TWI646966B - Process for preparing a crassocephalum crepidioides extract, extract prepared thereby and use of the extract - Google Patents

Abstract

The present invention provides a method of preparing a Showa sylvestris extract and an extract prepared thereby. The invention further relates to a pharmaceutical composition/combination comprising the extract of Showa grass. The use of the extract and the composition/combination comprising the extract for preventing or treating cancer is also provided.

Description

Method for preparing extract of Showa sylvestris, extract prepared thereby and use of the extract

The present invention relates to a method for preparing an herbal drug extract of Crassocephalum crepidioides and the use of the Showa sinensis extract for treating or preventing cancer in an individual in need thereof.

Cancer is the leading cause of death. Effective treatment has become a major focus of research in modern medicine, medicine, and academia. Conventional cancer treatments include surgery, chemotherapy, and target therapy. The tumor tissue is first removed by surgery. The remaining cells are then killed by chemotherapy and target therapy. However, this treatment is not enough for patients with terminal cancer. The survival rate and quality of life of patients with advanced cancer cannot be improved by conventional treatment methods.

Recently, it has been observed from clinical studies that the therapeutic effect of cancer patients receiving herbal medicine as adjunctive therapy (supportive therapy) is significantly improved, and herbal drugs can prevent or reduce the toxic side effects of chemotherapy to improve the effect of chemotherapy. And herbal drugs can enhance the patient's immune response and shorten the postoperative recovery time. In addition, herbal drugs have been found to be useful alone in treating patients who are not suitable for surgery and/or chemotherapy.

Showa sinensis is known as an edible plant which has been conventionally used as an herbal medicine for treating inflammatory diseases, hypertension, headache, vomiting, edema, constipation and the like.

Lie-fen Shyur et al. (US 8,048,455 B2) reveals the extract of Showa grass, and Compared with cisplatin (a chemotherapeutic agent), the extract of Showa sinensis has a better effect in inhibiting the growth of melanoma cells in C57BL/6J mice. Lie-fen Shyur et al. also found that the active ingredient contained in the extract of Showa gracilis is a galactolipid compound named 1,2-di-O-α-linidinyl-3-O-β-peri Galactose-sn-glycerol (dLGG), and dLGG inhibits the expression and production of iNOS, COX-2 and PEG2 in macrophage cell lines.

Lie-fen Shyur et al. (US 2014/356301 A1) further discloses a galactolipid-rich plant extract prepared by extracting a plant sample selected from the group consisting of a series of solvents: Taiwan white cabbage Subspecies ( Gynura divaricata subsp. formosana ) (Asteraceae), Murdannia bracteata (CBClarke) JKMorton ex DYHong (Dendrobium) and Crassocephalum rabens S. Moore (Asteraceae). The application also provides a composition for treating or preventing fulminant hepatitis and sepsis and related indications, and for skin whitening, comprising a plant extract or a purified product thereof, and a medicinal, healthy or food acceptable Vehicle.

Chien-Yung Lee (US 2004/0013748 A1) discloses a herbal combination for treating liver tumors and pancreatic cancer, which comprises Jiatonghao (Showa), Fuling, and the like.

YOKO ANIYA (WO 2008/105436 A1) discloses an extract of a plant naturally grown in Okinawa (i.e., Crassocephalum crepidioides S. Moore ). YOKO ANIYA found that the extract inhibits tumor necrosis factor-α and prostaglandin synthetase-2.

There is therefore a need to identify novel herbal extracts that can be used to treat cancer.

In one aspect, the invention relates to a method for preparing an extract of Showa sylvestris.

Another aspect of the invention relates to a showa extract obtainable from the process of the invention.

Yet another aspect of the invention relates to a composition comprising the extract of the Showa sylvestris.

Yet another aspect of the invention relates to a combination comprising the extract of the Showa sylvestris and a chemotherapeutic agent.

Another aspect of the invention provides a method for treating or preventing cancer in an individual in need thereof, comprising administering to the individual, the combination of the present invention, the extract of the present invention, alone or in combination with a chemotherapeutic agent. The invention also provides the use of the extract of Showa sylvestris or a composition/combination comprising the extract for treating or preventing cancer in an individual in need thereof.

Other aspects and advantages of the present invention will be apparent from the description and appended claims.

Fig. 1 shows an HPLC chart of the extract of Showa sylvestris of the present invention.

