KR101047755B1 - Antistress agent containing Dodol extract as active ingredient - Google Patents

Abstract

The present invention relates to an antistress agent containing an extract of Gynostemma pentaphyllum Makino belonging to Cucurbitaceae as an active ingredient.

The extradoldol extract according to the present invention has various physiological functions such as slowing of weight gain, reduction of grip strength and endurance and forced swimming, increase of catecholamine levels, and immune function. It can be usefully used in the development of antistress agent having a stress resistance enhancing action by showing a protective action and an improvement action against degradation phenomenon such as functions).

Stone stress

Description

Anti-stress agent containing dodol extract as an active ingredient {The pharmaceutical agents for the ameliorating effect on stress from the extracts of Gynostemma pentaphyllum}

The present invention relates to a novel medicinal use which is an antistress agent having a stress resistance enhancing action from the extract of Gynostemma pentaphyllum Makino belonging to the family Cucurbitaceae.

Doloe (Gynostemma pentaphyllum Makino, Gynostemmae Herba) is a perennial plant belonging to the foil and the (Cucurbitaceae), which is also called Vine cars as well as domestic, it is widely distributed in the region such as China, Japan, Southeast Asia, and Zenos theme (Gynostemma) About 30 species are known. Among them, pentapyllum species are widely distributed, and studies on species, ingredients, and efficacy have been widely conducted.

The main components of the stone are gypenosides, which are saponins-based compounds with a basic structure of dammarane, and about 100 species of phenenosides have been reported separately (Yoshikawa, K., Mitake, M.). , Takemoto, T. and Arihara, S. (1987) Studies on the constituents of Curcubitaceae plants.XVII.On the saponins constituents of Gynostemma pentaphyllum Makino (12) .Yakugaku. Zasshi 107: 355-360.).

In addition, the dodol contains phyllodulcin, a sweet flavor in the leaves, and contains vitamins (B ₁ , B ₂ , C), minerals, flavonoids, carotene, etc. in addition to saponin compounds. (Jiaogulan, China immortality herb, 1979, Blumert, M. and Liu, J., 1979, p. 12-20, Torchlight Publishing, Inc., Badger, Cal., USA.).

Dool et al. Have studied a variety of physiological activities with respect to ethanol or water extracts and zipenosides isolated therefrom. That is, the main physiological activity is the content reduction action, the antioxidant activity by vascular endothelial cell protection (Wang, ZJ and Luo, DH (2007) Antioxidant activities of different fractions of polysaccharide purified from Gynostemma pentaphyllum Makino.Carbohyd.Polymers 68: 54- 58 .; Huang, TH, Tran, VH, Roufogalis, BD and Li, Y. (2007) Gypenoside XLIX, a naturally occurring PPAR-alpha activator, inhibits cytokine-induced vascular cell adhesion molecule-1 expression and activity in human endothelial cells ... Eur J. Pharmacol 565: .. 158-165), cardiovascular function improvement action (Circosta, C., De Pasquale, R. and Occhiuto, F. (2005) cardiovascular effects of the aqueous extract of Gynostemma pentaphyllum Makino Phytomedicine 12:... .638 to 643), cholesterol lowering action (. Megallia, S., Aktanb, Fugen, Daviesc, NM and Roufogalisa, BD (2005) Phytopreventative anti-hyperlipidemic effects of Gynostemma pentaphyllum in rats J. Pharm pharmaceut Sci . 8: 507-515), immune function modulating action (Huang, WC, Kuo, ML, Li, ML, Yang, RC, Liou, CJ and Shen, JJ (2007) The extract of Gynostemma pentaphyllum enhanced the production of antibodies and cytokines in mice. Yankugaku Zasshi 127: 889-896 .), Inhibiting cancer cell growth (Hou, J., Liu, S., MA, Z., Lang, X., Wang, J., Wang, J.and Liang, Z. ( 1991) Effects of Gynostemma pentaphyllum Makino on the immunological functions of cancer patients.J. Trad. Chin.Med . 11: 47-52.), Antidiabetic action (Norberg, A., Hoa, NK, Liepinsh, E., Van) Phan D., Thuan, ND, J ㆆ rnvall, H., Sillard, R. and Ostenson, CG (2004) A novel insulin-releasing substance, phanoside, from the plant Gynostemma pentaphyllum.J . Biol. Chem . 279 41361- (41367.), Hepatic Function Protective Effects (Lin, JM, Lin, CC, Chiu, HF, Yang, JJ and Lee, SG (1993) Evaluation of the anti-inflammatory and liver-protective effects of Anoectochilus formosanus, Ganoderma lucidum and Gynostemma pentaphyllum in rats.Am. J. Chin.Med . 21: 59-69.), anti-inflammatory actions such as chronic bronchitis and gastric ulcers (Lin, JM, Lin, CC, Chiu, HF, Yang, JJ and Lee, SG (1993) Evaluation of the anti-inflammatory and liver-protective effects of Anoectochilus formosanus, Ganoderma luc idum and Gynostemma pentaphyllum in rats.Am. J. Chin.Med . 21: 59-69 .; Rujjanawate, C., Kanjanapothi, D. and Amornlerdpison, D. (2004) The anti-gastric ulcer effect of Gynostemma pentaphyllum Makino. Phytomedicine 11: 431-435.

