KR20020045288A - Immunopotentiating composition containing the extract of Rhodiola root - Google Patents

Abstract

PURPOSE: A composition containing a root extract of Rhodiola sachalinensis capable of immunopotentiating by increasing the expression of inducible nitrogen monoxide synthase (iNOS) of macrophage by synergism with interferon gamma(IFN-γ) is provided which can be used as an immunopotentiating agent such as an antimicrobial agent, anticancer agent, antitumor agent and antibacterial agent. CONSTITUTION: This immunopotentiating agent composition contains an effective amount of a root extract of Rhodiola sachalinensis to show an immunostimulating effect and additionally an effective amount of IFN-γ to activate macrophage. The composition also contains the root extract of Rhodiola sachalinensis in an amount of 1 to 1,000micron/ml. The immunostimulating effect is antibacterial, antitumor, antimicrobial or anticancer effect.

Description

Immunopotentiating composition containing the extract of Rhodiola root}

The present invention relates to a composition containing a root extract of Rhodiola sachalinensis , a novel substance that can be used as an immunostimulating agent, in particular an antibacterial, antibacterial, anticancer, and antitumor agent.

Rhodiola is a perennial herbaceous plant (蕭 培根; 中國 本草 圖鑑 (第 1 卷), 驪 江 出版社, Seoul, p314, 1994). It has fleshy roots and is used as an important medicinal site. . Hongkyungcheon is classified as pizza plant gate, Gyeongcheondae, and Hongkyungcheon by category, and there are 96 species worldwide. Especially, there are 70 species in China, and 34 species and 2 varieties in Xinjiang. have.

In addition, Hongkyeongcheon is a unique plant that has a low temperature, dryness, oxygen deficiency, strong ultraviolet light and strong adaptability even under bad conditions of 2,000 ~ 5,000m above sea level, where the temperature difference between day and night is severe. Therefore, it is known as one of the best health medicinal plants that rejuvenate after the discovery of ginseng and thorny ginseng, and overcome disease and poison.

Traditionally, Hong Kyung-cheon is an anti-stress agent (Korean Patent No. 98-8056), a hangover remover (Korean Patent No. 98-61433), a composition for treating diabetes, hypotension and fatigue (Korean Patent No. 94-23490) and It was used as a preventive agent of circulatory disease (Korean Patent Publication No. 99-71183).

On the other hand, nitrogen monoxide (hereinafter referred to as 'NO') is known to play a physiologically important role in the nervous system, immune system and blood circulation system (Mayer and Hemmens, 1997. Trends in Biochemical Science 22 , 477-481). From a cytobiological point of view, NO is known to mediate physiological phenomena in cells by binding to heme structure of guanylyl cyclase and increasing cGMP production (Murphy and Walker, 1998. Acta Physiologica Scandinavia 164 , 373-380).

In addition, NO synthesized by activated macrophages has a non-specific immune cytotoxic / cytostatic action (Knight, 2000. Annuals of Clinical and Laboratory Science 30 , 145). In macrophages, these non-specific immune functions are synthesized from L-arginine by inducible Nitric Oxide synthase (hereinafter referred to as iNOS). Induction of iNOS gene expression in macrophages requires a primary signal, interferon-γ (hereinafter referred to as 'IFN-γ'). Expression is then activated by secondary signals of bacterial tumor necrosis factor-α (TNF-α), lipopolyscharride (hereinafter referred to as "LPS") or pro-inflammatory cytokines. do. However, iNOS gene is not expressed when there is only a primary signal or a secondary signal, and can be expressed only when macrophages are simultaneously exposed to both signals, that is, by synergy of two signals.

NO induced by the expression of the iNOS gene in these macrophages shows an immune enhancing effect in the body, that is, antibacterial, antibacterial, antitumor and anticancer effects. Therefore, in the art, many studies have been made to increase the expression of iNOS gene and to use it as an antibacterial agent, an antibacterial agent, an antitumor agent, an anticancer agent, and the like.

Therefore, the present inventors also conducted a lot of research to find a substance that can enhance the immune function by increasing the expression of iNOS gene in macrophages, Rhodiola root extract increases the production of NO through induction of iNOS gene expression It has been found that the immune function can be enhanced, and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition containing Rhodiola chinensis root extract that can enhance immune function by increasing iNOS gene expression in macrophages.