The invention may be more readily understood by reference to the various embodiments of the invention, examples, and the accompanying drawings. All terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. It will be further understood that, unless explicitly defined herein, terms such as those defined in commonly used dictionaries should be interpreted consistently with their meaning in the context of the relevant art and should not be in an idealized or overly formal sense. Explanation. It is also understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting.

It must be noted that the singular forms "a", "the" Therefore, unless the context requires otherwise, the singular terms shall include the plural, and the plural terms shall include the singular.

Ranges are often referred to herein as "about" a particular value and/or to "about" another particular value. When the range is expressed in the above manner, it is included from a specific value and/or To the extent of another specific value. Similarly, when a value can be approximated by the term "about", it will be understood that it is another aspect of a particular value. It can be further appreciated that when referring to other endpoints and other endpoints themselves, the endpoints of each range are meaningful. As used herein, the term "about" means ±20%, preferably ±10%, more preferably ±5% and even more preferably ±1%.

Preparation

The present invention provides a method for preparing a Showa sylvestris extract, comprising the steps of: (a) contacting a Showa plant with 70% ethanol to obtain a suspension; (b) solid portion and liquid portion of the suspension Separating, and then collecting the liquid fraction to obtain a crude extract; (c) diluting the crude extract to a 60% ethanol extract; (d) the 60% ethanol extract and macroporous styrene divinylbenzene resin Mixing; (e) pouring the 60% ethanol extract and the macroporous resin mixture into the column; (f) washing the column with ethanol in a volume of about 2 to 10 times the column volume; (g) Dissolving the column with a solvent of ethanol/ethyl acetate or ethyl acetate, and collecting the eluted fraction at different time intervals; and (h) analyzing each of the eluted fractions and enriching the 1,2-di-O-α-linen The thiol-3-O-β-galacto-galactose-sn-glycerol (dLGG) and the lyophilized fraction containing phytol was identified as the extract of Showa sylvestris.

As used herein, the term "extract" refers to a concentrated preparation of essential components of the Showa sylvestris plant. The extract may be in the form of a liquid, extractum spissum, solid or powder.

As used herein, a Showa plant can be a whole plant or one or more parts thereof including, but not limited to, seeds, flowers, leaves, stems, and roots. In one embodiment of the invention, the Showa grass plant is a whole plant. In another embodiment of the invention, the Showa grass plant is a seed, flower, leaf or any combination thereof. In a preferred embodiment of the invention, the Showa grass plant It is dry and powdery.

In one embodiment of the invention, in step (a), the ratio of the weight of the dried plant to the volume of the 70% ethanol solvent is in the range of from about 1:1 to about 1:50, such as about 1:1, 1: 2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1: 27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49 or 1:50. In a preferred embodiment of the invention, the ratio is from about 1:1 to about 1:20. In another preferred embodiment of the invention, the ratio is about 1:10. Alternatively, the solid portion from step (b) can be treated under the contact conditions of step (a) to obtain another suspension. This iterative step can be carried out one or more times and all of the crude extracts obtained therefrom are mixed together prior to carrying out step (c).

In one embodiment of the invention, the crude extract obtained from step (b) can be concentrated into an extract or powder using any conventional concentration method for the solution, such as using a reduced pressure rotary evaporator.

In one embodiment of the invention, the 60% ethanol extract of step (c) can be obtained by directly diluting the crude extract with water. In a preferred embodiment of the invention, the crude extract obtained from step (b) is concentrated to an extract, and then the extract is diluted in 60% ethanol to prepare 60% ethanol of step (c). Extracts.

According to the present invention, the macroporous styrene divinylbenzene resin used therein is a styrene-divinylbenzene-based resin having pores having an average pore radius in the range of from about 45 Å to about 290 Å. The pore volume of the macroporous resin is about 1.5 ml/g, and the surface of the macroporous resin is an aromatic non-polar surface, and thus the surface is hydrophobic. In a preferred embodiment of the invention, the macroporous resin is Sepabeads resin or Diaion resin. In a further preferred embodiment of the invention, the macroporous resin is selected from, but not limited to, Sepabeads SP70 (Mitsubishi), Sepabeads SP710 (Mitsubishi), Sepabeads SP825 (Mitsubishi), Sepabeads SP850 (Mitsubishi), Sepabeads SP207 (Mitsubishi) , Sepabeads SP700 (Mitsubishi), Diaion HP20 (Mitsubishi), MCI Gel CHP20P (Sigma-Aldrich), Amberlite ® XAD ® -2 (Sigma-Aldrich) and ^{Amberlite ® XAD ® -4 (Sigma-} Aldrich). In an even more preferred embodiment of the invention, the macroporous resin is Sepabeads SP70 and the resin is pretreated with 10% ethanol.

