CN102451220A - A kind of Glycyrrhizae extract, its preparation method, its pharmaceutical composition and application - Google Patents

Abstract

The invention relates to a Grwood (Erthrophllum fordii Oliver) extract, a preparation method thereof, a pharmaceutical composition containing the extract, and application of the extract and the pharmaceutical composition in preparation of antitumor drugs. The invention extracts the extract with the anti-tumor effect by drying, crushing, extracting, concentrating and purifying the gelia repens leaves. The effective components of the extract comprise cassaine diterpenoid alkaloid compounds, and pharmacological experiments show that the extract has better anti-tumor activity in vitro.

Description

A kind of Glycyrrhizae extract, its preparation method, its pharmaceutical composition and application

technical field

The invention relates to an extract of Erthrophleum fordii Oliver, a preparation method thereof, a pharmaceutical composition containing the extract, and an application of the extract and the pharmaceutical composition in preparing antitumor drugs.

Background technique

Cancer is a frequently-occurring and common disease that seriously endangers human health. In 2001, the World Health Organization (WHO) reported that the incidence and mortality of cancer in the world increased by 22% compared with 1990, and will increase by about 50% in the next 20 years. In 2020, the number of cancer patients worldwide will reach 30 million and 10 million will die. Cancer is becoming the number one killer of human beings in the new century. Based on this, finding and discovering new drugs for the prevention and treatment of cancer, improving and improving people's health and quality of life has become an urgent problem for pharmaceutical researchers.

At present, chemotherapy is still the main method in the four major therapies of cancer surgery, radiotherapy, chemotherapy and biological therapy. Most of the mechanisms of action of clinically used drugs involve the nucleic acid metabolism of tumor cells, with DNA as the target. However, the serious toxicity of chemotherapy drugs and the incomplete killing of cancer cells have not been resolved. Therefore, continuing to find and develop new anticancer drugs is still the main research direction. In recent years, the progress made in the research of molecular oncology has provided many new and interesting approaches for tumor treatment, such as oncogenes and suppressor genes, signal transduction pathways, telomeres and telomerase, DNA topological heterogeneity, etc. Structase and so on are available new targets for anticancer drugs.

In the discovery of new targets, studying the signal transduction mechanism of tumor cells, selectively blocking the autocrine or paracrine signal transduction pathways of tumor cells, and destroying their self-controlled growth regulation mechanism is becoming an attractive research hotspot. Compared with classic cytotoxic anticancer drugs, signal transduction blockers have the advantages of strong selectivity, less toxic and side effects, and are not affected by cell resistance, especially for advanced tumors or metastatic cancers. Therefore, the study of tumor cell signal transduction mechanism has potential application value and significance.

The role of natural products as one of the sources of antitumor drugs cannot be ignored. Practice has proved that a considerable part of the discovered natural anticancer active ingredients are obtained from toxic plant resources. "Toxicity" also has a positive and beneficial side. Due to their strong biological activity, many poisonous plants are used as drugs, insecticides, fungicides, as well as for hunting animals and fishing, and become the main economic resources with special value. .

Therefore, finding compounds and active ingredients or components with novel anti-tumor mechanisms from poisonous plants has broad application prospects.

Erthrophleum fordii Oliver is a plant of the genus Erthrophleum in the family Leguminosae. It is an evergreen tree, usually about 1.2 meters in diameter, about 10 meters high, and some can reach 40 meters. There are 15 species in the world, distributed in tropical regions of Africa, tropical and subtropical regions of eastern Asia and northern Australia. (Flora of China, Science Press, Volume 39, Page 1177). There is only one species of this genus in my country, Erthrophleumfordii Oliver, also known as Chimuye and Dou Dengfeng, which are traditional herbal medicines in my country. Its bark and seeds can be used as medicine. It is pungent, flat, poisonous and enters the Heart Channel. The main function is to replenish qi and activate blood, and it can treat the symptoms of qi deficiency and blood stasis caused by heart qi deficiency, that is, heart palpitations, shortness of breath, fatigue, lower limbs and general edema, etc. Casain-type diterpene alkaloids are commonly found in the plants of the genus Goliath. At present, in China, except for the research on the chemical components and pharmacological activities of this plant by my research group, there are no other reports in the literature.