1 is a diagram showing the effect of nitrogen monoxide production by synergism of the Rhodiola root extract and IFN-γ at various concentrations in RAW264.7 cells. The difference is p <0.05).

Figure 2 is a diagram showing the effect of nitrogen monoxide production by synergism with IFN-γ according to the treatment time of the Rhodiola sachalinensis extract in RAW264.7 cells. Is p <0.05.)

Figure 3 shows the synergistic inhibitory effect of Rhodiola root extract and IFN-γ by nitric oxide synthase (NOS) and inducible nitric oxide synthase (iNOS) inhibitors in RAW264.7 cells (In the figure, * indicates that the difference in statistical NO production from the untreated sample is p <0.05, and ** indicates statistical NO production between the Rhodiola root extract (RSE) and the sample treated with IFN-γ only.) The difference is p <0.05).

Figure 4a is a diagram showing via RT-PCR that iNOS gene expression is increased by synergy of Rhodiola root extract and IFN-γ in RAW264.7 cells.

Figure 4b is a diagram showing the Northern blot analysis to increase the expression of iNOS gene by synergistic effect of Rhodiola root extract and IFN-γ in RAW264.7 cells.

In order to achieve the above object, the composition according to the present invention is characterized in that it contains the honggyeongcheon root extract as a substance that can induce the iNOS gene expression of macrophages, increase the production of NO, enhance the immune function .

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

In the present invention, the root extract of Rhodiola sachalinensis, which is traditionally descended using water or alcohol, or extracted by conventional extraction methods described in Chinese medicine or textbooks, induces the expression of iNOS gene and increases the production of NO. It is to find that the function can be enhanced, and to provide an immunostimulator composition using the same as an active ingredient.

The effective amount of the Honggyeongcheon root extract as an active ingredient in the immunostimulating agent of the present invention may be appropriately selected depending on the administration method, the type of formulation, the patient's age, the patient's weight, the patient's sensitivity and the condition of the disease, specifically limited Generally, but not always, the Rhodiola root root extract may be formulated to be administered at a concentration of 1 to 1,000 mg / kg body weight. Of course, the dose of the active ingredient may be an amount outside the above range.

Formulations for administering the honggyeongcheon root extract within the above range may have a conventional form, for example, may be used in the form of pills, capsules or drinks, medicines and the like. They may be administered for immune function through oral or various parenteral routes of administration, and are suitable for the dosage form and are already well known to those skilled in the art and various excipients, carriers or diluents which can be easily selected by those skilled in the art. It may contain.

On the other hand, in order to determine the effect of increasing the production of NO by the iNOS gene expression of the Rhodiola root extract, RAW264.7 cells, a murine macrophage line, were used. The reason is that RAW264.7 cells can be used to study the activation of macrophages in general and are widely used to study the activation of macrophages (Liu et al., 1999. Journal of Immunology 163 , 6606-). 6613).

According to the present invention, the red ginseng root extract can significantly increase NO production when treated with IFN-γ-treated RAW264.7 cells. As shown in FIG. 1, the treatment concentration of the Rhodiola sachalinensis extract which can efficiently increase NO production amount is 1 to 1000 µg / ml, and preferably 10 to 100 µg / ml. In addition, as shown in Figure 2, the Rhodiola chinensis root extract is treated for 6 hours or more, preferably 6 to 24 hours, NO production amount increases.

On the other hand, according to the present invention, when the honggyeongcheon root extract alone is used, NO production is not increased at all, and only when treating the honggyeongcheon root extract in the presence of IFN-γ, by their synergism (synergism) NO production can be increased. These results indicate that Rhodiola root extract can be used as a secondary activation signal of macrophages.

In addition, according to the present invention, the effect of increasing the amount of NO produced by the synergistic effect of Rhodiola sachalinensis extract and IFN-γ is N ^G MMA (N ^G -monomethyl-L-arginine) or iNOS inhibitor aminoguanidine (aminoguanidine; It may be reduced by the following "AG", and this result indirectly confirms that the effect of increasing the NO production of Rhodiola root extract is related to the iNOS gene expression increase. As a result of RT-PCR and Northern blot test as direct evidence that the increase of NO production of the Rhodiola sachalinensis extract induces the increase of iNOS gene expression, as shown in FIG. 4A and FIG. It can be seen that the extract increases the expression of the iNOS gene.

