Application and preparation of gypenoside B in antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs

Abstract

The invention belongs to the field of separation and extraction of Chinese medicinal materials, in particular to the invention, application and preparation of a new drug of gypenosanol B for antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs. The existing drugs represented by glucocorticoid drugs show anti-inflammatory, anti-shock, anti-viral and immunosuppressive therapeutic effects in many diseases, but at the same time, the side effects caused to patients are also very large, mainly the problem that long-term use of glucocorticoids causes adrenal atrophy, sugar and salt metabolism disorders and damage to the heart, liver, lungs and kidneys. The following scheme is proposed, and the preparation method thereof comprises the following steps: S1, taking 20kg of gypenosanol total saponins, dissolving them in methanol, mixing them with 40kg of silica gel, and evaporating the solvent; S2, applying a 100kg dry silica gel chromatography column, and eluting them with a chloroform-methanol gradient. The drug of gypenosanol B extracted by the invention is used for antagonizing the side effects of glucocorticoid drugs and the side effects caused by radiotherapy and chemotherapy, and has a definite therapeutic effect and is safe and non-toxic.

Description

Technical Field

[0001] The invention relates to the technical field of separation and extraction of traditional Chinese medicines, and in particular to the application and preparation of gypenoside B in antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs. Background Art

[0002] Tumors are currently the most serious disease that endangers human health. Early tumors generally have no clinical symptoms. Once clinical manifestations appear, they are usually in the middle and late stages and tumor metastasis has occurred. At present, the treatment of tumors is mainly through radiotherapy and chemotherapy, but radiotherapy and chemotherapy have certain side effects on the body and cannot kill all cancer cells. They can only kill cancer cells in proportion. Residual cancer cells develop quickly in the case of weak body and low immunity, forming new lesions. The effective ingredients extracted from Chinese herbal medicine are used to cooperate with radiotherapy and chemotherapy for comprehensive management, reduce the side effects of radiotherapy and chemotherapy, kill residual cancer cells, improve immunity, effectively control the development of the disease, and enable patients to significantly improve and control their condition in the shortest time, which can achieve the most ideal treatment effect.

[0003] Drugs represented by glucocorticoids have shown anti-inflammatory, anti-shock, antiviral and immunosuppressive therapeutic effects in many diseases, but at the same time they also cause great side effects to patients, mainly damage to the heart, liver, lungs and kidneys. Summary of the invention

[0004] The purpose of the present invention is to solve the problem that the drugs represented by glucocorticoid drugs in the prior art show anti-inflammatory, anti-shock, anti-viral and immunosuppressive therapeutic effects on many diseases, but at the same time cause great side effects to patients, mainly damage to the heart, liver, lungs and kidneys, and propose an application and preparation of gypenoside B in antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A preparation method of gypenoside B for antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs, comprising the following steps:

[0007] S1. Take 15kg-30kg of total saponins of Gynostemma pentaphyllum, dissolve them in methanol, mix with 30kg-50kg of silica gel, and evaporate the solvent;

[0008] S2, add 90-120kg dry silica gel chromatography column, elute with chloroform-methanol gradient, check with thin layer chromatography, collect and combine the eluate containing gypenosyl saponin B, and recover the solvent to obtain 5kg gypenosyl saponin B crude product;

[0009] S3, taking 90g-110g of crude gypenosan B, preparing a 200mg / ml solution with methanol, purifying by medium pressure preparative liquid chromatography, collecting gypenosan B components, and concentrating to obtain gypenosan B with a content of 50% to 90% or more;

[0010] S4. Dissolve gypenoside B with a content of 50% to 90% or more in a mixed organic solvent, and repeatedly recrystallize it, so that the purity can reach more than 96%.

[0011] Preferably, light and high temperature should be avoided during the entire preparation and purification process, and freeze-drying should be used.

[0012] Preferably, in S2, a 90kg-120kg dry silica gel chromatography column is applied and eluted with a gradient of chloroform-methanol (8:1→3:1).

