Rose extract, preparation method thereof and application thereof in preparing neuraminidase inhibitory medicine

Abstract

The present invention relates to the technical field of natural drug chemistry, and specifically discloses a rose extract, a preparation method thereof, and an application thereof in the preparation of a drug having a neuraminidase inhibitory effect. The preparation method of the rose extract comprises the following steps: taking roses, then extracting with ethanol, concentrating and drying the extract after the extraction is completed to obtain a rose ethanol extract, that is, the rose extract. Studies have shown that the rose extract prepared by the method of the present invention has an excellent neuraminidase inhibitory effect; its inhibitory effect on neuraminidase is significantly higher than that of oseltamivir carboxylate. Therefore, the rose extract of the present invention is used as an effective ingredient to prepare a drug having a neuraminidase inhibitory effect, which has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of natural drug chemistry, and in particular to a rose extract and a preparation method thereof and application of the rose extract in preparing a drug with neuraminidase inhibitory effect. Background Art

[0002] Neuraminidase (NA) is a key enzyme in influenza virus replication, transmission and pathogenesis. Its active site is highly conserved for both influenza A and B viruses, making it an ideal target for anti-influenza drug research. Currently, there are only four drugs targeting NA on the market, namely zanamivir, oseltamivir, panamivir and laninamivir. However, with the emergence of drug-resistant strains and drug toxicity problems, the discovery of new NA inhibitors has become an urgent need for drug research and development today.

[0003] Rose can be used as a Chinese herbal medicine, and has the effects of promoting blood circulation and removing blood stasis, strengthening the spleen and stomach, soothing the liver and relieving depression, and beautifying the skin. However, the prior art does not report that it has a neuraminidase inhibitory effect. Therefore, using rose as a raw material, developing an effective part with a neuraminidase inhibitory effect has important application value. Summary of the invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, the present invention first provides a method for preparing a rose extract.

[0005] The above technical problems to be solved by the present invention are achieved through the following technical solutions:

[0006] The present invention first provides a preparation method of rose extract, which comprises the following steps: taking roses, then extracting with ethanol, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract, that is, the rose extract.

[0007] The inventors surprisingly found in their research that the rose extract prepared by the method of the present invention has excellent neuraminidase inhibitory effect; its inhibitory effect on neuraminidase is significantly higher than that of the positive control drug oseltamivir carboxylate; therefore, the rose extract of the present invention is used as an effective ingredient to prepare a drug with neuraminidase inhibitory effect, which has important application value.

[0008] Preferably, the rose is a dried unopened rose bud.

[0009] Preferably, the usage ratio of rose to ethanol is 1 g: 8-15 mL.

[0010] Most preferably, the ratio of rose to ethanol is 1 g:10 mL.

[0011] Preferably, the extract is extracted by ultrasonic extraction, and the extraction time is 20 to 60 minutes.

[0012] Most preferably, the extraction time is 30 min.

[0013] Preferably, the ethanol refers to an ethanol aqueous solution with a volume fraction of 80 to 95%.

[0014] Preferably, the preparation method of the rose extract further comprises the following steps:

[0015] The rose ethanol extract is placed on a macroporous resin column, firstly eluted with an ethanol aqueous solution with a volume fraction of 24-26%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 24-26% is discarded; then eluted with an ethanol aqueous solution with a volume fraction of 48-52%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 50% is collected, and the rose extract is obtained after concentrating and drying.

[0016] Most preferably, the method for preparing the rose extract further comprises the following steps:

[0017] The rose ethanol extract is placed on a macroporous resin column, first eluted with an ethanol aqueous solution with a volume fraction of 25%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 25% is discarded; then eluted with an ethanol aqueous solution with a volume fraction of 50%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 50% is collected, and the rose extract is obtained after concentrating and drying.

[0018] The inventors further surprisingly found in the research that the rose extract obtained by further preparing the rose ethanol extract through the macroporous resin elution conditions of the present invention has a further significantly improved inhibitory effect on neuraminidase compared with the rose ethanol extract; it has a very excellent neuraminidase inhibitory effect; its inhibitory effect on neuraminidase is also significantly higher than that of the positive control drug oseltamivir carboxylate.

[0019] Here, the inventors need to explain that the macroporous resin elution conditions of the present invention are very critical. The rose extracts prepared under different macroporous resin elution conditions have huge differences in their inhibitory effects on neuraminidase. The inventors surprisingly found in a large number of experiments that only the rose extracts prepared under the macroporous resin elution conditions of the present invention can further significantly improve their inhibitory effects on neuraminidase compared with the rose ethanol extract; while the rose extracts prepared under other macroporous resin elution conditions cannot further significantly improve their inhibitory effects on neuraminidase compared with the rose ethanol extract.

