KR20170095762A - Drug discovery methods using plant developmental biology - Google Patents

Abstract

The present invention relates to a method for discovering a bioactive material based on effects using plants to discovery a new drug leading material by repeatedly experimenting screening based on developmental biological marker phenotype of plants. To this end, specific phenotype showed in plants when a specific material is treated to plants is screened as a candidate material for developing a new drug.

Description

Technical Field [0001] The present invention relates to a method of extracting physiologically active substances from plants,

The present invention relates to a method for finding a physiologically active substance based on an efficacy using a plant, which can simultaneously reduce cost and safety in screening candidates for new drug development. More specifically, the present invention relates to a method for separating an effective ingredient by using a specific phenotype appearing in plants as a marker when various crude extracts and fractions thereof are administered to, for example, Arabidopsis thaliana.

Plants have long been used as medicines in humans. This is because the small molecule compounds produced by plants are capable of healing human diseases.

The development of new drugs from a modern point of view has been tried and tested at a very last stage after injecting hundreds of millions of people. In contrast, our herbal medicine system has used plant materials directly in humans to empirically establish its action and side effects.

Such natural products including herbal medicine have been established after long experience, but since one herbal medicine contains hundreds to thousands of substances, it can not deny the existence of toxic substances along with the active ingredients. Especially when the toxic component is present in excess of the lethal dose together with the active ingredient, the medicinal product can not be used.

In addition, there is a high possibility that a medicinal ingredient exists in a trace amount in the medicinal herb. In this case, even if the component is successfully extracted, it may not be commercialized due to the problem of supply of the herbal medicine.

Therefore, it is desirable to lower the complexity of the active substance present in the herbal medicine or ultimately to use it as a single substance level. However, due to limitations such as lack of efficacy screening system or high price, It is very slow.

On the other hand, in the process of separating the active ingredient from the crude extract into a single substance through a pharmacological test, a number of pharmacodynamic experiments must be repeatedly performed. The activity test for separating the active substance is various, such as enzyme activity measurement, cell test, and animal bio experiment. However, these methods involve several limitations.

Among the existing methods, the most accurate method of determining the pharmacological effect is to carry out an animal biological experiment. However, in the case of isolating the active ingredient from the crude extract using a disease model mouse, it costs tens of millions of won, and the development cost up to the market stage reaches hundreds of billions of dollars. Due to the expense, we often fail to try and give up.

Due to these constraints, separating the active ingredients from traditional medicines requires large capital investments. However, another problem is that despite these large investments, the value of the new drug is not recognized in the final stage of clinical trials, and thus it is highly withdrawn. In other words, the possibility of success in new drug development is very low. The reason for this is that new drug candidates are overly toxic, have low efficacy, and have many side effects.

Therefore, it is essential to identify substances with low side effects and toxicity, including dramatic cost reduction. At the stage of new drug development, the final step of human-directed testing is essential, so even if that part can not be avoided, it should be able to reduce costs at least in the pre-stage.

People and plants have basic biology preserved. These biochemical identities are responsible for the survival of cells in many parts of the cell, such as cell division, chromosome replication, RNA synthesis, protein metabolism, synthesis of corresponding processes or amino acids, small RNA metabolism, and energy metabolism in mitochondria .

Thus, the plant system may be used prior to, or in place of, an animal biological experiment when isolating the active ingredient of the herbal medicine. At least if you are developing a common biological metabolic process, you might prefer to prioritize plant systems.

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method of screening a plant for the purpose of screening a plant in place of an experimental animal in consideration of the fact that basic biology is preserved in humans and plants, .

Specifically, the present invention aims at discovering a new drug-inducing substance by repeating screening based on the developmental biological marker phenotype of a plant. In particular, it is aimed to separate new drug substance from herbal medicines.

The present invention also aims to differentiate and standardize herbal medicines in a more quantitative and scientific way based on the medicinal properties.

The present invention also aims to discover physiologically active substances that regulate plant growth.

In order to achieve the above object, the present invention provides a method for screening candidate substances for the development of new drugs or herbicides using markers of a specific phenotype appearing on plants when a specific substance is treated on plants, Thereby providing a method for finding a physiologically active substance. Wherein said plant is preferably Arabidopsis, and is one of wild type, mutant, transgenic plants. The transgenic plant may be one in which a chimeric gene in which a gene of a human disease, a carcinogenic mutation gene of a human, a cosmetic gene, a herbicide resistance gene, or other functionally proven gene has been linked to a reporter gene has been introduced .

