TWI382845B - Cuscuta sp. extract and method for preparing the same and uses of cuscuta sp. - Google Patents

Description

Cuscuta extract and preparation method and application thereof

The present invention relates to a Cuscuta sp. extract and a preparation method thereof, in particular to a medicinal component-containing silk extract, a preparation method thereof, and an application of the medicinal component of the scorpion in extracting plants.

In the large-scale herbal process, it is usually necessary to plant a plant having a medicinal ingredient, and to harvest it after the growth period, and then perform steps such as drying, grinding, extracting, and concentrating to prepare a drug-containing component. Plant extracts. In the above process, a large area of land is required to plant the plant, and it is necessary to wait for the plant to mature after a certain period of time, and the plant must be replanted after harvesting. In addition, subsequent extraction and concentration requires complicated steps and consumes a large amount of organic solvent. Therefore, the traditional herbal process is quite time consuming and laborious, and the cost is high, the efficiency is low, and even environmental pollution is caused.

Cuscuta sp. is a parasitic Convolvulaceae plant with a special physiological structure. Since the cotyledons do not contain chloroplasts and cannot be used for photosynthetic nutrients, they must be entangled in the host plants, and inserted into the phloem of the host plant by the haustorium on the stalks of the stalks to connect with the phloem of the dodder itself. And maintain the life and growth by absorbing the nutrients of the host plant. In addition, dodder has no host specificity, it can be parasitic on various plants arbitrarily, and has self-parasitic self-parasitim ability, even parasitic on other parasitic plants (hyperparasitism). ability. For the parasitic properties of Cuscuta, see, for example, Birschwilks et al. Transfer of phloem-mobile substances from the host plants to the holoparasite Cuscuta sp. Journal of Experimental Botany . 57: 911-921 (2006), the contents of which are here for reference.

The inventors of the present invention have found that the dodder is parasitized on a plant having a medicinal component, and the medicinal component can be taken out from the plant and retained in the dodder, and the medicinal component can be obtained, and the medicinal component can be effectively extracted from the plant, thereby eliminating the aforementioned The complicated concentration steps of traditional herbal processes and other related disadvantages.

It is an object of the present invention to provide a method for preparing an extract of Cuscuta sp. comprising: a) parasiticly growing and growing on a medicinal plant; b) collecting the liquid in the dodder; And extracting the liquid using a solvent to obtain the extract; wherein the extract comprises at least one medicinal ingredient of the medicinal plant. Preferably, the medicinal plant is Camptotheca or Gynostemma pentaphyllum.

Another object of the present invention is to provide a dodder seed extract which is obtained by the above method.

It is still another object of the present invention to provide a method for extracting camptothecin and/or anti-blood cancer components from plants, comprising: I) parasitizing and growing on the plant; and II) collecting the liquid in the dodder; And III) purifying the liquid to obtain the camptothecin and/or anti-blood cancer component.

The detailed description of the present invention and the preferred embodiments thereof will be described in the following description.

The use of the terms "a", "an", and <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; In addition, in the present text, the term "medicinal ingredient" means any pharmacologically active ingredient such as a compound, a saccharide, an enzyme, an extract or the like.

Cuscuta sp. is a parasitic plant, and the stem of the stem can be inserted into the phloem of the host plant to absorb the nutrients of the host plant to survive. Generally, for the understanding of Dodder, it is only known that it has the characteristics of absorbing the nutrients of the host plant to maintain growth. The present inventors have found that the dodder is parasitic on a plant having a medicinal ingredient (ie, a medicinal plant), and the medicinal component can be extracted from the plant by the dodder, and the medicinal component is retained in the dodder to achieve the effect of natural concentration. To obtain botanical medicinal ingredients.

Accordingly, the present invention provides a method of preparing a dodder seed extract comprising: a) parasitizing and growing a dodder on a medicinal plant; b) collecting the liquid in the dodder; and c) extracting the liquid using a solvent And obtaining the extract; wherein the extract comprises at least one medicinal ingredient of the medicinal plant.