Therefore, when the Rhodiola root extract is treated in the presence of IFN-γ, NO production is increased through iNOS gene expression.

In addition, Knight, JA, Annuals of Clinical and Laboratory Science 30 , 14 5-158 (2000) showed that NO synthesized by increased iNOS expression in activated macrophages is a non-specific immune cytotoxicity / cytoproliferation inhibition ( It has been described that it can have a cytotoxic / cytostatic action to provide an immune enhancing effect, that is, antibacterial, antibacterial, antitumor and anticancer effect.

Thus, Rhodiola sachalinensis extract, which can increase the amount of NO synthesized by increasing iNOS expression of macrophages, can be used as an immune enhancing agent, i.e., but not limited to, antibacterial agents, antitumor agents, and anticancer agents. It can be used to enhance various immune functions of macrophages known in the art.

On the other hand, IFN- [gamma] is an immune function regulator secreted by T cells, which are cell-immunological function, IFN- [gamma] occurs naturally when a state of activation of immune function cells such as bacterial invasion occurs in the body. Thus, even when used alone, the Honggyeongcheon root extract may exhibit an enhanced immune function by synergistically with IFN-γ in the body. However, it is preferably used simultaneously with IFN-γ to further increase the synergy of increased iNOS expression.

On the other hand, the composition of the present invention is not particularly limited to the shape of the Rhodiola root root extract, it can be used as a liquid, solid.

Hereinafter, although an Example and drawing demonstrate this invention in detail, this invention is not limited by this.

Example

Reference Example 1 Preparation of Rhodiola sativus Root Extract

Rhodiola sachalinensis was purchased from Deokyu Pharmaceutical Co., Ltd. in Muju, Jeonbuk. 300 g of dried roots of Honggyeongcheon were extracted with distilled water at 100 ° C. for 7 hours. The extract was filtered through 0.45 μm filtration membrane and then lyophilized. At this time, the yield was 11.5g, the obtained honggyeongcheon extract was stored at -20 ℃.

Reference Example 2 Preparation of Liquid Extract

Prior to use, 1 g of dried extract was added to 10 ml of Hank's balanced salt solution; 0.185 g / l calcium chloride dihydrate, 0.1 g / l magnesium sulfate anhydride, 0.4 g / l potassium chloride, 0.06 g / l potassium phosphate monobasic anhydride, 0.35 g / l sodium bicarboxylic acid, 8.0 g / l sodium chloride, 0.05 g / l sodium phosphate dibasic anhydride and 1.0 g / l D-glucose) Filtration to prepare a liquid form.

Reference Example 3 Reagents Used in the Experiment

Murine recombinant interferon-γ (IFN-γ) was purchased from Genzyme (Munchen, Germany). Hank's balanced salt solution (HBSS), RPMI-1640, sulfanylamine, N- (1-naphthyl) -ethylenediamine dihydrochloride, polymyxin B, N ^G -monomethyl-L-azinine (N ^G MMA) and aminoguanidine (AG) were obtained from Sigma chemical company (St. Louis, MO, USA). RNA-isolation kits were purchased from Qiagen (Hilden, Germany) and Takara RNA PCR kit ver2.1 (TaKaRa RNA PCR kit ver2.1 (AMV)) was obtained from TaKaRa (Bohan, ROK). Fetal bovine serum (hereinafter referred to as 'FBS') and antibiotics were purchased from Gibco / RBL (Gaithersberg, MD). All reagents and media in tissue culture experiments were tested for endotoxin content using a colorimetric Limμlus amoebocyte lysate assay (test limit 10 pg / ml; Whittaker Bioproducts, Walkersville, MD). All of the reagents did not contain endotoxins. Tissue culture plates and 100 mm diameter petri dishes were purchased from Nunc (NAperville, IL).

REFERENCE EXAMPLE 4 Culture of RAW264.7 Macrophage Line

Rat macrophage lines, RAW264.7 cells, were obtained from the American Tissue Culture Collection (ATCC TIB-71, Rockville, MD). 10% heat-inactivated calf serum, 1% L-glutamine, 1% non-essential amino acids, 1% antibiotic / antibacterial (10,000 U / ml penicillin, 25 μg / ml amphotericin D or 10 mg / ml streptomycin), 1.5% sodium carbonate and 1% basic essential vitamins were added to the complete RPMI 1640 medium, which was incubated for 24 hours while maintaining a humidified 5% CO ₂ state at a temperature of 37 ° C.