[0013] The present invention also proposes an application of gypenosin B in antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs. The gypenosin B is used for antagonizing radiotherapy, chemotherapy and glucocorticoid drugs.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] my country has abundant Gynostemma pentaphyllum resources, which are widely distributed naturally and easy to cultivate artificially. Gynostemma pentaphyllum saponin B is a single active ingredient extracted from Gynostemma pentaphyllum. It not only has anti-tumor effects, but also has the effect of antagonizing the side effects of chemotherapy and glucocorticoid drugs in tumor treatment.

[0016] Radiotherapy and chemotherapy have certain side effects on the body. They cannot kill all cancer cells, but can only kill them in proportion. Residual cancer cells develop quickly when the body is weak and the immunity is low, forming new lesions. Research on Gynostemma Saponin B has confirmed that it can antagonize or reduce the side effects of radiotherapy and chemotherapy, kill residual cancer cells, enhance immunity, and effectively control the progression of the disease, so that the patient's condition can be significantly improved and controlled in the shortest time, achieving the most ideal treatment effect.

[0017] Gynostemma pentaphyllum saponin B can effectively reduce or alleviate the serious side effects caused by the use of glucocorticoid drugs. The most commonly used and representative glucocorticoid drugs in clinical practice include hydrocortisone, prednisolone acetate, dexamethasone, etc. Gynostemma pentaphyllum saponin B antagonizes the clinical side effects of the above drugs: 1. adrenal cortex function damage; 2. osteoporosis and spontaneous fractures; 3. Inducing and aggravating infections.

[0018] The drug of gypenosyl saponin B extracted by the invention is used for antagonizing the side effects produced by glucocorticoid drugs and radiotherapy and chemotherapy in anti-tumor treatment, has definite curative effect, is safe and has no toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The invention provides an application of gypenosan B in antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs and the structural formula of the prepared gypenosan B.

[0020] Figure 2 It is the structural formula of gypenoside B. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0022] A preparation method of gypenoside B for antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs, comprising the following steps:

[0023] Take 15kg-30kg of total saponins of Gynostemma pentaphyllum, dissolve it in methanol, mix it with 30kg-50kg of silica gel, evaporate the solvent, apply it to a 90-110kg dry silica gel chromatography column, elute it with a chloroform-methanol gradient, and check it with thin layer chromatography (using the upper layer of n-butanol-acetic acid-water (4:1:5) as the developing agent, and the color developer: 10% ethanolic sulfuric acid solution), collect and combine the eluent containing Gynostemma pentaphyllum saponin B, and recover the solvent to obtain 5kg of crude Gynostemma pentaphyllum saponin B.

[0024] Take 90g-110g of crude gypenosan B, prepare a 200mg / ml solution with methanol, purify it by medium-pressure preparative liquid chromatography, collect gypenosan B components, and concentrate to obtain gypenosan B with a content of more than 50% to 90%.

[0025] The purity of gypenoside B with a content of 50% to 90% or more is reached by dissolving it in a mixed organic solvent and repeatedly recrystallizing it. During the entire preparation and purification process, light and high temperature must be avoided and freeze drying must be used.

[0026] The present invention also proposes an application of gypenoside B in antagonizing the side effects of radiotherapy, chemotherapy and glucocorticoid drugs. Gynostemma pentaphyllum saponin B is used in antagonizing radiotherapy, chemotherapy and glucocorticoid drugs.

[0027] In this example, the structure of the compound Gynostemma pentaphyllum saponin B is determined.

[0028] Common name: Gynostemma saponin B

[0029] English name: gypenoside B

[0030] Chinese Pinyin: Jiaogulan Zaogan B

[0031] Structural formula and molecular formula

[0032] Structural formula: Figure 2

[0033] Molecular formula: C46H76O16

[0034] Molecular weight: 884.5

[0035] In this embodiment: the clinically effective dose for antagonizing radiotherapy and chemotherapy

[0036] The clinically effective dosage is 30mg~720mg / day / person, 2~3 times a day, in conjunction with the entire course of radiotherapy and chemotherapy. It can also be taken for a long time, which is safe and has no toxic side effects.

