CN102526146B - Pharmaceutical composition containing ginsenoside and cantharidin and use thereof - Google Patents

Abstract

The invention relates to the field of traditional Chinese medicine, and discloses a pharmaceutical composition containing ginsenoside and cantharidin and its application. The pharmaceutical composition containing ginsenoside and cantharidin according to the present invention comprises cantharidin or Chinese medicinal material extract containing cantharidin and ginsenoside or Chinese medicinal material extract containing ginsenoside, wherein said ginsenoside or containing ginseng The content of saponin in Chinese herbal extracts is sufficient to reduce the toxic and side effects of cantharidin. The pharmaceutical composition containing ginsenoside and cantharidin of the present invention obviously weakens the toxicity of cantharidin to the kidney, and the incidence of adverse reactions is obviously reduced, thereby greatly improving the safety of cantharidin use, and at the same time, it has the effect of improving the anti-inflammatory effect of cantharidin. The invention has the advantages of tumor effect, safety in clinical use, little toxic and side effects, and can be widely used in the preparation of medicines for treating various cancers.

Description

Pharmaceutical composition containing ginsenoside and cantharidin and use thereof

technical field

The invention relates to the field of traditional Chinese medicine, in particular to a pharmaceutical composition containing ginsenoside and cantharidin and its application.

Background technique

Mylabris, also known as Mylabris phalerataPallas or Mylabris cichorii Linnaeus, is the dry body of Mylabris phalerataPallas or Mylabris cichorii Linnaeus. Catch in summer and autumn, suffocate or burn to death, and dry in the sun. Mylabris is a kind of precious Chinese medicinal material. Li Shizhen, a great medical scientist in the Ming Dynasty, said in the masterpiece "Compendium of Materia Medica" that mylabris is pungent in nature, cold and poisonous. , breaking stones, blood accumulation, hurting human muscles, treating scabies, abortion, treating scrofula, clearing waterways, treating gonorrhea, treating malignant sores, fistulas, treating hernia, detoxifying boils, and treating rabies.

Cantharidin mainly contains cantharidin, monoterpene fats, resins, formic acid and pigments, among which cantharidin is its main active ingredient. Modern pharmacological studies have shown that it has an anti-tumor effect, and cantharidin can be administered 3-4 times to make the rabbit cancer tumor induced by tar disappear. In recent years, it has been found that cantharidin has a certain inhibitory effect on ascites-type liver cancer and reticulocyte sarcoma ARS in mice. Especially when the number of tumor cells inoculated with ascites-type tumors is small, it can be seen that the survival period of mice is prolonged. Cantharidin also has an antiviral effect. Feeding cantharidin 0.6-1.0 mg can treat acute Newcastle disease chicken plague, and the cure rate is over 90%. In addition, cantharidin has special effects on the treatment of skin diseases, vitiligo and stubborn tinea, and also has the effect of hair regeneration on alopecia areata, and has a good inhibitory effect on viral hepatitis.

However, cantharidin is highly toxic. Acute experiments on mice showed that the median lethal dose of intraperitoneal injection was 1.25 mg/kg; biopsies of the viscera showed pathological changes in various organs regardless of acute or subacute toxicity tests: myocardial fiber turbidity and swelling Obvious (especially in subacute test); liver cell turbidity and fatty degeneration; lung and spleen stagnation of blood with small focal hemorrhage; renal tubular epithelium turbidity and swelling. very big. The toxic and side effects of cantharidin clinical application mainly include burning sensation in the mouth and throat in the digestive system, nausea, vomiting or vomiting of blood samples, blood streaks, blood clots, abdominal cramps and other severe reactions, and there are different degrees of hematuria and toxic nephritis in the urinary system symptom. Skin, mucous membrane absorption poisoning, local blisters or congestion, burning pain and so on often occur. Most patients can recover after timely and effective treatment, but a small number of severely poisoned patients died due to acute renal insufficiency and systemic circulatory failure. Therefore, the clinical application of cantharidin and its extract and active ingredient cantharidin are greatly limited.

Contents of the invention

The present invention provides a pharmaceutical composition containing ginsenoside and cantharidin, which comprises cantharidin or Chinese medicinal material extract containing cantharidin and ginsenoside or Chinese medicinal material extract containing ginsenoside, wherein the ginseng The content of saponin or the extract of Chinese herbal medicines containing ginsenoside is sufficient to reduce the toxic and side effects of cantharidin.

Preferably, the pharmaceutical composition comprises ginsenoside and cantharidin, wherein the weight ratio of ginsenoside and cantharidin is 100:1 to 500:1, preferably 200:1 to 400:1, more preferably 300:1 is preferred.

Preferably, the ginsenosides are one or more of ginsenosides or ginsenoside monomers, such as ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, Re, Rg1, Rg2, Rh1, etc. One or more of the active components of the methane-type tetracyclic triterpene saponins. Preferably, the ginsenoside is one or more of ginsenoside Rb1, Rb2, Rc, Rb3, Rf, Rd, Re or Rg1.

As a preference, the pharmaceutical composition of the present invention comprises extracts of Chinese medicinal materials containing cantharidin and extracts of Chinese medicinal materials containing ginsenosides, wherein the content of the extracts of Chinese medicinal materials containing ginsenosides is sufficient to reduce the amount of cantharidin toxic side effects.

Preferably, the Chinese medicinal material extract containing ginsenosides is the extract of Araliaceae Panax genus plants such as ginseng, notoginseng, red ginseng, bamboo ginseng, American ginseng and other Chinese medicinal materials containing ginsenoside. More preferably, the Chinese medicinal material extract containing ginsenosides is total ginsenosides.