In the step (f) of the production method of the present invention, the concentration of the ethanol used may be from about 50% to 90%, preferably from about 70% to 90% and more preferably about 80%. The volume of ethanol used to wash the column is from about 2 times to 10 times the volume of the column, preferably from about 4 to 8 times and more preferably about 4 times.

In one embodiment of the preparation method of the present invention, the solvent used in the step (g) is ethanol/ethyl acetate, wherein the volume ratio of ethanol to ethyl acetate is from about 1:1 to 1:50, preferably about 1. From 1 to 1:30, more preferably from about 1:1 to 1:10, and even more preferably about 1:1. In another embodiment of the process of the invention, the solvent used in step (g) is ethyl acetate.

In step (h) of the preparation method of the present invention, the analytical method may be any known method for identifying dLGG. For example, HPLC and HPLC-MS.

In a preferred embodiment of the present invention, the extract of Showa sylvestris is at a residence time of 12.59 min, 12.893 min, 13.828 min, 14.158 min, 14.922 min, 15.455 min, and 16.478 min, respectively, when determined by HPLC under the following conditions. Has 7 main peaks:

String: Symmetry Shield C18, 5μm, 4.6×150mm

Temperature: environment

Dissolution: H ₂ O/CH ₃ CN gradient

Detection: UV 210nm

Pharmaceutical composition/combination

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a showa extract prepared by the production method of the present invention. The invention also provides a pharmaceutical combination comprising a therapeutically effective amount of a showa extract prepared by the method of the invention and a therapeutically effective amount of a chemotherapeutic agent. The pharmaceutical composition/combination can include a pharmaceutically acceptable carrier or vehicle.

As used herein, the term "therapeutically effective amount" refers to a sufficient amount of a herbal extract or compound to be administered to some extent to alleviate one or more symptoms of the disease or condition being treated. The result may be to alleviate and/or alleviate the signs, symptoms or causes of the disease or any other desired changes in the biological system.

The term "chemotherapeutic agent" as used herein refers to a functional agent having a property of inhibiting the growth or progression of a neoplasm, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma or leukemia. The inhibition of cancer metastasis or angiogenesis is often the nature of a chemotherapeutic agent. The chemotherapeutic agent can be a cytotoxic agent or a cytostatic agent. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and/or cell proliferation.

Non-limiting examples of chemotherapeutic agents include antimetabolites (eg, azathioprine, 6-mercaptopurine, 6-thioguanine, fludarabine, pentostatin, cladribine) , 5-fluorouracil (5FU), fluorouridine (FUDR), cytosine arabinoside (cytarabine), methotrexate, trimethoprim, pyrimidine methylamine and pemetrexazole (pemetrexed)); alkylating agents (eg cyclophosphamide, dichloromethyleneamine, uramustine, melphalan, chlorambucil, thiotepa) Thietepa)/chlorocamptothenic acid, ifosfamide, carmustine, lomustine, streptozocin, bubuan sulfate, dibromo Mannitol, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, tripotassium tetrachloride, procarbazine, altretamine, Dacarbazine, mitozolomide and temozolomide; anthracycline (eg daunorubicin) Cockroach (doxorubicin), epirubicin, idarubicin, and valrubicin; antibiotics (eg, actinomycin d, bleomycin, Mithramycin, anthramycin, streptozotocin, gramicidin D, mitomycin (eg mitomycin C), multiple carcinoma Duocarmycin (eg CC-1065) and calicheamicin; anti-mitotic agents (including, for example, maytansinoid, auristatin, dolastatin, hidden) Cryptophycin, vinca alkaloids (eg, vincristine, vinblastine, vindesine, vinorelbine) and taxanes (eg, paclitaxel) And docetaxel and colchicine; topoisomerase inhibitors (eg irinotecan, topotecan, amsacrine, etoposide) ), teniposide and mitoxantrone; and proteasome inhibitors ( Peptidyl acid).