The dodder used in the method of the present invention may be a dodder of various strains without any limitation. For example, a Cuscuta japonica Choisy var. japonica or a Japanese genus, such as Cuscuta japonica Choisy var. formosana (Hayata) Yunck., which is distributed among various filth strains distributed in Taiwan, may be used. , the most common and the easiest to get.

In step a) of the method of the present invention, the dodder can be parasitized on various medicinal plants, such as woody plants, herbaceous plants, ferns, and combinations thereof; preferably, parasitic in a group selected from the group consisting of Medicinal plants: Camptotheca acuminate , Gynostemma pentaphyllum , Morus alba L., Vinca rosea L., Lantana camara L., Kalanchoe pinnata , Duranta repens L., Cucurbita spp. , and combinations thereof.

In step b), the liquid in the phloem of the dodder is mainly collected to obtain the medicinal component in the medicinal plant. Any physical method can be employed to obtain the liquid. For example, the liquid can be separated from the silk by a centrifugal separation operation. If necessary, the collected silk seeds can be cut into small pieces and then centrifuged to improve the collection efficiency. Without being bound by theory, the inventors believe that since the dodder is parasitic on medicinal plants, a stem is produced on the stem and inserted into the phloem of the medicinal plant, so that the phloem of the dodder itself is connected to the phloem of the medicinal plant. And then extract the ingredients in the medicinal plants. However, since the medicinal component in the medicinal plant is not a component required for the growth of the dodder, it is concentrated and retained in the dodder, especially in the liquid of the phloem of the dodder.

In step c), the dodder liquid obtained in step b) can be extracted with a suitable solvent to obtain a dodder extract. For example, a solvent selected from the group consisting of water, methanol, ethanol, propanol, butanol, chloroform, and combinations thereof can be used. Preferably, a solvent selected from the group consisting of water, methanol, ethanol, propanol, chloroform, and combinations thereof is employed.

It may be necessary to use step (c) in addition to other suitable extraction means (such as ultrasonic vibration, etc.) to enhance the extraction effect. In addition, step c) may be repeated as needed to separate as much as possible the active ingredient and the inactive ingredient in the liquid of the dodder, and extract the desired pharmaceutical ingredient as much as possible.

In the step c), the liquid collected in the step b) may be dried to obtain a dried product before the extraction, and the dried product may be extracted using the above solvent.

In general, depending on the application form of the Cuscuta extract, the Cuscuta extract obtained in the step c) can be further dried as needed. For example, if the extraction solvent selected in the step c) is methanol or ethanol which is not skin irritating, and the obtained extract is to be directly applied to, for example, the skin, the extract is not additionally dried. However, if the silk extract of the present invention is to be administered orally, a drying step (for example, freeze-drying, concentration under reduced pressure, and/or gas introduction) may be utilized to remove the organic solvent in the silk extract. Avoid organic solvents that can harm your body.

In one embodiment of the method of the present invention, the dodder is parasitized on the camptotheca acuminata; thereafter, the liquid is separated from the grown dodder, and the liquid is extracted to obtain a dodder extract containing camptothecin. Preferably, the solvent used in the extraction step is methanol, and the amount of methanol is from about 30% by volume to about 99% by volume based on the total volume of methanol and the liquid; more preferably, the amount of methanol is about 60%. 5% by volume to about 99% by volume. Alternatively, the liquid may be first dried to obtain a dried product which is then extracted using methanol. The Cuscuta extract may be further purified and separated to obtain camptothecin for use in the preparation of a pharmaceutical composition. For example, purification separation can be carried out using High Performance Liquid Chromatography (HPLC) to obtain a high purity camptothecin.

Camptothecin is a compound present in Camptotheca acuminata having a chemical structure represented by the following formula (I) and found to have anticancer activity, for example, for treating digestive tumors and melanoma tumors.