Preferably, the Chinese medicinal material extract containing cantharidin is the extract of cantharidin.

As preferably, the Chinese medicinal material extract containing ginsenosides is total ginsenosides, and the Chinese medicinal material extract containing cantharidin is a cantharidin extract, wherein the weight ratio of total ginsenosides to the cantharidin extract is equivalent to 20: 1-100:1 crude drug ginseng: mylabris, that is, the pharmaceutical composition of the present invention, which comprises total ginsenosides and mylabris extract, wherein the weight ratio of total ginsenosides to mylabris extract is equivalent to 20:1 to 100: 1 crude drug ginseng: Mylabris.

Preferably, the weight ratio of total ginsenosides to mylabris extract is equivalent to 20:1 to 100:1 crude drug ginseng:mylabris, more preferably 50:1.

The invention also provides the application of the pharmaceutical composition in the preparation of medicines for treating various cancers. Wherein, the cancer may be liver cancer, cervical cancer and glioma.

The structures of cantharidin and ginsenoside monomers described in the present invention are well known to those skilled in the art, and can be obtained from commercial channels or prepared and identified by methods disclosed in the prior art. Wherein said ginsenoside monomers can be various ginsenoside monomers from any sources or mixtures thereof.

The cantharidin extract in the present invention refers to any form of extract containing cantharidin extracted from the cantharidin, including but not limited to the cantharidin extract prepared in the examples of the present invention. Likewise, the total ginsenosides refers to any form of total ginsenosides containing ginsenosides available to those skilled in the art, including but not limited to the total ginsenosides prepared in the examples of the present invention.

The present invention also provides a pharmaceutical preparation for treating cancer, which consists of an effective amount of the above pharmaceutical composition and pharmaceutically acceptable auxiliary materials.

The pharmaceutical composition containing ginsenoside and cantharidin according to the present invention comprises cantharidin or Chinese medicinal material extract containing cantharidin and ginsenoside or Chinese medicinal material extract containing ginsenoside, wherein said ginsenoside or containing ginseng The content of saponin in Chinese herbal extracts is sufficient to reduce the toxic and side effects of cantharidin. The pharmaceutical composition containing ginsenosides and cantharidin in the present invention significantly weakens the toxicity of cantharidin to the kidney, and the incidence of adverse reactions is significantly reduced, thereby greatly improving the safety of cantharidin in use, and at the same time, it has the effect of improving the antitumor effect of cantharidin. The invention has the advantages of safe clinical use, little toxic and side effects, and can be widely used in the preparation of medicines for treating various cancers.

Description of drawings

Fig. 1 shows the total ginsenosides HPLC-ELSD collection of illustrative plates prepared by embodiment 1;

为斑蝥提取物组，

为人参总皂苷与斑蝥提取物组合物组，**P＜0.01，与斑蝥提取物组相比；Fig. 2 shows the statistical diagram of the influence of embodiment 4 ginseng total saponins on mylabris extract inhibiting the proliferation of SMMC-7721 liver cancer cells, wherein,

is the mylabris extract group,

It is the composition group of total ginseng saponins and mylabris extract, **P<0.01, compared with the group of mylabris extract;

为斑蝥提取物组，

为人参总皂苷与斑蝥提取物组合物组，*P＜0.05，与斑蝥提取物组相比；Fig. 3 shows the statistical diagram of the effect of embodiment 4 ginseng total saponins on the mylabris extract inhibiting the proliferation of hela cervical cancer cells, wherein,

is the mylabris extract group,

It is the composition group of total ginseng saponins and mylabris extract, *P<0.05, compared with the mylabris extract group;

为斑蝥提取物组，

为人参总皂苷与斑蝥提取物组合物组，*P＜0.05，**P＜0.01，与斑蝥提取物组相比；Fig. 4 shows the statistical diagram of the influence of the total saponins of ginseng in Example 4 on the inhibition of U251 human glioma cell proliferation by the mylabris extract, wherein,

is the mylabris extract group,

For the composition group of total ginseng saponins and mylabris extract, *P<0.05, **P<0.01, compared with the group of mylabris extract;

为斑蝥提取物组，

为人参总皂苷与斑蝥提取物组合物组，*P＜0.05，*P＜0.01，与斑蝥提取物组相比；Fig. 5 shows the MTT experiment result diagram of the toxicity of the composition of total ginsenosides and mylabris extract to rat kidney cells (NRK) in embodiment 6, wherein,

is the mylabris extract group,

For the composition group of total ginseng saponins and mylabris extract, *P<0.05, *P<0.01, compared with the group of mylabris extract;

Fig. 6 shows the morphological observation figure that embodiment 7 total ginseng saponins causes the toxicity of normal rat kidney cell (NRK) to the mylabris extract, wherein, A is the total ginsenosides group; B is the normal control group; C is the total ginsenosides Composition group with Mylabris extract.

Detailed ways

The embodiment of the invention discloses a pharmaceutical composition containing ginsenoside and cantharidin and its application. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the products and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

Ginsenoside (Ginsenoside) is a kind of dammarane-type triterpene saponins, dammarane-type triterpene saponins, which mainly exist in ginseng medicinal materials, and widely act on the nervous system and immune system of the human body. The treatment of tumors and other difficult and miscellaneous diseases has shown unique and unique effects. There are more than 40 kinds of ginsenoside monomers, such as Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, Rg3, Rh1, Rh2 and Re, etc.