Various physiological modulations such as dodol have been described as adaptive effects (Yan YQ (chief ed.), (1996) Encyclopedia of Chinese Herbs, Vol 2, 1878-1882, China Pharmaceutical). University, China Medicine, Science and Technology Publisher, China.).

The living body responds to various mental and physical chronic stresses. The primary physiological response to stress is the hypothalamus (CRH)-anterior pituitary gland (ACTH)-glucocorticoids and the sympathetic-adrenal medulla (catecholamines: dopamine, norepinephrine, secrete epinephrine (epinephrine)] and represents a defense reaction against various kinds of stress (Ulrich-Lai, P. M and Engeland, WC (2005) Sympatho-adrenal activity and hypothalamic-pituitary- adrenal axis regulation. in Steckler, T. , Kalin, NH, Reul, JMHM (ed.). Handbook of stress and the brain, Part I, 419-436, Elsevier.).

In the excited state due to early stress, muscle tension increases and blood pressure rises, but chronic repetitive stress acts on the central nervous system, such as headache, migraine, fatigue, decreased cognitive function, decreased immune function, and neurological symptoms such as depression. takes place, such as endurance and reduced grip strength (Kovacs, KJ, Mikios, IH and Bali, B. (2005) Psychological and physical stressors. In Steckler, T., Kalin, NH, Reul, JMHM (ed.). Handbook of stress and the brain, Part I, 775-792, Elsevier.).

Acute and chronic stress adding methods using experimental animals vary according to the type of stress, and electric footshock stress is applied to stress adding methods including stress-induced pain / analgesia experiments (Kovacs, KJ, Mikios, .. IH and Bali, B. ( 2005) Psychological and physical stressors In Steckler, T., Kalin, NH, Reul, JMHM (. ed) Handbook of stress and the brain, Part I, 775-792, Elsevier;. Sorg , BA and Kalivas, PW (1991) Effects of cacaine and footshock stress on extracellular dopamine levles in the ventral striatum.Brain Res. 559: 29-36.).

Grip strength (grip forces) refers to muscle strength of the forelimbs and decreases when accompanied by anxiety, neurosis, depression, and muscle relaxant, and repetitive stress (electric footshock) causes painful noxious stimuli ( decrease the maximum grip strength (Saurez-Roca, H., Quintero, L., Arcaya, JL, Maixner, W. and Rao, SD (2006) Stress-induced and cutaneous hyperalgesia: ... Differential effect of milnacipran Physiol Beh 88: 82-87).. In addition, chronic stress reduces endurance (endurance, forced swimming test) ( Zhou, SR and Qiu, ZR (1990) A preliminary study on the effects of Gynostemma pentaphyllum on endurance, spontaneous motor activity and superoxide dismutase in mice. Asia. Pacific J. Pharmacol. 5 : 321-322.).

And stress increases the secretion of steroid hormones, which suppress immune responses by inhibiting the production of several cytokines produced from T cells and macrophage (Newton, R. ( 2008) Molecular mechanisms of glucocorticoid action: what is important? .Torax 55 : 603-613.). Dexamethasone, one of the steroid hormones, has been used as a model of stress-induced immune function reduction (Dhabhar, FS. (2002) Stress-induced augmentation of immune function-the role of stress hormones, leukocyte trafficking, and cytokines) Brain Behav Immun. 16: 785-798.).

In addition, reports of the reduction of immunity by stress are already known. It has been reported that immunity is suppressed by decreasing cytotoxic T lymphocytes (CTL) in vivo and decreasing the proportion of T cells that are CD4 + CD8 + during chronic heat stress (Hu, Y., Jin, H., Du, X., Xiao, C., Luo, D., Wang, B., She, R. (2007) Effects of chronic heat stress on immune responses of the foot-and-mouth disease DNA vaccination. DNA Cell Biol. 26 (8): 619-626.). It has also been reported that continued physical stress increased apoptosis, decreased lymphocytes function, and inhibited the release of several cytokines, resulting in decreased survival (Kiank, C., Entleutner, M). , F ㆌ rll, B., Westerholt, A., Heidecke, CD., Sch ㆌ tt, C. (2007) Stress-induced immune conditioning affects the course of experimental peritonitis.Shock . 27 (3): 305-311 .).

