Methanol extract of alcohol extraction total extract of overground whole herb of isodon amethystoides and application of methanol extract

Abstract

The invention discloses a methanol extract of alcohol extraction total extract of overground whole herb of isodon amethystoides and application of the methanol extract, the overground whole herb part of isodon amethystoides is taken to obtain 95% ethanol extraction total extract of the whole herb, the 95% ethanol extraction total extract passes through a macroporous resin chromatographic column, elution is carried out with 30%, 70% and 90% methanol respectively and sequentially, and all graded elution components are separated and collected. The isodon amethystoides herb methanol extract disclosed by the invention has an effect of resisting mycobacteria, which is expressed by inhibiting the growth activity of mycobacteria, including mycobacterium tuberculosis. The isodon amethystoides herb methanol extract disclosed by the invention can be used for preparing a medicine for resisting mycobacterium tuberculosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Chinese herbal medicine, more particularly to a methanol extract of alcohol extraction total extract of aboveground whole grass of Wangzaozi and application thereof. BACKGROUND

[0002] Tuberculosis (TB) is a major chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), and is the single pathogen infectious disease with the most deaths in the world, which has long threatened the global public health security. According to the Global Tuberculosis Report published by the World Health Organization in 2024, there were about 10.8 million new TB patients worldwide in 2023, with an incidence of 134 / 10 million, which increased by 4.6% compared with 2020. There were 1.25 million deaths from TB in 2023, which may exceed COVID-19 to become the main cause of death of single infectious disease. During this period, China estimated 741,000 new patients, with an incidence of 52 / 10 million and a mortality rate of 2.0 / 10 million, ranking third in the world. China is a high-burden country of TB and drug-resistant TB. Although China has made great efforts in the prevention and control of TB and has made great progress, the treatment and control of TB still have a long way to go.

[0003] Mycobacterium tuberculosis has strong infectivity and high pathogenicity. In the study of Mycobacterium, Bacillus Calmette-Guérin (BCG) is an important model, and Mycobacterium tuberculosis and BCG belong to slow-growing Mycobacterium. BCG is a live attenuated vaccine of Mycobacterium bovis, which is used to prevent tuberculosis. BCG is often used as a substitute strain to simulate in vitro bacteriostatic experiments of human tuberculosis bacillus due to its safety and genetic similarity with Mycobacterium tuberculosis. Through BCG, compounds with in vitro bacteriostatic activity can be quickly screened, providing experimental basis for the development of new anti-tuberculosis drugs.

[0004] Traditional Chinese medicine (TCM) has also played a certain role in the treatment of tuberculosis in the clinic, especially in the treatment of multidrug-resistant tuberculosis. Studies have shown that the combination of TCM and Western medicine treatment programs (such as different TCM compound prescriptions combined with anti-tuberculosis drug treatment) are superior to simple Western chemical therapy in sputum negative conversion rate, lesion absorption rate, cavity closure rate, and clinical comprehensive efficacy, and the incidence of adverse events in the combination of TCM and Western medicine treatment group is also lower than that in the control group. The related effects of TCM may depend on the direct bacteriostatic effect of alkaloids, cinnamic acid derivatives, quinones and terpenoids and other substances in Chinese herbal medicines, or by regulating the immune function of the host to improve the symptoms of patients. However, the stability of the active ingredients of natural product crude extracts as anti-tuberculosis preparations and the verification of efficacy and safety have not yet reached the standard of chemical drugs. Therefore, it is urgent to separate and purify the effective active components from the complex Chinese herbal medicine crude extract, and further discover and develop new drugs for the potential treatment of tuberculosis.

[0005] Radix Rabdosiae, the scientific name of Rabdosia rubescens, is a traditional Chinese herbal medicine in Suzhou, Anhui Province. The whole grass is used in clinical applications. The local people have used it for a long time to treat lung abscess, chronic bronchitis and other lung diseases. For many years, the characteristics of Radix Rabdosiae have gradually been valued, and some unique components of tetracyclic diterpenes have also been discovered and studied. However, there is no report on the use of Radix Rabdosiae for anti-mycobacterium tuberculosis. SUMMARY

[0006] The purpose of the present application is to provide a methanol extract of the alcohol extraction total extract of the above-ground whole grass of Radix Rabdosiae and its application, in order to solve the technical problems existing in the background art.