Example 1 NO Synthesis Effect of Rhodiola Radix Root Extract

The culture medium used in the experiment was treated with LPS inhibitor 100U / ml polymyxin B in order to exclude the possibility of increased gene expression by the activity of LPS, a secondary signal of iNOS gene expression.

The 264.7 cells cultured in Reference Example 3 were treated in a medium containing 10 U / ml of IFN-γ, and the liquid Rhodiola root extract of Reference Example 2 was treated for 24 hours at concentrations of 1, 10, 100 and 1000 µg / ml. Then, NO production amount of RAW264.7 cells was measured. NO production was determined by measuring the concentration of modified nitrites in contact with NO. As a control, the liquid Rhodiola root extract was treated without IFN-γ.

Meanwhile, the concentration of nitrite was measured by the Gries's reaction. That is, 100 μl / well of sample and the same amount of Gries solution (mixture of 1% sulfanylamide dissolved in 5% phosphoric acid solution and alpha-naphthylamine dissolved in distillate) are mixed, and at room temperature for 10 minutes. Reacted. Then, absorbance was measured at 540 nm using a Titertek ELISA Reader (Flow, Rockville, MD). The results are shown in FIG.

As shown in Figure 1, it can be seen that the production amount of NO increases with increasing the amount of the Rhodiola root extract in the liquid phase in the presence of IFN-γ. On the other hand, the increase in the amount of NO produced showed a difference in the amount of NO produced when the throughput of the extract was 1 µg / ml or more, and was significantly different from that when IFN-γ was not treated when 10 µg / ml or more. In addition, the amount of NO produced was saturated at 100 µg / ml or more. On the other hand, when only the liquid Rhodiola root extract was treated without IFN- [gamma], NO production was not increased at all.

Example 2 NO Synthesis Effect According to Rhodiola sativus Root Extract Treatment Time

RAW264.7 cells were treated with 100 μg / ml of the liquid Rhodiola root extract, and the culture was carried out in the same manner as in Example 1 except that the incubation time was varied to 0, 6, 12, and 18 hours. The results are shown in FIG.

As shown in Figure 2, the amount of NO produced is increased as the treatment time of the liquid honggyeongcheon root extract increases, it can be seen that the effective treatment time is more than 6 hours.

Example 3 Effect of NOS and iNOS Inhibitor on Rhodiola Root Extract

An LPS inhibitor poly town iksin B 100U / ㎖ adducted culture honggyeongcheon liquid in RAW264.7 cells in culture medium root extract 100㎍ / ㎖ and in a state in which process the IFN-γ 10U / ㎖, 2mM N G MMA or 100uM AG Further, and incubated in the same manner as in Reference Example 3 for 24 hours. Then, NO production amount was measured by the same method as described in Example 1. As a control, NO production was measured in culture medium that was not imposed with 2 mM N ^G MMA and 100 uM AG. The results are shown in FIG.

As shown in Figure 3, it can be seen that the NOS inhibitor N ^G MMA or the NO production significantly decreased the iNOS inhibitor AG when hayeoteul further added. These results indicate that Rhodiola root extract is involved in increasing the production of NOS by increasing the expression of iNOS gene.

Example 4 iNOS mRNA Expression Promotion Effect Test of Rhodiola Radix Root Extract by RT-PCR

RAW264.7 cells in culture medium supplemented with LPS inhibitor polymyxin B 100U / ml were treated with 10 μg / ml or 100 μg / ml of liquid Rhodiola root extract, IFN-γ 10U / ml, or both Incubated for 8 hours in the same manner as in Example 3. Cells were isolated from the culture medium and RNA was extracted using an RNA-separation kit. The extracted RNA was subjected to RT-PCR using TaKaRa RNA PCR kit. That is, cDNA was prepared by culturing a mixture of 1 mM dNTP, 1 U / μl RNase inhibitor, 0.25 U / μL Avian Myeloblastosis Virus (AMV) reverse transcriptase, 0.125 μM oligo-dT-adapter primer, and 1 μg RNA at 55 ° C. for 30 minutes. It was. It was then inactivated at 99 ° C. for 5 minutes to inhibit the activity of AMV reverse transcriptase in the mixture.

