An antiviral drug composition containing total saponins of Ganoderma lucidum and preparation method thereof

Abstract

The present invention belongs to the field of medical technology, and provides an antiviral pharmaceutical composition containing total saponins of snow ganoderma and a preparation method thereof. The pharmaceutical composition of the present invention is prepared by using total saponins of snow ganoderma and limonin as raw materials according to a certain weight percentage. Pharmacological experiments have shown that the pharmaceutical composition of the present invention improves the lung index and lung virus load of mice infected with influenza A virus, and increases the relative expression of SP‑A and SP‑D mRNA, showing significant synergistic effects, and has no toxic side effects, and has good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to an antiviral pharmaceutical composition containing total saponins of snow ganoderma and a preparation method thereof. Background Art

[0002] Ganoderma lucidum, also known as grass Ganoderma lucidum, is a wild plant in the plateau. The whole plant is used as medicine. It is sweet, bitter and cold in nature. It enters the liver, gallbladder and kidney meridians. The pharmacological effects of Ganoderma lucidum include anti-hypoxia, cardiovascular, anti-inflammatory, anti-tumor, antipyretic, analgesic, antibacterial, digestive and respiratory systems and hypoxia tolerance. Ganoderma lucidum contains a variety of chemical components such as alkaloids, saponins, coumarins, polysaccharides, flavonoids and 18 kinds of amino acids. Dawa Yangjin et al. studied the effect of total saponins of Ganoderma lucidum on gastrointestinal smooth muscle (Dawa Yangjin, Li Qiaoyun, Li Xia, et al. Effects of total saponins of Ganoderma lucidum on gastrointestinal smooth muscle [J]. Chinese Ethnomedicine, 2011, 20(13): 48-49). Dazhen et al. conducted an experimental study on the antioxidant effect of total saponins from Ganoderma lucidum (Dazhen, Li Qiaoyun, Xie Yunfei. Experimental study on the antioxidant effect of total saponins from Ganoderma lucidum [J]. Traditional Chinese Medicine and Clinic, 2013, 4(03): 26-29).

[0003] Limonin is a triterpenoid compound found in many plants of the Rutaceae and Meliaceae families. It is also one of the main components of traditional Chinese medicines such as Dictamni Cortex and Phellodendron Amurense. It has biological activities such as anti-tumor, anti-inflammatory, analgesic, neuroprotective, antibacterial, antiviral, antioxidant and liver protection.

[0004] There is no related report in the prior art on the antiviral effect of the pharmaceutical composition containing total saponins of Snow Ganoderma lucidum claimed in the present invention. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art, to develop and utilize drugs using modern drug research methods, to provide an antiviral drug composition containing total saponins of Snow Ganoderma with synergistic effects, and to provide a new antiviral drug composition for clinical use.

[0006] In the first aspect, the present invention provides an antiviral pharmaceutical composition containing total saponins of Rhodiola rosea, wherein the pharmaceutical composition consists of total saponins of Rhodiola rosea and limonin, and the weight ratio of the total saponins of Rhodiola rosea to limonin is 3-5:1.

[0007] Furthermore, the weight ratio of total saponins of Ganoderma lucidum to limonin in the pharmaceutical composition is 4:1.

[0008] Furthermore, the preparation method of the total saponins of Ganoderma lucidum is as follows: The powder of Ganoderma lucidum was extracted three times with six times the amount of 95% ethanol, the extracts were combined, concentrated and dried to obtain the ethanol extract of Ganoderma lucidum; the ethanol was evaporated from the residue, extracted three times with six times the amount of distilled water, the extracts were combined, concentrated and dried to obtain the water extract of Ganoderma lucidum; the water extract of Ganoderma lucidum was applied to a D101 resin column, rinsed with distilled water until the eluent was colorless, then eluted with 50% ethanol, the ethanol eluent was collected, concentrated and dried to obtain total saponins.

