KR100543355B1 - A Herb extract - Google Patents

Abstract

The present invention relates to an herbal extract, anti-tumor by extracting the herbal composition consisting of broiler, Jawan, Shiho, Dansam, Cheonoh, Osuyu, Ginseng, Gilgyeong, health, Seokchangpo, Hubak, Fuyeong, Hwangyeon, Johyup, Mugwort and Licorice It relates to an extract showing activity, hepatitis inhibitory activity and immunopotentiating activity.

Antitumor activity, hepatitis inhibitory activity, immune enhancing activity

Description

Herbal extract with immune enhancing activity {A Herb extract}

Figure 1 shows the results of MMP zymography using the hydrothermal extract of each herbal medicine using SK-Hep1 cell lines producing both MMP-9 and MMP-2 showed high inhibition of MMP-2 in broilers. Activity and inhibitory effect on MMP-9

1: control group 2: Seokchangpo

* 3: Husband 4: Broiler

5: thick 6

7: Fuling 8: Dansam

9: Cheon 10: Oh Su-yu

11: Jawan 12: Shiho

13; Street Scene 14: Ginseng

15: health

Figure 2 is the result of measuring the MMP inhibitory activity of the total ethanol extract (A) total hydrothermal extract (B).

Figure 3 is the observation of the effect on the cell death of HFH-T2 liver cancer cell line of the hot water tank extract.

A) normal liver cancer cell line, B) crude extract 10 ㎍ / ㎖ treated group,

C) 50 ㎍ / mL crude extract (cell death observed)

The present invention relates to a herbal extract, in more detail, each of the broiler, Jawan, Shiho, Dansam, Cheonoh, Osuyu, Ginseng, Gilgyeong, health, Seokchangpo, Hubak, Fuling, Hwangyeon, Johyup, mugwort and licorice It relates to an extract having antitumor activity, liver disease inhibitory activity and immunopotentiating activity.
Metastasis of cancer is the most characteristic of malignant tumors because cancer cells are spread from an early tumor to other parts of the body. Recent cancer research reports indicate that cancer-related deaths account for about 90% of cancer-related deaths, and cancer metastasis is an important cause of cancer deaths. Intensive research on the proliferation of tumor cells over the last few years has led to considerable understanding of the molecular mechanisms of cancer metastasis. Cancer metastasis results from complex interactions between cancer cells and their surroundings. Around the cancer cells is an extracellular matrix (ECM) and basement membrane, which are support structures that cancer cells cannot normally pass through.

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To overcome these extracellular barriers, cancer cells produce and secrete a variety of proteinases that can degrade ECM and basement membranes, especially matrix metalloproteinases (MMPs). In normal physiological environments, MMPs are tightly regulated by endogenous MMP inhibitors called tissue inhibitors of metalloproteinases (TIMPs). However, during cancer proliferation, invasion, and metastasis, the equilibrium between MMP and TIMP is broken and excess MMP is released. Therefore, MMP inhibitors are expected to be a new target to effectively prevent cancer invasion and metastasis, and many cancer researchers are focusing on developing MMP inhibitors as a new concept of anticancer treatment.

Therefore, many researchers are currently actively searching for a low molecular selective inhibitors for MMPs and researching synthetic designs, and the results of preclinical or clinical trials of chemically synthesized inhibitors and inhibitors isolated from natural products have been investigated. It has been shown to be a new and promising drug in the treatment of cancer. However, partial modifications of existing peptide structures are related to synthetic inhibitors, including their limited bioavailabilty, short half-life, lack of enzyme selectivity, and low solubility in aqueous solutions. . In view of the important physiological activity of MMPs, continuous administration of potent inhibitors that are less selective for certain MMPs can be expected to cause serious side effects. Several kinds of MMP inhibitors have been discovered in the development process from natural products such as plants and microorganisms, but there are many problems until the development of therapeutic drugs due to the poor enzyme inhibitory activity and strong cytotoxicity. As a way of overcoming these problems of the existing MMP inhibitors, the development of MMP inhibitors from Chinese herbal medicines, which have been demonstrated clinical efficacy in the private sector, is a very practical and effective strategy.