Korean Patent Registration No. 116040 is known to improve the stress function (antistress) effect by the mixture of Ogapi, larvae and Valerian, and Korean Patent Registration No. 535655 discloses the neuronal cell protective effect and antioxidant activity by the coltsfoot extract Neuroprotective effect by berberine-containing preparations (Shin Jung-Soo, Sang-Sun Lee, Eung-il Kim, Sung-Min Shim, and Lee Myung-gu. (1997) Effect of Berberine on blood catecholamine content in mice after restraint stress. Pharm. 7 : 81-85.).

As an antistress agent, mendecks (granules and tablets: a mixture of natural products such as ginseng root, ginseng, and hops) have been commercialized in Japan, and Ahn Shin-jung and ginseng are commercially available in Korea.

The inventors of the present invention examined stress physiological defenses against extraneous stress, while stress (electrical stimulation) -added stress resistance to all physiological functions exhibited by stress from experimental results by additional animals (mice and rats). Maintenance / increase action, grip force enhancement action, neurostable action and immune function improvement action) was confirmed and completed the present invention.

An object of the present invention is to provide a new medicinal use by extrapolation extract.

In order to achieve the above object, the present invention provides a novel medicinal use that is an antistress agent having a stress resistance enhancing action from the extract of Gynostemma pentaphyllum Makino belonging to the family Cucurbitaceae.

The dodol extract may be pulverized by drying the dodol leaves, and then chilled for about one day in an extract solvent of about 10 times, and then the extract extract may be obtained by a method such as filtration, concentration, and lyophilization. It does not specifically limit in selection. Extraction solvents include lower alcohols such as purified water, ethanol, methanol, isopropyl alcohol, n-butanol, polyhydric alcohols such as glycerol, propylene glycol, 1,3-butylene glycol, ethyl acetate, methyl acetate, benzene, n- Hydrocarbon solvents, such as hexane, diethyl ether, and dichloromethane, are mentioned, for example, One or two or more types of these solvents can be mixed and used for extraction.

The dodol extract may be extracted by using a liquid extract, pill, tablets, capsules, suspensions, syrups and the like to extract the extract using a liquid extract, soft extract, freeze-dried and spray drying method.

As described in the following experimental examples, the present invention is a stress (electro-stimulation method) -added to the stress physiological function exhibited by the stress from the experimental results of the experimental animals (mice and rats) (weight maintenance / increase action, Anti-stress action was confirmed by confirming that the grip force enhancing action and neurostable action) are shown.

The present invention can be administered in a variety of oral and parenteral formulations during actual clinical administration, with the most preferred route of administration being oral. In addition, when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are generally used are prepared.

Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like, which may include one or more excipients, for example microcrystalline cellulose, low-substituted hydroxypropyl cellulose, colloidal oxidation Silicon, calcium silicate, starch, calcium carbonate, sucrose or lactose, gelatin and the like can be mixed, and in addition to simple excipients, lubricants such as magnesium styrate talc can also be used. In addition, liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Etc. may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the dosage or dosage of the extradol extract according to the present invention varies depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the patient, and on an adult basis It is preferable to take a dosage of 1 mg / kg to 1000 mg / kg in divided doses once or several times a day.

On the other hand, dodol extracts have been reported to be very toxic and not toxic by repeated administration (Attawish, A., Chivapat, S., Phadungpat, S., Bansiddhi, J., Techadamrongsin, Y., Mitrijit, O., Chaorai, B. and Chavalittumrong, P. (2004) Chronic toxicity of Gynostemma pentaphyllum.Foteoterapia 75: 539-551.) Suitable for the development of therapeutic agents for chronic stress that require repeated administration.

Therefore, the extradoldol extract according to the present invention has various physiological functions such as weight loss, decrease in grip strength and endurance and forced swimming, increase in catecholamine levels and splenocyte proliferation. It can be seen that the anti-stressing and anti-stressing effect of the anti-stress of the immune system, such as suppression of the cytokine production ability, and the reduction of immune functions (immune functions). It can be usefully used for development.

Hereinafter, the present invention will be described in more detail.

Example 1 Preparation of Dodol Extract According to the Present Invention

20 kg of dodol leaves were dried and then chopped, extracted with 80% ethanol and concentrated by evaporation to obtain 2.1 kg of dodol extracts.

Experimental Example 1 Measurement of weight change, grip strength and endurance caused by stress

1. Purpose of Experiment

Chronic repetitive stress indicates anxiety, anxiety, loss of motivation and depression, resulting in weight loss, lower grip and endurance reactions. It is to confirm.