[0007] The technical scheme of the present application provides a methanol extract of the alcohol extraction total extract of the above-ground whole grass of Radix Rabdosiae. Specifically, the whole grass of the above-ground part of Radix Rabdosiae is taken, dried and pulverized;

[0008] Further, the above alcohol extract is concentrated under reduced pressure, and the alcohol extraction total extract of the above-ground whole grass of Radix Rabdosiae is obtained after drying at room temperature;

[0009] Further, the above alcohol extract is concentrated under reduced pressure, and the alcohol extraction total extract of the above-ground whole grass of Radix Rabdosiae is obtained after drying at room temperature;

[0010] Further, the alcohol extraction total extract is dissolved with methanol, and the solution is passed through a macroporous resin chromatographic column;

[0011] Further, 30%, 70% and 90% methanol are used to elute in sequence, and different fraction elution solutions are collected.

[0012] Thus, the methanol extract of the alcohol extraction total extract of the above-ground whole grass of Radix Rabdosiae is obtained, and finally a paste-like substance is obtained by evaporation and concentration at room temperature.

[0013] The beneficial effects of the technical solution of the present application are:

[0014] (1) Through the in vitro bacteriostatic test, the present application proves that the methanol extract of the alcohol-extracted total extract of the aerial whole grass of Prinsepia uniflora Batal has a significant inhibitory effect on BCG and shows a dose-dependent manner.

[0015] (2) Through the in vitro bacteriostatic half-inhibitory concentration (IC50) test, the present application proves that the methanol extract of different segments of the aerial whole grass of Prinsepia uniflora Batal has a BCG bacteriostatic effect, which shows that it may contain different BCG bacteriostatic components. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The in vitro bacteriostatic test of the methanol extract of the alcohol-extracted total extract of the aerial whole grass of Prinsepia uniflora Batal on BCG;

[0017] Figure 2 The half-inhibitory concentration (IC50) test of the methanol extract of the alcohol-extracted total extract of the aerial whole grass of Prinsepia uniflora Batal on BCG. DETAILED DESCRIPTION

[0018] The present application will be further described in detail below, and the embodiments of the present application are given for the convenience of illustration and description, and are not exhaustive or limit the present application to the disclosed forms. Many modifications and changes will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical application of the present application, and to enable those of ordinary skill in the art to understand the present application so as to design various embodiments with various modifications suitable for specific purposes.

[0019] Example 1, extraction and preparation of the methanol sample of the alcohol-extracted total extract of the aerial whole grass of Prinsepia uniflora Batal.

[0020] (1) Extraction of the aerial whole grass of Prinsepia uniflora Batal: dry the aerial whole grass of Prinsepia uniflora Batal into powder, add 500 ml of 95% ethanol (volume percentage) to every 100 grams of powder, and repeatedly extract at room temperature. After filtering the extract, evaporate and concentrate at 40°C, and then dry in a water bath at 45°C for about a week to obtain the extract, which is the alcohol-extracted total extract of the aerial whole grass of Prinsepia uniflora Batal. About 22 grams of total extract is obtained from every 100 grams of whole grass.

[0021] (2) Macroporous resin chromatography column: The macroporous resin was activated and packed into a column, and the column was equilibrated with 10% methanol. 150 grams of the total alcohol extract of the aerial whole plant of Evodia lepta was added to an appropriate amount of methanol and stirred until completely dissolved. Then, the sample solution was passed through the macroporous resin chromatography column. Elution was performed in sections with 30%, 70%, and 90% methanol, and the elution solutions were collected. Finally, the alcohol extract of the 30% methanol section (40.7152 g), the alcohol extract of the 70% methanol section (14.7118 g), and the alcohol extract of the 90% methanol section (16.2188 g) were obtained by drying under vacuum.

[0022] Example 2, Bacteriostatic experiment of the methanol extract of the total alcohol extract of the aerial whole plant of Evodia lepta on BCG.

[0023] (1) Experimental reagents and materials

[0024] Evodia lepta extract, sterile water, DMSO, 7H9 culture medium (BD DIFCO™ Middlebrook 7H9 Broth, item number 271310), OADC enrichment solution (Tube Middlebrook OADC Enrich, item number 211886), Tyloxapol (Sigma-Aldrich, item number T8761-50G), Alamar Blue reagent (Invitrogen™, item number DAL1100), EP tubes, 96-well plates, BCG (provided by the First Affiliated Hospital of the University of Science and Technology of China).