Compositions/combinations can be administered orally or parenterally to a patient in the form of a formulation, such as capsules, microcapsules, troches, granules, powders, dragees, pills, suppositories, injections, suspensions, and syrups. . Suitable formulations can be prepared by conventional methods using conventional, organic or inorganic carriers such as, for example, sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or carbonic acid. Calcium), binder (eg cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose or starch), disintegration Agents (such as starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calcium citrate), lubricants (such as magnesium stearate, light anhydrous Capric acid, talc or sodium lauryl sulfate), flavoring agents (such as citric acid, menthol, glycine or orange powder), preservatives (such as sodium benzoate, sodium bisulfite, parabens) Ester or propyl paraben), stabilizers (eg citric acid, sodium citrate or acetic acid), suspending agents (eg methylcellulose, polyvinylpyrrolidone or aluminum stearate), dispersing agents (eg hydroxy Propyl methylcellulose), a diluent such as water, and a base wax such as cocoa butter, white petrolatum or polyethylene glycol.

Practicality

The pharmaceutical compositions/combinations of the present invention are useful for treating or preventing cancer in an individual in need thereof.

As used herein, the term "treat, treating, treatment" means to cure, heal, alleviate, alleviate, alter, treat, ameliorate, ameliorate, affect a condition, symptom or predisposition of a condition, or reduce a condition or condition. For the purpose of the risk of symptoms or predisposition, the therapeutically active agent, composition, drug or combination is administered to an individual having the disease/condition or having the symptoms or predisposition thereof. For example, treating cancer refers to treatment that results in inhibition of cancer growth or cancer cell growth, regression of cancer growth (ie, reducing the size of detectable cancer), or disappearance of cancer.

As used herein, the term "individual" is intended to include mammals, primates, humans, and non-human animals. For example, an individual can be a patient with cancer (eg, a human patient or a veterinary patient). Unless otherwise indicated, the term "non-human animal" as used in the present invention includes all non-human vertebrate animals, such as non-human mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles. The term "cancer" refers to any of a variety of malignant neoplasms characterized by proliferation of cells that can invade surrounding tissues and metastasize to new body parts. Benign and malignant tumors are classified according to the type of tissue in which the tumor is found. For example, fibroids are tumors of fibrous connective tissue, and melanoma is an abnormal growth of pigment (melanin) cells. Malignant tumors derived from epithelial tissues (eg, skin, bronchi, and stomach) are called cancers. A malignant disease such as epithelial gland tissue found in the breast, prostate, and colon is called adenocarcinoma. Malignant growth of connective tissue (such as muscle, cartilage, lymphoid tissue, and bone) is called sarcoma. Lymphoma and leukemia are in A malignant disease produced in white blood cells.

In the case of neoplasms, cancer, tumor growth or tumor cell growth, inhibition can be assessed by: delayed onset of primary or secondary tumors, slowing of development of primary or secondary tumors, primary The incidence of sexual or secondary tumors is reduced, the severity of secondary effects of the disease is slowed or reduced, tumor growth arrests and tumor regression, and others. In extreme cases, complete inhibition is referred to herein as prevention or chemoprevention. In this context, the term "prevention" includes the complete prevention of a clinically apparent onset of neoplasia, or the prevention of a preclinically apparent stage of oncogenesis in an individual at risk. This definition also intends to prevent the transformation of pre-deterioration cells into malignant cells or to arrest or reverse the progression of malignant cells to pre-malignant cells. This includes prophylactic treatment of their pre-deteriorating cells in the context of developing a risk of neoplasia.

The following examples are provided to assist those skilled in the art in practicing the invention. </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Instance Example 1: Preparation of Showa Grass Extract

1.02 kg of dried Showa grass (whole plant) was ground and immersed in a 70% ethanol solution (10 times the total weight of the dried plants) for 24 hours, and then the plant extract was filtered under reduced pressure by a No. 1 filter paper. , the first filtrate was obtained. The solid residue was further impregnated with a 70% ethanol solution (5 times the total weight of the dried plants) for 24 hours, and then the plant extract was filtered under reduced pressure by a No. 1 filter paper to obtain a second filtrate. The first and second filtrates were mixed to obtain a crude extract of Showa sylvestris, and the crude extract was concentrated under reduced pressure to obtain 1.05 L of an extract having a weight of 213.69 g, a concentration of 203.51 mg/mL, and a yield of 20.95. %.

