring body weight, blood composition, blood sugar, serum lipids, various organ weights, and behavioral investigations.

It is an object of the present invention to provide an extract of Hyunsam ginseng excellent in antioxidant activity that inhibits cell aging of human fibroblasts and aging of mice and a composition comprising the same.

In order to achieve the above object, according to an embodiment of the present invention, it provides an extract of Hyunsam excellent in antioxidant activity and anti-aging effect.

Hereinafter, the present invention will be described in detail.

Hyunsam (玄 蔘) refers to the root of Hyunsam, a perennial herbaceous plant of Hyunsam, which is known to have the effect of inhibiting against Pseudomonas aeruginosa, expanding blood vessels, lowering blood pressure, and lowering blood sugar. If you use a small amount of mild cardiac effect if you use a large amount of Chinese medicine that causes heart poisoning. Hyunsam originates from folk medicine or has been used for other purposes in oriental medicine, but the anti-aging effect of Hyunsam is not known.

According to an embodiment of the present invention, Hyunsam extract excellent in antioxidant activity and anti-aging effect is provided.

The ginseng extract may be an extract extracted with an organic solvent such as water or alcohol. In addition, it may be a crude extract to which a general solvent extraction method is applied, and may also be a fraction purified by column chromatography. The method for producing an extract according to the present invention can be applied to all plant extraction methods known to those of ordinary skill in the art, for example, extraction with water, alcohol or mixed solvents, extraction with a bath or room temperature, etc. Including but not limited to.

When prepared as a medicament, it may be prepared by addition to one or more pharmaceutically acceptable carriers. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .

Extracts of the present invention can be administered orally during clinical administration and can be used in the form of general pharmaceutical formulations, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used Is prepared using.

The dosage of the composition may be applied differently depending on the age, sex, condition of the subject, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the drug used in combination. The daily dose that can be used without side effects is oral administration of 0.015 g of extract dried per kg of body weight. The invention also includes formulations of dosage units. The formulations may be present in individual dosage forms such as tablets, coated tablets, capsules, pills, suppositories, and ampoules. Solid preparations for oral administration include tablets, coated tablets, pills, powders, granules, capsules, and the like. Suspensions, solvents, emulsions, syrups, etc. are commonly used as simple diluents. In addition to phosphorus and liquid paraffin, various excipients may be included such as wetting agents, sweetening agents, fragrances, preservatives and the like.

The composition containing the present ginseng extract of the present invention can be added to the functional cosmetics or functional foods and the kind of such functional cosmetics or food is not particularly limited.

 EXAMPLE

Hereinafter, a preferred embodiment of the present invention. However, the following examples are only examples provided to aid the understanding of the present invention, and the present invention is not limited to the following examples.

Example 1: Preparation of Hyunsam Extract Composition

After grinding 100 g of dried ginseng finely, it is extracted with 70% alcoholic beverage ethanol for 2 hours at 50 ° C. and then dissolved in ethanol to remove ethanol components and concentrated by vacuum rotary evaporator. Thereafter, the extract was completely removed using a freeze dryer for 48 hours. One-tenth of the finished extract was dissolved in 10 ml of water as a stock solution (98 mg extract / ml water) and used in the following experiment.

In the following examples, the anti-aging effect, which has not yet been identified as the efficacy of Hyunsam, was identified in experiments using human fibroblasts in vitro and verified in experiments using ICR mice in vivo to prove the anti-aging effect of Hyunsam extract.

Example 2: Validation of anti-aging effect through cell culture

In this example, SA β-gal staining after the increase of cell division number and constant cell division after treatment of Hyunsam extract in order to determine how long the cellular senescence can be delayed by treating the cells with the Samsam extract composition of Example 1 The degree of aging was compared through (stained only in aged cells). Afterwards, the expression level of the aging marker protein was measured in order to confirm the anti-aging effect, and the SOD and catalase activity was measured to determine whether the anti-aging effect was related to the antioxidant effect. DNA damage was measured using immunochemistry to see the inhibitory effect of DNA damage by the ginseng extract.

end. Cell Culture of Human Embryonic Fibroblasts

The aging process was investigated through cell culture of human embryonic fibroblasts established at Hallym University, and the growth curve is shown in FIG. 1.