Ginsenosides all have a similar basic structure, and they are divided into two groups according to their glycoside structure: dammarane type and oleanane type. The dammarane type can be divided into two types: (1) Panaxadiol type-A type, the aglycone is 20(S)-protopanaxadiol, which contains the most ginsenosides, such as ginsenosides Rb1, Rb2, and Rb3 , Rc, Rd, Rg3, Rh2 and glycoside-based PD; (2) Panaxatriol type-B type, the aglycone is 20(S)-protopanaxatriol, including ginsenosides Re, Rg1, Rg2, Rh1 and glycosides base pt. In recent years, a large number of studies have found that under the action of biological enzymes, ginsenosides in the body are metabolized into rare active substances such as Rg3, Rg5, Rh2, Rh3, C-K, Rg2, Rh1, etc. Saponins, absorbed into the blood to play a therapeutic role. Therefore, rare active saponins such as Rg3, Rg5, Rh2, Rh3, C-K, Rg2, and Rh1 are the main active ingredients for ginsenosides to exert their health care functions.

The present invention finds that ginsenosides can significantly reduce the toxic effect of cantharidin on the kidney through a large number of compatibility screening studies, and at the same time enhance the anti-tumor effect of cantharidin, and find that ginsenosides from different sources have similar effects, such as those containing ginseng Araliaceae plants of saponins, such as ginseng, American ginseng, Panax notoginseng, etc., all contain active ingredients such as ginsenosides Rg2, Rh2, Rh3, Re and other dammarane-type tetracyclic triterpene saponins.

Therefore, the present invention provides a pharmaceutical composition comprising cantharidin or extracts of Chinese medicinal materials containing cantharidin and ginsenosides or extracts of Chinese medicinal materials containing ginsenosides, wherein the ginsenosides or The content of Chinese herbal medicine extract is sufficient to reduce the toxic and side effects of cantharidin.

In a specific embodiment, the pharmaceutical composition comprises ginsenoside and cantharidin, wherein the weight ratio of ginsenoside and cantharidin is 100:1 to 500:1, preferably 200:1 to 400 :1, more preferably 300:1.

Preferably, the ginsenosides are one or more of total ginsenosides or monomeric ginsenosides, such as ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, Re, Rg1, Rg2, Rh1, etc. One or more of the active components of the methane-type tetracyclic triterpene saponins. In a specific embodiment, the ginsenoside is a mixture of ginsenosides Rb1, Rb2, Rb3 and Rc. In a specific embodiment, the ginsenoside is a mixture of ginsenosides Rc, Rb2, Re and notoginsenoside NR4. In a specific embodiment, the ginsenosides are Rb1, Rb2, Rg1, 3-O-α-L-arabinofuranosyl (2-1)-α-L-rhamnopyranose (3-1) -β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-glucopyranoside and 3-O-α-L-furan Arabinose(2-1)-α-L-rhamnopyranose[-(4-1)-β-D-glucopyranose]-(3-1)-β-D-glucopyranosyl-alcohol Mixture of aranolic acid-28-β-D-glucopyranose-(6-1)-β-D-glucopyranosides. In a specific embodiment, the ginsenosides are ginsenosides Rf, Rb1, Rb2, Rd, Rg1, notoginsenoside NR4 and 3-O-α-L-arabinofuranoside (2-1)-α-L -Rhamnopyranose(3-1)-β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-glucopyranose ester Glycoside mixture. In another specific embodiment, the ginsenoside is a mixture of ginsenosides Rf, Rb1, Rb2, Rg1, Re and Rb3.

In another specific embodiment, the present invention provides a pharmaceutical composition, which comprises extracts of Chinese medicinal materials containing cantharidin and extracts of Chinese medicinal materials containing ginsenosides, wherein the extracts of Chinese medicinal materials containing ginsenosides The content is enough to reduce the toxic and side effects of cantharidin.

Preferably, the Chinese medicinal material extract containing ginsenosides is the extract of Araliaceae Panax genus plants such as ginseng, notoginseng, red ginseng, bamboo ginseng, American ginseng and other Chinese medicinal materials containing ginsenoside. In a specific embodiment, the Chinese medicinal material extract containing ginsenosides is total ginsenosides. HPLC-ELSD spectrum determination, the total ginsenosides include ginsenosides Rf, Rb ₁ , Rc, Rb ₂ , Rd, Rg ₁ , Re, 3-O-β-D-glucopyranosyl-oleanolic acid-28 -O-β-D-glucopyranose side, notoginsenoside NR ₄ , 3-O-α-L-arabinofuranoside (2-1)-α-L-rhamnopyranose (3-1) -β-D-glucopyranose-(4-1)-β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-pyranolic acid Glucopyranosylside, 3-O-α-L-arabinofuranos(2-1)-α-L-rhamnopyranose(3-1)-β-D-glucopyranosyl-oleanolic acid -28-β-D-glucopyranose-(6-1)-β-D-glucopyranose side, ginsenoside Rb ₃ 12 components, the total saponin content is greater than 80%.

Preferably, the Chinese medicinal material extract containing cantharidin is the extract of cantharidin.

In a specific embodiment, the Chinese herbal medicine extract containing ginsenosides is total ginsenosides, and the Chinese herbal medicine extract containing cantharidin is a cantharidin extract, wherein the weight ratio of total ginsenosides to the cantharidin extract is Equivalent to 20:1～100:1 crude drug ginseng: mylabris, that is, the pharmaceutical composition of the present invention comprises total ginsenosides and mylabris extract, wherein the weight ratio of total ginsenosides to mylabris extract is equivalent to 20: 1 to 100:1 crude drug ginseng: mylabris.

Preferably, the weight ratio of total ginsenosides to mylabris extract is equivalent to 20:1 to 100:1 crude drug ginseng:mylabris, more preferably 50:1.

The invention also provides the application of the pharmaceutical composition in the preparation of medicines for treating various cancers. Wherein, the cancer may be liver cancer, cervical cancer and glioma.