[0009] In a second aspect, the present invention provides a method for preparing the antiviral pharmaceutical composition containing total saponins of Snow Ganoderma lucidum described in the first aspect, comprising mixing the two active ingredients described in the first aspect.

[0010] In a third aspect, the present invention provides an antiviral pharmaceutical preparation containing total saponins of Snow Ganoderma lucidum, wherein the pharmaceutical preparation is prepared by mixing the pharmaceutical composition described in the first aspect with pharmaceutically acceptable excipients.

[0011] Furthermore, the pharmaceutical preparation is an oral preparation or an injectable preparation.

[0012] Furthermore, the oral preparation is a powder, tablet, pill, capsule, granule or oral liquid.

[0013] Furthermore, the oral preparation is a tablet.

[0014] In a fourth aspect, the present invention provides use of the antiviral pharmaceutical composition containing total saponins of Rhodiola rosea described in the first aspect or the antiviral pharmaceutical preparation containing total saponins of Rhodiola rosea described in the third aspect in the preparation of antiviral drugs.

[0015] Furthermore, the present invention also provides the use of the antiviral pharmaceutical composition containing total saponins of Rhizoma Ceratitis as described in the first aspect or the antiviral pharmaceutical preparation containing total saponins of Rhizoma Ceratitis as described in the third aspect in the preparation of anti-influenza A virus drugs.

[0016] Furthermore, the present invention also provides the use of the antiviral pharmaceutical composition containing total saponins of Rhizoma Ceratitis as described in the first aspect or the antiviral pharmaceutical preparation containing total saponins of Rhizoma Ceratitis as described in the third aspect in the preparation of anti-respiratory syncytial virus drugs.

[0017] Animal experiments show that the pharmaceutical composition of the present invention effectively improves the lung index and lung viral load of mice infected with influenza A virus, increases the relative expression of SP-A and SP-D mRNA, inhibits viral replication, and shows significant antiviral ability. The pharmaceutical composition of the present invention effectively improves the lung index and lung viral load of mice infected with respiratory syncytial virus, inhibits viral replication, and shows significant antiviral ability.

[0018] Compared with the prior art, the present invention has the following technical effects: The present invention discovers for the first time that the pharmaceutical composition composed of total saponins of Ganoderma lucidum and limonin has a significant synergistic effect in antiviral aspects, and provides a new antiviral pharmaceutical composition.

[0019] Pharmacological experiments have shown that the pharmaceutical composition of the present invention improves the lung index and lung virus load of mice infected with influenza A virus, and increases the relative expression of SP-A and SP-D mRNA, showing significant synergistic effects, has no toxic side effects, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Comparison of lung index among mice groups Compared with the model control group, * P<0.05, ** P<0.01; compared with the combination group, # P<0.05, ## P<0.01.

[0021] Figure 2 Comparison of viral load in lung tissue of mice in each group Compared with the model control group, * P<0.05, ** P<0.01; compared with the combination group, # P<0.05, ## P<0.01.

[0022] Figure 3 Comparison of relative expression of SP-A mRNA in the lungs of mice in each group Compared with the model control group, * P<0.05, ** P<0.01; compared with the combination group, # P<0.05, ## P<0.01.

[0023] Figure 4 Comparison of relative expression of SP-D mRNA in the lungs of mice in each group Compared with the model control group, * P<0.05, ** P<0.01; compared with the combination group, # P<0.05, ## P<0.01.

[0024] Figure 5 Comparison of lung index among mice groups Compared with the model control group, * P<0.05, **P<0.01; compared with the combination group, # P<0.05.

[0025] Figure 6 Comparison of viral load in lung tissue of mice in each group Compared with the model control group, * P<0.05, ** P<0.01; compared with the combination group, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0026] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0027] In the following examples, the total saponins of Ganoderma lucidum used were prepared by the following method.