[0037] In this embodiment, the clinically effective dosage for reducing the side effects of glucocorticoid drugs is

[0038] The clinically effective dosage is 30mg-720mg / day / person, 2-3 times a day. It can be taken for a long time in conjunction with the course of treatment of chemical drugs, and is safe and has no toxic side effects.

[0039] In this embodiment, the drug specification is: each unit contains 10 to 180 mg of gypenoside B.

[0040] In this embodiment, the drug dosage form is:

[0041] Gynostemma saponin B is administered orally in clinical use and is applicable to all oral preparations, such as: tablets (lozenges, sublingual tablets, enteric-coated tablets, chewable tablets, orodisintegrating tablets, effervescent tablets, dispersible tablets, sustained-release tablets, controlled-release tablets, etc.); capsules (enteric-coated capsules, soft capsules, sustained-release capsules, controlled-release capsules, etc.); granules (suspension granules, effervescent granules, enteric-coated granules, sustained-release granules and controlled-release granules, etc.); pills and sugar pills; powders; oral solutions (suspensions, emulsions); syrups, etc.

[0042] The technical solution of the present invention to solve the technical problem is: on the basis of the existing total saponin drugs of Gynostemma pentaphyllum, after scientific basic research on pharmacology and pharmacodynamics, a component with high content and strong activity, Gynostemma pentaphyllum saponin B, is screened out from 136 saponins of total saponins, and the mechanism of action of the pharmacology and pharmacodynamics of the compound is deeply studied. The results show that Gynostemma pentaphyllum saponin B is a natural selective peroxisome proliferator-activated receptor (PPAR)-α activator. The peroxisome proliferator-activated receptor PPAR-α is a nuclear hormone receptor, and its transcription level affects the function of fatty acids and their derivatives.

[0043] Gynostemma pentaphyllum saponin B can regulate the immune system in two directions, improve macrophage capacity, significantly increase the number of white blood cells, and at the same time increase the phagocytic function of white blood cells themselves, promote the secretion of interleukins in the body, and increase the production of serum immune proteins. The protection against neurotoxicity enables it to antagonize the toxic and side effects of chemotherapy and glucocorticoid drugs in tumor treatment.

[0044] The technical solution is to use modern extraction and purification technology to increase the purity of Gynostemma Saponin B to more than 90%, and all indicators meet the requirements and regulations of the first edition of the Chinese Pharmacopoeia.

[0045] In this embodiment, Gynostemma Pentaphyllum (Thunb) Makino. is a plant of the genus Gynostemma in the family Cucurbitaceae, and Gynostemma pentaphyllum saponin B is one of its main active ingredients. Studies have shown that Gynostemma pentaphyllum saponin B has pharmacological activities such as anti-tumor, lowering blood sugar and blood lipids, and enhancing immunity. A Gynostemma pentaphyllum saponin B compound with a dammarane structure is isolated and extracted from Gynostemma pentaphyllum, which is a natural peroxisome proliferator-activated receptor α (PPAR-α) agonist that can inhibit the activation of nuclear factor-κB. In vitro experiments have shown that Gynostemma pentaphyllum saponin B inhibits the induction of cytokines on the expression of vascular adhesion factor-1 (VCAM-1) in human endothelial cells through the PPAR-α pathway, and its activity is similar to that of Wy-14643. Enzyme-linked immunosorbent assay method was used to prove that Gynostemma pentaphyllum saponin B can inhibit the effect of TNF-α inducing the expression of VCAM-1 mRNA in human umbilical vein endothelial cells. Its effect of inhibiting LPS-induced THP-1 monocytes from expressing tissue factor is also through the PPAR-α pathway. PPAR has anti-proliferation, pre-apoptosis and differentiation-promoting functions, and therefore has a more comprehensive anti-cancer activity. PPAR is involved in the differentiation of pre-adipocytes into adipocytes and the differentiation of monocytes into macrophages. When PPAR and RXR ligands are present, bone marrow cells pre-differentiate into resting macrophages. When both are present continuously, PPAR can regress the differentiation of lipoma cells and trigger the differentiation of tumor cells into adipocytes.