Contents of the invention

One aspect of the present invention provides a kind of extract of Glycyrrhizae, which can be used to prepare antitumor drugs.

Another aspect of the present invention provides the preparation method of the above-mentioned extract of Glycyrrhiza officinale.

Yet another aspect of the present invention provides a pharmaceutical composition, which includes an effective dosage of the extract of Glycyrrhiza lanceolata as an active ingredient and a carrier commonly used in the field of pharmacy.

Yet another aspect of the present invention relates to the application of the extracts of gorgoniana and their compositions in the preparation of antitumor drugs.

In order to solve the technical problems of the present invention, the present invention takes the following technical solutions:

The present invention relates to a kind of extract that can be used for preparing antineoplastic medicine, and its preparation method is as follows:

(1) Weighing the leaves of Glycyrrhizae and extracting them with a solvent, and concentrating the extract to obtain Glycyrrhizae extracts.

The preferred Gemuyuan medicinal material is Gemuye. The leaves of Goliath are preferably dried and properly crushed to increase the contact area with the solvent and improve the extraction efficiency.

The extraction solvent can be water or various organic solvents; preferred solvents are alcohols or a mixture of water and alcohols; preferred alcohols include methanol, ethanol, isopropanol, butanol, etc., most preferably ethanol. The ethanol concentration can be 50-100% by volume; the preferred concentration is 70-100% by volume, and the most preferred ethanol concentration is 95% by volume.

The amount of solvent used during extraction is solvent volume:weight of the original drug=4-14:1 (liter/kg), preferably 6-12:1, more preferably 8-10:1.

Extraction can be performed statically or dynamically, preferably under dynamic conditions, such as stirring. In order to improve the extraction efficiency, means such as ultrasound can be used.

The extraction temperature can range from room temperature (eg, 20°C) to the solvent reflux temperature, preferably at the solvent reflux temperature.

The extraction can be carried out continuously or intermittently, and the intermittent extraction can be repeated 1 to 5 times, preferably 2 to 4 times, most preferably 3 times.

Each extraction time is 1-5 hours, preferably 1.5-4 hours, most preferably 2-3 hours.

After the extraction step is completed, the filtrates are combined, and the medicinal residues are filtered off. Then, the filtrate is heated and concentrated under normal pressure or reduced pressure to form a paste under dynamic conditions, preferably concentrated under reduced pressure. The resulting paste is the extract of gram wood.

A preferred method for preparing the extract of Goliath is as follows: Weigh the leaves of Golem, use 6 to 10 liters of 95% ethanol to reflux for 2 to 4 times per kilogram of Golem leaves, for 2 to 3 hours each time; combine the extracts, extract The liquid is concentrated under reduced pressure to obtain the extract, which is the extract of Gemu.

(2) In order to improve the efficacy of the drug and facilitate preparations and patients to take, the extract of the geranium extract obtained in step (1) can be further purified. The purification can be carried out by extractive separation steps, and/or column chromatography steps.

Preferably adopt extraction separation step, it specifically comprises:

Dissolve the Gemu extract obtained in step (1) in an alcoholic solvent, then mix the sample with diatomaceous earth, put it into a Soxhlet extractor, use organic solvents with small to large polarities to extract in sequence, and mix various organic The extracts from the solvents were concentrated separately to obtain various extracts.

Among them, preferred alcoholic solvents include methanol and ethanol, more preferably methanol.

The consumption of methyl alcohol when dissolving gemu extract is methanol volume: extract weight=1.5～2.5: 1 (liter/kg), preferably 2: 1.

The dissolved methanol solution is mixed with diatomite, and the amount of diatomite is extract weight: diatomite weight=1:4.5-5.5, preferably 1:5.

The organic solvents with small to high polarity can be selected from petroleum ether, ethyl acetate, and methanol; correspondingly, the concentrated extracts are the petroleum ether extract, ethyl acetate extract and methanol extract of Gemu respectively.