However, the concentration of camptothecin in the camptotheca acuminata is quite low, and the content (dry weight ratio) of the camptothecin is 0.01 to 0.1% by weight of the young leaves of Camptotheca acuminata; 0.012% by weight of the camphor tree; It is 0.05% by weight of Camptotheca acuminata; 0.02% by weight of Camptotheca acuminata; and 0.02% by weight of Camptotheca acuminata seeds. This can be reported by Zhang Shuhua, a researcher at the Forestry Research Institute, Yulin Group (Forestry Research News, Vol. 13, No) 4, 2006), the entire disclosure of which is hereby incorporated by reference. Therefore, if a large amount of camptothecin is obtained from the self-grass tree by the conventional plant extraction method, the yield is still quite limited even if it is expensive to carry out large-scale planting and extraction. However, according to the method of the present invention, camptothecin can be extracted economically and simply by the eucalyptus eucalyptus tree in the case where the preserved eucalyptus is substantially intact.

In another embodiment of the method of the present invention, the dodder is parasitized to Gynostemma pentaphyllum; thereafter, the liquid contained therein is separated from the grown dodder, and the liquid is extracted to obtain a dodder extract. Gynostemma pentaphyllum is known to have anti-tumor benefits, especially against blood cancer. Preferably, the solvent used in the extraction step is methanol, and the amount of methanol is from about 40% by volume to about 99% by volume based on the total volume of methanol and the liquid; more preferably, the amount of methanol is about 60%. 5% by volume to about 99% by volume. Here, the liquid may be first dried to obtain a dried product, which is then extracted with methanol. As shown in the appended examples, the dodder seed obtained by parasitizing the dodder seed in the method of the present invention has anti-blood cancer effect, and proves that the method of the present invention can effectively extract anti-blood cancer components from Gynostemma pentaphyllum, and provide the anti-blood cancer component. Cuscuta extract.

The present invention also provides a dodder seed extract which can be obtained by the above method of the present invention. Cassia extract containing different medicinal ingredients is provided depending on the medicinal ingredients contained in the host plant used. Wherein, when the host plant used is Hibiscus, the provided dodder extract contains camptothecin; and when the host plant is Gynostemma pentaphyllum, the provided dodder extract contains anti-blood cancer components.

According to the present invention, in addition to being used for the preparation of a medicinal ingredient, the dodder can also be used for detecting whether a medicinal ingredient is contained in a plant. Wherein, the dodder is parasitized on a host plant, and the dodder extract is obtained from the parasitic silk, and then the physiological activity test is performed on the dodder extract by testing whether the extract contains the desired pharmacologically active ingredient. It is known whether the host plant contains the pharmacologically active ingredient. Therefore, the present invention can perform active screening by a simple means, and it is not necessary to carry out a large number of planting and complicated extraction and concentration steps, which is advantageous for the detection of pharmaceutical ingredients and the development of new drugs.

The invention further provides a method for extracting camptothecin and/or anti-blood cancer component from a plant, comprising: I) parasitizing and growing on a plant; II) collecting the liquid in the dodder; and III) purifying The liquid is used to obtain the camptothecin and/or anti-blood cancer component. The method of the invention can be used to detect and further extract camptothecin and/or anti-blood cancer components in plants. Preferably, the plant of step I) is camptotheca or gynostemma, and in step III), the solvent obtained by step II) is extracted using a solvent to obtain an extract, wherein the solvent is selected From the following groups: water, methanol, ethanol, propanol, butanol, chloroform, and combinations thereof; more preferably, the solvent is methanol.

In step III), the camptothecin or anti-blood cancer component can be isolated by a suitable purification method for the preparation of a pharmaceutical composition. For example, to purify camptothecin, high performance liquid chromatography can be used.

The present invention utilizes dodder seeds to provide a medicinal ingredient and has a number of advantages. First, the method of the present invention only needs to collect the dodders parasitic on the host plant (ie, the medicinal plant) to obtain the desired medicinal component without consuming or damaging the host plant, thereby maintaining the integrity of the plant. Not restricted by host plants that are difficult to obtain or endangered. In addition, as long as the host plant is still in a growing state, the medicinal ingredient can be continuously produced by the method of the present invention, so that a large area of land is not required for large-scale and repetitive planting, and the plant is allowed to grow to maturity. The time of the period completely solves the shortcomings of traditional herbal preparation methods that must be replanted after plant harvesting. Furthermore, since the medicinal component obtained by the method of the present invention is naturally concentrated by the dodder, the complicated production steps such as drying, grinding, extraction, and concentration of the plant can be reduced, and the process of simplifying the process, saving the solvent and manpower can be achieved, and the effective saving can be achieved. Energy and land demand, reducing costs.