[0046] The anti-tumor mechanism of Gynostemma pentaphyllum saponin B is mainly manifested in anti-mutagenicity, cell cycle interference, immune enhancement, induction of tumor cell apoptosis, inhibition of autophagy of cancer cells, and induction of cancer cell reversal. The mechanism of antagonizing the side effects of chemotherapy and drugs is to enhance immune function and protect various organs. This has been proven in a large number of pharmacological and clinical trial data.

[0047] 1. Anti-tumor mechanism of action

[0048] 1. Anti-mutagenic effect: Gynostemma pentaphyllum saponin B can inhibit the growth of tumor cells in vivo and in vitro by blocking the mutagenic effect of certain mutagens in vivo. Using cyclophosphamide as a mutagen and the frequency of sister chromosome exchange after mutagenic treatment as an indicator, it is shown that the compound can significantly reduce the increased frequency of sister chromosome exchange caused by cyclophosphamide, proving that it has an anti-mutagenic effect. When 3-MC was used as an inducer, the activity changes of hepatocyte microsomal protein P448 (EROD) in vivo and in vitro after treatment were detected. It was found that after 3-MC induction, P448 (EROD activity decreased significantly with the increase of dose, confirming that the inhibition and regulation of metabolic enzymes and then participating in and affecting all metabolic enzyme systems in the body is one of the important mechanisms for inducing anti-mutagenicity.

[0049] 2. Interference with the cell cycle Gynostemma pentaphyllum saponin B has an effect on the cell cycle distribution of human liver cancer HepG2 cells. It is confirmed that after Gynostemma pentaphyllum saponin B acts on HepG2 cells, the cells undergo G / G phase arrest, preventing tumor cells from entering blood vessels and lymphatic vessels after the in situ tumor is separated, thereby inhibiting the invasion and metastasis of tumor cells. Gynostemma pentaphyllum saponin B can significantly increase the number of lymphocytes and macrophages around and within the S180 sarcoma, and stimulate the proliferation of fibroblasts around the tumor, so that the spleen mass of tumor-bearing mice carrying S180 sarcoma cells is significantly increased, the number of splenic white pulp is significantly increased, and the spleen volume is enlarged. The periarterial lymphoid sheath, marginal zone and lymphoid nodules are all significantly enlarged. By activating immune cells, the body's immunity is enhanced and tumor growth is inhibited. HepG2 cells transition to the S phase, inhibiting DNA replication. Flow cytometric analysis revealed that gypenoside B could induce an increase in the proportion of S-phase cells and a decrease in the number of G / G and G / M-phase cells in mouse leukemia L1210. Treatment of human prostate cancer cells PC-3 with gypenoside B revealed that gypenoside B could regulate the expression of cell cycle proteins, leading to cell cycle arrest in the S and G / M phases.

[0050] 3. Enhance immune function: Studies have shown that oral administration of Gynostemma Saponin B can enhance the immune function of patients with primary lung cancer after combined radiotherapy and chemotherapy. It can also protect normal cells from free radical damage by reducing lipid peroxidation and increasing the activity of superoxide dismutase in plasma, thereby inhibiting the growth and invasion of tumor cells. When Gynostemma Saponin B was intraperitoneally injected into mice transplanted with Lewis lung cancer cells, the immune response function of the mice was significantly improved, the total number of spleen cells increased, and the biological activity of NK cells and spleen cells was enhanced.