[0025] (2) Experimental steps

[0026] The methanol extract of the total alcohol extract of the aerial whole plant of Evodia lepta was dissolved in dimethyl sulfoxide (DMSO) to prepare a 200 mg / ml stock solution. The stock solution was diluted by two-fold serial dilution, and then added to the corresponding 96-well plates at 1 μl / well. Each extract was tested in triplicate, and the wells with DMSO (final concentration 1%) were used as controls.

[0027] The BCG in the logarithmic phase (8 ml of bacterial solution, 37°C static culture, OD600=0.2) was collected by centrifugation at 12000 rpm for 1 min at RT. The supernatant was discarded in a clean bench, washed twice with 7H9 culture medium, and finally resuspended with 2 ml of 7H9 culture medium for 20 min.

[0028] The OD600 of the supernatant was measured, and then diluted with 7H9 culture medium to OD600=0.36, and further diluted by 1000-fold.

[0029] The diluted bacterial solution was added to the corresponding positions of the above-mentioned 96-well plates at 99 μl / well. The blank control was 7H9 culture medium without bacterial solution.

[0030] After culturing at 37°C for one week, add 10 μl of Alamar Blue, a fluorescent indicator for detecting cell metabolic activity, to each well.

[0031] After incubation at 37℃ for 24 hours, 75% ethanol was added for inactivation. The results were then detected using an ELISA reader with an excitation wavelength of 540nm and an emission wavelength of 590nm. The data were recorded and statistically analyzed.

[0032] (3) Experimental results

[0033] Fluorescence values ​​from blank control wells (without cells) were used as background, and fluorescence values ​​from normal cell wells (without extract) were used as 100% viability controls. Fluorescence data from the experimental groups (cells under three different treatments) were converted to relative cell viability percentages using the following formula:

[0034] Cell viability (%) = (Experimental group fluorescence value - Blank control fluorescence value) / (Untreated group fluorescence value - Blank control fluorescence value) × 100

[0035] Calculate the mean and standard deviation (SD) of the three sets of experimental data, and plot a bar chart. Figure 1 ).

[0036] like Figure 1 As shown, the methanol extracts of the total extract of *Ziziphus jujuba* showed significant inhibitory effects on BCG activity, but the inhibitory effects varied considerably. The 30% and 70% methanol fractions of the total extract almost completely inhibited bacterial metabolic activity at concentrations ≥0.25 mg / ml; the 90% methanol fraction only completely inhibited BCG activity at extremely high concentrations ≥2 mg / ml.

[0037] The methanol extract of the total extract of the whole plant of *Ziziphus jujuba* described above was tested for its half-maximal inhibitory concentration (IC50) against BCG. The results are as follows: Figure 2 The calculation results showed that the half-inhibitory concentrations (IC50) of the methanol extracts of the whole herb alcohol extract (30%, 70%, and 90% methanol fractions) were 25.8 μg / ml, 33.2 μg / ml, and 102.9 μg / ml, respectively.

[0038] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described and explained in the present invention, unless otherwise specified or limited, shall be implemented according to conventional means in the art.

Claims

1. A methanol extraction method for the total extract of the whole aerial plant of *Ziziphus jujuba*, characterized in that: The methanol extract of the total extract of the whole plant of *Ziziphus jujuba* is a 95% ethanol extract obtained by fractional elution after the solution is passed through a macroporous resin chromatography column.

2. The methanol extraction of the total extract of the whole aerial plant of *Ziziphus jujuba* according to claim 1, characterized in that: The elution process is as follows: elution is performed sequentially using 30%, 70%, and 90% methanol.

3. The methanol extraction of the total extract of the whole aerial herb of *Ziziphus jujuba* according to any one of claims 1-2, characterized in that: Application of the methanol extract of the total extract of the whole plant of *Ziziphus jujuba* in the preparation of products for anti-tuberculosis mycobacterium.

4. The methanol extraction of the total extract of the whole aerial plant of *Ziziphus jujuba* according to claim 3, characterized in that: The anti-tuberculosis mycobacterial products include anti-tuberculosis drugs and other products.

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