Application of Astragalus Polysaccharide in Enhancing Curcumin's Anti-enterovirus 71 Type

Abstract

The application discloses a new medical application of astragaloside, in particular, a new medical application of astragaloside in enhancing the anti-enterovirus 71 type of curcumin. Researches show that EV71 infection causes the activity of human normal GES-1 cells to decrease, and the use of curcumin in the infected cells needs to reach a certain concentration to show obvious antiviral effect, and the use of astragaloside and curcumin in combination can show better antiviral effect under the use of lower concentration of curcumin, and the role of astragaloside in enhancing the anti-EV71 of curcumin is determined, and a foundation is laid for the combination of astragaloside and curcumin in preventing and treating hand-foot-mouth disease caused by EV71 infection.

Description

TECHNICAL FIELD

[0001] The present application relates to a new use of astragaloside in medicine, in particular to a new use of astragaloside for treating enterovirus 71 infection. BACKGROUND

[0002] Hand-foot-mouth disease is mainly caused by coxsackievirus A16 (CA16) and enterovirus 71 (EV71) infection, and severe patients are mostly caused by EV71 infection, which progresses rapidly and can show symptoms such as meningitis, encephalitis, myelitis, pulmonary edema, and circulatory disorders. The most dangerous is brainstem encephalitis, and a few cases are critically ill and can cause death, and surviving cases can have sequelae.

[0003] The vaccine for preventing hand-foot-mouth disease caused by EV71 infection was officially launched in the first half of 2016, and it is currently the only vaccine that can be used to prevent hand-foot-mouth disease. Vaccination of hand-foot-mouth disease vaccine can significantly reduce the prevalence of hand-foot-mouth disease, but the vaccination rate of the vaccine in China is not high. As of the end of 2021, the average cumulative vaccination rate nationwide was only 32% (calculated based on the 2014 birth population cohort), which is not enough to form a group immunity barrier. Hand-foot-mouth disease vaccination also has regional imbalances, with high vaccination rates in the eastern and developed regions and low vaccination rates in the western regions. Moreover, EV71 has genotype variation, and it is not clear whether the existing vaccine can provide protection.

[0004] There is currently no specific drug for treating hand-foot-mouth disease, and the main treatment is symptomatic treatment with interferon (IFN) and ribavirin (RBV). Therefore, finding an effective and specific anti-EV71 drug is a problem that we need to solve urgently.

[0005] Curcumin (Cur) is a yellow pigment extracted from the rhizomes of plants in the ginger family, such as turmeric. Existing studies have reported that it has the effects of inhibiting inflammatory response, antioxidant, and anti-rheumatoid.

[0006] Huang Hsing-I et al. found that Cur can play an anti-EV71 role in human colon cancer (HT29) cells at a concentration of 20 μM (Huang HI, Chio CC, Lin JY. Inhibition of EV71 by curcumin in intestinal epithelial cells. PLoS One. 2018 Jan 25; 13(1): e0191617.).

[0007] Similarly, 20 μM of Cur can also exert an anti-EV71 effect in Vero cells (Qin Y, Lin L, Chen Y, Wu S, Si X, Wu H, Zhai X, Wang Y, Tong L, Pan B, Zhong X, Wang T, Zhao W, Zhong Z. Curcumin inhibits the replication of enterovirus 71 in vitro. Acta Pharm Sin B. 2014 Aug; 4(4): 284-94.).

[0008] After EV71 infects human neuroblastoma (SH-SY5Y) cells, 20 µM of Cur can only exert a slight anti-EV71 effect, but 20 µM of Cur combined with 250 IU / mL of IFN-α can significantly inhibit the replication of EV71 (Wang Y, Dan K, Xue X, Chen B, Chen C. Curcumin assists anti-EV71 activity of IFN-α by inhibiting IFNAR1 reduction in SH-SY5Y cells. Gut Pathog. 2022 Feb 12; 14(1): 8.).