[0028] The powder of Ganoderma lucidum was extracted three times with six times the amount of 95% ethanol, the extracts were combined, concentrated and dried to obtain the ethanol extract of Ganoderma lucidum; the ethanol was evaporated from the residue, extracted three times with six times the amount of distilled water, the extracts were combined, concentrated and dried to obtain the water extract of Ganoderma lucidum; the water extract of Ganoderma lucidum was applied to a D101 resin column, rinsed with distilled water until the eluent was colorless, then eluted with 50% ethanol, the ethanol eluent was collected, concentrated and dried to obtain total saponins.

[0029] Example 1 Study on the antiviral effect of the pharmaceutical composition of the present invention on mice infected with influenza A virus 1. Experimental Methods 50 SPF-grade ICR mice, half male and half female, weighing 18-20 g, were fed with free access to water and food, and the room temperature was maintained at 20-25°C.

[0030] The mice were randomly divided into a normal control group, a model control group, a total saponin group of snow ganoderma, a limonin group, and a combination group, each group containing 10 mice, half male and half female. The mice were lightly anesthetized with isoflurane. Except for the normal control group, the other groups were infected with 15 LD50 H1N1 / PR8 influenza virus liquid by nasal drops. The normal control group used normal saline, and each mouse was given 35μL nasal drops. Starting on the day of infection, mice in each drug group were given drug treatment by gavage. The total saponin group of snow ganoderma was gavaged with 100mg / kg of total saponin of snow ganoderma each time, the limonin group was gavaged with 25.0mg / kg of limonin each time, and the combination group was gavaged with 50mg / kg of total saponin of snow ganoderma + 12.5mg / kg of limonin each time, once a day, for 4 days. The normal control group and the model control group were given 0.5% CMCNa.

[0031] 2. Index determination 2.1 Determination of lung index On the 5th day after infection, the mice were weighed, dissected, and their lungs were removed and weighed to calculate the lung index of the mice.

[0032] Lung index (%) = lung wet weight (g) × 100 / body weight (g) × 100%.

[0033] 2.2 Determination of lung viral load RT-PCR was used to detect the lung viral load and the relative gene expression of SP-A and SP-D in the lung.

[0034] 3. Statistical methods Graphpad Prism 9.0 software was used for data processing, and the measured data were expressed as mean ± standard deviation. The comparison among multiple groups was performed by one-way analysis of variance, and the comparison among groups was performed by Pairwise comparisons were performed, and P < 0.05 was considered statistically significant.

[0035] 4. Experimental Results 4.1 Lung index of mice in each group Compared with the normal control group, the lung index of mice in the model control group was significantly increased. Compared with the model control group, the combination group significantly reduced the lung index of virus-infected mice, and its effect of reducing the lung index of virus-infected mice was significantly better than the group using total saponins of snow ganoderma alone and the group using limonin alone.

[0036] The pharmaceutical composition of the present invention can significantly reduce the lung index of virus-infected mice. The two active ingredients, total saponins of snow ganoderma and limonin, show significant synergistic effects in reducing the lung index. Figure 1 .

[0037] 4.2 Virus load in lung tissue of mice in each group Compared with the normal control group, the viral load of the lung tissue of mice in the model control group was significantly increased. Compared with the model control group, the combination group significantly reduced the viral load of virus-infected mice, and its effect of reducing the viral load of virus-infected mice was significantly better than the group using total saponins of snow ganoderma alone and the group using limonin alone.

[0038] The pharmaceutical composition of the present invention can significantly reduce the viral load of virus-infected mice, and the two active ingredients of total saponins of snow ganoderma and limonin show significant synergistic effects in reducing viral load. Figure 2 .

[0039] 4.3 Results of SP-A and SP-D tests in lungs Compared with the normal control group, the relative expression of SP-A and SP-D mRNA in the lung tissue of mice in the model control group was significantly reduced. Compared with the model control group, the combination group significantly increased the relative expression of SP-A and SP-D mRNA in virus-infected mice, and its effect of increasing the relative expression of SP-A and SP-D mRNA in virus-infected mice was significantly better than the total saponins of snow ganoderma alone group and the limonin alone group.