Human fibroblasts show about 70 aging divisions and eventually cease cell growth.

To determine the anti-aging effect of Hyunsam extract, 1/100 (0.98 mg) of the extract of Example 1 was extracted when the number of continuously dividing human fibroblasts reached 50 times (the stage before the aging characteristics were shown). Extract Dried / ml Water The diluted extract was continuously treated with 1/10 of the medium (0.098 mg Extract Dried / ml Medium) to investigate whether it is possible to increase the number of fission cells of defined human fibroblasts. It described in 1.

Fission water when cells stop growing due to aging Herbal Medicine Cleavage Dongjeong Hyangcheon Palace, formerly the Samhan Medicinal Plant A in Daejo-gu (untreated) 6674696666666360

As shown in Table 1, it can be seen that it has an effect of delaying cell division arrest as compared to the control or other natural extracts. Cell division with a limited number of cleaved water to continue to process the ginseng extract to increase the number of cleavage can be a result that directly proves the anti-aging effect of Hyunsam extract.

I. Analysis of Acidic β-galactosidase (SA-β-gal) Expression, an Aging Marker

When the expression of acid β-galactosidase, an enzyme that is expressed only in aging cells and tissues, is stained using a substrate colored with X-gal, staining around the nucleus is colored around the nucleus in aging human fibroblasts. It can be seen that. If the extract had anti-aging effects, it would be much less stained in the same fission water. SA-β-gal staining at cleaved number 63 is shown in FIG. 2.

Using the above experimental method, staining cells treated with 1/100 dilution (0.98 mg extract dried / ml water) and untreated control cells of the present ginseng extract undiluted solution of Example 1 can obtain the results shown in Table 2.

Percentage of SA-β-gal Stained Positive Cells at Cell Cleavage 63 Herbal Medicine % Of positive cells Dongjeong Hyangcheon Palace, formerly the Samhan Pharma A 2016192021252340

In the results of Table 2, it can be seen that the expression of acidic β-galactosidase, which is an aging marker protein, was decreased because of aging of the cells, which was delayed by anti-aging when treated with Hyunsam extract.

All. Protein Expression of Aging-related Genes

In order to investigate the expression of p21, a gene that is activated during aging, cells were cultured under the above conditions in the culture of aging human fibroblasts, and then the cells were harvested from cleaved water 54 and cleaved water 66 to obtain PBS (Phosphate Buffered) Saline) was washed twice, protein extracts were prepared and proteins were separated by SDS polyacrylamide gel electrophoresis and analyzed by Western blot using p21 antibody and ECL staining. Hyunsam extract used in the experiment is an aqueous solution (0.98 mg extract / ml water) diluted 1/100 of the extract of Hyunsam extract of Example 1. The analysis results are shown in FIG. 3.

As shown in FIG. 3, the expression of p21, an aging marker factor protein, was not observed in the aged cells (population doubling number 54) treated with the present ginseng extract, but not in the control cells. In the dividing water (population doubling number 66), a slight (approximately double) expression of p21 protein was observed in the cells treated with Hyunsam extract.

This result confirms the degree of aging inhibition by Hyunsam extract. It can be seen that Hyunsam extract reduces the expression of p21 protein, which is an aging marker protein, compared to the control by delaying or inhibiting aging.

la. DNA damage analysis

The extent of DNA damage caused by oxidative stress, known to increase in aging cells, was compared using immunoohistochemical staining. Fibroblasts added with Hyunsam extract and non-fibroblasts were compared using immunostaining chemical methods using an antibody against hydroxy guanine. Hyunsam extract used in the experiment is a dilution of 1/100 dilution of the extract of Hyunsam extract of Example 1. The results are shown in FIG. In Figure 4 it can be seen that the number of stained cells further increased in the group not treated with Hyunsam extract. These results indicate that Hyunsam extract reduces DNA damage, a marker of aging, and indicates that the degradation of DNA damage slows aging of fibroblasts.

hemp. Antioxidant Enzyme Activity

The activity of antioxidant enzymes directly indicates cell damage caused by oxidative stress. The results of measuring the activity of the antioxidant enzyme catalase (catalase) in fibroblasts treated with Hyunsam extract are shown in FIG. 5, and the results of measuring the superoxide dismutase (SOD) activity are shown in FIG. 6. Hyunsam extract used in the experiment is a composition diluted 1/100 of the Hyunsam extract stock solution of Example 1.