[0009] The above documents disclose that Cur can exert an anti-EV71 effect in different cells infected with EV71, but it is obvious that the drug dose of Cur is large. SUMMARY

[0010] The purpose of the present application is to overcome the shortcomings of the prior art and provide an application of astragaloside for enhancing the anti-enterovirus 71 effect of curcumin.

[0011] Astragaloside IV (AST-IV) is the quality standard of astragalus injection and the effective active ingredient of astragalus, and has the effects of anti-inflammatory, immune enhancement, and antioxidant.

[0012] The present application has found that astragaloside can enhance the anti-EV71 effect of curcumin, and when astragaloside and curcumin are used in combination, only a small dose of curcumin is needed to achieve its anti-EV71 effect.

[0013] Therefore, the present application first provides the application of astragaloside in the preparation of a drug for treating hand-foot-mouth disease.

[0014] Further, the application also provides application of the astragaloside IV in the preparation of a medicine for treating EV71 infection.

[0015] Further, the application also specifically provides application of the astragaloside IV in the preparation of a medicine for treating hand-foot-mouth disease caused by EV71 infection.

[0016] More specifically, based on the phenomenon that the astragaloside IV can enhance the protective effect of curcumin on cytotoxicity caused by EV71 infection discovered by the application, the application provides application of the astragaloside IV in the preparation of a medicine for treating hand-foot-mouth disease caused by EV71 infection in cooperation with curcumin.

[0017] In particular, the application also further provides application of a medicine composition of the astragaloside IV and curcumin in the preparation of a medicine for treating hand-foot-mouth disease caused by EV71 infection.

[0018] In the medicine composition, the dose molar ratio of the astragaloside IV to the curcumin is 1:2-2.5.

[0019] More preferably, in the medicine composition, the dose molar ratio of the astragaloside IV to the curcumin is 1:2-2.3.

[0020] Further, the application also provides a medicine for treating hand-foot-mouth disease caused by EV71 infection, which contains the medicine active ingredient astragaloside IV and curcumin or a pharmaceutically acceptable salt thereof, and further contains a pharmaceutically acceptable carrier or excipient.

[0021] In the medicine for treating hand-foot-mouth disease caused by EV71 infection, the medicine active ingredient astragaloside IV is specifically used to enhance the anti-EV71 effect of another medicine active ingredient curcumin, so as to effectively reduce the use dose of curcumin.

[0022] The application adopts the CCK-8 method to detect the protective effect of 3.5 μM AST-IV combined with 7.5 µM Cur on cytotoxicity caused by EV71 infection, further adopts the Western blot method and the TCID 50 Method to detect the anti-EV71 effect of 3.5 μM AST-IV combined with 7.5 µM Cur, and it is proved by the cell experiment that, when 7.5 µM Cur and 3.5 μM AST-IV are used to intervene in the GES-1 cells infected by EV71, a better anti-EV71 effect can be achieved to protect the GES-1 cells from the damage effect of cytotoxicity caused by EV71. When Cur is used alone, 7.5 µM Cur has no anti-EV71 effect, and only Cur greater than or equal to 20 µM can reflect the anti-EV71 effect.

[0023] Therefore, the AST-IV combined with Cur has low drug dosage, good pharmacological effect and low toxicity, indicating good medicinal prospects. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a cell toxicity detection result after AST-IV and Cur intervene GES-1 cells.

[0025] Figure 2 is an influence of different concentrations of Cur intervention on proliferation activity of EV71 infected GES-1 cells.

[0026] Figure 3 is a morphological change of EV71 infected GES-1 cells after different concentrations of Cur intervention.

[0027] Figure 4 is a Western blot detection of VP1 protein level expression in cells after different concentrations of Cur intervention.

[0028] Figure 5 is a TCID 50 detection of virus titer in supernatant of cells after different concentrations of Cur intervention.

[0029] Figure 6 is an influence of Cur and AST-IV co-intervention on morphological change of EV71 infected GES-1 cells.

[0030] Figure 7 is an influence of Cur and AST-IV co-intervention on proliferation activity of EV71 infected GES-1 cells.