The invention is further illustrated by the following specific embodiments. The embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[Example 1] Preparation of medicinal ingredients in plants

Experiment A, preparation of extracts of Japanese dodders parasitic on the eucalyptus

First, about 15 cm of Cuscuta japonica Choisy var. japonica was taken and parasitized on Camptotheca acuminate and allowed to grow on the camptotheca. During the parasitic period, a straw is produced on the stem of the Japanese stalk, and inserted into the phloem of the camptothelium to absorb nutrients. After at least 10 days of parasitization of Cuscuta in Japan, the new tissue of the Japanese Cuscuta was taken and cut into pieces of about 1 cm, which was placed in a centrifuge tube and centrifuged (DENVILLE 260D, purchased from DENVILLE Scientific). After centrifugation at a speed of 10,000 rpm for 5 minutes, the liquid obtained by centrifugation was collected to obtain a phloem sap of Japanese scorpion. Next, the liquid is dried to obtain a dried product, and the dried product is extracted with methanol to obtain a Japanese dodder extract containing a component of the phloem of the camptotheca.

Experiment B, measuring the content of camptothecin in Japanese dodder extract

The Japanese Cuscuta extract prepared in Example 1 was analyzed for the camptothecin in the extract by High Performance Liquid Chromatography (HPLC, L-2455, available from HITACHI). content. The analytical conditions of the liquid chromatograph are: chromatographic column: KANTO Chemical Company, RP-18 GP Aqua 250-4.6 (5 μm); detection wavelength: 254 nm; mobile phase: acetonitrile (40%)-30 Ammonium acetate (60%); injection volume: 10 μl; flow rate: 1 ml/min; elution time: 10 minutes. The camptothecin standard (purchased from Sigma-Aldrich Chemical Co.; C9911-100MG) was subjected to HPLC comparative analysis with the extract to determine the camptothecin concentration in the extract, and the results are shown in Fig. 1.

The results of the HPLC analysis showed that about 3 μg of camptothecin (i.e., 3 wt/vol% (3 μg/100 μl)) was contained per 100 μl of Japanese dodder phloem sap.

The above results show that the preparation method of the present invention can efficiently and economically prepare camptothecin compared to the conventional planting method.

[Example 2] Detection of medicinal components of plants using dodder Experiment C. Preparation of an extract of Japanese dodder, which is parasitic on Gynostemma pentaphyllum

First, about 15 cm of Japanese dodder was used, which was parasitized on Gynostemma pentaphyllum and allowed to grow on Gynostemma pentaphyllum. During the parasitization, the stalk of the Japanese stalk is produced and inserted into the phloem of Gynostemma pentaphyllum to absorb nutrients. After at least 10 days of parasitization of the dodder, the new tissue of the dodder was taken and cut into pieces of about 1 cm, and then placed in a centrifuge tube, centrifuged (DENVILLE 260D, purchased from DENVILLE scientific) at 10,000 rpm. After centrifugation for 5 minutes at a speed of /min, the liquid obtained by centrifugation was collected to obtain a phloem sap of Japanese scorpion. Next, the liquid is dried to obtain a dried product, and the dried product is extracted with methanol to obtain a Japanese dodder extract containing a component of the phloem of Gynostemma pentaphyllum.