2. Add stress and animal model

The animals were mice (ICR male, weight 25-30 g), rats (SD, male, 250-280 g), and mice (C57BL / 6) under 12 hours of day and night cycles and at a temperature of 23 ± 2 ° C. The animals were bred, using a method such as Sog (Sorg, BA and Kalivas, PW (1991) Effects of cacaine and footshock stress on extracellular) using an electric shock (electric footshock, Shock generator, Seil Electric Co. Daejeon) to the experimental animals. dopamine levles in the ventral striatum.Brain Res. 559: 29-36.). Electric footshock (EF; duration and interval, 10 seconds / 3 minutes; 2 mA; duration 21) in experimental animals (mice / rats). (See Fig. 1) The stress addition in the experiment of improving immune function using Mice (C57BL / 6) was performed 2-3 hours after oral administration of the extradol extract from the 4th day after the start of the oral administration of the extradol extract. (Once a day, 3 minutes for 10 seconds; 15 days for stress).

In this experiment, the dodol extract prepared in <Example 1> was set to 50 mg / kg, and the 21-day administration of 50 mg / kg / day oral administration did not show toxic effects on mice and rats. Dodol extracts were administered orally (once daily at 50 mg / kg) 2-3 hours prior to stress and stress (once daily, 10 seconds apart, 2 mA for 3 minutes; duration 21 days).

The subjects were treated with the extradol extract (hereinafter referred to as the 'dodol administration group'), the stress-added group (hereinafter referred to as the 'stress addition group'), and the control group without administration of the extradol extract and the stress-free group ( Hereinafter referred to as 'control group'.

3. Protective effect against slowing weight gain by stress

After stress addition, the experimental animal (ICR mice, male, 25-30 g) was administered the extradol extract (50 mg / kg, orally, once daily, 21 days) according to the present invention, and added EF stress to body weight. The change was measured after the start of the experiment compared to the day 1 control group, and the results were expressed as means ± SEM (n = 8-10) as the change in body weight and increase and decrease (* p <0.05, ** p <0.01, ANOVA followed by Tukey's test) is illustrated in FIGS. 2A and 2B.

As can be seen in Figures 2a and 2b, the control group gained weight normally by 28.5% body weight of the 21st control group, the stress-added group increased 13.1% body weight of the 21st control group compared to the control group Significantly slowed. In addition, the dodol-administered group according to the present invention increased 24.1% of the 21-day control group showed a slowdown in weight gain compared to the control group, but did not show weight loss.

It can be seen from the results of Figures 2a and 2b that the weight is relatively reduced by the addition of stress for a long time, that the administration of the extradol extract indicates a protective action against the slowing of weight gain due to stress.

4. Protective action against the reduction of grip force by stress according to the present invention

Repetitive mental and physical stress causes muscle hyperalgesia, leading to painful nociception, which reduces maximum grip strength (Saurez-Roca, 2000). H., Quintero, L., Arcaya, JL, Maixner, W. and Rao, SD (2006) Stress-induced and cutaneous hyperalgesia:... Differential effect of milnacipran Physiol Beh 88: 82-87).. Induction of muscle hyperalgesia suggests that norepinephrine and serotonin play an important role in the central nervous system (Tsuda, A., Tanaka, M., Ida, Y., Tsujimaru, S., Ushijima, I., Nagasaki, N. (1986) Effects of preshock experience on enhancement of rat grain noradrenaline turnover induced by psychological stress.Pharmacol.Biochem.Behav . 24: 115-119.).

Therefore, in this experiment, in order to confirm the stress resistance enhancing effect of the extradol extract, the extradol extract (50 mg / kg, oral, once daily, administered for 21 days) was applied to experimental animals (ICR mice, male, 25-30 g). Dosage was added and EF stress was added and grip force changes were compared with the Day 1 control group. Biometric measurements were performed by Saurez-Roca, H., Quintero, L., Arcaya, JL, Maixner, W. and Rao, SD (2006) Stress-induced and cutaneous hyperalgesia: Differential effect of milnacipran Beh Physiol 88:.... were measured to 82-87), the change of the grip according to the stress are shown in the decrease of the grip 3a and 3b, in comparison with the control based on the start date. Experimental results shown in Figures 3a and 3b is shown by means ± SEM (n = 8-10) (* p <0.05, ANOVA followed by Tukey's test).

As can be seen in Figures 3a and 3b, the grip strength of the control group increased with weight gain during the measurement period (12.5% increase on the 21st day), but the stress added group decreased the grip strength according to the stress addition (day 16.4 % Reduction), the extradolent group gradually increased grip force (15.2% grip force increase on Day 21) despite stress.

These results indicate that the extradol extract exhibits a protective action against the reduction of grip force by stress addition, and the maintenance / increase action of the grip force by administration of the extradol extract may be related to the defense action of weight loss. .

5. Endurance Measurement

Chronic stress reduces endurance (forced swimming test), so the effect of stress-added endurance by the extra stone extract administration according to the present invention was tested.