On the other hand, there are a number of herbs that have been used in the treatment of cancer through long experience. Several cancer researchers have made significant progress in the development of anticancer drugs from these drugs. Taxol, which has been evaluated as the best anticancer agent developed so far and is particularly effective for ovarian cancer, has been separated from taxus , an anticancer drug commonly used in China, and DNA topoisomerase. It is well known that camptothecin, a topoisomerase-I inhibitor, has been isolated from Camptotheca acuminata , and vincristine and vinblastine have been isolated from catharanthus roseus . . However, since they have been explored and developed in terms of differentiation and proliferation of cancer cells, they have strong clinical toxicity, have significant side effects in the treatment of patients, and are not applicable to long-term cancer treatment. Until now, there are few studies for developing a therapeutic agent of the concept of cytostatic therapy, which inhibits cell proliferation by preventing cancer invasion, metastasis, and neovascularization of cancer cells. In this context, the development of MMP inhibitors by biochemical approaches from herbal medicines could be an example of the development of new anticancer drugs.

So far reported, type IV collagenase (collagenase or gelatinaes) MMP-2 and MMP-9 is closely associated with cancer metastasis. The main components of ECM and the basement membrane, which are the biggest barriers to cancer metastasis, are type IV collagen, and MMP-2 and MMP-9 are gelatin-like and collagen-like in the catalytic domain. Therefore, it contains a fibronectin-like domain and has substrate specificity for type IV collagen. In addition, MMP-2 and MMP-9 have been shown to be involved in metastasis of cancer in human cancer cell line cultures and experimental animal models.

In order to detect cell lines secreting type IV collagenase, various tumor cell lines such as HT1080, C32TG, HepG2 T98G, MIA-PaCa2, UAcc62, U937, G361 were used. Check the degree of induction of IV collagenase. Enzymatic activity measurement of gelatinases involves radiolabeled gelatin (3H-acetylated gelatin) or photosynthetic peptides (fluorogenic MMP-2 / MMP-9 sustrate, DNP-Pro-Leu-Gly-Met-Trp-Ser-). Arg-OH) is used to measure the radioactivity or fluorescence of the degradation products, and gelatin zymography using active protein electrophoresis. Among them, gelatin zymography is relatively simple, and enzyme activity can be measured with a small sample amount. In addition, since the approximate molecular weight is known, it is convenient to identify the secreted MMP.

In general, most herbal medicines are stored and distributed in a dry state, so it should be noted that they are infected with viruses, bacterial fungi, etc. during the storage period. It is also important to note that no other ingredients have been introduced, and special considerations require accurate appraisal of herbal ingredients. When extracting the dried plant material, cut it to a suitable size, and then, using a mortar and pestle, grinder, etc. to completely powder the organic solvent and hot water extraction. The dried material is first extracted with methanol or ethanol. Alcohol can extract the components in the cell by breaking the cell membrane, but the intracellular components cannot be eluted with a nonpolar solvent such as ether or carbon trichloride.

In the early stages of exploration, a small amount is used as an exploratory sample without completely separating and purifying the active substance. In this experiment, gelatin geography was slightly modified and used to search for MMP inhibitors. First, when preparing acrylamide gel, a separation gel containing 1.0 mg / ml of gelatin, which is a substrate, was prepared. Serum-free serum of SK-Hep1 was added to SDS sample buffer solution (4% SDS, 125 mM Tris-Cl (pH 6.8), 10% glycerol). After electrophoresis with 50mM Tris-HCl (pH 7.5) buffer containing Triton X-100, washed twice for 30 minutes to remove SDS and then culture buffer solution (50mM Tris-HCl, pH 7.5), 10 min CaCl 2, 0.01% NaN 3) again washed for 20 min. After mixing the solvent extract and the culture buffer solution for geography, and incubating at 37 ° C. for 20 hours, the gel was dyed with 2.5% gel and 0.1% Coomasie brilliant blue, followed by decolorization. It was judged that there was an MMP inhibitor activity that the resolution was markedly reduced.

In recent years, the treatment and prevention of cancer using the pharmacological activity of the herbal medicine has been important. For example, polysaccharides extracted from bamboo leaves are effective against celiac cancer in mice implanted with Sarcoma-180 [Ikekawa, T. et al. (1968) Gann 59: 155-157]. The activity is due to an indirect host-mediated immune-enhancing effect, in particular the activity of polysaccharides present in basidiomycetes cell walls is known to be important [Tanaka, T. et al. (1965) Gann, 56: 529-536; Hamuro, J. et al. (1978) Cancer Res., 38: 3080-3085; Maeda, Y. and Chihara, G., (1971) Nature, 229: 634-635; Komatsu, N. et al. (1969) Gann. 60: 137-144; Oh, G. T. et al. (1992) Arch. Pharm. Res., 15: 379-381.