Experiment D, preparation of extracts of Japanese dodders parasitic on eucalyptus

First, take about 15 cm of Japanese dodder, parasitize it on the eucalyptus ( Ficus microcarpa ) and grow it on the eucalyptus. During the parasitization, a straw is produced on the stem of the Japanese stalk and inserted into the phloem of the eucalyptus to absorb nutrients. After at least 10 days of dodder parasitization, the new tissue of the dodder was taken and cut into pieces of about 1 cm, which was placed in a centrifuge tube and centrifuged (DENVILLE 260D, purchased from DENVILLE Scientific) at 10,000 rpm. After centrifugation for 5 minutes at a minute speed, the liquid obtained by centrifugation is collected to obtain a phloem sap of Japanese scorpion. Next, the liquid is dried to obtain a dried product, and the dried product is extracted with methanol to obtain a Japanese dodder extract containing a component of the phloem of the eucalyptus.

Experiment E. Detecting the medicinal components of the host plant Gynostemma pentaphyllum and Eucalyptus

In this experiment, human bone marrow cancer cell line HL-60 (Human Leukemia cell line-60) (purchased from the Food Industry Research Institute) was used for cell cytotoxicity and apoptosis induction (apotosis) test to screen from the experiment. C and the Japanese Cuscuta extract prepared in Experiment D, thereby identifying whether Gynostemma pentaphyllum or Eucalyptus contains a drug component resistant to blood cancer.

First, the Japanese scorpion extract prepared in Experiment C and Experiment D was dissolved in dimethyl sulfoxide, respectively, and prepared at a concentration of 0 to 40 μg/ml as a sample for blood cell physiological activity test. 1×10 ⁵ human bone marrow blood cancer cell line HL-60 was seeded in a 24-well plate, and the volume of each well was 1 ml, and a sample with a concentration of 0 to 40 μg/ml was administered to the well. in. After culturing the 24-well plate in a 37 ° C incubator containing 5% carbon dioxide for 48 hours, the suspension cells in each well were collected and transferred to a special tube for fluorescence activated cell sorter (FACS) to centrifuge. The machine (DENVILLE 260D, purchased from DENVILLE scientific) was centrifuged at 900 rpm for 5 minutes, the medium was removed, and the cells were washed twice with Dulbecco's phosphate Buffered Saline (D-PBS) and 100 was added. 500 microliters of dye (propidium iodide, concentration 4 μg/ml)), protected from light for 30 minutes at 4 ° C, and finally flow cytometry (FACSCanto, purchased from Becton Dickinson) American Business Co., Ltd.) detects the survival rate of human bone marrow blood cancer cells in treated suspension cells. The results are shown in Figure 2.

The experimental results show that the extract of Japanese dodder, which is parasitic on Gynostemma pentaphyllum, has the effect of poisoning human bone marrow blood cancer cell line HL-60, and can poison 50% of human bone marrow blood cancer cell line HL-60 at a concentration of 15 μg/ml; The extract of Japanese Cuscuta, which is parasitic on eucalyptus, is non-toxic to kill human bone marrow blood cancer cells HL-60.

Next, 1 x 10 ⁵ human bone marrow blood cancer cell line HL-60 was seeded in a 24-well plate, and 15 μg/ml of the extract sample was administered to each well, and the above-mentioned culture centrifugal washing was repeated. The procedure was followed by cell cycle analysis by flow cytometry (FACSCanto, purchased from Becton Dickinson). The results are shown in Figures 3 and 4.

As a result, the extract induced about 10% of human bone marrow blood cancer cell line HL-60 to undergo apotosis, and therefore, it was confirmed that Gynostemma pentaphyllum contains a drug component resistant to blood cancer.

On the other hand, the extract of Japanese dodder, which is parasitic on eucalyptus, does not poison the human bone marrow blood cancer cell line HL-60, and as shown in Fig. 3 and Fig. 4, the extract does not significantly induce human bone marrow blood cancer. The effect of cell HL-60 on apoptosis. Therefore, tests have shown that eucalyptus does not contain medicinal components that are resistant to blood cancer.

The results of Experiment E show that the method of the present invention can be used to efficiently and conveniently screen and/or extract medicinal ingredients in plants.