The present invention also provides a method for standardizing an efficacy-based herbal medicinal product using a plant, wherein the herbicide is administered to a plant and a grade for the medicinal herb is determined based on a specific phenotype appearing in the plant. Herein, the plant may be a transgenic plant into which a chimeric gene having a reporter gene linked to a promoter of a gene sensitive to a specific herb extract is introduced. In this case, the degree of expression of the reporter gene can be quantitatively analyzed to determine the grade for the herbal medicine.

The present invention has the advantage of cost reduction and safety at the same time in screening candidates for new drug development.

In addition, there is an advantage in that it is possible to quickly and inexpensively find the active ingredient of a natural product, which is already marketed, including conventional herbal extracts, which have already been proved to be effective, and to find a mode-of-action easily .

It can also be used to demonstrate the safety and efficacy of new drugs.

In addition, if the degree of physiological activity of a specific natural substance is confirmed, it will lead to the development and commercialization of a product having a high content of the substance, and it is expected to increase the income of the region and farmers producing the product.

In addition, since the present invention is basically based on botanical understanding, it can lead to the development of basic botanical science. In other words, you can understand how a plant responds to new materials and unfolds its survival strategy. This understanding will enable us to acquire new knowledge that has not been known until now, because plants can test various genetic pools or flexibility that have been stored in the wild for many years.

Further, the present invention can be applied to the standardization of herbal medicines. Standardization requires a standard. However, although the surface component of the herbal medicine is known, precise standardization is impossible because the active ingredient is unknown. After accurately describing the phenotype of a plant that responds to a specific medicinal herb, you can set a grade for the medicinal herb based on this phenotype. This has the effect of promoting the scientificization of Chinese medicine and the industrialization of Oriental medicine.

In addition, the physiologically active substance selected according to the present invention has an effect of regulating the growth of the plant, so that it can be used in an agricultural aspect. For example, when the substance has a hitting effect inhibiting plant growth, It can be used as a herbicide which inhibits the growth of weeds.

In addition, the present invention can lead to the development of new high function transgenic plants, which can contribute greatly to the development of advanced agriculture, especially based on plant factories.

1) A photographs of cotyledons and hypocotyls and roots of Arabidopsis thaliana plants 3 days after germination, B) Pictures of root tips and roots of Arabidopsis, C) Photographs of Arabidopsis adults growing in soil, D) Is a picture of a single adult.
FIG. 2 is a diagram illustrating a process of extracting a bioactive substance based on a plant marker phenotype.
Figure 3 illustrates the process of securing fractions from crude extracts.
FIG. 4 is a diagram illustrating a screening method using transgenic plants.
FIG. 5 is a diagram illustrating a method of standardizing herbal medicines.
Figure 6 is a schematic illustration of an understanding of the mechanism of action of the active ingredient.
FIG. 7 is a graph showing the absorbance of crude extract according to PC1 and PC2 of Principal Component Analysis. FIG.
Fig. 8 is a photograph showing the response of the Arabidopsis thaliana plant to taxol and camptothecin.
Fig. 9 is a photograph showing the roots of the Arabidopsis thaliana control roots and roots treated with taxol.
FIG. 10 is a photograph of a Arabian pole showing a state in which a leaf surface rosette is changed from three terraces to one topography during taxol treatment.
11 is a graph showing changes in hypocotyl length growth of Arabidopsis thaliana plants depending on the concentrations of taxol and camptothecin.
Fig. 12 is a photograph showing the reaction of Arabidopsis thaliana plants to the blood-boosting fish bath and its constituent medicaments.
FIG. 13 is a photograph showing the reaction of Arabidopsis thaliana plants against the nucleus acuminata and its constituent drugs.
14 is a photograph showing the response of Arabidopsis thaliana plants to the empirical area and its constituent medicaments.
Fig. 15 is a photograph showing the reaction of Arabidopsis thaliana plants with other anticancer medicinal materials (bamboo shoots, ganoderma, lobules, mistletoe).
FIG. 16 is a graph showing the number of side weeds of the Arabidopsis thaliana plants appearing in response to herbal medicines and each prescription 1 / 5X diluent. * Means statistical significance (n> 8, p <0.05 Student's t-test)
Fig. 17 is a graph showing the number of side roots of Arabidopsis thaliana plants according to the concentration of the nuclerite root, rhubarb, and stalk. The horizontal axis indicates the dilution factor.
Fig. 18 is a root photograph of the Arabidopsis thaliana showing the response of roots to the concentration of ginseng crude extract.
FIG. 19 is a photograph of a Arabidopsis leaf showing a state in which the color of the colon is changed to reddish brown in response to the emergence.

