Over-ground whole herb extract of isodon amethystoides and application of above-ground whole herb extract

Abstract

The invention discloses an overground whole herb extract of isodon amethystoides and application thereof, the overground whole herb part of isodon amethystoides is taken, the obtained extract is characterized by being alcohol extracted total extract of the whole herb, the alcohol extracted total extract is respectively and sequentially extracted by petroleum ether, ethyl acetate and n-butyl alcohol, and respective extracted parts and residual water-soluble parts are separated. The isodon amethystoides herb 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 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 kind of Wangzaozi aboveground whole herb extract and its application. BACKGROUND

[0002] Tuberculosis, also known as "phthisis", is a chronic infectious disease caused by Mycobacterium tuberculosis. The disease mainly occurs in lung tissue, trachea, bronchus and pleura, and can also invade liver, kidney, brain, lymph nodes and other organs. Tuberculosis has a long history and has long been a threat to human health. For the prevention and treatment of tuberculosis, although vaccines and some drug treatment methods have been developed, the situation remains severe in recent years, with high incidence and mortality. According to the Global Tuberculosis Report released by the World Health Organization in 2024, there were about 10.8 million new tuberculosis cases worldwide in 2023, with an incidence rate of 134 / 10 million, up 4.6% from 2020. There were 1.25 million deaths from tuberculosis in 2023, which may exceed COVID-19 to become the leading cause of death from a single infectious disease. During this period, China estimated that there were 741,000 new cases, an incidence rate of 52 / 10 million, and a mortality rate of 2.0 / 10 million, ranking third in the world. According to the national epidemiological sampling survey, the national treatment success rate for tuberculosis is high, but the treatment cost for drug-resistant tuberculosis is high. High-risk groups include people over 60 years of age, diabetes patients, and immunosuppressed groups. The incidence rate in western regions is significantly higher than that in eastern regions, and about 10% of the counties (districts) are high-prevalence areas. It can be seen that it is still of great practical significance to continuously develop drugs against tuberculosis.

[0003] Mycobacterium tuberculosis has strong infectivity and high pathogenicity. In the study of Mycobacterium, Bacillus Calmette-Guérin (BCG) and Mycolicibacterium smegmatis are the main models. Mycobacterium tuberculosis and BCG belong to slow-growing Mycobacterium. Compared with Mycobacterium tuberculosis, which divides once every 18-24 hours, the doubling time of Mycolicibacterium smegmatis is only 3-5 hours, which facilitates rapid data acquisition in the laboratory. Therefore, Mycolicibacterium smegmatis is an ideal substitute model for tuberculosis research and is commonly used for anti-tuberculosis drug research and development, gene function analysis, and bacterial physiological mechanism research. BCG is a live attenuated vaccine of Mycobacterium bovis and is used to prevent tuberculosis. BCG is often used as a substitute strain to simulate in vitro bacteriostatic experiments of human Mycobacterium tuberculosis due to its safety and genetic similarity to 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] Although the western medicine treatment of tuberculosis has been relatively routine, the western medicine components have great side effects and are prone to produce drug-resistant strains, and the treatment of drug-resistant tuberculosis is costly. The Tuberculosis Prevention and Control Center of the China Disease Control and Prevention Center mentioned in the five goals of the Global Tuberculosis Epidemic Ending Implementation Strategy in 2023-2030 that quality-assured and reasonably-priced tuberculosis products should be easily available. Therefore, it is a reasonable choice to develop new Chinese herbal medicine preparations for treating tuberculosis. Chinese herbal medicine has a long history of treating tuberculosis. By tonifying qi (such as Huangqi and Renshen), nourishing yin (such as Maidong and Tian-dong), and regulating immune function, the patient's constitution is improved. At the same time, the western medicine for treating tuberculosis is prone to cause liver damage and gastrointestinal discomfort, and Chinese medicine can relieve adverse reactions by soothing the liver and regulating qi (Chaihu) and invigorating the spleen and stomach (Fuling), thereby improving treatment compliance. Some Chinese herbal medicines such as Baibu and Baiji can relieve cough and hemostasis, and Dong-wengxue and Yuzhu can relieve chills and night sweats, thereby improving the quality of life of patients. Nowadays, Chinese herbal medicine has a defect in treating tuberculosis, which is that it needs to be used in combination with western medicine for treating tuberculosis (such as Rifampicin and Isoniazid), and cannot be used alone to replace conventional treatment. However, Chinese herbal medicine has a long history and a wide source, and it is possible to screen effective components from Chinese herbal medicine for treating tuberculosis and develop new preparations for treating tuberculosis, which has great potential.