In a specific embodiment, the ginsenoside is a mixture of ginsenosides Rb1, Rb2, Rb3 and Rc, and its combination with cantharidin has a synergistic effect on cantharidin on SMMC-7721 liver cancer cells. In a specific embodiment, the ginsenoside is a mixture of ginsenosides Rc, Rb2, Re and notoginsenoside NR4, and its combination with cantharidin has a synergistic effect on cantharidin on Hela cervical cancer cells. In a specific embodiment, the ginsenosides are Rb1, Rb2, Rg1, 3-O-α-L-arabinofuranosyl (2-1)-α-L-rhamnopyranose (3-1) - Mixtures of β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-glucopyranosides in combination with cantharidin The compound has a synergistic effect on cantharidin on U251 glioma cells. In a specific embodiment, the ginsenosides are ginsenosides Rf, Rb1, Rb2, Rd, Rg1, notoginsenoside NR4 and 3-O-α-L-arabinofuranoside (2-1)-α-L -Rhamnopyranose(3-1)-β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-glucopyranose ester The mixture of glycosides and its combination with cantharidin have attenuated the toxicity of cantharidin in normal kidney cells of NRK rats. In another specific embodiment, the ginsenoside is a mixture of ginsenosides Rf, Rb1, Rb2, Rg1, Re and Rb3, and its composition with cantharidin has an effect on cantharidin on QSG-7701 human normal liver cells. Attenuation, synergistic effect on cantharidin in SMMC-7721 liver cancer cells and H22 liver cancer mice.

The structures of cantharidin and ginsenoside monomers described in the present invention are well known to those skilled in the art, and can be obtained from commercial channels or prepared and identified by methods disclosed in the prior art. Wherein said ginsenoside monomers can be various ginsenoside monomers from any sources or mixtures thereof.

The cantharidin extract in the present invention refers to any form of extract containing cantharidin extracted from the cantharidin, including but not limited to the cantharidin extract prepared in the examples of the present invention. Likewise, the total ginsenosides refers to any form of total ginsenosides containing ginsenosides available to those skilled in the art, including but not limited to the total ginsenosides prepared in the examples of the present invention.

The pharmaceutical composition of the present invention can be prepared into various conventional preparations through conventional techniques in the art or the methods disclosed in the present invention and pharmaceutically acceptable adjuvants. Therefore, the present invention also provides a pharmaceutical preparation for treating cancer, which consists of an effective amount of the above pharmaceutical composition and pharmaceutically acceptable auxiliary materials. The preparations can be tablets, capsules, dropping pills, pills, powders, injections, suspensions, granules or freeze-dried powder injections and the like. The formulations containing the composition of the present invention can be administered in a suitable manner in the following ways: oral administration, spray inhalation, rectal administration, nasal cavity administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous administration , intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, wherein oral, intramuscular, intraperitoneal or intravenous administration is preferred.

The dose and method of use of the pharmaceutical composition of the present invention depend on many factors, including the patient's age, body weight, sex, natural health status, nutritional status, activity intensity of the compound, time of administration, metabolic rate, severity of the course of the disease, and the treating physician subjective judgment. Those skilled in the art can easily determine the dosage and method of use of the pharmaceutical composition according to the above factors.

The pharmaceutical composition containing ginsenoside and cantharidin according to the present invention comprises cantharidin or Chinese medicinal material extract containing cantharidin and ginsenoside or Chinese medicinal material extract containing ginsenoside, wherein said ginsenoside or containing ginseng The content of saponin in Chinese herbal extracts is sufficient to reduce the toxic and side effects of cantharidin. The pharmaceutical composition containing ginsenosides and cantharidin in the present invention significantly weakens the toxicity of cantharidin to the kidney, and the incidence of adverse reactions is significantly reduced, thereby greatly improving the safety of cantharidin in use, and at the same time, it has the effect of improving the antitumor effect of cantharidin. The invention has the advantages of safe clinical use, little toxic and side effects, and can be widely used in the preparation of medicines for treating various cancers.

In order to further understand the present invention, the present invention will be described in detail below in conjunction with examples.

Embodiment 1: Preparation of total ginsenosides

20kg ginseng, cut into small pieces of 1-2cm, add 160L 50% ethanol to reflux and extract twice, the first time for 3 hours, the second time for 1.5 hours, combine the extracts, centrifuge at 4500rpm for 20 minutes, take the supernatant and filter it with filter paper, collect The filtrate is used for later use.

Take 5kg of the processed AB-8 resin, put it into a stainless steel chromatography column with a diameter of 30cm, the diameter-to-height ratio of the resin bed is 1:10, wash with water, filter the obtained ginseng extract and load it at ^a speed of 1mL/min.cm Twice, static adsorption for 12h. First wash 4BV with water and discard; then wash 3BV with 30% ethanol and discard; finally elute with 75% ethanol at a speed of 2-3mL/ ^min.cm2 , collect the eluate, concentrate and dry to obtain total ginsenosides 50g.

Embodiment 2: The HPLC-ELSD pattern determination of total ginseng saponins

The total ginsenosides prepared in Example 1 were taken for HPLC-ELSD spectrum determination. Chromatographic column: Kromasil C18 (250mm×4.6mm, 5.0μm). Mobile phase: A is acetonitrile, B is water, gradient elution program: 0~10min, 5%A~20%A; 10~35min, 20%A; 10~35min, 20%A~29%A; 36~ 70min, 29%A; 70～100min, 47%A. Flow rate: 1mL·min-1; Column temperature: 35°C. Injection volume: 20 μL. Evaporative light scattering detector: T=40°C, P=0.35MPa, the carrier gas is high-purity nitrogen. The test results are shown in Figure 1.