[0040] The pharmaceutical composition of the present invention can significantly increase the relative expression of SP-A and SP-D mRNA in virus-infected mice. The two active ingredients, total saponins of snow ganoderma and limonin, show significant synergistic effects in increasing the relative expression of SP-A and SP-D mRNA in virus-infected mice. Figure 3 , 4 .

[0041] When mice are infected with influenza virus through the nose, the virus stimulates alveolar epithelial cells, triggers the body's immune response, forms an inflammatory factor storm, leads to pulmonary capillary leakage and inflammatory cell infiltration, pulmonary edema, alveolar wall thickening and even collapse, forms lung consolidation, and significantly increases lung weight. During influenza virus infection, the levels of SP-A and SP-D decrease, which may seriously aggravate the invasion of the virus and may also affect the host cytokine pattern induced by the virus.

[0042] Compared with the normal control group, the lung index and viral load of the mice in the experimental model control group were significantly increased, and the relative expression of SP-A and SP-D mRNA was significantly reduced. After the influenza virus-infected mice were treated with the pharmaceutical composition of the present invention, the lung index and lung viral load were significantly reduced, and the relative expression of SP-A and SP-D mRNA was increased. The pharmaceutical composition of the present invention inhibited the replication of the virus and showed significant antiviral ability.

[0043] Example 2 Study on the antiviral effect of the pharmaceutical composition of the present invention on mice infected with respiratory syncytial virus 1. Experimental Methods 50 SPF-grade ICR mice, half male and half female, weighing 18-20 g, were fed with free access to water and food, and the room temperature was maintained at 20-25°C.

[0044] The mice were randomly divided into a normal control group, a model control group, a total saponin group of Ganoderma lucidum, a limonin group, and a combination group. Each group contained 10 mice, half of which were male and half were female. The mice were lightly anesthetized with isoflurane. Except for the normal control group, the other groups were nasally dripped with 10 TICD 50RSV-long virus droplet infection (100L / mouse), the normal control group mice were given an equal volume of normal saline. Starting from the day of infection, each group of mice was given drug treatment by gavage, the snow ganoderma total saponin group was gavaged with 100mg / kg snow ganoderma total saponin each time, the limonin group was gavaged with 25.0mg / kg limonin each time, and the combination group was gavaged with 50mg / kg snow ganoderma total saponin + 12.5mg / kg limonin each time, once a day, for 4 days, and the normal control group and the model control group were given 0.5% CMCNa.

[0045] 2. Index determination 2.1 Determination of lung index On the 5th day after infection, the mice were weighed, dissected, and their lungs were removed and weighed to calculate the lung index of the mice.

[0046] Lung index (%) = lung wet weight (g) × 100 / body weight (g) × 100%.

[0047] 2.2 Determination of lung viral load Lung viral load was detected using RT-PCR.

[0048] 3. Statistical methods Graphpad Prism 9.0 software was used for data processing, and the measured data were expressed as mean ± standard deviation. The comparison among multiple groups was performed by one-way analysis of variance, and the comparison among groups was performed by Pairwise comparisons were performed, and P < 0.05 was considered statistically significant.

[0049] 4. Experimental Results 4.1 Lung index of mice in each group Compared with the normal control group, the lung index of mice in the model control group was significantly increased. Compared with the model control group, the combination group significantly reduced the lung index of virus-infected mice, and its effect of reducing the lung index of virus-infected mice was significantly better than the group using total saponins of snow ganoderma alone and the group using limonin alone.

[0050] The pharmaceutical composition of the present invention can significantly reduce the lung index of mice infected with respiratory syncytial virus. The two active ingredients of total saponins of snow ganoderma and limonin show significant synergistic effect in reducing the lung index. Figure 5 .