The cDNA obtained above was amplified by PCR using a DNA thermal cycler (Perkin Elmer, Cetus, Norwalk, CT). PCR reaction conditions are as follows.

Primers for PCR for iNOS

Positive primer: 5'-TTTGGAGCAGAAGTGCAAAGTCTC-3 '

Negative primer: 5'-GATCAGGAGGGATTTCAAAGACCT-3 '

Primers for PCR for β-actin

Positive primer: 5'-TTGTAACCAACTGGGACGATATGG-3 '

Negative primer: 5'-GATCTTGATCTTCATGGTGCTAGG-3 '

0.8mM iNOS PCR primer or β-actin primer, 1X PCR buffer, 0.2mM dNTP (solution containing 0.05 mM each of dATP, dTTP, dGTP and dCTP) and 0.03U Taq polymerase (Taq polymerase) 30 μl of distilled water was mixed at the concentration, and PCR was performed by the following procedure.

After PCR, 10 μl of the sample was electrophoresed on a 1% agarose gel containing EtBr (ethidium bromide), and photosensitive to UV to identify DNA bands. The results are shown in Figure 4a.

As shown in Figure 4a, when only IFN-γ treatment, the expression of iNOS is very little, only when the Rhodiola root extract was not expressed at all. However, when the IFN-γ and Rhodiola root extracts were treated simultaneously, iNOS expression was increased. On the other hand, the results of β-actin indicates that the mRNA samples used to perform RT-PCR for each sample is the same. These results show that the Rhodiola root extract of the present invention acts as a secondary activator for iNOS expression of macrophages. That is, for the expression of iNOS gene, IFN-γ and Rhodiola root extract should be treated simultaneously.

Example 5 iNOS mRNA Expression Promoting Effect Test of Rhodiola Radix Root Extract by Northern Blot Analysis

The base sequence of the probe used in the Northern blot is composed of sense (Sense; 5'-GGCCTTGGCTCC AGCATGTAC-3 ') and antisense (Antisense; 5'-GTTCTGGATGAGAGCGGCAGC-3'). And the 562 bp iNOS DNA fragment probe was used for the RT-PCR method of Example 4.

On the other hand, in the same manner as in Example 4, experimental and control groups were set and RNA was separated from each group. Then, 15-20 μg of RNA sample was taken, denatured with formaldehyde / formamide, and nylon membrane using capillary action in the presence of 20X SSC (3.0M sodium chloride, 0.3M sodium citrate, pH 7.0). Transferred to. After pre-hybridizing, [α- ³² P] dCTP labeled iNOS DNA fragment probe with 1 × 10 cpm / ml activity, 5X SSC, 10 × Denhardt's solution, 50% form Hybrid formation was carried out at 42 ° C. for 20 hours with a mixed solution of amide, 10 mM Na ₂ HPO ₄ (sodium phosphate dibasic) and 0.5% sodium dodecyl sulfate (SDS). The nylon membrane was then removed with excess radiation through a three step wash with a wash solution of 2X SSC / 0.5% SDS, 1X SSC / 0.5% SDS and 0.2X SSC / 0.5% SDS. Then, after drying the nylon membrane, the degree of radiolabeling was confirmed by photosensitizing the X-ray film. The results are shown in Figure 4b.

As a result, as shown in Figure 4b, it could be confirmed through Northern blot analysis that the honggyeongcheon root extract increased the iNOS gene expression as in Example 4.

As described above, the honggyeongcheon root extract according to the present invention can increase the amount of NO production by increasing the expression of iNOS gene of macrophages by synergy with IFN-γ, and thus, the composition according to the present invention May be used as an immunostimulating agent, such as antibacterial agent, antitumor agent, anticancer agent.

Claims (4)

Immunostimulator composition characterized in that it contains Rhodiola sachalinensis root extract in an amount effective to exhibit an immune enhancing effect. 2. The immunopotentiator composition according to claim 1, wherein said composition further contains an amount of interferon gamma (IFN- [gamma]) effective to activate macrophages. The immunostimulator composition according to claim 1 or 2, wherein the immune enhancing effect is an antibacterial, antitumor, antibacterial or anticancer effect. The immunostimulator composition according to claim 1 or 2, wherein the extract of Rhodiola savona root is contained in an amount of 1 to 1000 µg / ml.