[0020] The present invention also provides a rose extract prepared by the above preparation method.

[0021] The present invention also provides an application of the rose extract in preparing a neuraminidase inhibitor.

[0022] The present invention also provides an application of the rose extract in preparing a medicine having a neuraminidase inhibitory effect.

[0023] Beneficial effects: The present invention provides a novel method for preparing rose extract; studies have shown that the rose extract prepared by the method of the present invention has excellent neuraminidase inhibitory effect; its inhibitory effect on neuraminidase is significantly higher than that of oseltamivir carboxylate. Therefore, the rose extract of the present invention as an effective ingredient for preparing a drug with neuraminidase inhibitory effect has important application value. DETAILED DESCRIPTION

[0024] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0025] Example 1 Preparation of Rose Extract

[0026] Take dried unopened rose buds, and then use ethanol (95% ethanol) for ultrasonic extraction. After the extraction is completed, the extract is concentrated and dried to obtain a rose ethanol extract, that is, the rose extract;

[0027] The dosage ratio of rose buds to ethanol is 1 g:10 mL; the temperature of the ultrasonic extraction is 50° C., the ultrasonic power is 1500 W, and the frequency is 25 KHz; and the ultrasonic extraction time is 30 min.

[0028] Example 2 Preparation of Rose Extract

[0029] (1) taking dried unopened rose buds, and then using ethanol (95% ethanol) for ultrasonic extraction, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract; wherein the ratio of rose buds to ethanol is 1 g: 10 mL; the temperature of the ultrasonic extraction is 50° C., the power of the ultrasound is 1500 W, and the frequency is 25 KHz; the ultrasonic extraction time is 30 min;

[0030] (2) The rose ethanol extract is loaded onto a D101 macroporous resin column (the weight of the D101 macroporous resin filler in the macroporous resin column is 40 times the weight of the rose ethanol extract); firstly, eluted with 5 times the column volume of a 25% ethanol aqueous solution, and discarded the eluate eluted with the 25% ethanol aqueous solution; then, eluted with 5 times the column volume of a 50% ethanol aqueous solution, collected the eluate eluted with the 50% ethanol aqueous solution, and concentrated and dried to obtain the rose extract.

[0031] Example 3 Preparation of Rose Extract

[0032] (1) taking dried unopened rose buds, and then using ethanol (80% ethanol) for ultrasonic extraction, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract; wherein the ratio of rose buds to ethanol is 1 g: 8 mL; the temperature of the ultrasonic extraction is 40° C., the power of the ultrasound is 1000 W, and the frequency is 40 KHz; the ultrasonic extraction time is 60 min;

[0033] (2) The rose ethanol extract is loaded onto a D101 macroporous resin column (the weight of the D101 macroporous resin filler in the macroporous resin column is 40 times the weight of the rose ethanol extract); firstly, eluted with 3 times the column volume of an ethanol aqueous solution with a volume fraction of 24%, and discarded the eluate eluted with the 24% ethanol aqueous solution; then, eluted with 5 times the column volume of an ethanol aqueous solution with a volume fraction of 48%, collected the eluate eluted with the 48% ethanol aqueous solution, and concentrated and dried to obtain the rose extract.

[0034] Example 4 Preparation of Rose Extract

[0035] (1) taking dried unopened rose buds, and then using ethanol (95% ethanol) for ultrasonic extraction, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract; wherein the ratio of rose buds to ethanol is 1 g: 15 mL; the temperature of the ultrasonic extraction is 60° C., the power of the ultrasound is 1200 W, and the frequency is 25 KHz; the ultrasonic extraction time is 20 min;

[0036] (2) The rose ethanol extract is loaded onto a D101 macroporous resin column (the weight of the D101 macroporous resin filler in the macroporous resin column is 40 times the weight of the rose ethanol extract); firstly, eluted with 3 times the column volume of an ethanol aqueous solution with a volume fraction of 26%, and discarded the eluate eluted with the 26% ethanol aqueous solution; then, eluted with 5 times the column volume of an ethanol aqueous solution with a volume fraction of 52%, collected the eluate eluted with the 52% ethanol aqueous solution, and concentrated and dried to obtain the rose extract.

[0037] Comparative Example 1 Preparation of Rose Extract

[0038] (1) taking dried unopened rose buds, and then using ethanol (95% ethanol) for ultrasonic extraction, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract; wherein the ratio of rose buds to ethanol is 1 g: 10 mL; the temperature of the ultrasonic extraction is 50° C., the power of the ultrasound is 1500 W, and the frequency is 25 KHz; the ultrasonic extraction time is 30 min;

[0039] (2) placing the rose ethanol extract on a D101 macroporous resin column (the weight of the D101 macroporous resin filler in the macroporous resin column is 40 times the weight of the rose ethanol extract); eluting with 5 times the column volume of a 25% ethanol aqueous solution, collecting the eluate eluted with the 25% ethanol aqueous solution, and concentrating and drying to obtain the rose extract.