It can be seen from the results of Example 1 and Example 2 that the dodder extract prepared by the method of the present invention contains the medicinal component of the host plant, so the preparation method of the dodder extract of the present invention can be used for large-scale production of various medicinal plants. A pharmaceutical ingredient, for example, a plant secondary metabolite, a compound, a saccharide, a peptide, a protein or an enzyme. In addition, the method of the present invention can provide a sample of a silkworm extract containing a host plant component for physiological activity testing, thereby efficiently screening the active ingredients in the plant, and is therefore quite advantageous for the development of new drugs.

The above embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be as set forth in the scope of the patent application described hereinafter.

Figure 1 shows the HPLC analysis of the camptothecin standard and the extract of the Japanese scorpion parasitized by Camptotheca acuminata;

Figure 2 is a statistical graph showing the poisoning effect of the extract of Japanese scorpion, which is parasitic on Gynostemma pentaphyllum, on human bone marrow blood cancer cell line HL-60;

Figure 3 is a flow cytometric analysis of the effects of the extract of Japanese dodder, which is parasitic on Gynostemma pentaphyllum or Eucalyptus, on the cell cycle of human myeloid blood cancer cell line HL-60;

Figure 4 is a statistical bar graph showing the effect of the extract of Japanese Cuscuta, which is parasitic on Gynostemma pentaphyllum or Eucalyptus, on the cell cycle of human myeloid blood cancer cell line HL-60.

Claims (17)

A method of preparing an extract of a medicinal plant, comprising: a) parasitizing and growing a medicinal plant on a medicinal plant; b) collecting a liquid in the stalk; and c) extracting the liquid using a solvent, Obtaining the extract; wherein the extract comprises at least one medicinal ingredient of the medicinal plant. The method of claim 1, wherein the dodder is Japanese Cuscuta japonica . The method of claim 1, wherein the medicinal plant is selected from the group consisting of woody plants, herbs, ferns, and combinations thereof. The method of claim 1, wherein the medicinal plant is selected from the group consisting of Camptotheca acuminate , Gynostemma pentaphyllum , Morus alba L., Vinca rosea L., and Lantana ( Lantana camara L.), Kalanchoe pinnata , Duranta repens L., Cucurbita spp. , and combinations thereof. The method of claim 1, wherein the medicinal plant is a camptothecin and the medicinal component is camptothecin. The method of claim 1, wherein the medicinal plant is Gynostemma pentaphyllum, and the extract is for use against blood cancer. The method of any one of claims 1 to 6, wherein the solvent selected from the group consisting of water, methanol, ethanol, propanol, butanol, chloroform, and combinations thereof is used in step c). The method of claim 7, wherein the solvent is methanol. An extract of a medicinal plant obtainable by a method comprising the steps of: a) parasitizing and growing a medicinal plant; b) collecting the liquid in the dodder; and c) using a The liquid is extracted by a solvent to obtain the extract; wherein the extract contains at least one medicinal ingredient of the medicinal plant. The extract of claim 9, wherein the solvent selected from the group consisting of water, methanol, ethanol, propanol, butanol, chloroform, and combinations thereof is used in step c). The extract of claim 10, wherein the solvent is methanol. The extract of any one of clauses 9 to 11, wherein the medicinal plant is a camptothecin and the medicinal component is camptothecin. The extract of any one of clauses 9 to 11, wherein the medicinal plant is Gynostemma pentaphyllum, and the extract is for use against blood cancer. A method for extracting camptothecin and/or an anti-blood cancer component from a plant, comprising: I) parasitizing a dodder on a plant and growing thereon; II) collecting a liquid in the dodder; and III) purifying the liquid to obtain The camptothecin and/or anti-blood cancer component. The method of claim 14, wherein the plant is Hibiscus or Gynostemma pentaphyllum. The method of claim 15, wherein the step (III) comprises using a solvent to extract the liquid obtained in the step II) to obtain an extract, wherein the solvent is selected from the group consisting of water, methanol, ethanol, Propanol, butanol, chloroform, and combinations thereof. The method of claim 16, wherein the plant is a camptothecin, and the extract is subjected to high performance liquid chromatography in step III) to obtain camptothecin.