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

1. Plant to be tested

The Arabidopsis (Fig. 1), a dicotyledon belonging to the cabbage family, has been an intensive subject of plant developmental biology research for the past 30 years and has received numerous innovative discoveries ( http://www.arabidopsis.org ) The published genomic sequences and genetic maps made it possible to understand systems biology based on molecular genetics and genomics.

Through the accumulated researches of many years, it is possible to understand the general growth of the Arabidopsis thaliana, the development of root and stem, the development of the stem, the development of the flower stem, the development of the leaf, the regulation of flowering, And mechanisms to respond to environmental stresses were understood at the genetic level.

The Arabidopsis plant has the advantage of being able to cultivate in limited space because the size of the seed is very small and the vegetative plant is also very small compared with other plants. High-throughput screening (HTS) is possible because the size of the Arabidopsis thaliana plants is so small that one individual can be administered to each well of a 96-well plate and the drug tested.

Also, the growth period is very short, and it takes about 4 weeks from the metamorphosis to the adult and 6 weeks from the metamorphosis to the seeding, which corresponds to the plant with the shortest cycle among seed-bearing plants. Therefore, it is advantageous to cultivate large scale in the laboratory and observe the dynamics.

It can be cultivated in various forms such as liquid, agar solid medium, soil, hydroponic cultivation depending on the analysis requirement. The present invention is based on the investigation of the reaction after culturing the Arabidopsis thaliana plants in a liquid culture medium containing a natural product for a certain period of time after germination.

2. How to identify physiologically active substances based on plant marker phenotype

If the phenotype of the plant induced by the active ingredient is established, the physiologically active substance can be extracted based on this phenotype.

FIG. 2 is a diagram illustrating a process of extracting a bioactive material based on a plant marker phenotype. As shown in FIG. 2, the present invention relates to a method for detecting a specific growth reaction (i.e., a marker phenotype) after culturing a plant in a medium containing a natural product or a compound, Repeated fractions and efficacy tests can be used to isolate a single substance that causes marker phenotypes.

3. Isolation of physiologically active components

There are two main ways to search for a bioactive substance from a crude extract or fraction as a single component.

First, it is the approach by stepwise fractionation.

The crude extract is systematically fractionated according to solubility, molecular weight, etc., and the activity test is carried out to reduce the complexity of the natural product and ultimately to separate the single substance having the drug effect.

Figure 3 is a diagram illustrating the process of securing fractions from crude extracts based on solubility differences. As shown in FIG. 3, the solvent is changed from a polar solvent (methanol) close to water to a nonpolar solvent (nucleic acid or chloroform) to obtain a fraction, and a substance dissolved in the solvent is tested. When activity is high in a particular fraction, the fraction may be further separated according to molecular weight, etc., and then subjected to an activity test to separate a single substance.

Second, it separates directly the substances that penetrate into the roots.

All small molecule materials were separated from the control and treatment roots and compared with the control and experimental groups. The fractions are analyzed by FT-IR, broadband absorbance check, HPLC, MS / MS. If a particular substance difference appears, the substance will be the primary active substance to be analyzed.

4. Development of an automated quantitative analysis system that responds to the active ingredients of a particular natural product

One of the effects of this technology is to provide a standardization technique for herbal medicine. This requires the development and use of systems that can more quantitatively measure the response to natural products.

For example, the following method can be used for quantitative comparison according to expression of a reporter gene. For example, a microarray or RNA-seq is performed to digest Arabidopsis thaliana plants with extracts and then to identify genes that respond to them. We develop a chimeric gene Promoter-Lucicerase or Promoter-Venus (GFP) reporter system that binds a reporter gene with a promoter of a gene whose expression is induced for a specific extract. By introducing this reporter system into the Arabidopsis thaliana, a transgenic plant can be produced and quantitative analysis using this plant can be attempted.