[0005] Rabdosia rubescens, also known as Rabdosia rubescens, is a plant of the Lamiaceae family, which is a traditional Chinese medicine used by local people in Suzhou, Anhui Province. The whole grass is often used in clinical application. The local people have long used it to treat lung abscess, chronic bronchitis and other lung diseases. Over the years, the characteristics of Rabdosia rubescens have gradually been valued, and some unique components of tetracyclic diterpenes have been discovered and studied. However, there is no report on the use of Rabdosia rubescens for anti-tuberculosis Mycobacterium tuberculosis. SUMMARY

[0006] The purpose of the present application is to provide a Rabdosia rubescens aboveground whole grass alcohol extract and its application, in order to solve the technical problems existing in the background art.

[0007] The technical solution of the present application provides a Rabdosia rubescens aboveground whole grass alcohol extract. The preparation method of the Rabdosia rubescens aboveground whole grass alcohol extract is ethanol solvent immersion method. Specifically, the whole grass of the aboveground part of Rabdosia rubescens is taken, dried and pulverized.

[0008] Further, the aboveground whole grass powder of Rabdosia rubescens is extracted with new 95% ethanol at room temperature to obtain an extract.

[0009] Further, the above alcohol extract is concentrated under reduced pressure, and the Rabdosia rubescens aboveground whole grass alcohol extract is obtained after drying at room temperature.

[0010] Further, the alcohol extract is dissolved with water, and sequentially extracted with solvents with weak to strong polarity.

[0011] Further, extract with petroleum ether, take the petroleum ether segment after repeated extraction.

[0012] Further, extract with ethyl acetate, take the ethyl acetate segment after repeated extraction.

[0013] Further, extract with n-butanol, take the n-butanol segment after repeated extraction.

[0014] Further, take the water segment after extraction.

[0015] Thus, the alcohol extraction total extract of the aerial whole plant of Paliurus hudsonius, the petroleum ether segment, the ethyl acetate segment, the n-butanol segment and the water segment of the alcohol extraction total extract are obtained. Each extraction segment is extracted in order from weak to strong according to the polarity of the extraction solvent, so that the similar and compatible components in the alcohol extraction total extract are separated, forming a more refined combination of components. The alcohol extraction total extract of the aerial whole plant of Paliurus hudsonius and each extraction segment thereof are finally evaporated and concentrated at room temperature to obtain a paste.

[0016] The beneficial effects of the technical scheme of the present application are:

[0017] (1) Through in vitro bacteriostatic test, the present application proves that the alcohol extract of the aerial whole plant of Paliurus hudsonius provided by the present application has a significant inhibitory effect on Mycobacterium smegmatis and BCG, and shows a dose-dependent effect. The subsequent extraction segments, including the petroleum ether segment, the ethyl acetate segment, the n-butanol segment and the water segment, also have different degrees of inhibitory effect on Mycobacterium smegmatis and BCG.

[0018] (2) Through in vitro half-inhibitory concentration (IC50) test, the present application proves that the alcohol extract of the aerial whole plant of Paliurus hudsonius and each extraction segment thereof have different Mycobacterium smegmatis and BCG bacteriostatic effects, showing that they may contain different Mycobacterium smegmatis and BCG bacteriostatic components. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the bacteriostatic experiment of the alcohol extract of the aerial whole plant of Paliurus hudsonius on Mycobacterium smegmatis in the present application;

[0020] Figure 2 It is the half-inhibitory concentration (IC50) test of the alcohol extract of the aerial whole plant of Paliurus hudsonius on Mycobacterium smegmatis in the present application;