[0051] Gynostemma pentaphyllum saponin B can increase the number of lymphocytes, improve the activity of natural killer cells and the production of serum hemolysin in mice, enhance the phagocytic function of peritoneal macrophages in mice, improve the ability of spleen cells to secrete antibodies, enhance the body's own killing of in situ tumor cells, and on the other hand, promptly remove tumor cells that enter blood vessels and lymphatic vessels after being separated from the in situ tumor, thereby inhibiting the invasion and metastasis of tumor cells. Through the detection of the changes in the total number of spleen lymphocytes and NK cell activity in Lewis lung cancer tumor-bearing mice after administration, it was confirmed that Gynostemma pentaphyllum saponin B can stimulate the increase in the number of spleen lymphocytes and the enhancement of NK cell activity in tumor-bearing mice, thereby killing tumor cells, and confirmed that the anti-tumor effect of Gynostemma pentaphyllum saponin B is related to its regulation of the body's immune function. The activity of Gynostemma pentaphyllum saponin B on the immune system is mainly reflected in the enhancement of nonspecific immunity, humoral immunity, cellular immunity, the effect on lymphocyte transformation and interleukin 2 (IL-2) secretion, and the increase of natural killer cells (NK). Gynostemma pentaphyllum saponin B can significantly enhance nonspecific immune function.

[0052] 2. Side effects of resistance to radiotherapy and chemotherapy

[0053] Various radiotherapy methods and chemotherapy drugs are toxic to actively growing tissues, and have varying degrees of bone marrow suppression and digestive tract reactions. Experiments have shown that Gynostemma pentaphyllum saponin B can effectively antagonize or reduce the side effects of radiotherapy and chemotherapy.

[0054] 1. Bidirectional regulation of the immune system

[0055] Gynostemma saponin B can enhance macrophage ability, significantly increase the number of white blood cells, while increasing the phagocytic function of white blood cells themselves, promoting the secretion of interleukins in the body, and increasing the production of serum immune proteins.

[0056] Gypenosin B 50mg / kg was administered to mice for 10 consecutive days, which significantly increased the spleen weight and significantly increased the body weight compared with the control group. It can significantly promote the clearance rate of colloidal carbon in the blood by the mononuclear macrophage system and improve the phagocytic function of mononuclear cells. The 30mg / kg dose can also increase the weight of the thymus. Gypenosin B 50mg / kg was administered to normal mice for 10 consecutive days, which had a biphasic regulatory effect on spleen weight, that is, it increased the spleen weight (mg / 10g body weight) less than the median, and reduced the spleen weight greater than the median; it had no significant effect on the thymus weight (mg / 10g body weight) less than the median, and it could also significantly reduce the thymus weight greater than the median. Gypenoside B 200mg / kg or 400mg / kg was administered to mice for 10 consecutive days, which could significantly counteract the weight loss of thymus and spleen caused by cyclophosphamide, and could reduce the significantly increased total white blood cell count and lymphocyte percentage to a level close to that of the control group; and increase the decreased neutrophil percentage to a level close to that of the control group; the thymus in the model group was atrophied and the spleen weight increased. After medication, the thymus index recovered and exceeded that of the control group, and the spleen index also returned to normal; the serum lysozyme content in the model group was significantly reduced, indicating that the body's anti-infection ability was reduced, and the lysozyme content in the medication group was significantly increased and exceeded that of the control group. Gavage of 60mg / kg or 100mg / kg of Gynostemma pentaphyllum saponin B to mice for 7 consecutive days can significantly increase the number of peripheral blood white blood cells, and enhance the chemiluminescence peak when phagocytosing yeast polysaccharide. At the same time, the luminescence index (peak luminescence / white blood cell count) is also significantly improved: gavage of GPs80mg / kg or 120mg / kg to mice with immunosuppression induced by prednisone acetate for 7 consecutive days can significantly increase the number of white blood cells, peak luminescence and luminescence index. Modern pharmacological studies have shown that Gynostemma pentaphyllum saponin B can not only increase the white blood cell count of normal animals, but also restore the white blood cell count of some animals with low white blood cell counts, and enhance the phagocytic function of normal or low-functioning white blood cells.