In other words, the expression level of the reporter gene in the regional or developmental stepwise extracts can be grasped, and the degree of induction of expression by the chemical fraction of the extract can be grasped to determine the intensity of the specific physiologically active substance.

5. Identification of mechanism of action of physiologically active ingredient

The genetic understanding of the mechanism of action of bioactive substances at the target gene level has several important implications. First, botany has its own meaning. It is a very interesting field of pure botanical research that the defense mechanism of this plant when the foreign small molecule substance is administered to the Arabidopsis thaliana plant. Evolutionally, it can provide clues to understand how the plant reacts to the external environment.

Traditional genetic methods are used to find these mechanisms. Approximately 200,000 individuals of the EMS-treated mutant population are grown on media supplemented with the natural product.

After a certain period of growth, a susceptible mutant or susceptible mutant that does not exhibit a specific marker phenotype may be isolated. Mutations can be identified through mutagenesis several times to remove mutations that are not related to the marker phenotype, and then whole genome resequencing can be used to identify which genes are mutagenized and become resistant. Because Arabidopsis is a model plant, genome sequences and functional genomics have been developed, so that understanding of the genetic mechanisms can be done easily.

It is also possible to obtain a mechanism of action in humans. If a specific biological process is found to be involved in Arabidopsis, it will be possible to deduce it from human biology. If, as in the case of camptothecin, the activity of the plant's topoisomerase I is found to be affected, it may explain the mechanism centered on the gene involved in human cell division.

6. Screening method using transgenic plants

FIG. 4 is a diagram illustrating a screening method using transgenic plants. Transgenic plants can be prepared by cloning genes other than the human disease target gene, human carcinogenic mutation gene, cosmetic gene, herbicide resistance gene, and other functionally proven genes exemplified in Fig. 4 and introducing them into Arabidopsis thaliana plants. These genes will be chimeric genes that function in conjunction with reporter genes that produce photochemically easy-to-detect substances. For example, Luciferase is easy to detect the activity, so it combines human disease gene with Luciferase to make chimeric gene and introduce it into plants to produce transformant. By applying various natural products to this transformant, the reaction can be quantitatively investigated and analyzed.

7. How to standardize herbal medicine

When a plant phenotype induced by the active ingredient is established, the herb medicine can be standardized based on this phenotype, and the herb medicine collected at different regions or times can be classified based on the phenotype of the plant, that is, .

FIG. 5 is a diagram illustrating a method of standardizing herbal medicines. Analyzes of reporter activity assays, marker phenotypes reactive with natural products, metabolite patterns, and RNA-seq data can be used to comprehensively assess the amount of active ingredients contained in the medicinal product being tested.

8. Understanding the mechanism of action of active ingredients

Figure 6 is a schematic illustration of an understanding of the mechanism of action of the active ingredient. It is based on the Arabidopsis genetics to find the target gene of the active substance and to understand the biological process. Once this process is understood in plants, this process can be applied to human biology.

<Experimental Example>

1. Preparation of Hot Water Extract from Traditional Chinese Medicines

In order to prepare extracts that have already proven their efficacy in traditional use, they have followed the hot water extraction method instead of organic solvents. The medicines were purchased at Kyungdong market in Gyoung - dong.

First, the dried medicinal herbs were cut into small pieces and then put into a glass bottle together with 10 ml of distilled water per 1 g of herbal medicine. The glass bottle inlet was loosely closed with a lid, placed in a high-pressure sterilizer and extracted at 110 ° C for 1.5 hours.

After high pressure extraction, the glass bottle was placed in an oven at 50 ° C and slowly cooled. In order to obtain the hot-water extract, the liquid component was placed in a 50 ml falcon tube by tilting the glass bottle. After centrifugation at 2650 rpm for 10 minutes to remove solids and suspended particles, the supernatant was transferred to a new tube. The thus obtained extract was used as a stock solution (1X) to separate the active ingredients.

2. Analysis of the differences according to the absorbance of the hot-water extract

To investigate whether hydrothermal extracts of medicinal herbs contain various natural products spectrophotometrically and to distinguish them from other medicinal materials due to such differences, the absorbance of the extracts was examined at different wavelengths.