[0021] Figure 3 It is the bacteriostatic experiment of the alcohol extract of the aerial whole plant of Paliurus hudsonius on BCG in the present application;

[0022] Figure 4 It is the half-inhibitory concentration (IC50) test of the alcohol extract of the aerial whole plant of Paliurus hudsonius on BCG in the present application. DETAILED DESCRIPTION

[0023] The application is explained in further detail below, with examples given for the purpose of illustration and description only and are not intended to be exhaustive or to limit the application to the forms disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. Embodiments are chosen and described in order to provide the best illustration of the principles of the application and its practical application, and to enable one of ordinary skill in the art to understand the application for various embodiments with various modifications as are suited to the particular use contemplated.

[0024] Example 1, extraction and preparation of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou.

[0025] (1) Extraction of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou: dry the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou into powder, take 100 grams, add 500 milliliters of 95% ethanol (volume percentage), repeatedly extract at room temperature. Filter the extract, evaporate and concentrate at 40°C, dry in a water bath at 45°C for about a week to obtain the extract, which is the total extract of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, about 22 grams of total extract per 100 grams of whole plant.

[0026] (2) Extraction: take 22 grams of the total extract of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou and add to a 500 milliliter separatory funnel, add 200 milliliters of water to stir into a suspension. Then, add petroleum ether 200 milliliters each time for extraction, extract 3 times, and concentrate the extract at 40°C to obtain the petroleum ether segment extract, dry the petroleum ether segment extract in a water bath at 45°C for about a week to obtain the petroleum ether segment extract of the total extract of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, about 2.9 grams; then add ethyl acetate 200 milliliters each time for extraction, extract 3 times, and concentrate the extract at 40°C to obtain the ethyl acetate segment extract, dry the ethyl acetate segment extract in a water bath at 45°C for about a week to obtain the ethyl acetate segment extract of the total extract of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, about 1.9 grams; then add n-butanol 200 milliliters each time for extraction, extract 3 times, and concentrate the extract at 40°C to obtain the n-butanol segment extract, dry the n-butanol segment extract in a water bath at 45°C for about a week to obtain the n-butanol segment extract of the total extract of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, about 1.8 grams; and concentrate the remaining extract at 40°C to obtain the water segment extract, dry the water segment extract in a water bath at 45°C for about a week to obtain the water segment extract of the total extract of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, about 2.1 grams.

[0027] Example 2, antibacterial experiment of the extract of the whole plant of the aerial part of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou on Mycobacterium smegmatis.

[0028] (1) Experimental reagents and materials

[0029] Extract of the whole plant of Wangzaozi, sterile water, DMSO, 7H9 medium (BD DIFCO™ Middlebrook 7H9 Broth, No. 271310), OADC enrichment solution (Tube Middlebrook OADC Enrich, No. 211886), Tyloxapol (Sigma-Aldrich, No. T8761-50G), Alamar Blue reagent (Invitrogen™, No. DAL1100), EP tube, 96-well plate, Mycobacterium smegmatis (provided by University of Science and Technology of China).

[0030] (2) Experimental steps

[0031] The extract of the whole plant of Wangzaozi was dissolved in dimethyl sulfoxide (DMSO) except for the water extraction section, and the water extraction extract was dissolved in sterile water to prepare a mother liquor of 100-400 mg / ml. The above-mentioned Wangzaozi extract mother liquor was diluted by two times in concentration gradient successively, and then added to the corresponding 96-well plate, 1 μl / well, and each extract was tested in triplicate. The well with DMSO (final concentration 1%) was used as a control.

[0032] The Mycobacterium smegmatis cultured to the logarithmic phase (4 ml 7H9 medium, 37°C, 200 rpm, 2d, OD600=1) was collected by centrifugation at 12000 rpm for 1 min at RT. The supernatant was discarded in the super-clean bench, washed twice with 7H9 medium, and finally resuspended with 2 ml 7H9 medium, and stood for 20 min.

[0033] The OD600 of the supernatant was measured, and then diluted with 7H9 medium to OD600=0.2, and further diluted by 1000 times.