[0051] 4.2 Virus load in lung tissue of mice in each group The normal control group rats did not show the expression of viral load in lung tissue when infected. Compared with the model control group, the combination group significantly reduced the viral load of virus-infected mice, and its effect of reducing the viral load of virus-infected mice was significantly better than the group using total saponins of snow ganoderma alone and the group using limonin alone.

[0052] The pharmaceutical composition of the present invention can significantly reduce the viral load of mice infected with respiratory syncytial virus, and the two active ingredients of total saponins of snow ganoderma and limonin show significant synergistic effects in reducing the viral load. Figure 6 .

[0053] Compared with the normal control group, the lung index and viral load of mice in the experimental model control group increased significantly. After the mice infected with respiratory syncytial virus were treated with the pharmaceutical composition of the present invention, the lung index and lung viral load were significantly reduced. The pharmaceutical composition of the present invention inhibited the replication of the virus and showed significant antiviral ability.

[0054] The above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description, and the obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. An antiviral pharmaceutical composition containing total saponins of Ganoderma lucidum, characterized in that: The pharmaceutical composition consists of total saponins of Ganoderma lucidum and limonin, and the weight ratio of the total saponins of Ganoderma lucidum to limonin is 3-5:

1.

2. The antiviral pharmaceutical composition containing total saponins of Ganoderma lucidum according to claim 1, characterized in that: The weight ratio of total saponins of snow ganoderma to limonin in the pharmaceutical composition is 4:

1.

3. The antiviral pharmaceutical composition containing total saponins of Ganoderma lucidum according to claim 1, characterized in that: The preparation method of the total saponin of Ganoderma lucidum is as follows: extract the Ganoderma lucidum powder three times with six times the amount of 95% ethanol, combine the extracts, concentrate and dry to obtain the Ganoderma lucidum ethanol extract; evaporate the ethanol from the residue, extract three times with six times the amount of distilled water, combine the extracts, concentrate and dry to obtain the Ganoderma lucidum water extract; apply the Ganoderma lucidum water extract to a D101 resin column, rinse with distilled water until the eluent is colorless, then elute with 50% ethanol, collect the ethanol eluent, and concentrate and dry to obtain the total saponin.

4. An antiviral pharmaceutical preparation containing total saponins of Ganoderma lucidum, characterized in that: The pharmaceutical preparation is prepared by mixing the pharmaceutical composition according to any one of claims 1 or 2 with pharmaceutically acceptable excipients.

5. The antiviral pharmaceutical preparation containing total saponins of Ganoderma lucidum according to claim 4, characterized in that: The pharmaceutical preparation is an oral preparation or an injection preparation.

6. The antiviral pharmaceutical preparation containing total saponins of Ganoderma lucidum according to claim 5, characterized in that: The oral preparation is powder, tablet, pill, capsule, granule or oral liquid.

7. The antiviral pharmaceutical preparation containing total saponins of Ganoderma lucidum according to claim 6, characterized in that: The oral preparation is a tablet.

8. Use of the antiviral pharmaceutical composition containing total saponins of Rhizoma Sativae according to any one of claims 1 or 2 or the antiviral pharmaceutical preparation containing total saponins of Rhizoma Sativae according to claim 4 in the preparation of antiviral drugs.

9. Use of the antiviral pharmaceutical composition containing total saponins of Rhizoma Ceratitis according to any one of claims 1 or 2 or the antiviral pharmaceutical preparation containing total saponins of Rhizoma Ceratitis according to claim 4 in the preparation of anti-influenza A virus drugs.

10. Use of the antiviral pharmaceutical composition containing total saponins of Rhizoma Sativae according to any one of claims 1 or 2 or the antiviral pharmaceutical preparation containing total saponins of Rhizoma Sativae according to claim 4 in the preparation of anti-respiratory syncytial virus drugs.

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