It can be seen from Figure 1 that the total ginsenosides prepared in Example 1 of the present invention include ginsenosides Rf, Rb ₁ , Rc, Rb ₂ , Rd, Rg ₁ , Re, 3-O-β-D-glucopyranosyl-zi Aranolic acid-28-O-β-D-glucopyranose side, notoginsenoside NR ₄ , 3-O-α-L-arabinofuranoside (2-1)-α-L-rhamnopyranose (3-1)-β-D-glucopyranose-(4-1)-β-D-glucopyranose--oleanolic acid-28-β-D-glucopyranose-(6-1) -β-D-glucopyranose, 3-O-α-L-arabinofuranos(2-1)-α-L-rhamnopyranose(3-1)-β-D-glucopyranose Base-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-glucopyranoside and ginsenoside Rb ₃ 12 components, the total saponin content is greater than 80%. Among them, the structural formula of each index component is as follows:

Embodiment 3: the preparation of mylabris extract

Weigh 20g of mylabris powder, pour it into a 1000mL round bottom flask, add 250mL of acetone, shake intermittently at 30°C for 2h, and extract twice. The extracts were combined, concentrated under reduced pressure, washed with 95% ethanol:petroleum ether (1:1.5) until the washing liquid was colorless, and a near-white crude product was obtained, then about 5mL of acetone was added, heated to dissolve, filtered while hot, and the filtrate was placed at 4°C Natural crystallization gave 50 mg of white transparent needle crystals, yield 0.25%. The product was detected by liquid chromatography and gas chromatography, and no impurity peaks were found, indicating that the product had a purity greater than 99.5%.

Example 4: Inhibitory effect of the ginsenoside composition of the present invention on tumor cells

Select SMMC-7721 liver cancer cells, Hela cervical cancer cells, and U251 glioma cells respectively, and use the MTT method to measure the inhibitory effect of the ginsenoside composition of the present invention on tumor cells.

1. Inhibitory effect of the composition of total ginseng saponins and mylabris extract on tumor cells

The cell densities of SMMC-7721 liver cancer cells, Hela cervical cancer cells, and U251 glioma cells were adjusted to 1.0×10 ⁵ /mL, and seeded in 96-well plates, 100 μL per well. Tumor cell administration treatment method: the concentrations of the Mylabris extract prepared in Example 3 and the total ginsenosides prepared in Example 1 are 5 μg/mL+1540 μg/mL, 2.5 μg/mL+770 μg/mL, 1.25 μg/mL+385 μg /mL, 0.625μg/mL+192.5μg/mL, 0.3125μg/mL+96.25μg/mL. No drug was added and an equal amount of PBS buffer was used as a negative control. Three parallel wells were set up for each group, and culture continued. After 48 hours of drug administration, add 5 mg/mL MTT10 μL to each well, lightly shake the culture plate, incubate in an incubator for 4 hours, discard the supernatant, add 100 μL of dimethyl sulfoxide, and shake at room temperature for 10 minutes to dissolve it completely. The OD492 value was measured in the instrument. Experiments were repeated three times. The results are shown in Figures 2-4.

As can be seen from Figures 2 to 4, when the concentrations of the mylabris extract prepared in Example 3 and the total ginsenosides prepared in Example 1 were 5 μg/mL+1540 μg/mL and 2.5 μg/mL+770 μg/mL, the total ginsenosides can significantly Improve the effect of cantharidin on the proliferation of SMMC-7721 liver cancer cells, Hela cervical cancer cells and U251 human glioma cells.

2. The inhibitory effect of the composition of ginsenoside monomer and cantharidin on tumor cells

The cell densities of SMMC-7721 liver cancer cells, Hela cervical cancer cells, and U251 glioma cells were adjusted to 1.0×10 ⁵ /mL, and seeded in 96-well plates, 100 μL per well. Tumor cell drug treatment method: three concentrations of monomer T1, T2, T3 (250, 125, 62.5 μg/mL); two concentrations of cantharidin B1, B2 (1.25, 2.5 μg/mL); three concentrations of monomer Concentration and two concentrations of cantharidin were combined respectively; no drug was added and an equal amount of PBS buffer was used as a negative control. Three parallel wells were set up for each group, and culture continued. After 48 hours of drug administration, add 10 μL of 5 mg/mL MTT to each well, lightly shake the culture plate, incubate in an incubator for 4 hours, discard the supernatant, add 100 μL of dimethyl sulfoxide, and shake at room temperature for 10 minutes to dissolve it completely. Measure the OD492 value in the standard instrument. Experiments were repeated three times. The results are shown in Tables 1-3.

The effect of table 1 ginsenoside composition on the proliferation of SMMC-7721 liver cancer cells

Table 2 Effect of ginsenoside composition on proliferation of Hela cervical cancer cells

Table 3 Effect of ginsenoside composition on U251 glioma cell proliferation

From the results in Table 1, it can be seen that the inhibitory rate of ginsenosides in the range of 62.5-250 μg/ml to SMMC-7721 cells is in a significant dose-effect relationship, and ginsenosides Rb1, Rb2, Rc, and Rb3 can significantly increase the concentration of cantharidin at 1.25 μg/mL and 2.5 μg/mL inhibitory effect (P<0.01), and q>0.85; ginsenosides Rf, Rd, Rg1 significantly increased the inhibitory effect of cantharidin at 2.5 μg/mL, and q>0.85. The results showed that ginsenosides Rb1, Rb2, Rc, Rb3, Rf, Rd and Rg1 had synergistic effects on cantharidin in inhibiting SMMC-7721 liver cancer cells.