Endurance was measured using a rats using a forced swimming test (50 cm cylinder with depth-diameter angle, 32 ± 2 ° C water temperature, 12 sec potential) (Zhou, SR and Qiu, ZR (1990) A preliminary study on the effects of Gynostemma pentaphyllum on endurance, spontaneous motor activity and superoxide dismutase in mice.Asia.Pacific. J. Pharmacol. 5 : 321-322.

As can be seen in Table 1, endurance by forced swimming was 6.08-6.58 minutes in the control group and decreased with stress in the stressed group (swimming time: 6.13 minutes on Day 1, 5.92 minutes on Day 3, 5.78 minutes on day 7, 5.75 minutes on day 10, 5.58 minutes on day 14; n = 8-10), and the extradolent group showed endurance enhancing effect (swimming time: 6.10 minutes on day 1, 3rd) 6.58 minutes for Day 7, 6.91 minutes for Day 7, 7.03 minutes for Day 10, 7.15 minutes for Day 14; n = 10-12; about 28.1% enhancement on Day 14.

Experimental Example 2 Catecholamine Content Determination

Since the living body responds to various stresses in favor of glucocorticoids and catecholamines, etc., and the defense reaction, the effect of the extradol extract according to the present invention on the stress-induced catecholamine content in blood and brain was examined.

1. Effect of Dodol Extract on Stress-induced Catecholamine Content in Blood

The experimental animals (ICR mice, male, 25-30 g) were administered extradol extract (50 mg / kg, oral, once daily, 21 days), added EF stress (day 21), and blood was collected. HPLC method (Mitsui, A., Nohta, H., Ohkura, Y. (1985) High-performance liquid chromatography of plasma catecholamines using 1,2) to determine the content of catecholamines (norepinephrine, epinephrine) in the blood serum -diphenylethylenediamine as precolumn fluorescence derivatization reagent. J. Chromatogr . 344: 61-70. TCA (0.3 M, 300-500 μl) and 300 pmol of isoproterenol are added to the blood, homogenized and centrifuged. The supernatant is pretreated using Toyopak (IC-SP S, Na ⁺ form, 0.5 ml of resin) and the eluate is subjected to fluorescence derivatization. The final reaction solution was quantified by catecholamine content by HPLC method is shown in Figure 4a, 4b. 4A and 4B show the results as means ± SEM (3 tests) (* p <0.05, ** p <0.01, ANOVA followed by Tukey's test).

As can be seen in Figures 4a and 4b, the content of norepinephrine and epinephrine in blood was significantly increased in the stress-added group compared to the control group (norepinephrine 75.3%, epinephrine 80.6% increase), this increase in the dodol administration group It showed a reducing effect (norepinephrine 17.1%, epinephrine 17.3% decrease).

2. Effect of extradolescent extract on stress-induced catecholamine content in brain

In addition, an extra-dodol extract (50 mg / kg, oral, once daily, administered for 21 days) was administered to experimental animals (ICR mice, male, 25-30 g), and EF stress (day 21) was added. Then, the brain was extracted, and the catecholamines (dopamine, norepinephrine, and epinephrine) in the brain were measured by the HPLC method, and are shown in FIGS. 5A, 5B, and 5C. 5a, 5b and 5c are shown in means ± SEM (three tests) (* p <0.05, ** p <0.01, ANOVA followed by Tukey's test).

As can be seen in Figures 5a, 5b and 5c, the content of catecholamines in the brain was significantly increased in the stress-added group compared to the control group (dopamine 83.5%, noepinephrine 17.5%, epinephrine 95.0% increase), in the dodol administration group It showed a reducing effect (dopamine 19.5%, no epinephrine 12.5%, epinephrine 5.0% reduction).

3. Review

Increasing the content of catecholamine in the blood and brain is thought to be more stressed on the 21st day than in the 21 days of continuous stress. The catecholamine in the blood is derived from the adrenal medulla and acts on cardiovascular function. Acts on the excitatory response of the central nervous system. Therefore, the increase in catecholamines due to stress is an excitatory response to stress (elevation of heart rate, blood pressure, and mental excitability), and the decrease in catecholamine content by the administration of extradol extracts may have a lowering / stable effect on all excitatory reactions. It is believed to be able. In particular, the reduction of norepinephrine content in the brain by the extradol extract may affect the pain-reducing action caused by stress-induced noxious stimulation.

Experimental Example 3 Measurement of Immune Function Recovery Decreased by Dexamethasone Administration

Since the administration of dexamethasone (dexamethasone), which is one of the steroid hormones, has been used as a model for lowering immune function due to stress, the extradolescent extract according to the present invention can restore the immune function (spocyte cell proliferation ability, cytokine recovery ability, etc.) reduced by dexamethasone. The number of rats was examined using mice receiving dexamethasone.