The extracts were diluted with distilled water and each dilution was dispensed into 3 or 6 replicates on a 6 or 12 well cell culture plate (SPL, Korea), and the absorbance was measured using an Epoch Microplate Spectrophotometer (BioTek, US). At this time, distilled water used as a diluent was used as a control group. The absorbance was measured at intervals of 5 nm from 200 nm to 995 nm and statistical analysis was performed using the absorbance of the distilled water from the absorbance of the extract using the limit value.

Principal component analysis (PCA) was performed using R package to show the difference between medicinal materials in two - dimensional graph.

FIG. 7 is a graph showing the absorbance of Crude Extract according to PCA and PC2 of PCA. Natural products derived from the same medicinal materials are relatively closely distributed, and natural products derived from different medicinal herbs are classified into 2 It is shown that they are distributed in different areas in the dimension space.

Therefore, unknown natural products can be identified by this method, and can be used as a tool for standardization in terms of authenticity of natural products and inclusion of active ingredients according to proximity. In other words, this distribution pattern can be used for the standardization of the medicinal ingredients and the natural medicines.

3. Culture of Arabidopsis thaliana

After sterilization of the wild seedling, Columbia seed, it was allowed to stand at 4 ℃ for one day to increase the germination rate. MS agar The seeds of Arabidopsis thaliana seedlings sown on solid medium were germinated at 22 ℃ for 16 hours / night for 8 hours under long-day conditions and grown for 2 ~ 3 days.

The crude herbal extract solution was diluted at a ratio of 0.5X, 0.25X, and 0.05X, and 1ml of MS liquid medium and distilled water were appropriately blended so that the total volume became 2ml. Each dilution was dispensed into 3 wells of a 12 well cell culture plate, and then 5 plants of Arabidopsis thaliana were added to each well, and the plate was placed on a stirrer and cultured at 100 rpm for 3 days. After 3 days, the growth of the seedlings was analyzed.

The analysis of the reaction of Arabidopsis thaliana plants against the anticancer agent was carried out in the same manner except that the anticancer agent was administered instead of the herbal medicine extract.

The length of root, the number of root tips, and the length of hypocotyls were measured to distinguish the developmental biological differences that the Arabidopsis thaliana plants develop in response to anticancer drugs and extracts. The measurement of the length was made using ImageJ software (http://rsb.info.nih.gov/ij/) as the image of the photo file.

4. Reaction of Arabidopsis thaliana plants to FDA-approved anticancer drugs

(1) Selection of anticancer drugs

To test whether the growth of Arabidopsis thaliana could be used as a marker phenotype, we investigated the Arabidopsis response to Taxol (chemical name: Paclitaxel) and camptothecin, an anticancer drug of US FDA approved anticancer drugs .

Taxol is produced in Taxus plants and is known to act on tubulin and inhibit chromosomal migration during cell division. In contrast, camptothecin is produced in the bark and stem of Camptotheca acuminata and is known to act on DNA topoisomerase I to inhibit DNA supercoiling relaxation function. In other words, we could confirm the usefulness of this method by observing whether two different anticancer drugs cause different phenotypes in plants.

(2) With Taxol Changes in Root Length and Side Root Development of Arabidopsis thunbergii According to the Concentration of Camptothecin

Fig. 8 is a photograph showing the appearance of Arabidopsis thaliana plants treated with taxol and camptothecin.

As can be seen from FIG. 8, when Taxol was treated, the growth of root and hypocotyl was inhibited in proportion to the concentration of DMSO treatment. However, the generation of side root was not greatly affected. Whether treated or not, the number of side root was about five.

In contrast, camptothecin significantly suppressed the production of side root. In the case of camptothecin, the inhibition of the side branching was suppressed at a concentration as low as 0.05 uM. It is shown that anticancer drugs having different action mechanisms induce different growth patterns in plants, and suggests that it is possible to screen efficacy-based substances with these growth patterns as markers.

(3) Changes in roots of Arabidopsis thaliana plants treated with taxol

Fig. 9 is a photograph showing the roots of the Arabidopsis thaliana control roots and roots treated with taxol. The photographs marked with Mock show the control treated with water, and the photos marked with Taxol show the treated after treatment for 3 days at 2 uM. The roots treated with taxol are more dense between root and root than the control, and the epidermal cells are opaque. This is because the epidermal cells of the root are separated from the tissue and have an atypical static appearance. This phenomenon is a specific phenotype of Taxol, which shows that the drug induces a distinctive feature in the growth of the plant, and this feature can be used as a marker phenotype.