[0057] 2. Protection against neurotoxicity

[0058] The study of the protective effect and molecular mechanism of oxaliplatin-induced peripheral neurotoxicity in rats showed that gypenoside B can improve the behavioral changes of oxaliplatin-induced peripheral neurotoxicity in rats under temperature and mechanical stimulation, and its mechanism is related to the upregulation of NGF levels and Nrf2 signaling pathway.

[0059] Male SD rats were randomly divided into normal control group, model control group, low-dose, medium-dose and high-dose groups of gypenosin B. Except for the normal control group, all other groups were intraperitoneally injected with oxaliplatin 4 mg / kg. At the same time, the model control group was gavaged with 5% glucose as the vehicle, and the low-dose, medium-dose and high-dose gypenosin B were gavaged with 50, 100 and 200 mg / kg, respectively. The behavioral changes of rats under temperature and mechanical stimulation were regularly detected, and the rats were killed after 40 days of administration. ELISA was used to detect the content of NGF in plasma, and Western blot was used to detect the expression levels of Nrf2 and its downstream NQO-1 and HO-1 proteins in L4-5 dorsal root ganglia. Results Compared with the normal control group, the model control group showed obvious behavioral changes and decreased plasma NGF. Western blot detection showed that the expression levels of Nrf2 and its downstream NQO-1 and HO-1 proteins were significantly decreased. Administration of gypenosin B significantly improved the behavioral changes in rats, indicating that low, medium, and high doses of gypenosin B could upregulate the levels of Nrf2 and its downstream NQO-1 and HO-1.

[0060] 3. Reduce the side effects of glucocorticoid drugs

[0061] Long-term studies have shown that the combination of Gynostemma Saponin B and some hormone drugs with large toxic side effects not only does not affect the efficacy, but can antagonize the side effects. The experiment used hormone drugs represented by prednisone acetate (hereinafter referred to as prednisone) and Gynostemma Saponin B to study its anti-inflammatory, anti-shock, anti-toxic and immunosuppressive effects and antagonize the side effects of serious damage to the heart, liver, lungs and kidneys, and obtained satisfactory results.

[0062] 1. Anti-adverse reactions of prednisone

[0063] 1.1 Antagonistic effect of gypenoside B on prednisone-induced adrenal atrophy in mice

[0064] method:

[0065] ICR mice, 77 males, weighing 22.7±1.1 g, were randomly divided into six groups;

[0066] ①Blank control group: ②Gynostemma pentaphyllum saponin B 360mg / kg group; ③Prednisone 30mg / lkg group: ④Prednisone 7.5mg / kg+Gynostemma pentaphyllum saponin B 90mg / kg group: ⑤Prednisone 15mg / kg+Gynostemma pentaphyllum saponin B 180mg / kg group: ⑥Prednisone 30mg / kg+Gynostemma pentaphyllum saponin B 360mg / kg group. The animals were weighed and gavaged once a day for 9 consecutive days. On the 10th day, the animals were killed, the bilateral adrenal glands were removed, fixed with formalin solution for paraffin sections, and the changes in the tissue structure of each group were observed under a microscope. The experimental results are as follows:

[0067] 1. In the blank control group of 12 cases, the shape and arrangement of cells in the zona glomerulosa, zona fasciculata and zona reticularis of the cortical area were normal, without obvious lesions. The cells in the medullary area also showed no degeneration, necrosis or hemorrhage. There was no inflammatory cell infiltration and division phase in the entire suprarenal parotid tissue.

[0068] 2. Gynostemma pentaphyllum saponin B 360 mg / kg group: 12 cases, the results were basically the same as those of the blank control group.