Other pharmacological activities of herbal medicines are known to be immune enhancing, anticoagulant and digestive system [Wagner, H. (1989) Planta Medica 55: 235-241; Wagner, H. (1990) Pure Appl. Chem. 62: 1217-1222; Hodges, L.C. Et al. (985) Carbohyd. Polym. 5: 141-154; Kiyohara, H. et al. (1996) Planta Medica 62: 14-19]. In addition, alginic acid of Sargassum fulvellum and sulfulated galactofucan of Sargassum kjellmanianum have anti-tumor activity through the host [Yamada, H. et al. (1987) Agric. Biol. Chem. 51, 785-790; Shimizu, S. et al. (1988) Biochem. Biophys. Res. Comm. 150, 335-341], Porphyra yezoensis polysaccharides have activating activity of phagocytic cells in vitro and in vivo [Shimizu, S. et al. (1989) J. Am. Oil. Chem. Soc., 66, 237-241], Gracilaria verrucosa polysaccharides enhance the phagocytic activity of mice [Shinmen, Y. et al., (1989) Appl. Microbiol. Biotechnol. 31, 11-16.

In addition, the fat removal activity and anticoagulant activity in blood vessels of physiologically sulphide polysaccharides have been known [Nakagawa, K. et al. (1972) Carbohyd. Res., 21: 420-426; Hatanaka, K. et al. (1987) J. Med. Chem. 30, 810-814], dextran sulfate, pentosan, polysulfate in polysaccharides [Erik De Clercq. (1989) J. Antimicrob. Chemother., 23: 35-46.22], lentinan ulfate [Hatanaka, K. et al. (1989) Jpn. J. Cancer Res. 80: 95-98.23], ribofuranan sulfate (Hatanaka, K. et al. (1991) J. Carbohy. Chem., 10: 681-690.24], fucan sulfate (Nishino, T. et al., (1991) Agric. Biol. Chem. 55: 791-796, and galactan sulfate (Nagumo, T. et al. (1988) Kitasato Arch. of Exp. Med., 61: 59-67] have an effect on cytotoxicity induced by human immunodeficiency virus, in particular the activity of ribofuranan sulfate depends on the degree of sulfation. .

Plants with such pharmacological activity are evaluated to exhibit immunopotentiating and antitumoral activity by promoting host-defense activity and macrophage activity. In addition, since only a few drugs have been clinically recognized as effective against immune system activation and anti-tumor, many researchers have been searching for effective drugs for anti-tumor.

On the other hand, scheduled apoptosis is a major cause of hepatitis and causes acute hepatitis and chronic hepatitis due to hepatocyte destruction by Fas-FasL binding [Feldmann, G. (1997) J. Hepatol. 36: 1-11. In other words, predetermined apoptosis is developed by binding Fas (or CD95, APO-1), a tumor necrosis factor receptor protein, with FasL, its ligand [Nagata, S. And Golstein, P. (1995) Science 267: 1449-1456, resulting in hepatitis or diabetes [Chervonsky, A. V. et al. Cell 89: 17-24; Griffith, T. S. et al. (1995) Science 270: 1189-1192; Bellgrau, D. (1995) Nature 377: 630-632. Therefore, the material showing cell death inhibitory activity by Fas-FasL media can be usefully used as a therapeutic agent for hepatitis.

Herbal medicine consisting of broiler, Jawan, Shiho, Dansam, Cheonoh, Osuyu, Ginseng, Gilgyeong, Health, Seokchangpo, Hubak, Bokryeong, Hwangyeon, Johyup, Mugwort and Licorice of the present invention is a dry herb. Although it is limited to a combination prescription with other medicines, it has been used in the past as a herbal medicine mainly for clearing detoxification, and is known to be particularly effective in the treatment of inflammatory diseases and cervical cancers related to obstetrics and gynecological diseases (Huh, Jun Bo). However, there is no known research on the antitumor activity and the hepatitis inhibitory activity of the herbal components.