(4) Changes in leaf surface texture of Arabidopsis thaliana plants treated with taxol

FIG. 10 is a photograph of a Arabian pole showing a state in which a leaf surface rosette is changed from three terraces to one topography during taxol treatment.

Plants make numerous hairs on the surface of the leaves to protect themselves from insects. In the case of Arabidopsis, the hairs are branched into three. However, as shown in FIG. 10, when taxol was treated, the branches of the squamous cells changed from the normal three-point type to the one-point type compared to the control group. The development of the soma is related to the development of the root hair, and its molecular mechanism is well known.

(5) Taxol and According to the concentration of camptothecin , Change in hypocotyl length growth

11 is a graph showing changes in hypocotyl length growth of Arabidopsis thaliana plants depending on the concentrations of taxol and camptothecin. Statistical analysis was carried out to investigate the degree of hypocotyl growth. As can be seen in FIG. 11, the growth of hypocotyls was inhibited at an overall significant level.

5. Response of Arabidopsis thaliana plants to medicinal plants

(1) Selection of medicinal herbs to be surveyed

Based on the results of the above anticancer drug response, we decided to test herbal medicines with similar functions. First of all, we selected 'traditional medicine' (Moon et al., 2006), which is a prescription known to have anticancer activity in general, and ' Some of their constituents and some other anti-cancer medicines were the subjects of the primary investigation.

(2) Subject to prescription and composition of medicinal herbs

[Table 1] Prescribing and constituting medicinal herbs for investigated medicinal herbs are as follows.

After extracting crude extracts from each prescription and herbal medicines, a culture diluted 1/5 was used as the initial concentration. A 0.5 ml crude extract was added to 1.5 ml of 0.5X MS medium to prepare a culture.

[Table 1]

(3) Response of Arabidopsis thaliana plants to crude extracts

Fig. 12 is a photograph showing the reaction of the Arabidopsis thaliana plant to the blood-shrimping bath and its constituent medicinal plants, Fig. 13 is a photograph showing the reaction of the Arabidopsis thaliana plant to the thalamic nutrient bath and its constituent medicinal materials, Fig. 15 is a photograph showing the response of the Arabidopsis thaliana plant to other medicinal herbs (bamboo shoots, ganoderma, lobules, mistletoe). Fig.

As can be seen from Figs. 12 to 15, the roots were generally short in length and the formation of side roots was inhibited. Some roots were browned. However, the inhibition of root growth was weak in some medicines, and the length extension of hypocotyls was generally controlled in the same direction as root growth. In other words, growth of hypocotyl was suppressed when root length was inhibited.

It is suggested that the prescription used as an anticancer agent in oriental medicine and its constituent medicines generally inhibit the side root formation, suggesting the possibility of separating the new material from the marker of the side root formation inhibitory phenotype.

It is known that Arabidopsis root of Arabidopsis is formed by repetitive cleavage after acquiring cleavage ability of root cells (Dubrovsky et al., 2000). Therefore, it is expected that medicinal herbs that inhibit the production and growth of side roots will contain substances that generally inhibit cell division.

(4) Statistical analysis of side-root production

In order to quantitatively analyze the reaction of each herb medicinal material, the number of side root was measured in more than 12 seedlings by each treatments. As shown in Fig. 16, it was found that many herbal medicines inhibit the formation of side root, although there is a difference in degree. However, some herbal medicines such as chrysanthemums did not have a great influence on the formation of side root, or the side of root of small root accelerates the formation of side root.

(5) Measurement of the concentration of IR50

As a representative example of the inhibition of side root formation, the concentration of crude extract required to reduce the number of side roots by half compared with the control was obtained by further analyzing the seedling root, rhubarb and stopper. When the crude extracts were freeze-dried, they were 24 mg / ml in the nucleation bath, 30 mg / ml in the rhubarb, and 3 mg / ml in the stool. Based on the statistical analysis, the concentrations of crude extracts required to inhibit side-rooting by half were very low, 74 ug / ml, 77 ug / ml, and 11 ug / ml, respectively (Fig. 17) . These results suggest that plant response is very specific and very sensitive system.