[0031] Figure 8 is a Western blot detection of VP1 protein level expression in cells after Cur and AST-IV co-intervention.

[0032] Figure 9 is a TCID 50 detection of virus titer in supernatant of cells after Cur and AST-IV co-intervention.

[0033] In the figure: compared with the control group, p <0.0001; compared with the EV71 infection group, p <0.01, p <0.001. EMBODIMENT

[0034] The specific embodiments of the present application are described in further detail below in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present application, so that those skilled in the art can well understand and utilize the present application, and are not intended to limit the protection scope of the present application.

[0035] The production process, experimental method or detection method involved in the embodiments of the present application, if not specifically stated, are all conventional methods in the prior art, and their names and / or abbreviations belong to conventional names in the art, which are very clear and explicit in the related application field. Those skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the conventional conditions or the conditions recommended by the manufacturer.

[0036] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application do not have special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art.

[0037] Example 1: Toxicity detection of AST-IV and Cur intervention on GES-1 cells.

[0038] Logarithmic growth period GES-1 cells were taken and inoculated on 96-well plates, and GES-1 cells were intervened with AST-IV at final concentrations of 3.5 µM, 7 µM, 10.5 µM and 14 µM and Cur at final concentrations of 7.5 µM, 10 µM, 15 µM, 20 µM and 25 µM for 24 h as different experimental groups, and un-intervened GES-1 cells were used as a control group, and pure culture medium was used as a blank group.

[0039] 2 h before the end of the culture, 20 µL of CCK8 reagent was added to each well, and the absorbance A value of each well of cells was determined at 450 nm with a microplate reader after the end of the culture. The proliferation activity of cells was detected by CCK-8 method.

[0040] Cell survival rate / %=(experimental group A value-blank group A value) / (control group A value-blank group A value)×100%.

[0041] The results are shown in Figure 1 a) AST-IV at concentrations of 3.5 µM, 7 µM, 10.5 µM and 14 µM had no effect on the proliferation activity of GES-1 cells; b) Cur at concentrations of 7.5 µM, 10 µM, 15 µM, 20 µM and 25 µM also had no effect on the proliferation activity of GES-1 cells.

[0042] Example 2: Protective effect of Cur on EV71-infected GES-1 cells.

[0043] 1×104 GES-1 cells were seeded in 96-well plates, and then 0 µM, 7.5 µM, 10 µM, 15 µM, 20 µM and 25 µM of Cur were added respectively for 2 h, followed by EV71 infection for 24 h to form the EV71 infection group, the EV71+7.5 µM Cur group, the EV71+10 µM Cur group, the EV71+15 µM Cur group, the EV71+20 µM Cur group, and the EV71+25 µM Cur group, a total of 6 experimental groups.

[0044] Meanwhile, GES-1 cells without EV71 infection and Cur intervention were used as the control group, and pure culture medium was used as the blank group.

[0045] Two hours before the end of EV71 infection, 20 μL of CCK8 reagent was added to each group, and the absorbance A value was measured at 450 nm using an enzyme marker until the end of culture. The cell survival rate was calculated to detect the cell viability.

[0046] The detection results are shown in Table 1. Figure 2 Compared with the control group, the viability of EV71-infected cells was significantly reduced. Compared with the EV71 infection group, the cell viability did not increase significantly after 7.5 µM, 10 µM and 15 µM of Cur were used to intervene EV71-infected cells, while the cell viability increased significantly after 20 µM and 25 µM of Cur were used to intervene EV71-infected cells.

[0047] The above results show that only Cur with a concentration of 20 µM or more has a significant protective effect on the cytotoxicity caused by EV71 infection.

[0048] Furthermore, 1×10 6 GES-1 cells in the logarithmic growth phase were seeded in 6-well plates, and then divided into the control group, the EV71 infection group, the EV71+7.5 µM Cur group, the EV71+10 µM Cur group, the EV71+15 µM Cur group, the EV71+20 µM Cur group and the EV71+25 µM Cur group according to the same treatment method described above. After 2 h of intervention with Cur at different concentrations, EV71 infection was performed for 24 h, and the morphological changes of GES-1 cells in different groups were observed under an inverted microscope and photographed.