From the results in Table 2, it can be seen that the inhibitory rate of ginsenosides to Hela cells in the range of 62.5-250 μg/mL is in a significant dose-effect relationship, and wherein ginsenoside Rb1 significantly increases the inhibitory effect of cantharidin at 1.25 μg/mL and 2.5 μg/mL (P< 0.01), and q>0.85; ginsenosides Rc, Rb2, Re, notoginsenoside NR4 significantly increased the inhibitory effect of cantharidin at 2.5 μg/mL, and q>0.85, the results showed that ginsenosides Rb1, Rc, Rb2, Re and Notoginseng saponin NR4 has a synergistic effect on cantharidin inhibiting Hela cervical cancer cells.

From the results in Table 3, it can be seen that the inhibitory rate of ginsenosides to U251 cells in the range of 62.5-250 μg/mL showed a significant dose-effect relationship. 1)-α-L-rhamnopyranose (3-1)-β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β- D-glucopyranosylside significantly increased the inhibitory effect of cantharidin at 1.25 μg/mL and 2.5 μg/mL (P<0.01), and q>0.85; 3-O-β-D-glucopyranosyl-olean Fruit acid-28-O-β-D-glucopyranoside and ginsenoside Rb3 significantly increased the inhibitory effect of cantharidin at 1.25 μg/mL, and q>0.85; notoginsenoside NR4 could significantly increase the inhibitory effect of cantharidin at 2.5 μg /mL inhibition. The results showed that the inhibitory rate of ginsenosides in the range of 62.5-250 μg/mL to U251 cells showed a significant dose-effect relationship. α-L-rhamnopyranose(3-1)-β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-pyranose Glucopyranoside, ginsenoside Rb3 and notoginseng saponin NR4 have synergistic effect on cantharidin inhibiting U251 glioma cells.

Example 5: In vivo anti-tumor effect of the ginsenoside composition of the present invention

Get the total ginsenosides prepared in Example 1 of different concentrations and the compatibility of the mylabris extract prepared in Example 3, investigate its influence on the Kunming mice transplanted with _H22 tumors, and evaluate the synergistic effect of total ginseng saponins on the mylabris extract in vivo . The specific operation is as follows: resuscitate H ₂₂ cells, adjust the number of cells to 1×10 ⁷ with physiological saline, take KM mice for passage three times, and finally wipe the abdomen of mice with 75% alcohol under aseptic conditions, take out the ascites, adjust The number of cells was 1×10 ⁷ , and 0.2 mL was inoculated into the right axilla of each mouse. 24 hours after inoculation, they were randomly divided into groups, and the animals were administered continuously for 10 days, and the animals were sacrificed 1 hour after the last administration. No drug was added and an equal amount of PBS buffer was used as a blank control. Three parallel sets were set up for each group. The statistical results are shown in Table 4

Table 4 The effect of total ginseng saponins on the inhibition of mylabris extract on the tumor weight of H22 mice

Note: *P<0.05, *P<0.01, compared with the cantharidin group.

As can be seen from the results in Table 4, compared with the blank control group, the mylabris extract can significantly reduce the mouse tumor weight, and the inhibition rate is 53.48%. rate, inhibition rate were 64.31% and 74.31%.

Embodiment 6: In vitro cytotoxicity test of the ginsenoside composition of the present invention

1. In vitro toxicity test of the combination of ginseng total saponins and mylabris extract on normal renal cells

The normal kidney cells of NRK rats were selected, and the in vitro cytotoxic effect of the composition of the total ginsenosides prepared in Example 1 and the Mylabris extract prepared in Example 3 was determined by the MTT method. Administration treatment method: the concentrations of the Mylabris extract prepared in Example 3 and the total ginsenosides prepared in Example 1 are 5 μg/mL+1540 μg/mL, 2.5 μg/mL+770 μg/mL, 1.25 μg/mL+385 μg/mL , 0.625 μg/mL+192.5 μg/mL, 0.3125 μg/mL+96.25 μg/mL. Three parallel wells were set up for each group, and culture continued. After 48 hours of drug administration, add 10 μL of 5 mg/mL MTT to each well, lightly shake the culture plate, place it in an incubator and incubate for 4 hours, discard the supernatant, add 100 μL of dimethyl sulfoxide, shake at room temperature for 10 minutes, and dissolve it completely. The OD ₄₉₂ value was determined by a microplate reader. Experiments were repeated three times. The statistical results are shown in Figure 5.

The results in Figure 5 show that total ginsenosides have a significant dose-effect relationship on the inhibition rate of normal kidney cells in NRK rats within the concentration range of 31.25-250 μg/mL, indicating that total ginsenosides have an attenuating effect on NRK in rat normal kidney cells extracted from mylabris .

2. In vitro normal kidney and liver cell toxicity test of ginsenoside monomer and cantharidin combination

NRK rat normal kidney cells and QSG-7701 human normal liver cells were selected, and the in vitro cytotoxicity of the ginsenoside composition was determined by the MTT method. The specific operation is as follows: select NRK rat normal kidney cells and QSG-7701 human normal liver cells at the cell density of NRK rat normal kidney cells and QSG-7701 human normal liver cells respectively, and use the MTT method to measure the in vitro cytotoxicity of the ginsenoside composition effect. The specific operation is as follows: adjust the cell density of NRK rat normal kidney cells and QSG-7701 human normal liver cells to 1.0×10 ⁵ /mL, respectively, and inoculate them in a 96-well plate, 100 μL per well. Cell administration treatment method: three concentrations of monomer T1, T2, T3 (250, 125, 62.5 μg/mL); two concentrations of cantharidin B1, B2 (1.25, 2.5 μg/mL); three concentrations of monomer Combined with two concentrations of cantharidin, respectively; no drug plus an equal amount of PBS buffer as a negative control. Three parallel wells were set up for each group, and culture continued. After 48 hours of drug administration, add 10 μL of 5 mg/mL MTT to each well, lightly shake the culture plate, place it in an incubator and incubate for 4 hours, discard the supernatant, add 100 μL of dimethyl sulfoxide, shake at room temperature for 10 minutes, and dissolve it completely. The OD ₄₉₂ value was determined by a microplate reader. Experiments were repeated three times. See Tables 5 and 6 for statistical results.