As shown in FIGS. 5 and 6, the catalase activity in the group treated with Hyunsam extract was significantly increased (more than 2 times in cleaved water 54 and more than 15% in cleaved water 66), but the activity of SOD was almost increased. It did not appear. This result suggests that SOD is not deficient in aging fibroblasts, but catalase is deficient and induced by Hyunsam extract and is associated with the anti-aging efficacy of Hyunsam extract.

Example 3: Identification of anti-aging effect of Hyunsam extract through experimental animals

In the present embodiment, the effectiveness of Hyeonsam extract as an anti-aging material was examined through in vivo experiments, and at the same time, the presence or absence of side effects was determined to determine safety.

end. Changes in Body Weight of Mouse During Hyunsam Administration

Experimental animals were lined with ICR mice maintained in the Department of Laboratory Animals, Hallym University. The age of the mice used in the experiment was administered for 6 months using 16 months of age. During the experiment period, the environment was maintained at 23 ± 2 ° C and 55 ± 10% relative humidity. The animals were kept in cages for 4 hours under artificial lighting for 12 hours. Feed and drinking water for experimental animals were supplied freely. In order to verify the aging inhibitory and anti-aging effects of Hyunsam extract in the mouse thus secured 100 times dilution of the extract of Hyunsam extract of Example 1 and ingested instead of water. The control group consumed water only. In order to maintain the freshness of the Hyunsam extract administered, it was replaced every two days, and administered for 6 months. The control diet was water only.

Body weight was measured for a certain time (10 AM) until the end of the experiment, the results are shown in FIG. As shown in FIG. 7, the body weight of 16-month-old mice was 38.1 ± 5.4 g in the control group and 37.6 ± 5.7 g in the 100-fold diluted Hyunsam extract. After 6 months of administration, Hyunsam extract group showed 41.2 ± 7.4 g, which showed less weight loss than 31.0 ± 4.7 g of control. It can be seen that Hyunsam extract has the effect of overcoming weight loss due to aging in vivo by preventing the weight loss of the mouse with aging.

I. Changes in Total Momentum of Mice by Hyunsam Extract

The amount of momentum of the mouse was measured using Motility-Meter 2012 (RHEMA_LABORTELHNIK, Germany) to measure the total amount of exercise for 1 hour. This method measures the amount of movement of the mouse by quantifying the distance the mouse has moved for 1 hour using Motility-Meter. This method can exclude the improvement of experimental data by repetitive learning, which can be a problem in animal experiments. . Using this method, a certain time (between 10 and 17:00 am) was measured at 1 month intervals until the end of the experiment.

In 16-month-old ICR mice (control), the total amount of momentum during one hour was 816 ± 341 and that of 22 months after birth was 439 ± 308. On the contrary, in the group administered the composition diluted 100-fold distilled extract of Hyunsam extract of Example 1 (hyeonsam extract-administered group), the total amount of exercise was 749 ± 354 at 22 months of age but the total amount of exercise was 924 ± 344 even though the age increased. 23.1% increased from the beginning of the experiment (Fig. 8).

In the above results, the increase in the amount of exercise of mice treated with Hyunsam extract can be said to directly demonstrate the anti-aging effect of mice by Hyunsam extract.

All. Changes in Total SOD (superoxide Dismutase) Activity in the Liver

The results of measuring the activity of SOD in the liver after administration of a composition obtained by diluting 100% of the ginseng extract stock solution of Example 1 (hereinafter referred to as “hyun ginseng extract”) for 16 months after birth for six months are shown in Table 3 below.