The inventors of the present invention, while studying the activity of the herbal extract of the composition, which is a traditional natural herbal, confirmed the immuno-enhancing and anti-tumor activity such as B-lymphocyte stimulating activity and phagocytic stimulating activity on the extract, In the present invention, it was found that the extracted polysaccharides have the effect of inhibiting the binding of Fas-FasL, which is the cause of hepatocellular inflammation, and inhibiting the expression of FasL in hepatocellular apoptosis.

Accordingly, it is an object of the present invention to provide an extract having antitumor, infection suppression and immunopotentiating activity extracted from a herbal medicine.

It is also an object of the present invention to provide a dietary supplement comprising the extract as an active ingredient.

In order to achieve the above object, the present invention obtains the hot water extract from the herbal medicine traditionally used mainly in the field of herbal medicine and ethnic medicine in Korea, and fractionated and purified it to test its activity by inhibiting cell death, anticancer activity and It was confirmed to have immunopotentiating activity.

The present invention is anti-tumor activity, liver disease suppression activity extracted from herbal medicine consisting of broiler, Jawan, Shiho, Dansam, Cheonoh, Osuyu, Ginseng, Gilgyeong, health, Seokchangpo, Hubak, Bokryeong, Hwangyeon, Johyup, Mugwort and Licorice The present invention relates to a herbal extract having immuno-enhancing activity, and the medicinal herbs constituting the herbal medicine can be used in the same amount.

In addition, the present invention is prepared by crushing the crude drug in water, aqueous solution or buffer solution, and then extracting the precipitate after centrifugation with hot water extract and has a herbal extract of hot water having antitumor activity, liver disease suppression activity and immunopotentiating activity. .

The present invention also relates to an anti-tumor agent, a liver function-related disease treatment agent and an immunopotentiator pharmaceutical composition containing the herbal extract as an active ingredient. The liver function disease is characterized by inflammation, liver failure, alcoholic hepatitis, environmental hepatitis, viral hepatitis, stress hepatitis, fatty liver disease and the like caused by hepatocellular death mediated by Fas-FasL interaction.

The pharmaceutical composition can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the extract of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. Is prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in one or more herbal extracts. (Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the extract is 200 to 600 mg / kg, preferably 200 to 500 mg / kg, may be administered 1 to 3 times a day. If the dose is less than 200mg / kg the effect is insignificant, if it exceeds 600mg / kg the increase in the expected effect on the dosage is not economical.

In addition, it can provide a health supplement with the extract of the present invention as an active ingredient.

The present invention will be described in detail by the following examples.

The following examples illustrate the present invention in detail, and the content of the present invention is not limited by the examples.

Example 1: Herbal Medicine Extraction

500 g of dried herbal medicine consisting of broiler, Jawan, Shiho, Dansam, Cheonoh, Osuyu, Ginseng, Gilkyung, Health, Seokchangpo, Hubak, Bokryeong, Hwangyeon, Johyup, Mugwort and Licorice are chopped and ground in 50mM PBS (pH 7.2) solution. Afterwards, the mixture was centrifuged at 4 ° C. and 15,000 × g for 20 minutes. Thereafter, 5 g of the sample was added, and 10 times the amount of methanol was added thereto, followed by heating and leaching at 70 ° C. for 3 hours, followed by filtration (Oriental filter No. 1).

Example 2: MMP-9 Inhibitory Activity (MMP Zymography) and Apoptosis Inducing Activity of Herbal Extracts>

The dried herbal medicine (hereinafter, used in combination with the trade name "Misamjeong" in the detailed description of the invention) is chopped, pulverized in 50mM PBS (pH 7.2) solution, and centrifuged at 4 ° C and 15,000Xg for 20 minutes. It was. Thereafter, 5 g of the sample was added, 10 times the amount of methanol was added, and the mixture was filtered and leached by heating at 70 ° C. for 3 hours (Oriental filter No. 1), and the filtrates were collected and used for the MMP-9 inhibition experiment.