The time for reflux extraction of petroleum ether, ethyl acetate and methanol is 40 to 50 hours, preferably 48 hours.

The consumption of sherwood oil, ethyl acetate, methanol is respectively 5～10 liters, preferably 8 liters.

Conventional techniques in the art can be used for concentration, such as heating concentration under normal pressure or reduced pressure, thin film evaporation, etc., preferably concentration under reduced pressure.

A preferred purification method comprises the steps of: dissolving the Gemu extract obtained in step (1) in methanol of 2 times the amount (liter/kg); the diatomite mixing sample of the gained methanol solution and 5 times the weight of the extract, Put in a Soxhlet extractor; reflux extraction with 8 liters of petroleum ether, ethyl acetate, and methanol for 48 hours; concentrate under reduced pressure and evaporate to dryness to obtain the petroleum ether extract, ethyl acetate extract, and methanol extract of Gemu .

Pharmacological studies have shown that the ethyl acetate extract of Gemu has the best medicinal effect.

(3) In order to improve the efficacy of the unit dose, reduce the amount of medicine used by patients, and facilitate the quality control of the product, the extract prepared in step (2) can be further purified. Purification can be performed by column chromatography, such as various types of exchange column or adsorption column chromatography, to obtain effective fractions.

Preferred adsorption columns include normal and reverse phase silica gel, alumina, cellulose, polyamide, activated carbon columns, and macroporous resin columns. A preferred gel column is a hydroxypropyl sephadex column.

Most preferred are polyamide columns.

The amount of adsorbent used in the adsorption chromatography is 10 to 200 times, preferably 10 to 30 times, the weight of the sample.

Eluent can use eluent commonly used in this area, such as water, organic solvent, and their mixture, and described organic solvent comprises methanol, ethanol, acetone, formamide, dimethylformamide etc.; Preferred eluent Including water, alcohols and mixtures of water and alcohols, the preferred alcohol is ethanol.

The weight ratio (liter/kg) of the amount of elution solvent to the adsorbent is preferably 3-20:1, more preferably 10-15:1, more preferably 12:1.

The extract obtained by chromatographic separation can be freeze-dried or vacuum-dried into dry powder, or the concentrated liquid can be directly spray-dried into dry powder, or refined and concentrated by membrane technology, and then into extract or dry powder.

A preferred method for purifying the extract obtained in step (2) is to separate the ethyl acetate extract obtained in step (2) on a polyamide column. It specifically includes the following steps: dissolving the ethyl acetate extract in water, filtering and adding to a chromatographic column equipped with 30-60 mesh polyamide fillers of 10-30 times the weight, followed by water, 25-35% ethanol , 55～65% ethanol and 90% ethanol carry out gradient elution, the consumption of every eluent and the weight ratio (liter/kg) of polyamide are 10～15: 1, preferably 12: 1; The collected eluents are concentrated to dryness respectively to obtain water extract, 25-35% ethanol extract, 55-65% ethanol extract and 90% ethanol extract. Pharmacological studies have shown that water extract has the best efficacy, followed by 25-35% ethanol extract.

(4) In order to further obtain effective components and facilitate the quality control of the product, the extract prepared in step (3) can be further purified.

The water extract and 25-35% ethanol extract obtained in step (3) can be separated and purified by column chromatography, and optional column chromatography includes exclusion chromatography and adsorption column chromatography.

A preferred size exclusion chromatography is gel chromatography. Preferred adsorption columns include forward and reverse phase silica gel, alumina, cellulose, polyamide, activated carbon columns, and macroporous resin columns.

Most preferred are Sephadex chromatography and reverse phase silica gel.

In the hydroxypropyl-sephadex chromatography, the dosage of the hydroxypropyl-sephadex chromatography is 20-100 times of the sample weight, preferably 30-60 times.

The eluent used in the hydroxypropyl dextran gel chromatography can be an eluent commonly used in the art, such as water, organic solvents, and mixtures thereof, and the organic solvents include methanol, ethanol, acetone, ethyl acetate , chloroform, dichloromethane and petroleum ether; preferred eluents include water, alcohols and mixtures of water and alcohols, the preferred alcohols being methanol.