[0069] 3. Prednisone 30 mg / kg group: 11 cases, the cells of the zona glomerulosa in the cortical area were basically normal, and the lesions of the zona fasciculata were obvious. Among them, 4 cases had vacuolar degeneration of cells, 5 cases had focal disorder, unclear cell boundaries, different nuclei of different sizes, dark staining, no degeneration and necrosis of the zona reticularis cells, 2 cases had hemorrhage, no obvious lesions in the medulla area, and no inflammatory cell infiltration and division phase were found in the entire adrenal tissue.

[0070] 4. Prednisone 7.5mg / kg + Gynostemma pentaphyllum saponin B 90mg / kg group, a total of 15 cases, the results were basically the same as the blank control group.

[0071] 5. In the prednisone 15mg / kg + gypenosylcera pentaphyllum saponin B 180mg / kg group, in 13 cases, the cells of the zona glomerulosa in the cortex were arranged regularly, the cells of the zona reticularis had no degeneration and necrosis, 3 cases of zona fasciculata cells had vacuolar degeneration, 3 cases of zona fasciculata focal degeneration, disordered arrangement, unclear cell boundaries, different nuclei sizes, dark staining, no obvious lesions in the medulla, and no inflammatory cell infiltration and mitosis were found in the entire adrenal tissue.

[0072] 6. Prednisone 30mg / kg + Gynostemma saponin B 360mg / kg group, a total of 14 cases, cortical area: zona reticularis cells were normal, zona fasciculata lesions were more obvious, 2 cases of cells had vacuolar degeneration, 6 cases had focal disorder, cell boundaries were unclear, nuclei were of different sizes, dark staining, zona reticularis cells had no degeneration and necrosis, 2 cases had hemorrhage, no obvious lesions in the medulla area, no inflammatory cell infiltration and division phase were found in the entire adrenal tissue.

[0073] Summary: Compared with the blank group, the Gynostemma Saponin B group was basically the same as the control group, the prednisone group had obvious lesions, the Gynostemma Saponin B + prednisone low-dose group was basically consistent with the blank control group, the Gynostemma Saponin B + prednisone medium-dose group had mild lesions, the Gynostemma Saponin B + prednisone high-dose group had obvious lesions, and compared with the Gynostemma Saponin B + prednisone group, the Gynostemma Saponin B + prednisone high-dose group had obvious lesions relative to the Gynostemma Saponin B group, and the Gynostemma Saponin B prednisone high-dose group had slightly less lesions relative to the prednisone group.

[0074] 1.2 Effects of prednisone on the weight of the adrenal glands, vitamin C and cholesterol content in mice

[0075] method:

[0076] ICR mice, male, weighing 22.13±1.83g, were randomly divided into six groups, namely: ① blank control group, which were gavaged with the same volume of tap water; ② Gynostemma saponin B 720mg / kg group; ③ Prednisone 60m group; ④ Gynostemma saponin B + prednisone low-dose group, prednisone 15mg / kg + Gynostemma saponin B 180mg / kg group; ⑤ Gynostemma saponin B + prednisone medium-dose group, prednisone 30mg / kg + Gynostemma 360mg / kg group; ⑤ Gynostemma saponin B + prednisone high-dose group, prednisone 60mg / kg + Gynostemma saponin B 720mg / kg group. The drug was administered once a day for 9 consecutive days. One hour after the last administration, the animals were weighed and killed. The adrenal glands on both sides were quickly removed and weighed for wet weight. The left adrenal gland was homogenized and the vitamin C content was determined by the 2.4-dinitrobenzene trap method. The right adrenal gland was extracted with ethanol and acetone and the cholesterol content was determined by the ferrous-sulfuric acid colorimetric method. The significance of the differences between the groups was determined by the t test.