The mice used in the experiment were 6-week-old male C57BL / 6 species purchased from Orient Co., Ltd. and used for the experiment after 1 week of acclimatization, and in the semi-SPF system of Chungbuk National University Laboratory Animal Support Center. Breeding. The dodol extract was dissolved in PBS and then orally administered at 30 and 90 mg / kg daily for 21 days, and dexamethasone was dissolved in drinking water at 20 μg / mL from day 17 of the dodol extract administration and freely supplied for 4 days.

1. Splenocyte Proliferation

Splenocytes were isolated from mouse spleen, erythrocytes were removed with ACK lysis buffer, 1 × 10 ⁶ cells / well was added to 96-well microtiter plates, and then concanavalin A (Con A, 1 μg / mL) and anti-CD3 monoclonal. Antibody (mAb, 100 ng / mL) or lipopolysaccharide (LPS, 100 ng / mL) was added to each concentration and cultured mixed. After 54 hours of incubation, 0.5 μCi of [ ³ H] -thymidine was added to each well, followed by further culture for 18 hours, and the cells were collected on a glass filter using an automatic cell harvester. It was. The amount of [ ³ H] -thymidine present in the glass filter was measured with a microbetacounter (Wallac, USA) by adding a scintillation cocktail and the experimental results are shown in FIG. 6. The experimental results shown in FIG. 6 are represented by means ± SD (* p <0.05, ** p <0.01 compared to control levels (ANOVA followed by Tukey's test)).

As can be seen in Figure 6, the splenocytes isolated from the mice orally administered dexamethasone using drinking water is the splenocytes isolated from the normal control group to the extent of proliferation by stimulation of LPS, anti-CD3 mAb and Con-A Was reduced by 20-30% from the proliferative capacity of LPS, anti-CD3 mAb, and Con-A, and the reduced splenocyte proliferation was significantly increased by oral administration of 90 mg / kg of extracellular extracts per day. It was confirmed that the increase.

2. Cytokine Production Capacity Recovery

To determine the efficacy of extradol extracts on the recovery of cytokine production inhibited by dexamethasone administration, mice were orally administered twice a day for 21 days, and dexamethasone (20 μg / mL) was used as drinking water. It was supplied daily.

To 500 μl of blood isolated from mouse venous vein, 2 ml of RPMI-1640 containing 2.5 IU / ml of heparin, penicillin, and streptomycin were added to the polypropylene tube. (100 μg / mL) was added followed by incubation for 48 hours. After incubation, the polypropylene tube was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was separated. The amount of TNF-α and IL-1β present in the supernatant was determined by ELISA kit (BD Biosciences) for each cytokine. Analysis was shown in Figures 7a and 7b. 7A and 7B are shown as means ± SD (* p <0.05, ** p <0.01 compared to control levels (ANOVA followed by Tukey's test).

As can be seen in Figure 7a, when LPS is added to the whole blood culture medium of the blood separated from dexamethasone and normal mice, respectively, the concentration of TNF-α secreted dexamethasone compared to the normal group ( DEX) was reduced in the experimental group, but the oral extract was orally administered at 90 mg / kg / day and returned to almost normal level.

In addition, as can be seen in FIG. 7B, IL-1β secretion was also decreased in the experimental group fed dexamethasone compared to the normal group when LPS was added at a concentration of 100 μg / mL, but decreased by the supply of dexamethasone. Secretion of IL-1β was restored to the level similar to that of the normal group by oral administration of the extradol extract.

The results of 7a and 7b mean that the immune function lowered due to stress can be restored to the normal level by administration of the extradol extract.

3. Confirmation of antigen-specific CTL induction activity

In vitro cytotoxic T lymphocyte (CTL) is reduced during chronic heat stress, so the effect of the extradol extract according to the present invention on the induction of OVA-specific cytotoxic T lymphocyte (CTL) (antigen-specific T of the extradol extract) Cell activation efficacy).

The extradoldol extract according to the present invention was orally administered (30, 90 mg / kg) to C57BL / 6 mice for a total of 21 days, and ovalbumin (OVA) was injected into the vein on day 14 of the extradolent administration. On day 7 after OVA was administered intravenously, target cells of OVA-specific cytotoxic T lymphocytes (CTL) were injected. Target cells were cultured syngeneic spleen cells with OVA [257-264] peptide (1 μM) and labeled with fluorescent material CFSE. Eighteen hours after microvenous administration of target cells, the method described by Lee et al. (Lee, YH., Lee, YR., Im, SA., Park) was used to isolate the spleen and lymph nodes of mice and to kill the target cells. , SI., Kim, KH., Gerelchuluun, T., Song, S., Kim, K., Lee, CK. (2007) Calcineurin inhibitors block MHC-restricted antigen presentation in vivo.J Immunol. 179 : 5711-5716 8) and measured by FACS Canto II (Becton-Dickinson) as shown in Figures 8a and 8b. 8a and 8b are shown by means ± SD (* p <0.05, ** p <0.01 compared to control levels (ANOVA followed by Tukey's test).