[0034] The diluted bacterial solution was added to the corresponding position of the above-mentioned 96-well plate, 99 μl / well. The 7H9 medium without bacteria was used as a blank control.

[0035] After 24 h of culture at 37°C, the fluorescence indicator Alamar Blue for cell metabolic activity detection was added, 10 μl / well.

[0036] After 15 h of incubation at 37°C, the instrument was detected with an enzyme-labeled instrument, the excitation wavelength was 540 nm, the emission wavelength was 590 nm, the instrument received 590 nm fluorescence value data was recorded, and statistical analysis was performed.

[0037] (3) Experimental results

[0038] The fluorescence value of the blank control well without cells was taken as the background, and the fluorescence value of the normal cell well without extract was taken as 100% survival rate control. The fluorescence value data of the experimental group (3 groups of different treated cells) was converted to the relative cell viability percentage according to the following formula:

[0039] Cell viability (%) = (experimental group fluorescence value - blank control fluorescence value) / (untreated group fluorescence value - blank control fluorescence value) x 100

[0040] The average value and standard deviation (SD) of 3 groups of experimental data were calculated, and a column chart was drawn Figure 1 ).

[0041] As Figure 1 shown, all the whole plant alcohol extracts of Wangjiazi and the subsequent extraction segment extracts have significant inhibitory effect on the activity of M. smegmatis, but the inhibition degree is different. Among them, the total extract, petroleum ether segment, ethyl acetate segment and other extracts in the whole plant alcohol extract can almost completely inhibit the metabolic activity of the bacteria at a concentration of ≥0.5 mg / ml; the n-butanol segment and water segment extracts have a bacterial activity of less than 80% at a concentration of ≥0.5 mg / ml; and the inhibitory ability of all extracts of the whole plant alcohol extract increases with the increase of concentration. The inhibitory ability of all extracts on the activity of M. smegmatis shows concentration dependence in the range of 0.125 mg / ml to 4 mg / ml.

[0042] The total extract, petroleum ether segment and ethyl acetate segment extracts of the whole plant alcohol extract with strong inhibitory effect on the activity of M. smegmatis were selected for half-inhibitory concentration (IC50) test on M. smegmatis, and the results are shown in Figure 2 . The calculation results show that the half-inhibitory concentration (IC50) of the total extract, ethyl acetate segment and petroleum ether segment extracts of the whole plant alcohol extract is 156.1 μg / ml, 79.8 μg / ml and 26.4 μg / ml, respectively.

[0043] Example 3, BCG antibacterial experiment of the whole plant extract of Wangjiazi.

[0044] (1) Experimental reagents and materials

[0045] Wang jujube extract, sterile water, DMSO, 7H9 medium (BD DIFCO™ Middlebrook 7H9 Broth Cat. No. 271310), OADC enrichment solution (Tube Middlebrook OADC Enrich Cat. No. 211886), Tyloxapol (Sigma-Aldrich Cat. No. T8761-50G), Alamar Blue reagent (Invitrogen™ Cat. No. DAL1100), EP tubes, 96-well plates, BCG (provided by the First Affiliated Hospital of the University of Science and Technology of China).

[0046] (2) Experimental steps

[0047] The BCG cultured to 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 medium, and finally resuspended with 2 ml of 7H9 medium, and stood for 20 min.

[0048] The OD600 of the supernatant was measured, and then diluted with 7H9 medium to OD600=0.36, and further diluted 1000-fold.

[0049] The diluted bacterial solution was added to the corresponding positions of the above 96-well plate, 99 μl / well, and the blank control was 7H9 medium without bacterial solution.

[0050] After incubation at 37°C for one week, the fluorescence indicator Alamar Blue for cell metabolic activity detection was added, 10 μl / well.

[0051] After incubation at 37°C for 24 h, 75% ethanol was added for inactivation, and the data was recorded and statistically analyzed by using an enzyme-labeled instrument with excitation wavelength of 540 nm and emission wavelength of 590 nm.