The weight ratio (liter/kg) of the amount of elution solvent to the adsorbent is 2-8:1, preferably 4:1.

The amount of adsorbent used in the reversed-phase silica gel column is 20-100 times, preferably 30-60 times, the weight of the sample.

The eluent used in the reversed-phase silica gel column can be an eluent commonly used in the art, such as water, organic solvents, and mixtures thereof. Described organic solvent comprises methanol, acetonitrile. Methanol is preferred.

The weight ratio (liter/kg) of the amount of elution solvent to the adsorbent is 10-20:1, preferably 15:1.

The detection of the chemical components of each fraction can be carried out by using the method of spraying a color developing agent after thin layer chromatography or the method of high performance liquid chromatography/ultraviolet detection.

As the adsorbent for thin layer chromatography, common adsorbents in this field can be used, for example: alumina, silica gel, diatomaceous earth, polyamide, cellulose, starch and the like. It is preferably silica gel, most preferably silica gel GF ₂₅₄ . As the developing agent, low-boiling organic solvents and their mixtures commonly used in this field are mainly used. The low-boiling organic solvents include: petroleum ether, n-hexane, benzene, diethyl ether, methylene chloride, chloroform, ethyl acetate, acetone, ethanol, methanol and the like. A mixed developer of chloroform and methanol is preferred. The chromogenic agent mainly uses the chromogenic agent and universal reagent of alkaloid compound commonly used in this field. Alkaloid chromogenic reagents include bismuth potassium iodide reagent, mercury potassium iodide, iodine-potassium iodide reagent, etc. General chromogenic reagents include sulfuric acid, sulfuric acid/vanillin, phosphomolybdic acid or silicotungstic acid, etc. Bismuth potassium iodide reagent is preferred.

The wavelength of high performance liquid chromatography/ultraviolet detection is selected at 210-230nm, preferably 220nm. Multiple wavelength scans are also possible.

The obtained extract can be freeze-dried or vacuum-dried into dry powder, or the concentrated liquid can be directly spray-dried into dry powder, or refined and concentrated by membrane technology, and then made into extract or dry powder.

The preferred separation and purification step is to dissolve the water extract and 25-35% ethanol extract respectively in methanol and filter, then add to 30-60 times the weight of hydroxypropyl dextran gel column, using methanol as eluent , the weight ratio (liter/kg) of the consumption of methanol and gel is 4:1. After each fraction was detected by thin layer chromatography or high performance liquid chromatography, it was combined and concentrated to a reversed-phase silica gel column of 30 to 60 times the weight, and gradient elution was carried out with methanol/water as the eluent. The ratio of methanol:water was from 5 :95 to 90:10, the weight ratio (liter/kg) of the amount of eluent to reversed-phase silica gel for each gradient is 15:1. The collected fractions are combined and enriched to obtain the total alkaloids after being detected by thin layer chromatography or high performance liquid chromatography.

The present invention relates to any one of the extracts obtained in the above steps (1) to (4). The above extracts all include the total alkaloids obtained in step (4). The active ingredients of the geranium extract of the present invention include casaine-type diterpene alkaloid compounds. The above-mentioned extract can be freeze-dried or vacuum-dried into dry powder, or the concentrated liquid can be directly spray-dried into dry powder, and then made into various dosage forms.

The present invention relates to a pharmaceutical composition, which comprises any one of the extracts obtained in the above steps (1) to (4) and a pharmaceutically acceptable carrier.

The present invention also relates to a pharmaceutical composition containing the extract of the present invention as an active ingredient and conventional pharmaceutical excipients or adjuvants. Usually the extract of the present invention accounts for 0.1-95% of the total weight of the pharmaceutical composition.

The present invention also provides a pharmaceutical composition, which includes the effective dosage of the extract of licorice root as the active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be prepared according to methods known in the art. When used for this purpose, if necessary, the extract of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to make a suitable administration form that can be used as human medicine or veterinary medicine or dosage form.

The extract of the present invention or the pharmaceutical composition containing it can be administered in the form of a unit dose, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal cavity, oral mucosa, eye, lung, skin, vagina, Peritoneal, rectal, etc., preferably oral administration.