Yields of each medicinal herb obtained by hot water extraction are shown in Table 1, and the results of MMP inhibitory activity experiment using hot water extract of each medicinal herb using SK-Hep1 cell line producing both MMP-9 and 2 As shown in Fig. 1, the MMP inhibitory activity of broilers and hunch was the strongest. In broilers, high inhibitory activity against MMP-2 and inhibitory effect against MMP-9 were also observed. In particular, the inhibitors of MMP-9 and MMP-2 showed high inhibitory effects. In the case of broilers and thick gourds showing inhibitory activity against MMP-9 and MMP-2, the results of MMP inhibitory activity experiments after 50, 100, 300, 500 ㎍ / mL change were 50 as shown in FIG. The total ethanol extract at the concentration of ㎍ / ㎖ showed a significant decrease in both MMP-2 and MMP-9. In contrast, the total hydrothermal extract showed similar results to the total ethanol extract at the concentration of 300 µg / ml.

For reference, the effects on the cancer cell death by the hydrothermal extract of each herbal medicine is as shown in FIG. Treatment of the hot-water tank extract showed that cell death of HFH-T2 was induced. In particular, in the normal liver cancer cell line (A), a slight cancer cell line death was observed in the crude extract 10 ㎍ / ㎖ treated group (B), but in the crude extract 50 ㎍ / ㎖ treated group (C) it was observed that most of the hepatocellular carcinoma cell death induced .

In the same way as ethanol extract, hot water extract showed the strongest inhibitory activity of herbal medicine. Inhibition of MMP-2 and MMP-9 by the concentrations of the hot water extract of medicinal herbs showed that the ethanol extract showed higher inhibitory activity of MMP-2 and 9 even at the concentration of 50㎍ / ㎖ than the hot water extract. It was found that the activity of MMP-2 was almost suppressed at the concentration of.

Example 3 Inhibitory Effect of Extracts on Cell Death of Hepatocytes by Fas-FasL Mediation

In order to confirm the therapeutic effect of inhibiting cell death and hepatitis caused by Fas-FasL mediated galenical extract of the present invention, after the invitation liver (hepatocyte primary) separation of the mice were incubated (2 x 10 ⁵ cm per ^5). In addition, ball and cultured by adding the NIH3T3 fibroblasts ⁽⁵ x 10 5 per ⁵ cm) that do not express or express FasL. In this case, zVAD.fmk, a key enzyme and caspase inhibitor of cell death by Fas-FasL binding, and caspase-/-and caspase-3- were used as control mice [Hughes, D]. P and Crispe, IN (1995) J. Exp. Med. 182: 1395-1401. The viability of hepatocytes was observed under a microscope after staining with 3 uM of propidium iodide (PI) for 10 minutes, and normal hepatocytes were classified as PI-negative viable cells.

When hepatocytes were co-cultured with FasL-expressing fibroblasts as described above, mouse hepatocytes died within 24 hours, but most of the hepatocytes cultured with the control fibroblasts survived. Furthermore, when the liver cells were simultaneously cultured with FasL-expressing fibroblasts, transient bleb formation began and DNA cleavage was observed. However, the addition of the caspase inhibitor zVAD.fmk did not cause the death of hepatocytes (see Table 1). ). This shows that FasL is the cause of cell death of hepatocytes, and that caspase inhibited by zVAD.fmk is involved in cell death.

On the other hand, when hot water bath extract was added at a concentration of 50 mg / 15ml, cell death by FasL expression-NIH3T3 was reduced by about 40% or more to improve hepatocyte viability. This result seems to be due to the blockage or inhibition of Fas receptor binding of hepatocytes by FasL produced by fibroblasts, thereby preventing the transmission of signal of cell death to Fas receptor (Table 2). In addition, the protective effect of the extract was found to be more effective in hepatocytes isolated from caspase-3 deficient mice than wild-type hepatocytes, confirming that cell death by Fas-FasL mainly mediates caspase-3 (Table 3). .

On the other hand, various fractions of the extract showed a strong inhibitory effect in crude extracts by assaying the inhibitory effect (PI-negative cells) on the cell death of hepatic cells during fibroblasts and co-culture for 24 hours (Table 4).

Example 4: Chemical Composition of Extracts

In order to investigate the chemical properties of the extract was performed as follows. Quantification of total sugars was carried out using the phenol-sulfuric acid method [Dubois, M. et al. (1956) Anal. Chem. 28: 350-354, and the reducing sugars were used in the Nelson-Somogyi method [Nelson, NJ and Somogyi, M. (1952) J. Biol. CHem. 195: 19-23] and glucose was used as a standard. Protein quantitation is based on Low Serum Albumin (BSA), Lowry method [Lowry, HO, et al. (1951) J. Biol. Chem., 193: 265-275], and the hydrolysis of the polysaccharides was carried out in a screwcap vial for 6 hours at 100 ℃ with 2M sulfuric acid. After hydrolysis it was neutralized with barium carbonate (BaCO ₃ ) and separated in Amberlite IR-120 (H ⁺ form) column. Partial hydrolysis of polysaccharides to remove galactofuranosyl residues was performed with 25 mM sulfuric acid (10 ml) at 100 ° C. for 4 hours, neutralized with 0.5 M NaOH and dialyzed with distilled water.

