KR19990057560A - Ginseng extract having a method of increasing the content of harmful oxygen scavenging enzyme content and antioxidative effect of ginseng - Google Patents

Abstract

The present invention can be used as an anticancer agent for extracting panaxdiol and panaxtriol in each ginseng region and assaying the ability to induce antioxidant enzyme expression according to the saponin ratio. To ensure that. The present invention divides ginseng for raw materials into the fuselage part, the United States, Central America, semi, and semi-semiconductor and extracted by each part of the ginseng X A (ginseng body parts), X B (Ginseng ginseng and Central America), X C (Ginseng Semi ), And the combination ratio (P / T) of panax diol and panaxtriol to X D (Ginseng Semi) and cloned transcription control sites of the SOD1 gene and chloramphenicol acetyl Treated with transeptase (CAT) structural genes and transformed into transgenic animal cell lines (HepG2 cells), irradiated with saponin D and C fractions from semi- and semi-areas, respectively, with P / T of 1.8 or higher and free of oxygen It relates to assay methods to confirm that they contribute to increased expression of enzymes and to the use of X and irradiated saponins extracted from the above semi-semi-sememis as anticancer agents. According to the present invention, there is an effect that can be used industrially anti-cancer drug and xironponin from semi and sesame.

Description

Ginseng extract having a method for increasing the effect of harmful oxygen scavenging enzyme content and antioxidative effect of ginseng

The present invention relates to a method for searching for an appropriate expression inducing agent of NOx and irradiated saponin with a specific saponin content composition.

Ginseng has been known to have tumor suppression effects and chemical carcinogen inhibitors and metastasis inhibition by various experiments (Brekhman, II and I. V Dardymov, 1969. Ann. Reu. Pharm. 9, 419-426; Sato, K., Mochizuki, M., Saiki, I., Yoo, YC, Samukawa, K. and Azuma, I., 1994 Biol Pharm Bull 17, 635-639). Ginseng has also been reported to protect tissue damage caused by stress conditions in living tissues (Wagner, H.N. and Liu, X., The International Textbook of Cardiology. Pp140. Pergamon Press, New York, 1987). However, it is not yet known which pathway plays what role. However, the anti-cancer effect of ginseng is thought to be due to the active component of ginseng saponin to remove the harmful oxygen formed in the early stage of cancer development by inducing the expression of antioxidant enzyme (SOD) gene. have.

SOD is a coenzyme of copper and zinc, which is present in large quantities in the cytoplasm and converts superoxide into hydrogen peroxide and oxygen. The transcription control region is a DNA nucleotide sequence that is responsible for the transcription of a gene and is measured and displayed in the opposite direction from the transcription initiation region and is generally regarded as a region of about 1 kb in length. In this region, various transcription regulators combine to perform their functions.

In particular, the antioxidant enzyme (Peroxide Dismutase: SOD1) is an enzyme that acts as an early defense mechanism of cellular damage caused by O2 radicals, and converts free radicals into H2O2 and shows antioxidant defense against tissue destruction or cell transformation by oxidation. (Coyle, JT and Puttfarcken, P. 1993. Science 262, 689-695; Fridovich, I., 1975. Ann. Reu. Biochem. 44 147-159) Another harmful substance, hydrogen peroxide, occurs as a by-product. It induces oxidative stress in cells, like harmful oxygen, causing cell aging and cancer. In order to remove such hydrogen peroxide, another homeostasis enzyme, catalase, is present in the cell to protect the cell from oxidative stress by converting hydrogen peroxide, which is a cytotoxic substance, into water and oxygen (Nakamura, H., Yamamoto). , M., GOT o, k., Osumi, t., Hashimoto, T. and Endo, H., 1989. Gene 79, 279-288). On the other hand, beta carotene, tocopherol, uric acid, glutathione, siruloplasmin, transferrin and albumin are known as enzymes and antioxidants for removing active oxygen in cells. However, there are few reports verifying at the molecular level whether these components directly induce an increase in SOD1 gene content.

As mentioned above, ginseng is known to cause an increase in harmful oxygen scavenging enzymes in the human body, but until now, the specific homeostasis effect according to the compositional ratio of panax diol and panax triol has not been tested, and the effective saponins obtained in trace amounts There is a problem in formulating an effective ratio of ginseng saponin is not industrially used as an anticancer agent.

The present invention verifies the effect of ginseng saponin fraction known to have no side effects in vivo on the molecular level to verify the specific composition ratio of saponins suitable for the expression of homeostasis enzyme by verifying at the molecular level the specific saponin composition ratio It is to obtain a ginseng extract that can contribute simply to the expression of antioxidant enzymes without artificially adjusting the composition ratio of saponin by finding and extracting specific parts of ginseng having.