The administration route of the extract of the present invention or the pharmaceutical composition containing it can be injection administration. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection and acupoint injection.

The dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage form can be solution (including true solution and colloid solution), emulsion (including oil-in-water type, water-in-oil type and double emulsion), suspension, injection (including water injection, powder injection and infusion), eye drops medicaments, nasal drops, lotions and liniments, etc. The solid dosage form can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules formulations, powders, pellets, dripping pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, gels, pastes, etc.

The extract of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

In order to make the unit dosage form into tablets, various excipients known in the art, including diluents, binders, wetting agents, disintegrants, lubricants, glidants, can be widely used. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, iso Propanol, etc.; binders can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, arabic mucilage, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose , cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and structural acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, lauryl Sodium sulfonate; Lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

In order to formulate a dosage unit into a pellet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, macrogolglyceride laurate, kaolin, talc, etc.; binders such as Gum Arabic, phoenix gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecylsulfonate, methylcellulose, ethyl cellulose etc.

In order to formulate the administration unit into a suppository, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.

In order to form a dosage unit into a capsule, the extract of the present invention as an active ingredient is mixed with the above-mentioned various carriers, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule. The active ingredient extract of the present invention can also be made into microcapsules, suspended in an aqueous medium to form a suspension, and can also be packed into hard capsules or made into injections for application.

For example, the extract of the present invention is made into injection preparations, such as solutions, suspension solutions, emulsions, and freeze-dried powder injections. This preparation can be aqueous or non-aqueous, and can contain one and/or more A pharmaceutically acceptable carrier, diluent, binder, lubricant, preservative, surfactant or dispersant. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like. In addition, in order to prepare isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the preparation for injection, and in addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. These excipients are commonly used in the art.

In addition, colorants, preservatives, fragrances, correctives, sweeteners or other materials can also be added to the pharmaceutical preparations, if necessary.

In order to achieve the purpose of medication and enhance the therapeutic effect, the extract or pharmaceutical composition of the present invention can be administered by any known administration method.

The dosage of the extract pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, personality and individual reaction of the patient or animal, the route of administration, and the number of administrations , therapeutic purposes, so the therapeutic dosage of the present invention can vary widely. Generally speaking, the dosages of the pharmaceutical ingredients in the present invention are well known to those skilled in the art. According to the actual amount of drug contained in the final preparation of the extract composition of the present invention, it can be adjusted appropriately to meet the requirement of its therapeutically effective dose, so as to complete the preventive or therapeutic purpose of the present invention. The daily suitable dose range of the extract of the present invention: the dosage of the extract of the present invention is 0.001～150mg/Kg body weight, preferably 0.1～100mg/Kg body weight, more preferably 1～60mg/Kg body weight, most preferably 2～100mg/Kg body weight 50mg/Kg body weight. The above-mentioned dosage can be administered in a single dosage form or divided into several dosages, such as two, three or four dosages, depending on the clinical experience of the administering physician and the dosage regimen including the use of other therapeutic means. The total dosage required for each treatment may be divided into multiple doses or administered in a single dose. The extract or pharmaceutical composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs and the dosage is adjusted.

The technical advantage that the present invention has is:

1. It is a pure natural ingredient.

2. It has obvious anti-tumor activity in vitro.

3. The medicinal material is easy to obtain, the extraction process is simple, and it is suitable for large-scale industrial production.

Description of drawings

Figure 1: Flowchart of the preparation of the extract of graminaceus.

Detailed ways

The following examples and pharmacological activity experiments are used to further illustrate the present invention, but this does not imply any limitation to the present invention.

The preparation method of embodiment 1 Gemu extract

Weigh 5.2kg of dry gramme leaves, pulverize, and extract 3 times with 95% ethanol under reflux, each time for 3 hours, and the extract is concentrated under reduced pressure to obtain 806g extract (extraction rate 15.5%). The medicinal extract (670g) that is dissolved in methanol part mixes sample with 3.5 kilograms of diatomaceous earth, is packed in the Soxhlet extractor, extracts with 8 liters of sherwood oil, ethyl acetate and methanol successively respectively, obtains sherwood oil extract (90g ), ethyl acetate extract (45g) and methanol extract (532g) extract.