Determination of SOD Activity from Liver of ICR Mice after Administration of Hyunsam for 6 Months Group (N = 2) Units / g wet weight tissue Unit / mg of total protein Control 1,118 ± 110.7 9.2 ± 0.3 Hyunsam extract administration group 1,099 ± 120.7 8.5 ± 2.8

As shown in Table 3, the activity per gram of tissue was 1,118 ± 110.7 in the control group and 1,099 ± 120.7 in the present ginseng extract group, and there was no difference between the control group and the control group. The SOD activity per mg of protein showed a similar trend, showing no significant difference of 9.2 ± 0.3 in the control group and 8.5 ± 2.8 in the present ginseng extract group. These results support in vitro experiments and show that senescence inhibition and retardation by brown ginseng extract is not due to SOD activity.

la. Stability test and long-term weight change

After 16 months of administration of the composition obtained by diluting 100% of the ginseng extract stock solution of Example 1 (hereinafter referred to as "hyun ginseng extract) for 16 months after birth, the weight-to-weight of the heart, liver, lung, kidney, and spleen was measured. It is shown in Table 4.

 Weight Comparison of Organs after Administration of Hyunsam Extract for 6 Months Group (N = 2) organ organ weight / animal weight × 100 ratio Control Heart (g) 0.9 ± 0.1 Liver (g) 6.1 ± 0.5 Spleen (g) 1.0 ± 0.0 Lungs (g) 0.9 ± 0.2 kidney Left (g) 1.0 ± 0.3 Right (g) 0.9 ± 0.1 Hyunsam Extract Heart (g) 1.0 ± 0.3 Liver (g) 4.8 ± 0.2 Spleen (g) 0.4 ± 0.0 Lungs (g) 1.0 ± 0.2 kidney Left (g) 1.1 ± 0.1 Right (g) 1.1 ± 0.1

As shown in Table 4, there was no statistically significant difference in body weight ratio between the control group and the administration group, supporting the findings of the stability test. In addition, visual inspection was performed by general autopsy, but no abnormal organ was found.

hemp. Molecular biological analysis of in vivo anti-aging effects

Western blot of protein expression of p27 (increase in aging), a marker of aging, was administered to 12-month-old ICR mice for 3 months and 6 months. As a result of analysis by the three-month treatment group did not have any effect, but in the six-month administration group, p27 expression was reduced in the treatment of Hyunsam extract (Fig. 9).

These results indicate that ginseng has the effect of delaying aging not only in in vitro cell culture but also in vivo.

As described above, the present invention uses functional extracts for anti-aging, functional foods and anti-aging using extracts that can delay or anti-aging anticipated that the demand from the domestic ginseng produced in Korea in the aging society is expected to increase greatly. By developing and supplying materials derived from it, it is possible to prevent aging delays and senile diseases. In addition, it will be possible to increase the incomes of farmers who produce Hyunsam and to provide competitive high-income crops suitable for Korean farmers in the market open system.

All simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

1 is a diagram showing a growth curve of human embryonic fibroblasts.

Figure 2 is a photograph of acid β-galactosidase (SA-β-gal) stained cells in the number 63 of human fibroblasts.

Figure 3 is a diagram showing the change in p21 protein expression of human fibroblasts in the control and the group administered with Ginseng extract.

Figure 4 is a diagram showing the results of immunochemical analysis using anti-hydroxy guanine antibody in the cell culture in the control group and Hyunsam extract administration group.

Figure 5 is a view showing the results of catalase (catalase) activity in the fibroblasts of the control group and Hyunsam extract administration group.

Figure 6 is a view showing the results of the measurement of superoxide dismutase (SOD) activity in the fibroblasts of the control and the administration of the present ginseng extract.

Figure 7 is a view showing the weight change of the ICR mouse in the control group and Hyunsam extract administration group.

8 is a view showing the change in the amount of exercise of ICR mice in the control group and Hyunsam extract administration group.

9 is a diagram showing the change of p27 expression in the brain of ICR mice in the control and the group administered with Ginseng extract.

Claims (5)

Hyunsam 70% ethanol extract with excellent anti-aging effect. delete Anti-aging or inhibiting pharmaceutical composition comprising 70% ethanol extract according to claim 1. delete delete