425 g of the extract was diluted with 12 L of 60% ethanol to obtain a crude extract. The crude extract was added to a container containing macroporous resin SP70 (Mitsubishi) pretreated with 10% ethanol. in. The contents of the container (including the resin and the diluted extract) were stirred and then allowed to stand overnight. Pour the contents into the column. The dissolution rate of the column is controlled at 1.5 times to 2 times the volume of the column per hour. The first part of the solution is the non-adsorbed sample. The column was then dissolved by a gradient method using a solvent of 80% ethanol and ethanol: ethyl acetate (1:1), respectively. The volume of each solvent is 4 times the volume of the column. The fraction which was dissolved by ethanol:ethyl acetate (1:1) and rich in dLGG was collected, concentrated and dried to obtain 45.82 g of a dried product (Cr-E03) in a yield of 10.78%.

Example 2: Analysis of the components of the extract of Showa sylvestris

HPLC conditions

String: Symmetry Shield C18, 5μm, 4.6×150mm

Temperature: environment

Dissolution: H ₂ O/CH ₃ CN gradient

Detection: UV 210nm

The chromatogram of Showa sinensis extract (Cr-E03) is shown in Figure 1. After the Cr-E03 was aligned with the standard and LCMS-APCI (+), it was confirmed that the peak (5) was dLGG ([M ⁺ H] ⁺ : 613, 595 and 335), and the peak (6) was phytol (MW). =296.5).

Example 3: Showa grass extract inhibits growth of MKN45 cancer cells

MKN45 cells were cultured in RPMI-1640 medium (Sigma-Aldrich) containing 5% FBS (Gibco ^®) in the. 6 x 10 ³ cells were added to each well of a 96-well plate. Each well contained 180 μL of the above medium. The plates were incubated for 4 hours at 37 ° C, and then 20 μL of different concentrations of crude extract, Cr-E03 and dLGG were added to each well of the plate. The test was repeated 3 times for each concentration. After further incubation at 37 ° C for 48 hours, the medium in the wells was removed and 180 μL containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 5% of -2-(4-sulfophenyl)-2H-tetrazolium (MTS) FBS medium was added to the wells. After reacting at 37 ° C for 1 hour, the absorbance of the wells at 490 nm was measured by an ELISA reader (Model 680 microplate reader, Bio-Rad). The IC ₂₅ , IC ₅₀ and IC ₇₅ values of the crude extract, Cr-E03 and dLGG were obtained by calculation using GraphPad Prism 5 software (GraphPad Software, Inc.), respectively. The IC ₂₅ , IC ₅₀ and IC ₇₅ values were defined as concentrations at which the test samples reached 25%, 50%, and 75% of the maximum inhibition of MKN45 cell growth, respectively. The results are shown in Table 1.

As can be seen from Table 1, the crude extract, Cr-E03 and dLGG all inhibited the growth of MKN45 cells.

Example 4: Effect of Combination of Showa Extract and 5-Fluorouracil (5-FU) or Epirubicin on Inhibition of Growth of MKN45 Cancer Cells

Different concentrations of each crude extract, Cr-E03 and dLGG were combined with different concentrations of 5-fluorouracil (5-FU) and epirubicin. The test was performed on the same procedure as in Example 3, and the combined combination index (CI) was also obtained by calculation of the GraphPad Prism 5 software. The results are shown in Table 2. The CI theorem was first provided by Chou T. C. and Talalay P. in 1984 (Advances in Enzyme Regulation, 22: 27-55, 1984). According to the CI theorem, CI = 1 indicates a combination with additive/cumulative effects; CI < 1 indicates a combination with synergy; and CI > 1 indicates a combination with antagonistic effects.

As shown in Table 2, the crude extract and Cr-E03 were combined with 5-FU or epirubicin, respectively, and synergistic effects were achieved in inhibiting the growth of MKN45 cells. However, no synergistic effect was observed with the combination of dLGG and 5-FU and epirubicin, respectively.

Example 5: Effect of combination of Cr-E03 and Cr-E03 with 5-FU or epirubicin on inhibition of growth of A549 lung cancer cells and Colo205 colon cancer cells

The effect of Cr-E03 and Cr-E03 in combination with 5-FU or epirubicin inhibiting the growth of A549 and Colo205 cancer cell lines was determined based on methods similar to those of Examples 3 and 4.