It was confirmed from Table 5 that most of the components of the crude extracts were saccharides (93.7%) and contained trace amounts of protein and sulfur. From these results, the function as an antiviral agent by sulfur which exists in the herbal extract of this invention is also anticipated.

Example 5: B-lymphocyte stimulating activity of the extract

To examine B-lymphocyte stimulatory activity against crude extracts, experimental animals were obtained from spleen extraction experiments when female mice (C57BL / 6XC3H) F1 (B6C3F1) were purchased from the Biotechnology Research Institute and weighed 17-22 g. Sheep red blood cells (sRBC) were purchased from Korea Medium Company (Seoul), guinea pig complement and RPMI 1640 medium from Gibco BRL (Grand Island, NY, USA), and lipopolysaccharide (LPS) from Sigma The product was used.

In vitro activation and lymphocyte production (AFC) assay of lymphocytes are as follows. Splenocytes were cultured in RPMI 1640 medium (containing 10% fetal bovine serum) to adjust the cell number to 5x10 ⁶ cells / ml, and then 0.5 ml per well (4 per treatment group) 48-well plates (Costar) After transfer to, sample or LPS (25 μg / ml, Sigma) is added. For in vitro stimulation, the plates are incubated with shaking at a 37C Bellco (Bellco Biotech., Vineland, NJ, USA) stainless steel tissue incubator.

Antibody production was measured 2 days after the culture, AFC for TNP (trinitrophenyl) -hapten treated sRBC was measured by Jerne plaque assay and the number of cells using a hemacytometer. In this case, the antibody-producing cell number was expressed as an activity value, and the specific activity was expressed as follows. That is, it was based on the treatment of 25 μg / ml lipopolysaccharide (LPS), which is generally used as a control with high antibody production, as a control. The amount of LPS was used as a control since the activity of LPS was saturated at nearly 25 μg / ml.

Table 2 shows the results of assaying 2 mg of crude extract for polyclonal antibody production in B57 lymphocyte stimulatory activity in C57BL / 6XC3H mice.

Example 6: Assay of Antitumor Activity of Extracts

The anticancer activity of the polysaccharides in the extract was assayed by the following method. Sarcoma 180 cells (0.1 ml, 7 × 10 ⁶ cells) were implanted subcutaneously in the right groin of rats at 4 weeks old ICR mice. Experimental samples were dissolved in PBS at an appropriate concentration, autoclaved, and transplanted near the inner peritoneum, followed by daily injection (2 mg / 100 g) for 10 days after 24 hours. After observing all the rats for 5 weeks, the tumor growth inhibition rate was calculated from the following equation by killing the rats, resecting the tumors, and weighing them.

Inhibition Rate (%) = 100 (A-B) / A

A is the mean value of the tumor weight of the control group and B is the mean value of the tumor weight of the experimental group. Complete regression is the ratio of rat numbers showing complete regression to the number of mice.

As a result, the crude extract showed a very high inhibitory activity of 48.5% with 2 mg / 100 g of antitumor activity in rats implanted with Sarcoma 180 solid tumor (Table 7).

Example 7: B-lymphocyte stimulating activity assay of extract

The crude extract was immunostimulated for 1.0 mg / ml in 0.1 mg / ml of each sample, showing 89% activity of the LPS group as a control (Table 8).

<Example 8: Macrophage-stimulating activity of the extract >

Hot water extract was administered after dissolving in PBS for abdominal cavity and oral diet. Forced intake for oral diet and extraction for PEC isolation and phagocytic activity of the spleen.

Splenic macrophage separation of the spleen was performed by gently crushing the tissue, then single cellizing the tissue with Tris buffer-ammonium chloride solution and passing through a cell extractor (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Remaining cells were thoroughly washed with HBSS and rediluted with RPMI medium (10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, containing 100 g / ml streptomycin, RPMI-FCS). Diluted cells were made to adhere to plastic containers and cultured phagocytes to RPMI-FCS-HEPES.