[0040] Comparative Example 2 Preparation of Rose Extract

[0041] (1) taking dried unopened rose buds, and then using ethanol (95% ethanol) for ultrasonic extraction, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract; wherein the ratio of rose buds to ethanol is 1 g: 10 mL; the temperature of the ultrasonic extraction is 50° C., the power of the ultrasound is 1500 W, and the frequency is 25 KHz; the ultrasonic extraction time is 30 min;

[0042] (2) loading the rose ethanol extract onto a D101 macroporous resin column (the weight of the D101 macroporous resin filler in the macroporous resin column is 40 times the weight of the rose ethanol extract); first eluting with 5 times the column volume of a 50% ethanol aqueous solution, discarding the eluate eluted with the 50% ethanol aqueous solution; then eluting with 5 times the column volume of a 75% ethanol aqueous solution, collecting the eluate eluted with the 75% ethanol aqueous solution, and concentrating and drying to obtain the rose extract.

[0043] Comparative Example 3 Preparation of Rose Extract

[0044] (1) taking dried unopened rose buds, and then using ethanol (95% ethanol) for ultrasonic extraction, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract; wherein the ratio of rose buds to ethanol is 1 g: 10 mL; the temperature of the ultrasonic extraction is 50° C., the power of the ultrasound is 1500 W, and the frequency is 25 KHz; the ultrasonic extraction time is 30 min;

[0045] (2) loading the rose ethanol extract onto a D101 macroporous resin column (the weight of the D101 macroporous resin filler in the macroporous resin column is 40 times the weight of the rose ethanol extract); first eluting with 5 times the column volume of a 75% ethanol aqueous solution, discarding the eluate eluted with the 75% ethanol aqueous solution; then eluting with 5 times the column volume of a 95% ethanol aqueous solution, collecting the eluate eluted with the 95% ethanol aqueous solution, and concentrating and drying to obtain the rose extract.

[0046] Experimental Example 1 In vitro neuraminidase (NA) inhibitory activity assay

[0047] (1) Solution preparation

[0048] NA buffer solution: used to dissolve freeze-dried NA. Contains 0.1mol / L K3PO4, 0.025mol / L KCl, 0.26mg / mL BSA, and the pH is adjusted to 6.0 with hydrochloric acid.

[0049] MES buffer solution: contains 32.5mmol / L MES, 4mmol / L CaCl2, and the pH is adjusted to 6.5 with Tris base.

[0050] NA solution: 25U NA is dissolved in 8mL NA buffer solution, and then divided into 8 tubes of 1mL 3.125U / mL NA solution using EP tubes, and stored at -20℃. When using, take the aliquoted NA solution for experiment, dilute it to 0.015U / mL with MES buffer solution, and use the prepared NA solution only on the same day.

[0051] MUNANA solution: Use MES buffer solution to prepare a 0.1mmol / L MUNANA solution.

[0052] Samples to be tested: first dissolved with DMSO to prepare a stock solution of a certain concentration. When used, take a certain amount of the stock solution and dilute it with MES buffer solution to a working solution of a certain concentration.

[0053] The samples to be tested are the rose extracts prepared in Examples 1 and 2 and Comparative Examples 1 to 3 respectively;

[0054] Oseltamivir carboxylate solution (positive control): first dissolve in DMSO to prepare a stock solution of a certain concentration. When used, take a certain amount of the stock solution and dilute it with MES buffer solution to a working solution of a certain concentration.

[0055] Stop buffer solution: used to terminate the enzymatic reaction. Contains 0.1g / mL glycine and 25% ethanol.

[0056] (2) The inhibitory activity of rose extract on NA was determined by fluorescence method.

[0057] NA activity assay was performed in a 96-well plate. The sample addition method is shown in Table 1. The specific operation method is as follows: the 96-well plate contains 40 μL of diluted NA solution and 20 μL of fluorescent substrate solution. In each test well, the final concentration of NA is 0.006 U / mL, and the final concentration of MUNANA is 0.02 mmol / L.