(6) Response of Arabidopsis thaliana plants to other medicinal plants

6-1. Effect of Ginseng Extract on the Root Growth of Arabidopsis thaliana

FIG. 18 is a root photograph of the Arabidopsis thaliana showing the response of roots to the concentration of ginseng crude extract.

As shown in Fig. 18, when the Arabidopsis thaliana was cultured under various concentrations, the total root length was shortened according to the concentration, and the number of side root was increased only at a specific concentration. Specifically, at the low concentration of 0.05X, the side root was developed and the root hair was densely formed. At 0.25X, the number of side root was increased but the root length was decreased. At the highest concentration of 0.5X, the growth of side root was suppressed and the length of main muscle was also shortened.

At 0.25X and above, the growth inhibition of roots is remarkably shorter than that of the water-treated control, indicating that the components of ginseng have a negative effect on the growth of Arabidopsis. This reaction is attributed to the ginseng component, which may be used as a marker phenotype for separating certain active ingredients from ginseng. .

6-2. Arabic pole On Yu plants  Felicity Crude extract  Influence observation

FIG. 19 is a photograph of a Arabidopsis leaf showing a state in which the color of the colon is changed to reddish brown in response to the emergence.

Fig. 19A shows the control and B shows the color change after treating the grass extract. It was confirmed that the color of the base of leaf area changed from green (A) to reddish brown (B). This is a specific reaction that appears in the shoot and can be used as a marker phenotype when the substance inducing this reaction is separated from the shoot.

<References>

Cragg GM, Grothaus PG, Newman DJ (2009) Impact of natural products on developing new anti-cancer agents. Chem Rev 109: 3012-3043

Ishida T, Kurata T, Okada K, Wada T (2008) A genetic regulatory network in the development of trichomes and root hairs. Annu Rev Plant Biol 59: 365-386

Newman D, Cragg G (2009) Natural products in medicinal chemistry. Bioorg Med Chem 17: 2120

Schmidt BM, Ribnicky DM, Lipsky PE, Raskin I (2007) Revisiting the ancient concept of botanical therapeutics. Nat ChemBiol 3: 360-366

Moon NY, Kim DC, Baek SH (2006) Effect of Hyulbuchukotang on the inhibition of proliferation of uterine leiomyoma cells and cell apoptosis. Journal of Oriental Obstetrics and Gynecology 19 (2): 186-198

Claims (6)

By establishing a specific phenotype that appears in plants when the plant is cultured in a medium containing the active ingredient and when the plant is cultured in a medium containing the herb extract, by contrasting the specific phenotype of the plant with the marker, And screening for a candidate substance for development. The method according to claim 1, wherein the plant is a Arabidopsis thaliana. The method according to claim 1 or 2, wherein the plant is any one of wild type, mutant, and transgenic plants. [Claim 4] The plant according to claim 3, wherein the transgenic plant is a transgenic plant into which a chimeric gene in which a gene of a human disease, a carcinogenic mutation gene of a human, or a cosmetic gene is linked to a reporter gene A method for discovering physiologically active substances based on efficacy using. [Claim 3] The method of claim 2,
A. Establishing a specific phenotype as a marker in the Arabidopsis thaliana when the Arabidopsis thaliana plant is germinated and cultured in medium containing the active ingredient for a certain period of time;
B. selecting a herbal medicine ingredient which is expected to contain the medicinal ingredient by comparing the specific phenotype appearing in Arabidopsis thaliana with a marker when the medicinal herb plant is germinated and cultured in a medium containing the herbal medicine extract for a certain period of time;
Wherein the method comprises the steps of: [Claim 6] The method according to claim 5,
C. When the selected herbal extracts were fractionated and the Arabidopsis thaliana plants were cultured for a certain period of time in the medium containing the respective fractions, the specific phenotype appearing on the Arabidopsis thaliana was compared with the marker, Selecting fractions to be separated;
D. fractionating the selected fractions and repeating step C, thereby reducing the complexity of the medicinal natural substance contained in the medicinal plant extract and accessing the medicinal ingredient;
Wherein the method further comprises the step of extracting the physiologically active substance from the plant.

Images