Table 5 Effects of ginseng monomer compounds on normal kidney cell proliferation in NRK rats

Table 6 Effects of ginseng monomer compounds on QSG-7701 human normal liver cell proliferation

It can be seen from the results in Table 5 that the inhibitory rate of ginsenosides in the concentration range of 62.5-250 μg/mL to NRK rat normal kidney cells showed an obvious dose-effect relationship, in which ginsenosides Rf, Rb1, Rb2, Rd, Rg1, notoginsenoside NR4 and 3-O-α-L-arabinofuranosyl(2-1)-α-L-rhamnopyranose(3-1)-β-D-glucopyranosyl-oleanolic acid-28-β -D-glucopyranose-(6-1)-β-D-glucopyranoside significantly reduced the inhibitory effect of cantharidin at 1.25 μg/mL and 2.5 μg/mL; 3-O-α-L-furan arabic Sugar (2-1)-α-L-rhamnopyranose (3-1)-β-D-glucopyranose-(4-1)-β-D-glucopyranosyl-oleanolic acid- 28-β-D-glucopyranose-(6-1)-β-D-glucopyranoside significantly reduced the inhibitory effect of cantharidin at 1.25 μg/mL, and all P<0.05, q<0.85; ginsenoside Rb3 obviously reduces the inhibitory effect of cantharidin at 2.5 μ g/mL, q＜0.85, the result shows that ginsenoside Rf, Rb1, Rb2, Rd, Rg1, notoginseng saponin NR4, 3-O-α-L-arabinofuranoside ( 2-1)-α-L-rhamnopyranose (3-1)-β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)- β-D-glucopyranose, 3-O-α-L-arabinofuranos(2-1)-α-L-rhamnopyranose(3-1)-β-D-glucopyranose- (4-1)-β-D-glucopyranosyl-oleanolic acid-28-β-D-glucopyranose-(6-1)-β-D-glucopyranoside and ginsenoside Rb3 pair Cantharidin inhibits renal cell NRK and has an attenuating effect.

It can be seen from the results in Table 6 that the inhibitory rate of ginsenosides in the concentration range of 62.5-250 μg/mL to QSG-7701 human normal liver cells showed an obvious dose-effect relationship, among which ginsenosides Rf, Rb1, Rc, Rb2, Rg1, Re, Rb3 Significantly reduced the inhibitory effect of cantharidin at 1.25 μg/mL and 2.5 μg/mL, the results showed that ginsenosides Rf, Rb1, Rc, Rb2, Rg1, Re and Rb3 had attenuated effect on cantharidin inhibiting QSG-7701.

Embodiment 7: Observation of the toxicity morphology of normal rat kidney cells (NRK) caused by ginsenoside composition of the present invention to cantharidin

Under the microscope, the normal renal cells adhered to the wall, were irregular, and the cell morphology was normal. The renal cells of the cantharidin extract group (1.25 μg/mL) prepared in Example 3 became round, the adherent cells decreased, and the cell morphology changed significantly. After the total ginsenosides prepared in Example 1 of the present invention and the cantharidin extract (385 μg/mL+1.25 μg/mL) prepared in Example 3 were combined, the cell morphology improved significantly, and the results are shown in FIG. 6 .

Example 8: Toxic apoptosis experiment of normal rat kidney cells (NRK) induced by cantharidin by the ginsenoside composition of the present invention

Using a 24-well plate, adjust the normal kidney cell concentration of NRK rats to 4×10 ⁵ , inoculate the total ginsenosides prepared in Example 1 and the mylabris extract prepared in Example 2 respectively according to Table 7, and receive 500 μL per well, each group There are three parallel holes. After 48 hours of administration, the cells in each group were collected, rinsed twice with PBS, discarded the supernatant, stained according to the instructions of the V-FITV/PI double-stained apoptosis kit (purchased from Nanjing Jiancheng Biology Co., Ltd.), and performed flow cytometry. The statistical results are shown in Table 7.

Table 7 Effect of total ginsenosides on cantharidin-induced apoptosis of normal rat kidney cells

group Complete cells (%) Apoptotic cells (%) dead cells (%) normal control group 95.63±0.65 3.97±0.51 0.40±0.26 Mylabris extract (0.625μg/mL) 77.10±4.24 ^△ 21.40±6.36 ^△ 0.60±0.28 Mylabris Extract (1.25μg/mL) 65.97±3.05 ^△△ 33.37±4.11 ^△△ 0.53±0.40 Mylabris Extract (2.5μg/mL) 58.55±1.91 ^△△ 36.45±2.76 ^△△ 0.90±0.14 Mylabris extract + total ginsenosides (1.25μg/mL+0.385mg/mL) 90.30±0.20** 8.55±0.92** 0.73±0.25 Mylabris extract + total ginsenosides (1.25μg/mL+0.77mg/mL) 8670±283 ^△ * 12.40±3.82* 0.45±0.35

Note: *P<0.05, **P<0.01, compared with the cantharidin (1.25ug/mL) group; ^△ P<0.05, ^△△ P<0.01, compared with the normal group.