The ethyl acetate extract was separated by polyamide (30～60 mesh, 800g) column chromatography, and was successively eluted with 10 liters of water, 30% ethanol, 60% ethanol and 90% ethanol to obtain the water extract (PA, 20g ), 30% ethanol extract (PB, 7.6g), 60% ethanol extract (PC, 5.1g) and 90% ethanol extract (PD, 1.6g). Component PA and component PB were purified by SephadexLH-20 column chromatography and ODS column chromatography respectively, and combined to obtain the total alkaloid ET-Ery (17.8g). The flow chart of extraction and purification is shown in Figure 1.

pharmacological test

Test Example 1: Determination of Antitumor Activity of Extracts in Vitro

experimental method:

Culture tumor cells with normal growth, inoculate them into 96-well plates at 1×10 ⁴ cell/mL (100 μL per well), and culture them in a 5% CO ₂ incubator at 37°C for 24 hours, then add the tested drugs, and re-screen After administration and culture for 5 days, the culture medium was discarded, and 100 μL of 0.04% MTT (prepared in culture medium) was added to each well, and cultured for 4 hours under the same conditions. The culture medium was discarded, and 150 μL/well of DMSO was added. After mixing, measure wavelength 570nm, reference wavelength 450nm, colorimetrically record the light absorbance, and calculate the inhibition rate of the drug to cancer cell growth according to the following formula: tumor cell growth inhibition rate=(control A-administration A)/A×100 %. Make a standard curve and calculate IC ₅₀ .

Judgment standard re-screening to calculate IC ₅₀ , for crude product, IC ₅₀ > 50μg/mL is invalid; 50μg/mL > IC ₅₀ > 5μg/mL is weak; 5μg/mL > IC ₅₀ > 0.5μg/mL is effective ; IC ₅₀ <0.5μg/mL is strong. For monomeric compounds, IC ₅₀ > 10 μM is invalid; 10 μM > IC ₅₀ > 1 μM is weak; 1 μM > IC ₅₀ > 0.1 μM is effective; IC ₅₀ < 0.1 μM is strong.

Table 1 shows the results of the in vitro anti-tumor activity of the 95% ethanol total extracts of the leaves of Lemongrass and other extracts. See Table 2 for the in vitro antitumor activity assay results of the components and total alkaloids of the ethyl acetate extract of Lemongrass leaves.

The cytotoxic activity determination result of table 1 grid leaf 95% ethanol total extract and other extracts

Note: HCT-8: human colon cancer cells; Bel-7402: human liver cancer cells; BGC-823: human gastric cancer cells; A549: human lung adenocarcinoma cells;; A2780: human ovarian cancer cells

The results showed that the ethyl acetate extract of Gemu had different inhibitory activities on different tumor cells.

Table 2 The cytotoxic activity determination results of each component and total alkaloids of the leaf ethyl acetate extract

Note: HCT-8: human colon cancer cells; Bel-7402: human liver cancer cells; BGC-823: human gastric cancer cells; A549: human lung adenocarcinoma cells; A2780: human ovarian cancer cells

The results showed that the total alkaloids enriched from the ethyl acetate extract showed different inhibitory activities on different tumor cells, among which the inhibitory activity on human lung adenocarcinoma was the strongest.

Claims (16)