The present invention reveals the action of harmful oxygen scavenging enzyme according to the irradiated ponine content of each part of ginseng, and the anti-cancer effect of ginseng has a specific saponin composition ratio that actively removes harmful oxygen formed in vivo at the early stage of cancer development. By confirming the site by molecular genetic method and extracting the specific site of the above-mentioned ginseng ginseng was made industrially available as an expression inducing agent of antioxidant enzymes.

1 is a recombinant gene carrier in which a transcriptional regulatory region of a harmful oxygen scavenging enzyme (SOD1) gene and a structural gene of chloramphenicol acetyl-transferase (CAT) is fused to a hepatic cell (HepG2 cell), and then the checkonin of Table 2 The fraction showing the inducible effect by treating A, B, C, and D is a graph showing the results obtained by measuring CAT activity.

According to the present invention, the ginseng for raw materials is divided into the fuselage part, the Americas, the Central America, the semi, and the semi-semester, and used as a sample for extracting each part of the ginseng, and the test XA (Ext A: ginseng body part is used). ), XB (Ext B: Ginseng and Central America), XC (Ext C: Ginseng semi), XD (Ext D: Ginseng semi-sem), and ginseng extracted from the manufactured X Investigating the inducible effects of ROS-inducing enzymes on four types of irradiated saponin (X, A, B, C, D,) containing the main active ingredient saponin, the difference in the content ratio between diol saponins and triol saponins The present invention was completed by finding out the effects of X- and D-C in remarkably excellent effects of induction of harmful oxygen scavenging enzymes.

Ginseng saponin has the basic structure of panaxana diol and panaxanatriol, which is a triterpene compound of dammarane, and according to the type of sugar, the type of sugar and the number of sugars, It is divided into saponin and panaxtriol saponin. In the present invention, saponin samples for each ginseng site having different content ratios of diol saponins and triol saponins were prepared and used in the experiment (see FIGS. 1 and 2).

CAT activity analysis is a method of measuring transcription regulation by selecting the chloramphenicol acetyl-transferase (CAT), which is a antibiotic antibiotic inhibitor gene of bacteria, as a surrogate gene, and measuring the transcriptional regulation by the size of the activity. The recombinant gene carrier prepared in front of the gene was introduced into the animal cell line and CAT activity was measured.

Hereinafter, the present invention will be described in detail.

The present invention is a saponin that promotes the transcription of harmful oxygen scavenging enzyme (SOD1) gene in ginseng saponin extracted by various parts of ginseng, and induces an effect of increasing the content of harmful scavenging enzyme according to the content ratio of diol (panaxadiol). It is organized by way of identification.

The present invention also relates to the irradiated saponin D and the saponin C fraction having saponin composition with an excellent effect of promoting the transcription of the antioxidant enzyme SOD1 gene in ginseng saponins extracted by various parts of ginseng.

In addition, the present invention also relates to a fusion vector of a transcriptional regulatory region of CAT (SOD1) and CAT (chloramphenicol acteyl transferase) structural gene prepared to verify the antioxidant efficacy of ginseng saponin.

The present invention also relates to saponin treatment experiments for measuring transcriptional regulation of antioxidant enzymes introduced into animal cell lines to screen the antioxidant efficacy of ginseng saponin.

In addition, the present invention includes a recombinant DNA of ginseng body part extract X A, ginseng extract and B, Ginseng semi extract X C and ginseng semi-ex extract X D as X of the irradiated testponin samples. It is also related to the method of treating the animal cell line but the activity concentration of each X in the case of SOD1 concentration of 100㎍ / ㎖.

The present invention also relates to a method characterized in that a sample of ginseng saponin is introduced into an animal cell line into which SOD1 recombinant DNA is introduced, followed by treatment for 36 hours after 36 hours of introduction of recombinant DNA.

According to the present invention, since the effect of increasing the oxygen scavenging enzyme according to the saponin composition of ginseng and the antioxidant effect of each part of the ginseng is verified, the required effect is obtained by using the X part of the ginseng having the antioxidant effect as an anticancer agent. Industrially, there is an effect that can be used to manufacture anticancer drugs.

Example

Hereinafter, an example of the present invention will be described in detail with reference to Examples.

Materials and methods

end. Samples and Reagents

The special reagents used in this experiment were all molecular biological grades, restriction enzymes and denatured enzymes were purchased from NEB, various reagents were purchased from SIGMA, and DNA separation kit was obtained from Qiargen. ). The ginseng material for extracting 4 kinds of irradiated saponins with different content ratios of Panax diol saponins and Panax triol saponins from Ginseng was provided by Korea Ginseng and Tobacco Research Institute.