It was found that Cr-E03 alone effectively inhibited the growth of A549 cancer cell lines with an IC ₅₀ value of 75.6 μg/mL.

The inhibitory effects of Cr-E03 in combination with 5-FU and epirubicin on the growth of A549 and Colo205 cell lines are shown in Table 3.

The results in Table 3 show that the combination of Cr-E03 with 5-FU or epirubicin provides an additive effect on the inhibition of A549 cells, and the combination of Cr-E03 and epirubicin provides strong synergy in inhibiting Colo205 cells. effect.

It is to be understood that the above-described embodiments and specific examples, while indicating the preferred embodiments of the invention Various changes and modifications of the present invention will become apparent to those skilled in the art. All such modifications as would be apparent to those skilled in the art are intended to be included within the scope of the following claims.

Claims (19)

A method for preparing an extract of Showa sylvestris comprising the steps of: (a) contacting a Showa plant with 70% ethanol to obtain a suspension; (b) separating the solid portion of the suspension from the liquid portion, And then collecting the liquid portion to obtain a crude extract; (c) diluting the crude extract to a 60% ethanol extract; (d) mixing the 60% ethanol extract with the macroporous styrene divinylbenzene resin; (e) pouring the 60% ethanol extract and the macroporous resin mixture into the column; (f) washing the column with ethanol in a volume of about 2 to 10 times the column volume; (g) using ethanol / Ethyl acetate or ethyl acetate solvent is dissolved in the column, and the eluted fraction is collected at different time intervals; and (h) each dissolved fraction is analyzed and will be enriched with 1,2-di-O-α-linidinyl The -3-O-β-galacto-galactose-sn-glycerol (dLGG) and the lyophilized fraction containing phytol was identified as the extract of Showa sylvestris. The method of claim 1, wherein the Showa grass plant is a whole plant. The method of claim 1, wherein the Showa grass plant is selected from the group consisting of a seed, a flower, a leaf, or any combination thereof. The method of any one of claims 1 to 3, wherein the Showa grass plant is dry and powdered. The method of claim 4, wherein in step (a), the ratio of the weight of the dried and powdered plant to the volume of the 70% ethanol solvent ranges from about 1:1 to about 1:50. The method of claim 5, wherein in step (a), the ratio of the weight of the dried and powdered plant to the volume of the 70% ethanol solvent is about 1:10. The method of claim 1, wherein the macroporous resin is selected from the group consisting of Sepabeads SP70 and Sepabeads SP710. The method of claim 1 or 7, wherein the macroporous resin is Sepabeads SP70. The method of claim 1, wherein the solvent used in the step (g) is ethanol/ethyl acetate, and wherein the volume ratio of ethanol to ethyl acetate is about 1:1. A showa grass extract obtainable by the method of any one of claims 1 to 9. The extract of Showa grass according to claim 10, when it is determined by HPLC under the following conditions, has seven main residence times at 12.59 min, 12.893 min, 13.828 min, 14.158 min, 14.922 min, 15.455 min, and 16.478 min, respectively. Peak: Column: Symmetry Shield C18, 5μm, 4.6×150mm Temperature: Environmental Dissolution: H ₂ O/CH ₃ CN Gradient Detection: UV 210nm A pharmaceutical composition comprising a therapeutically effective amount of a Showa grass extract as claimed in claim 10 or 11 and a pharmaceutically acceptable carrier. A pharmaceutical combination comprising a therapeutically effective amount of a Showa grass extract and a chemotherapeutic agent according to claim 10 or 11. The pharmaceutical combination of claim 13, wherein the chemotherapeutic agent is selected from the group consisting of an antimetabolite, an anthracycline, and a proteasome inhibitor. The pharmaceutical combination of claim 13 or 14, wherein the chemotherapeutic agent is 5-fluorouracil (5-FU) or epirubicin. Use of a Showa grass extract of claim 10 or 11 for the manufacture of a medicament for treating cancer in an individual in need thereof. Use of the Showa sylvestris extract of claim 10 or 11 for the manufacture of a medicament for the treatment of cancer in a subject in need thereof in combination with a chemotherapeutic agent. The use of claim 17, wherein the chemotherapeutic agent is selected from the group consisting of an antimetabolite, an anthracycline, and a proteasome inhibitor. The use of claim 17 or 18, wherein the chemotherapeutic agent is 5-fluorouracil (5-FU) or epirubicin.