[0049] Figure 3Compared with the control group, the number of GES-1 cells infected by EV71 was significantly reduced, the cells were shrunk, and the number of suspended cells was significantly increased. Compared with the EV71 infection group, after the intervention of 7.5 µM, 10 µM, and 15 µM Cur on the EV71 infected cells, the cell morphology did not change significantly, but after the intervention of 20 µM and 25 µM Cur on the EV71 infected cells, the number of cells was significantly increased, and the cell morphology was restored. The above results also showed that 20 µM and 25 µM Cur had a significant protective effect on the cell damage caused by EV71 infection.

[0050] After the morphological observation of the above-mentioned cells in each group except the control group, the cells were collected in a 1.5 mL EP tube, 60 µL of cell lysis solution was added to each tube to extract protein, VP1 protein was separated by 12% SDS-PAGE gel, and the level of VP1 protein in the cells was detected by Western blot.

[0051] The results are shown in Table 2. Figure 4 Compared with the EV71 infection group, after the intervention of 7.5 µM, 10 µM, and 15 µM Cur on the EV71 infected cells, the level of structural protein VP1 in the cells did not change significantly, but after the intervention of 20 µM and 25 µM Cur on the EV71 infected cells, the level of VP1 protein in the cells was significantly reduced, indicating that 20 µM and 25 µM Cur can inhibit the replication of virus in the cells.

[0052] RD-A cells at a concentration of 1×10 4 were inoculated in a 96-well plate and cultured in a 37°C, 5% CO2 cell incubator overnight. The supernatant of the cells in each group except the control group was collected and diluted by 10 -1 ~ 10 -11 times in turn. The diluted virus was added to the 96-well plate, one column for each dilution, a total of 8 wells, and 11 dilutions were added to 11 columns, 100 µL was added to each well, and a non-infected control group was set up at the same time.

[0053] The 96-well plate was placed in the incubator for continuous culture, and the results were observed and recorded every 12 h, continuously for 4-7 days. Reed-Muench method was used to calculate the results: distance ratio=(percentage of more than 50% lesion rate-50%) / (percentage of more than 50% lesion rate-percentage of less than 50% lesion rate), lgTCID 50 = difference between distance ratio and logarithm of dilution + logarithm of dilution of more than 50% lesion rate.

[0054] The results are shown in Table 2. Figure 5As shown, compared with the EV71 infection group, the virus titer in the supernatant of the cells after the intervention of 7.5 μΜ, 10 μΜ, 15 μΜ Cur on the EV71 infected cells had no obvious change, but the virus titer in the supernatant of the cells after the intervention of 20 μΜ, 25 μΜ Cur on the EV71 infected cells was obviously reduced. The above results showed that 20 μΜ, 25 μΜ Cur could inhibit the release of the virus.

[0055] Example 3: Protective effect of the co-action of Cur and AST-IV on EV71 infected GES-1 cells.

[0056] 1 × 10 6 cells / mL of GES-1 cells in the logarithmic growth phase were inoculated in a 6-well plate and divided into a control group, an EV71 infection group, an EV71 + AST-IV group, an EV71 + Cur group and an EV71 + AST-IV + Cur group.

[0057] First, 3.5 μΜ AST-IV was added to the EV71 + AST-IV group, 7.5 μΜ Cur was added to the EV71 + Cur group, and 3.5 μΜ AST-IV and 7.5 μΜ Cur were added to the EV71 + AST-IV + Cur group, and then the GES-1 cells were intervened for 2 h in advance, and then the cells in each group except the control group were infected with EV71 for 24 h. The morphological changes of the GES-1 cells in different groups were observed under an inverted microscope, and photographs were taken.