[0058] For evaluating the inhibitory activity of the sample to be tested, the sample to be tested was first dissolved in DMSO to prepare a stock solution with an initial concentration of 50 mg / mL, and then diluted to an initial concentration working solution of 160 μg / mL. Finally, by the half-dilution method, a series of gradient working solutions with concentrations of 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL were obtained. The final concentrations of the above working solutions after adding to the wells were 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL, respectively. 40 μL of the prepared sample solution to be tested was added to the well, followed by 40 μL of NA solution. After incubation at 37°C for 30 minutes, 20 μL of MUNANA solution was added to the well, and after reacting for 10 minutes, 100 μL of stop solution was added to stop the enzymatic reaction. Each experiment was repeated three times. The fluorescence value was read by an instrument with the excitation wavelength set at 360 nm and the emission wavelength set at 460 nm. The data were processed with GraphPad Prism 8 to calculate the IC 50 The fluorescence value measured at the end of the reaction is I, and the inhibition rate of each concentration is calculated by the following formula:

[0059]

[0060] Table 1 Different experimental groups

[0061]

[0062]

[0063] According to the above test steps (1) and (2), with linalool as the positive control, the results are shown in Table 2.

[0064] Table 2 The results of the experimental study on the inhibition of neuraminidase by the rose extract of the present invention

[0065]

[0066] From Table 2, the IC 50 The experimental results can draw the following conclusions:

[0067] (1) The rose extract prepared in Example 1 has an IC50 The value is significantly smaller than that of the positive control drug oseltamivir carboxylate; this indicates that the rose extract prepared by the method of the present invention has excellent neuraminidase inhibitory effect; and its inhibitory effect on neuraminidase is significantly higher than that of oseltamivir carboxylate.

[0068] (2) The rose extract prepared in Example 2 has an IC 50 The value is further significantly smaller than the rose extract prepared in Example 1, and is also significantly smaller than the positive control drug oseltamivir carboxylate; this indicates that the rose extract obtained by further preparing the rose ethanol extract through the macroporous resin elution conditions of the present invention has a further significantly improved inhibitory effect on neuraminidase compared with the rose ethanol extract; it has a very excellent neuraminidase inhibitory effect; its inhibitory effect on neuraminidase is also significantly higher than that of the positive control drug oseltamivir carboxylate.

[0069] (3) The rose extracts prepared in Comparative Examples 1 to 3 had an IC 50 The value was not improved compared with the rose extract prepared in Example 1, but was reduced, even greatly reduced; and its IC 50 The value is also greater than or even significantly greater than the positive control drug oseltamivir carboxylate; this shows that: the macroporous resin elution conditions of the present invention are very critical, and the rose extracts prepared under different macroporous resin elution conditions have huge differences in the composition and content of the active ingredients contained therein, which leads to huge differences in their inhibitory effects on neuraminidase. The above experimental results show that only the rose extract prepared under the macroporous resin elution conditions of the present invention can further significantly improve its inhibitory effect on neuraminidase compared with the rose ethanol extract, and can prepare a rose extract with very excellent inhibitory effect on neuraminidase; while the rose extract prepared under other macroporous resin elution conditions cannot further significantly improve its inhibitory effect on neuraminidase compared with the rose ethanol extract.

Claims

1. A method for preparing a rose extract, characterized in that: The method comprises the following steps: taking roses, then extracting with ethanol, and after the extraction is completed, concentrating and drying the extract to obtain rose ethanol extract, that is, the rose extract.

2. The method for preparing the rose extract according to claim 1, characterized in that: The rose is a dried unopened rose bud.

3. The method for preparing the rose extract according to claim 1, characterized in that: The dosage ratio of rose to ethanol is 1g:8-15mL; Most preferably, the ratio of rose to ethanol is 1 g:10 mL.

4. The method for preparing the rose extract according to claim 1, characterized in that: The extract is extracted by ultrasonic extraction, and the extraction time is 20 to 60 minutes; Most preferably, the extraction time is 30 min.

5. The method for preparing the rose extract according to claim 1, characterized in that: The ethanol mentioned above refers to an ethanol aqueous solution with a volume fraction of 80 to 95%.

6. The method for preparing the rose extract according to claim 1, characterized in that: The following steps are also included: The rose ethanol extract is placed on a macroporous resin column, firstly eluted with an ethanol aqueous solution with a volume fraction of 24-26%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 24-26% is discarded; then eluted with an ethanol aqueous solution with a volume fraction of 48-52%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 50% is collected, and the rose extract is obtained after concentrating and drying.

7. The method for preparing the rose extract according to claim 6, characterized in that: The following steps are also included: The rose ethanol extract is placed on a macroporous resin column, first eluted with an ethanol aqueous solution with a volume fraction of 25%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 25% is discarded; then eluted with an ethanol aqueous solution with a volume fraction of 50%, and the eluate eluted with the ethanol aqueous solution with a volume fraction of 50% is collected, and the rose extract is obtained after concentrating and drying.

8. The rose extract prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the rose extract according to claim 8 in the preparation of a neuraminidase inhibitor.

10. Use of the rose extract according to claim 8 in the preparation of a medicament having a neuraminidase inhibitory effect.

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