As can be seen in Figures 8a and 8b, in vivo CTL assay confirmed the activation effect of OVA-specific T cells of the extradol extract in normal mice, OVA-specific CTL induction in the control group administered OVA (100 ㎍ / mice) The activity was 51% in the spleen and 49% in the lymph node, and the CTL-induced activity was increased to 73% in the spleen and 69% in the lymph node.

These results demonstrate that the administration of the extradolescent extract according to the present invention has an immune enhancing effect of enhancing antigen-specific CTL induction in normal mice.

Experimental Example 4 Measurement of Recovery of Immune Function Degraded by Addition of Stress

The effects of dodol extracts on immune function recovery / improvement in thymus and spleen during stress (weight of thymus and spleen, cell number recovery, cell proliferation, cytokine recovery, OVA-specific CTL recovery, etc.) were examined.

Mice were subjected to oral dosing extract (10, 30, 50 mg / kg) once a day for 20 days in mice, and the stress was added from day 4 from the start of oral dosing extract using an electric shock device. Oral administration of extra-dodol extracts was performed 2-3 hours (once a day, 3 minutes every 10 seconds; 15 days of stress period).

The thymus and spleen of the mice were separated and red blood cells were removed with ACK lysis buffer (150 mM NH ₄ Cl, 1 M KHCO ₃ , 0.1 mM Na ₂ EDTA, pH 7.2-7.4), followed by 5% FBS and 0.1% NaN _3. After washing with added PBS (staining buffer), suspended in 50 μL staining buffer to which 1 μg of anti-CD16 / CD32 monoclonal antibody (clone 2.4G2) was added and incubated for 20 minutes on ice to induce the antibody Fc receptor. Non-specific binding was used and 50 μL of pre-diluted antibody solution was added, incubated for 20 minutes in an ice bath, and the unbound antibody was removed by washing twice with staining buffer. Thymic cells were double stained with anti-CD4 and anti-CD8, and spleen cells were double stained with anti-CD4 and anti-CD8 or anti-CD11b and anti-CD11c. The monoclonal antibodies used in the experiments, anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD11b (clone M1 / 70) and anti-CD11c (clone N418), were identified as Pharmingen (San Diego). , CA) was used for the experiment. In the case of a biotinylated antibody, 50 μL of PE-conjugated avidin was added and incubated in an ice bath for 10 minutes, followed by washing twice. After staining, the cells were fixed by adding 300 µl of PBS added with 1% ρ-formaldehyde, and then analyzed with 10,000 live cells using FACS Canto II (Becton-Dickinson), respectively. b, FIG. 11A, b, c, FIG. 12, FIG. 13A, b, and FIG. The experimental results shown in FIGS. 9, 10A, b, 11A, b, c, 12, 13A, b, and 14 are represented by means ± SD (* p <0.05, ** p <0.01 compared to control levels (ANOVA followed by Tukey's test).

1. Recovery of thymus and spleen weight

As can be seen in Figure 9, the addition of electrical stimulation stress significantly reduced the size of the spleen and thymus of the mouse 45-50%, 20-50% significantly increased when the extra stone extract according to the present invention .

2. Thymic Cell Number Recovery

As can be seen in Figures 10a and 10b, the analysis of the cellularity of the thymus and spleen cells isolated from the mice, in the thymus significantly reduced the T cells CD4 + CD8 + by stress, the extradol extract according to the present invention Oral administration at 50 mg / kg increased significantly.

3. Spleen Cell Recovery

11a, 11b, and 11c in the splenocytes CD4 + or CD8 + T cells significantly reduced by stress, it can be seen that significantly increased when oral administration of the extra-dodol extract of the present invention 50 mg / kg, Figure In 11c, CD11b + and CD11c + cells were analyzed in splenocytes. As a result, the CD11b + cells were significantly reduced by stress, and the extradolescent extract of the present invention was significantly increased when orally administered at 50 mg / kg.

4. Recovery of Splenocyte Proliferation Capacity

In addition, the splenocytes isolated from the mice subjected to electrical stimulation stress from FIG. 12 decreased by 30% from the proliferative capacity of the splenocytes isolated from the control group when stimulated with Con-A. It was confirmed that the reduced splenocyte proliferation ability was significantly increased by 35% by oral administration of the extradoldol extract according to the present invention 90 mg / kg / day.

5. Cytokine Production Capacity Recovery

As can be seen in FIG. 13, when LPS was added at 100 μg / mL to whole blood cultures of blood isolated from stress-stimulated mice and normal mice, the secretion of TNF-α was higher than that of the control group. Although it was reduced in the stress-adjusted group, the dodol-administered group according to the present invention recovered to a level similar to normal (FIG. 13A), and IL-1β secretion was decreased in the stress-added group compared to the control group when LPS was added at a concentration of 100 μg / mL. It was confirmed that, but the dodol administration group according to the present invention did not change (Fig. 13b).