[0052] (3) Experimental results

[0053] The fluorescence value of the blank control well without cells was taken as the background, and the fluorescence value of the normal cell well without extract was taken as 100% survival rate control. The fluorescence value data of the experimental group (3 groups of different treated cells) was converted to the relative cell viability percentage according to the following formula:

[0054] Cell viability (%) = (experimental group fluorescence value - blank control fluorescence value) / (untreated group fluorescence value - blank control fluorescence value) x 100

[0055] The average value and standard deviation (SD) of the 3 groups of experimental data were calculated, and a bar graph was drawn. Figure 3

[0056] As​Figure 3 As shown in the figure, all of the alcohol extract of the whole plant of Ziziphus ju Jub and the subsequent extractive segment extract have a significant inhibitory effect on the activity of BCG, and the inhibitory effect increases with the increase of the concentration of the extract, but the inhibitory effect is quite different. The total extract of the alcohol extract of the whole plant, the petroleum ether segment extract, the ethyl acetate segment extract and other extracts almost completely inhibit the metabolic activity of the bacteria at a concentration of ≥0.25 mg / ml; the n-butanol segment extract has an average bacterial activity of less than 40% at a concentration of ≥2 mg / ml; and the water segment extract has an average bacterial activity of less than 70% at a concentration of ≥1 mg / ml.

[0057] The total extract of the alcohol extract of the whole plant, the petroleum ether segment extract and the ethyl acetate segment extract which have a strong inhibitory effect on the activity of BCG are selected to test the half-inhibitory concentration (IC50) of BCG, and the results are as shown in the figure. Figure 4 The calculation results show that the half-inhibitory concentration (IC50) of the total extract of the alcohol extract of the whole plant, the ethyl acetate segment extract and the petroleum ether segment extract is 33.4 μg / ml, 31.6 μg / ml and 27.7 μg / ml respectively.

[0058] Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art and related fields without creative labor should belong to the protection scope of the present application. The structures, devices and operation methods not specifically described and explained in the present application, such as without special description and limitation, are implemented according to the conventional means in the art.

Claims

1. An extract of the entire above-ground plant of *Ziziphus jujuba*, characterized in that: The whole plant extract of jujube (Ziziphus jujuba) is available as an alcohol extract.

2. The extract of the entire above-ground plant of *Ziziphus jujuba* according to claim 1, characterized in that: The above-ground whole herb extract of *Ziziphus jujuba* is a 95% ethanol extract.

3. The extract of the entire aerial plant of *Ziziphus jujuba* according to any one of claims 1-2, characterized in that: The above-ground whole herb alcohol extract of *Ziziphus jujuba* was obtained by petroleum ether extraction to obtain the petroleum ether fraction of the above-ground whole herb alcohol extract of *Ziziphus jujuba*.

4. The extract of the entire aerial plant of *Ziziphus jujuba* according to any one of claims 1-2, characterized in that: The above-ground whole herb alcohol extract of *Ziziphus jujuba* was extracted with ethyl acetate to obtain the ethyl acetate fraction of the above-ground whole herb alcohol extract of *Ziziphus jujuba*.

5. The extract of the entire aerial plant of *Ziziphus jujuba* according to any one of claims 1-2, characterized in that: The above-ground whole herb ethanol extract of *Ziziphus jujuba* was extracted with n-butanol to obtain the n-butanol fraction of the above-ground whole herb ethanol extract of *Ziziphus jujuba*.

6. The extract of the entire aerial plant of *Ziziphus jujuba* according to any one of claims 1-2, characterized in that: The water-soluble portion of the above-ground whole herb alcohol extract of *Ziziphus jujuba* after extraction is the aqueous fraction of the above-ground whole herb alcohol extract.

7. The extract of the entire aerial plant of *Ziziphus jujuba* according to any one of claims 1-6, characterized in that: Application of the ethanol extract of the whole plant of *Ziziphus jujuba* and its subsequent extracts in the preparation of products for anti-tuberculosis mycobacterium.

8. The extract of the entire above-ground plant of *Ziziphus jujuba* according to claim 7, characterized in that: The anti-tuberculosis mycobacterial products include anti-tuberculosis drugs and other products.

Images