Ginseng Production

1) Selection of sample by part of raw ginseng

The ginseng for raw materials was divided into fuselage parts, rice, Central America, semi, and semi-semesters and used as extract samples for each part of ginseng. Considering that the composition ratio of Panaxanadiol saponin and Panaxanatriol saponin were similar as well as their contents, the extracts were prepared by extracting the same amounts of weight mix ratio (50:50).

2) Extract Extraction Manufacturing

The ginseng body parts of raw ginseng, each part consisting of American and Central American, semi and semi-seed are separated and extracted by adding ethyl alcohol to the sample ginseng for each part, and the extract is filtered and concentrated under reduced pressure. It was concentrated to the range to obtain X A from ginseng fuselage, X B from ginseng and Latin America, X C from ginseng semi, and X D from ginseng semi-semi. Irradiated saponins of the aforementioned four species showed different ratios of diol saponins and triol saponins.

Fabrication of SOD-CAT Fusion Vectors

SOD-CAT fusion vectors were prepared based on the DNA base sequences and the recognition sites of transcriptional regulators. (Kim, Y., H. H. Y. Yoo, G. Jung, J-Y. Kim and H. Rho, 1993. Gene 133, 267-271). Remove thymidine kinase promoter from BamHI and XhoI from pBLCAT2 (Luckow, B., and G. Schutz, 1987. Nucleic Acids Res. 15, 5490) vector containing CAT structural genes PRSP-1633 prasmid was prepared by inserting a 1.7 kb DNA fragment containing the transcriptional regulatory region of the enzyme.

Cell culture and maintenance

All animal cell lines were maintained at 5 ° C. CO2 and 37 ° C. and contained Dulbecco containing the antibiotics penicillin (100 units / ml), streptomycin (100 μg / ml) and 10% fetal calf serum. Cultured with modified Eagle's medium (Dulbecco's modified Eagle's medium: DMEM).

Transformation of Animal Cell Lines

Transformation was performed using the calcium sulfate method using BES [N, N-bis (2-hydroxyethyl) -2-aminoethane sulfonic acid] (Chen, C. and H. Okayama, 1988. Bio Techniques 6, 632-638). One day before transformation, 5 ml 10 cells per 10 cm plate with 10 ml complete culture, mix DNA (10 µg) with 50 µl of 2.5M CaCl2, and 500 µl 2xBBS (50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 6.95) After 10-20 minutes at room temperature, the cells were transformed.

Promoter strength measurement by CAT activity measurement

CAT activity measurements were cited by Goman et al. (Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Mol. Cell. Biol. 2, 1044-1051). After 48 hours after transformation, the cells were harvested, completely freed in 100 μl of cold 250 mM Tris-Cl (pH 8.0), and then frozen and thawed three times. β-galactosidase activity was measured in advance to calibrate the efficiency of transformation and to measure CAT activity (Sambrook, J., EF Fritsch, and T. Maniatis, 1989. Molecular Cloning; a laboratory manual. 2nd ed. cold Spring Harbor Laboratory, New York.) CAT reaction solutions were prepared together and prepared in tubes, and acetyl-CoA was dissolved in an amount sufficient to react. 20 μl of the cytosolic fraction boiled at 65 ° C. for 15 minutes was added to each CAT reactor, mixed, reacted at 37 ° C. for 1 hour, 1 ml of ethyl acetate was added, and chloramphenicol was extracted. Ethyl acetate was evaporated over 1 hour using a dryer (Speed-Vac evaporator) and then developed by thin film chromatography (TLC) plates. CAT activity was measured by exposing the TLC plate to an X-ray film.

Results and Discussion

end. Manufacture of ginseng by region

Raw ginseng was divided into fuselage parts, rice, Central America, semi, and semi-semi by each part and used as sample for extracting each part of ginseng. When red ginseng was used as such ginseng component, saponin content and composition of X prepared from raw ginseng for each part were shown in Table 1 below. The irradiated fraction of whole saponin, the main active ingredient of ginseng, was extracted from X Saponin content and composition of the four samples are shown in Table 2.

TABLE 1

Saponin Content and Composition of Ext Samples

(Unit:% of X in buildings)

* PD: Protopanaxadiol Saponin (Rb1 + Rb2 + Rc + Rd)

PT: ProtoPanaxatriol Saponin (Re + Rf + Rg1)

TABLE 2

-Saponin Content and Composition of Irradiated Phoponin Samples

(Unit:% of building irradiated ponin)

* PD: Protopanaxadiol Saponin (Rb1 + Rb2 + Rc + Rd)

PT: ProtoPanaxatriol Saponin (Re + Rf + Rg1)

I. Expression Analysis of Rat SOD1 Gene

-1633 bp transcriptional regulatory region of the HOD1 gene was placed in front of the chloramphenicol acetyl-transferase (CAT) structural gene to introduce these plasmid DNAs into HepG2 cells and correct the transformation efficiency. Promoter activity was compared by measuring CAT activity.