1. a preparation method of Gemia extract, it is characterized in that comprising the following steps: weighing Gemia leaf and extracting with solvent, extracting solution obtains Gemia extract after concentrating, and wherein said solvent is selected from alcoholic solvent or water Mixture with alcoholic solvents. 2. The preparation method according to claim 1, wherein said alcohols are selected from methanol, ethanol, isopropanol and butanol. 3. according to the preparation method of claim 1, it is characterized in that described method comprises the following steps: weigh gram leaf, every kilogram gram leaf uses 6-10 liters of 90% ethanol to reflux 2～4 times, each 2 ~ 3 hours; combine the extracts, and concentrate the extracts under reduced pressure to obtain the extract, which is the extract of Gemma. 4. The preparation method according to any one of claims 1-3, characterized in that the preparation method further comprises an extraction and separation step. 5. according to the preparation method of claim 4, wherein said extracting and separating step comprises: dissolving the extract of Rhizoma japonica obtained in any one of claims 1-3 in an alcoholic solvent, mixing the sample with diatomaceous earth and loading into the Soxhlet extractor, sequentially use organic solvents with small to large polarities to reflux extraction, and concentrate the extracts of various organic solvents to obtain the extracts of each organic solvent, wherein the alcohol solvent is selected from methanol or ethanol , the organic solvent is selected from petroleum ether, ethyl acetate and methanol. 6. according to the preparation method of claim 5, comprise the steps: the medicinal extract that any one of claim 1～3 obtains is dissolved in methanol, the consumption of methyl alcohol and the weight ratio of medicinal extract are 1.5～2.5: 1 liter/ kg; mix the resulting methanol solution with diatomaceous earth and place it in a Soxhlet extractor. The weight ratio of diatomite to extract is 4.5 to 5.5:1; Reflux extraction for 48 hours respectively; the amount of petroleum ether, ethyl acetate and methanol is 5-10L; the obtained extracts are respectively concentrated and evaporated to dryness under reduced pressure to obtain the petroleum ether extract, ethyl acetate extract and methanol extract of Gemu things. 7. according to the preparation method described in any one of claim 4-6, it is characterized in that described method further comprises a column chromatography step, and used chromatographic column is selected from normal phase silica gel column, reversed phase silica gel column, Alumina column, cellulose column, activated carbon column, macroporous resin column, polyamide column, hydroxypropyl dextran gel column; the eluent used is selected from water, methanol, ethanol, acetone, dichloromethane, Chloroform, ethyl acetate or mixtures thereof. 8. The method according to claim 7, wherein the chromatographic column is preferably a polyamide column, and the eluent is preferably water, ethanol and mixtures thereof. 9. The method according to claim 8, comprising the steps of: dissolving the ethyl acetate extract obtained in claim 5 or 6 in water and filtering and loading the sample to 30～60 mesh polyamide fillers with 10～30 times of weight In the chromatographic column, gradient elution is carried out in sequence with water, 25-35% ethanol, 55-65% ethanol and 90% ethanol, and the weight ratio of the amount of each eluent to polyamide is 10-15 : 1 liter/kg; then each part of the eluate collected is concentrated to dryness respectively, and the water extract, 25-35% ethanol extract, 55-65% ethanol extract and 90% ethanol extract of Degemu things. 10. according to the preparation method of claim 7, it is characterized in that on the basis of described method also further comprise the step of gel column chromatography and reverse phase column chromatography, wherein gel column chromatography uses hydroxypropyl dextran Gel column, reversed phase column chromatography using reversed phase silica gel column. 11. The method according to claim 10, comprising the steps of: dissolving the water extract obtained in any one of claims 7-9 and the 25-35% ethanol extract respectively in methanol and filtering, adding to 30-60 times Weight of the hydroxypropyl dextran gel column, with methanol as eluent for elution. After being detected by thin layer chromatography or high performance liquid chromatography, the fractions were combined and concentrated, and then loaded onto a reversed-phase silica gel column with a weight of 30 to 60 times, and gradient eluted with methanol/water as the eluent. Fractions are combined after being detected by thin layer chromatography or high performance liquid chromatography, and concentrated to obtain the extract of Goliath, which is the total alkaloids of Golem. 12. The extract of Glycyrrhizae obtained by the preparation method according to any one of claims 1-11. 13. The gorgonian extract according to claim 12, characterized in that the active ingredients of the gerbera extract include casain-type diterpene alkaloid compounds. 14. A pharmaceutical composition, which is characterized in that it comprises the extract of Glycyrrhizae as claimed in claim 12 or 13 and a pharmaceutically acceptable carrier as an active ingredient. 15. The application of the extract of gorgoniana according to claim 12 or 13 in the preparation of antitumor drugs. 16. The application of the pharmaceutical composition according to claim 14 in the preparation of antitumor drugs.

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