[0058] The results are shown in Table 1. Figure 6 Compared with the control group, after the GES-1 cells were infected with EV71, the number of cells was obviously reduced, the cells were shrunk, and the number of suspended cells was obviously increased. Compared with the EV71 infection group, the number of cells and the cell morphology in the EV71 + 3.5 μΜ AST-IV group and the EV71 + 7.5 μΜ Cur group intervened alone had no obvious change, but the number of cells in the EV71 + 3.5 μΜ AST-IV + 7.5 μΜ Cur group co-acting obviously increased, and the cell morphology recovered.

[0059] 1 × 10 4 cells / mL of GES-1 cells were inoculated in a 96-well plate and divided into a control group, an EV71 infection group, an EV71 + AST-IV group, an EV71 + Cur group and an EV71 + AST-IV + Cur group.

[0060] EV71+AST-IV group was added with AST-IV at a concentration of 3.5 µM, EV71+Cur group was added with Cur at a concentration of 7.5 µM, and EV71+AST-IV+Cur group was added with AST-IV at a concentration of 3.5 µM and Cur at a concentration of 7.5 µM. The GES-1 cells were intervened for 2 h in advance, and then infected with EV71 for 24 h, except for the control group.

[0061] 2 h before the end of EV71 infection, 20 μL of CCK8 reagent was added to each group, and the absorbance A value was measured at 450 nm with a microplate reader until the end of culture. The cell survival rate was calculated to detect cell viability.

[0062] The results are shown in Figure 7 Compared with the control group, the viability of EV71-infected cells decreased significantly. Compared with the EV71 infection group, the cell viability of the EV71+3.5 µM AST-IV group and the EV71+7.5 µM Cur group did not increase significantly, but the cell viability of the EV71+3.5 µM AST-IV+7.5 µM Cur group increased significantly.

[0063] The above results show that the combination of 3.5 µM AST-IV and 7.5 µM Cur has a significant protective effect on cell toxicity caused by EV71 infection.

[0064] The cells in each group after the above morphological observation were collected in a 1.5 mL EP tube, 60 µL of cell lysis solution was added to each tube to extract protein, VP1 protein was separated by 12% SDS-PAGE gel, and Western blot was used to detect the level of VP1 protein in the cells.

[0065] The results are shown in Figure 8 Compared with the EV71 infection group, the level of structural protein VP1 in the EV71+3.5 µM AST-IV group and the EV71+7.5 µM Cur group did not change significantly, but the level of VP1 protein in the EV71+3.5 µM AST-IV+7.5 µM Cur group decreased significantly. The above results show that the combination of 3.5 µM AST-IV and 7.5 µM Cur can inhibit the replication of virus in cells.

[0066] 1 × 10 4 RD-A cells at a concentration of 1 × 10 -1 ~ 10 -11Dilute 10 times. Add the diluted virus into 96-well plates, inoculate one column for each dilution, 8 wells in total, 11 dilutions are added into 11 columns respectively, 100 μL is added into each well, and set up non-inoculated control group at the same time.

[0067] Put the 96-well plates into the incubator for continuous culture, observe and record once every 12 hours, continuously observe for 4-7 days, and calculate the virus titer by using Reed-Muench method.

[0068] The results are shown in Table 1. Figure 9 As shown in Table 1, compared with the EV71 infection group, the virus titer in the supernatant of the EV71+3.5 μM AST-IV group and the EV71+7.5 μM Cur group has no obvious change, but the virus titer in the supernatant of the EV71+3.5 μM AST-IV+7.5 μM Cur group is obviously reduced. The above results also show that the combination of 3.5 μM AST-IV and 7.5 μM Cur can inhibit the release of virus.

[0069] The above embodiments of the present application do not describe all the details, and the present application is not limited to the above described embodiments. Various changes, modifications, replacements and variations made to the embodiments by those skilled in the art without departing from the principles and purposes of the present application shall be included in the protection scope of the present application.

Claims

1. The application of a pharmaceutical composition consisting of astragaloside A and curcumin in the preparation of a drug for the synergistic treatment of hand-foot-mouth disease caused by EV71 infection, wherein the dosage of astragaloside A is 3.5 μM and the dosage of curcumin is 7.5 μM.

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