PEC extracts from the periphery of the extract with 5 ml Hank's Balance Solution (HBSS) after intraperitoneal and oral intake, and centrifuged to wash the cell mass twice with HBSS, followed by HBSS (25mM-HEPES (-2-hydroxyethyl-peperazine). It was dissolved in 1 ml of -N-2-ethane sulfonic acid) The number of PECs thus obtained was determined using an F-300 cytometer (Medical Electronics, Kobe, Japan).

The phagocytic activity of PEC is measured by culturing in fluorescent microparticles. In other words, PEC (1 × 10 ⁵ ) was centrifuged, rediluted in 100 μl of HBSS-HEPES, and then diluted 100-fold with HBSS-HEPES to Fluoresbrite carboxylate microspheres (2.0 m; Polyscience, Warrington, PA, USA) was incubated for 1 hour at 37 ℃ to 20μl. 2 ml of cold PBS (containing 3 mM ethylenediaminetetraacetic acid disodium salt (EDTA-PBS)) was added to the culture to stop the reaction, and the cell masses were collected to collect the EPICS-Profile II flow-cytometer, Coulter, Hialeah , FL, USA) was used to measure phagocytic activity.

For chemical luminescence activity measurement, the cells were counted by trypan blue staining using the F-300 microcell measuring instrument so that the phagocytic density of PEC and spleen was 4 x 10 ⁵ / ml and RPMI- After adjusting with FCS-HEPES, 500 μl of cell solution contained 20 μl of 0.2% luminol (luminol, 5-amino-2,3-dihydro-1,4-phthalazine-dione, Sigma, Co., USA) Transfer to polystyrene cuvettes. After 15 minutes of mixing each cuvette gently, it was measured using a luminometer (luminometer, Multi-biolumat LB9505; Berthold, Wildbad, Germany). Activity was expressed as total activity for 15 minutes.

To measure the effect on the phagocytic activity of the spleen, extracts were administered intraperitoneally in 10 mice and three of them were isolated on 3, 6, and 18 days after administration of the temporary chemiluminescence. It was measured (Table 9). The chemical luminescence in the mice receiving the extract was higher than that of the control group.

PEC of the mice administered with the extract of 5 mg per mouse showed the highest chemical luminescence. The production of phagocytic phagocytes in these mice was found to be dose dependent. The B cell stimulatory activity of the extract was measured in polyclonal antibody-producing cells in C57BL / 6XC3H mice, which was the highest at 5 mg administration (Table 10) and showed B-cell stimulatory activity.

Example 9: Toxicity of Hot Water Extract and Effect on Blood Leak Enzyme>

In order to use the extracted hot water extract clinically, it should not be toxic, and the effects on toxicity and hepatocellular enzymes were examined. To evaluate the lethal dose of intraperitoneal administration of hydrothermally extracted polysaccharides to normal rats (5 rats in group 1), no death occurred at 5 mg / kg or less for 5 days. In addition, intraperitoneal administration of 950 mg / kg had no effect on S-GOT, S-GPT, S-Alp after 24 hours (Table 11).

As described above, the herbal extract of the present invention has antitumor activity, hepatitis inhibitory activity, immunopotentiation activity and diabetes treatment effect, and thus can be used as antitumor agent, therapeutic agent for liver function, therapeutic agent for immunoactivator and diabetes treatment agent. .

Claims (4)

Herbal extract with immune enhancing activity, characterized in that it is extracted from herbal medicine consisting of broiler, Jawan, Shiho, Dansam, Cheonoh, Osuyu, Ginseng, Gilgyeong, Health, Seokchangpo, Hubak, Bokryeong, Hwangyeon, Johyup, Mugwort and Licorice . [Claim 2] The herbal extract having immuno-enhancing activity according to claim 1, wherein the herbal extract is prepared by crushing the herbal medicine in water, an aqueous solution or a buffer solution, and then extracting the precipitate after centrifugation with hot water. A pharmaceutical composition for immuno-enhancing comprising the herbal extract of claim 1 or 2 as an active ingredient. The pharmaceutical composition for immunopotentiation according to claim 3, wherein the effective dose of the extract is 200 to 600 mg / kg.

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