6. OVA specific CTL recovery

Figure 14 shows the results of confirming the activation efficacy of OVA-specific T cells of the extra-dol extract according to the present invention in vivo CTL assay, OVA-specific CTL induction activity in the stress-added group administered OVA (100 μg / mice) spleen 34% in the lymph node and 32% in the lymph node. The CTL-induced activity in the extradolose group was significantly increased by 10% in the spleen and 17% in the lymph node. have.

These results demonstrate that the extradol extract according to the present invention has an immunopotentiating effect of restoring antigen-specific CTL induction even in mice subjected to electrical stimulation.

Therefore, these results indicate that the immune function in the thymus and spleen reduced by the electrical stimulation stress is restored to the normal level by the oral administration of the extra stone extract.

Figure 1 shows a schematic diagram of the experimental method according to the present invention.

Figure 2 shows the effect of the extra-doldol extract (GP) according to the present invention on the weight change of the animals with electrical stimulation stress (mice),

  Figure 2a is a graph showing the weight change of the stressed animals,

  2b is a graph showing the weight change rate of stressed animals over time.

Figure 3 shows the effect of the extra-doldol extract (GP) according to the present invention on the grip force change of the animals with electrical stimulation stress (mice),

  Figure 3a is a graph showing the change in grip force of the stressed animals,

  Figure 3b is a graph showing the rate of change over time grip stressed animals.

Figure 4 shows the effect of the extradolescent extract (GP) according to the present invention on the blood catecholamine content of the electrical stimulation stress added animals (mice),

  Figure 4a is a graph showing the change in the content of norepinephrine in the blood of stressed animals,

  Figure 4b is a graph showing the change in the epinephrine content in the blood of stressed animals.

Figure 5 shows the effect of the extra-dodol extract (GP) according to the present invention on the catecholamine content in the brain of the electrical stimulation stress added animals (mice),

  Figure 5a is a graph showing the change in the dopamine content in the brain of stressed animals,

  Figure 5b is a graph showing the change in the content of norepinephrine in the brain of stressed animals,

  Figure 5c is a graph showing the change in the epinephrine content in the brain of stressed animals.

Figure 6 shows the efficacy of the extradolescent extract (GP) according to the present invention on the recovery of splenocyte proliferation inhibited by the administration of dexamethasone.

Figure 7 shows the efficacy of the extra-dodol extract (GP) according to the present invention on the recovery of cytokine production capacity inhibited by dexamethasone administration,

  Figure 7a is a graph showing the effect on the recovery of TNF-α production inhibited by dexamethasone administration,

  Figure 7b is a graph showing the effect on the recovery of IL-1β production capacity inhibited by dexamethasone administration.

Figure 8 shows the effect of the extra-dodol extract (GP) according to the invention on the OVA-specific cytotoxic T lymphocyte (CTL) induction,

  Figure 8a is a representative figure showing the degree of death of target cells in the spleen and lymph nodes of the mouse,

  8b graphically shows the rate of target cell death.

Figure 9 shows the efficacy of the extra stone extract (GP) according to the present invention on the recovery of thymus and spleen weight reduced by electrical stimulation stress.

Figure 10 shows the efficacy of the extradolescent extract (GP) according to the present invention on the recovery of thymic cell number reduced by electrical stimulation stress,

  Figure 10a is a graph showing the number of thymic cells isolated from the mouse,

  10b is a graph showing the number of thymic cells stained with anti-CD4 and anti-CD8.

Figure 11 shows the efficacy of the extradolescent extract (GP) according to the present invention on the recovery of splenocyte numbers reduced by electrical stimulation stress,

  Figure 11a is a graph showing the number of splenocytes isolated from mice,

  Figure 11b is a graph showing the number of splenocytes stained with anti-CD4 and anti-CD8,

  11c is a graph showing the number of splenocytes stained with anti-CD11b and anti-CD11c.

Figure 12 shows the efficacy of the extradolescent extract (GP) according to the present invention on the recovery of the splenocyte proliferation ability inhibited by electrical stimulation stress.

Figure 13 shows the efficacy of the extradolescent extract (GP) according to the present invention on the recovery of cytokine (cytokine) production capacity inhibited by electrical stimulation stress,

  Figure 13a is a graph showing the effect on the recovery of stress-inhibited TNF-α production capacity

  Figure 13b is a graph showing the effect on the recovery of IL-1β production capacity inhibited by stress.

Figure 14 shows the efficacy of the extradolescent extract (GP) according to the present invention on the recovery of OVA-specific cytotoxic T lymphocytes (CTL) inhibited by electrical stimulation stress.

Claims (1)

Immune function restoring agent that is lowered by stress containing Gynostemma pentaphyllum Makino extract as an active ingredient.

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