All. Diol-based probeponin assays induce increased antioxidant enzyme SOD1 gene expression

In order to investigate the expression of SOD1 genes caused by ginseng components, 48 hours after transfection using pRSP-1633, a plasmid having the longest transcription control site, HepG2 cells were investigated. B, probeponin C, and probeponin D were treated at 100 μg / ml, respectively, and 0.1% ethanol was treated as a control. Then 22 hours incubation on the medium containing blood. 1-1.5-fold in the experimental group treated with irradiation-ponin A (PD / PT = 1.55), 1.5-2.0-fold in the experimental group treated with irradiation-ponin B (PD / PT = 1.72), and irradiation-ponin C (PD / PT = 2.25) In the experimental group treated with, it was 1.-1.5 times, and the experimental group treated with irradiation-ponin D (PD / PT = 2.61) showed 2.5-3.5 times harmful oxygen scavenging enzyme induction effect (FIG. 3).

As described above, a recombinant gene carrier fused with a transcriptional regulatory site of a toxic oxygen scavenging enzyme (SOD1) gene and a structural gene of chloramphenicolacetyl-transferase (CAT) was introduced into HepG2 cells, and these ginseng saponin fractions were then introduced. According to Figure 3 showing the results of detecting the fractions showing the inducible effect by treating each of them through CAT activity measurement, SOD1 gene is induced by the treatment of irradiated with (pon) (A), (B), (C), (D) In particular, it shows that the SOD1 gene inducible effect of irradiated phosphonine D and C fractions extracted from the semi-semi and semi-area, respectively, is excellent. It can be seen that the X in the semi site can be used as an anticancer agent. Noxious oxygen scavenging enzyme (SOD1) was not inhibited by ginseng body irradiated saponin (A) and ginseng dam and central American irradiated saponin (B), which reaffirms the antioxidant enzyme-inducing effect of diol saponins. It can be seen that.

la. Argument

The specific inducibility of the SOD1 gene in four of the irradiated fractions of saponin, the main active ingredient of ginseng, was dependent on the saponin content and composition ratio. In particular, the higher the ratio of the content of diol saponin was found to be significantly higher induction effect of toxic oxygen scavenging enzyme through the experiment. Semi-semiconductor and semi-molecular parts with high relative content ratio of diol saponin were found to be the main samples that induce the increase of the number of SOD1, especially the sites containing large amounts of Rb1 and Rb2. Based on this, the effective PD / PT ratio of the irradiated saponin was about 1.8%, which is 80% of the ratio of the test group C (2.25), the semi-extracted irradiated saponin of ginseng. Fractions containing diol saponins prepared in ginseng bodies and in the Americas and Central America do not inhibit the transcription of SOD1 genes, which enables direct use of diol saponins at various sites in ginseng, which separates the components of a single probeponin. Considering the difficulty of the process and the limited yield, the ginseng can be industrialized as an anticancer agent because it provides an important meaning of providing usefulness as an antioxidant enzyme inducer of diol fraction.

An object of the present invention is to examine the antioxidant efficacy according to the composition ratio of ginseng saponin and to examine the saponin composition of each region of ginseng to be used to increase the amount of harmful oxygen X extracted from a special site of ginseng as industrial anticancer agent This is to make it available.

Claims (4)

Containing an investigational phosphonine that promotes the transcription of the NOx depletion enzyme (SOD1) gene, which is extracted from semi and semi-semi of ginseng with ethyl alcohol and has a compounding ratio (PD / PT) of panax diol and panaxtriol of 1.8 or more. Ginseng X. Promotes the transcription of the NOx depletion enzyme (SOD1) gene with a blending ratio (PD / PT) of panax diol and panaxtriol of 1.8 or more extracted from ethyl extract from semi and semi-semi of ginseng. Investigative phonine. A method for analyzing the antioxidant efficacy of ginseng saponins using a fusion vector between a transcriptional regulatory region of a toxic oxygen scavenging enzyme (SOD1) gene and chloramphenicol acetyl-transferase (CAT) structural genes. Concentration of 100 g / ml extract of ginseng was extracted in animal cells transformed using a plasmid prepared from a fusion vector of a toxic scavenging enzyme (SOD1) gene and chloramphenicol acetyl-transferase (CAT) structural gene. Method to assay the efficacy of inducing the expression of the antioxidant enzyme gene for the x by each part of ginseng.