The experimental results showed that total ginsenosides (0.385, 0.77mg/mL) significantly reduced the apoptosis of NRK cells induced by the mylabris extract, and the apoptosis rate decreased from 33.37% to 8.55%.

Example 9: Inhibitory effect of the ginsenoside composition of the present invention on cantharidin-induced nephrotoxicity in rats

The rat kidney injury model was induced by intraperitoneal injection of cantharidin (0.14mg/kg) for 15 consecutive days, and ginsenoside was intraperitoneally injected 1 hour before cantharidin, with doses of 1.6mg/kg and 3.2mg/kg. After the last administration 1h, put into metabolic cage and collect 24h urine, measure urine volume and urine protein content in urine, investigate the inhibitory effect of ginsenoside composition of the present invention on the kidney injury of SD rats caused by cantharidin, the result See Table 8.

Table 8 Effect of total ginsenosides on cantharidin-induced nephrotoxicity in rats

Note: *P<0.05, **P<0.01, compared with the cantharidin (1.25ug/mL) group; ^△△ P<0.01, compared with the normal control group.

As can be seen from the results in Table 8, cantharidin can increase the urine protein content of rats, and the content of urea nitrogen and creatinine in the blood is increased, and total ginseng saponins can obviously reduce the urine protein content, blood urea nitrogen and creatinine caused by cantharidin, indicating that Ginsenosides can reduce the renal toxicity caused by cantharidin.

Embodiment 10: the preparation of tablet of the present invention

Formula (according to 1000 tablets):

Cantharidin 1g

Ginsenoside Rb1 300g

Preparation method: Take the above formula amount of cantharidin and ginsenoside Rb1 and mix evenly, add appropriate amount of starch, ethyl cellulose, lactose, cross-linked povidone, add to a high-efficiency wet granulator and stir at 6 speeds, high-speed cutting and pre-mixing 3min. Add 250 g of 95% ethanol into the high-efficiency wet granulator, stir at 6 speeds, and cut at high speed for 3 minutes. The air inlet temperature is 50±5°C and dried for 15-25 minutes. Sieve through a 1.5mm sieve, add magnesium stearate and micro-powder silica gel in the prescribed amount, mix well, press into tablets, pack, and inspect.

Embodiment 11: the preparation of capsule of the present invention

Formula (according to 1000 capsules):

Spotted weed extract 5g

Total Ginseng Saponins 600g

Preparation method: take the cantharis extract and total ginsenosides of the above formula, add appropriate amount of calcium carbonate, mannitol, pregelatinized starch, and sodium carboxymethyl starch, add to a high-efficiency wet granulator and stir at 6 speeds, high-speed cutting and pre-mixing 3min. Add about 200g of 5% pregelatinized starch slurry into the high-efficiency wet granulator, stir at 6 speeds, and cut at high speed for 2 minutes. The air inlet temperature is 50±5°C and dried for 15-20 minutes. Sieve through a 1.5mm sieve, add magnesium stearate and micro-powder silica gel in the prescribed amount, mix well, fill capsules, pack, and inspect to obtain the product.

Embodiment 12: the preparation of capsule of the present invention

Formula (according to 1000 capsules):

Spotted weed extract 5g

Total Ginseng Saponins 600g

Preparation method: take the cantharis extract and total ginsenosides of the above formula, add appropriate amount of calcium carbonate, mannitol, pregelatinized starch, and sodium carboxymethyl starch, add to a high-efficiency wet granulator and stir at 6 speeds, high-speed cutting and pre-mixing 3min. Add about 200g of 5% pregelatinized starch slurry into the high-efficiency wet granulator, stir at 6 speeds, and cut at high speed for 2 minutes. The air inlet temperature is 50±5°C and dried for 15-20 minutes. Sieve through a 1.5mm sieve, add magnesium stearate and micro-powder silica gel in the prescribed amount, mix well, fill capsules, pack, and inspect to obtain the product.

The descriptions of the above embodiments are only used to help understand the application and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (7)

1. a pharmaceutical composition, the Chinese crude drug extract that it comprises Cantharidin or contains Cantharidin and ginsenoside or the Chinese crude drug extract that contains ginsenoside, described ginsenoside is one or more in ginsenoside Rb1, Rb2, Rc, Rb3, Rf, Rd, Re or Rg1, and the content of wherein said ginsenoside or the Chinese crude drug extract that contains ginsenoside is enough to reduce the toxic and side effects that Cantharidin produces.

2. a pharmaceutical composition, it comprises the Chinese crude drug extract that contains Cantharidin and the Chinese crude drug extract that contains ginsenoside, described ginsenoside is one or more in ginsenoside Rb1, Rb2, Rc, Rb3, Rf, Rd, Re or Rg1, and the content of the wherein said ginsenoside's of containing Chinese crude drug extract is enough to reduce the toxic and side effects that Cantharidin produces.

3. pharmaceutical composition according to claim 1, it comprises ginsenoside and Cantharidin, and the weight ratio of wherein said ginsenoside and Cantharidin is 100:1～500:1.

4. according to pharmaceutical composition described in claim 1 or 2, the wherein said Chinese crude drug extract that contains Cantharidin is Mylabris extract.

Described in claim 1 to 4 any one pharmaceutical composition for the preparation for the treatment of various cancers medicine in purposes.

Described in claim 1 to 4 any one pharmaceutical composition for the preparation for the treatment of various hepatocarcinoma, cervical cancer and glioma medicine in purposes.

7. a pharmaceutical preparation for the treatment of cancer, is characterized in that, pharmaceutical composition described in claim 1 to 4 any one of effective dose and pharmaceutically acceptable adjuvant, consists of.

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