[0077] The experimental results are as follows:

[0078] Compared with the control group, the weight gain, adrenal wet weight and index, vitamin C content and cholesterol content of the 720mg / kg group of gypenosan B were not significantly different; while the weight gain of the 60mg / kg prednisone group was significantly slowed down, the adrenal wet weight and index were significantly reduced, and the vitamin C content and cholesterol content were significantly increased: the weight gain of mice in the three dose groups of gypenosan B + prednisone was also slowed down, but the degree was less than that of simple prednisone, the adrenal index did not change significantly, the vitamin C content did not increase significantly, and the cholesterol content was significantly increased, but lower than that of the simple prednisone group. Compared with the 60mg / kg prednisone group of the same dose, the adrenal wet weight of the high-dose gypenosan B prednisone group was significantly increased, and the vitamin C and cholesterol contents were significantly reduced.

[0079] Summary: The above results indicate that Gynostemma pentaphyllum saponin B can effectively counteract prednisone-induced adrenal atrophy in mice.

[0080] 1.3 Effects on rat liver glycogen, blood glucose, serum total protein, lipase, serum potassium, sodium, and calcium

[0081] method:

[0082] 68 SD rats, half male and half female, weighing 186.2±26.7g, were randomly divided into 6 groups, namely: ① blank control group; ② gypenosan B 240mg / kg group; ③ prednisone 20mg / kg group; ④ gypenosan B + prednisone low-dose group (gypenosan B 60mg / kg + prednisone 5mg / kg); ⑤ gypenosan B + prednisone medium-dose group (gypenosan B 120mg / kg + prednisone 10mg / kg); ⑥ gypenosan B + prednisone high-dose group (gypenosan B 240mg / kg + prednisone 20mg / kg). The drug was administered once a day for 9 consecutive days. One hour after administration on the 9th day, blood and liver were collected under anesthesia. Blood sugar was measured by o-toluene plate method, liver glycogen was measured by Hui Han method, blood pool-total protein was measured by double shrinkage method, serum lipase was measured by turbidimetry, serum Na+ was measured by pyrophoric acid turbidimetry, serum Ca2+ was measured by methyl vanillin blue colorimetry, and serum K+ content was measured by flame photometer method. The significance of the difference between the groups was determined according to the t test results.

[0083] Summary: The experimental results show that compared with the blank control group, except for the significant increase in blood sugar and serum sodium in the prednisone 20mg / kg group, there were no significant changes in blood sugar and serum Na+ in the other experimental groups: there were no significant changes in glycogen, serum total protein, lipase, K+ and Ca+2 in the experimental group. In addition, the serum sodium in the 20mg / kg prednisone group was significantly higher than that in the gypenoside B + prednisone high-dose group, indicating that gypenoside B has the effect of counteracting the increase in serum sodium caused by the high-dose use of prednisone.

[0084] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A use of gypenoside B in the preparation of a drug for antagonizing the side effects of prednisone, characterized in that: The side effect of the prednisone drug is adrenal atrophy; The structural formula of the gypenoside B is: The preparation of gypenoside B comprises the following steps: S1. Take 15kg-30kg of total saponins of Gynostemma pentaphyllum, dissolve it in methanol, mix it with 30kg-50kg of silica gel, and evaporate the solvent; S2, add 90kg-120kg dry silica gel chromatography column, elute with chloroform-methanol gradient, check with thin layer chromatography, collect and combine the eluate containing gypenoside B, and recover the solvent to obtain 5kg gypenoside B crude product; S3, taking 90g-110g of crude gypenosan B, preparing a 200mg / ml solution with methanol, purifying it by medium-pressure preparative liquid chromatography, collecting gypenosan B components, and concentrating to obtain gypenosan B with a content of 50% to 90%; S4, dissolving 50% to 90% of gypenoside B in a mixed organic solvent, and repeatedly recrystallizing, the purity can reach more than 96%; Among them, light and high temperature must be avoided during the entire preparation and purification process, and freeze-drying must be used.

2. The use according to claim 1, characterized in that In the S2, the gradient elution was performed with chloroform-methanol 8:1→3:1.

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