Water extraction extract of overground whole herb of isodon amethystoides and application of water extraction extract

Abstract

The invention discloses a common rabdosia leaf above-ground whole herb water extraction extract and application thereof, the above-ground whole herb part of common rabdosia leaves is taken, the obtained extract is characterized by being a whole herb water extraction total extract, petroleum ether and ethyl acetate are respectively and sequentially used for extraction, and the respective extracted 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, and more particularly to a water extract of the whole plant of Evonymus alata Buch.-Ham and an 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 worldwide, which has long threatened the global public health security. In recent years, the increase of multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) cases has brought great challenges to traditional treatment methods. This type of tuberculosis has poor treatment effect on conventional antibiotics, long course of treatment, large side effects, and low cure rate, and new treatment strategies are urgently needed to intervene. In addition, HIV co-infection, the increase of the aging population and the problem of urban floating population have further complicated the spread of TB.

[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 the in vitro bacteriostatic experiment 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] In recent years, domestic and foreign traditional Chinese medicine research institutions have begun to combine with modern biotechnology to explore the molecular mechanism of the effect of Chinese medicine ingredients on tuberculosis bacillus and the clinical efficacy of Chinese medicine combined with western medicine. For example, the multi-center clinical trial of "integrated traditional Chinese and western medicine treatment of drug-resistant tuberculosis" has shown certain efficacy and has been included in the diagnosis and treatment specifications in some areas. However, the promotion of traditional Chinese medicine in TB prevention and treatment still faces challenges, including insufficient standardized research, limited clinical evidence system, and quality control problems of traditional Chinese medicine materials. Therefore, it is urgent to separate and purify effective active components from complex Chinese herbal medicine crude extracts to further discover and develop new drugs for potential treatment of tuberculosis.

[0005] Rabdosia lophanthoides Hara, scientific name Rabdosia lophanthoides, is a plant of the Labiatae Rabdosia genus, a folk traditional Chinese medicine in Suzhou, Anhui Province, and is clinically used for treating lung abscess, chronic bronchitis and other lung diseases. Over the years, the characteristics of Rabdosia lophanthoides have gradually attracted attention, and some unique components of tetracyclic diterpenes have also been discovered and studied. However, there is no report on the use of Rabdosia lophanthoides for anti-mycobacterium tuberculosis. SUMMARY

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

[0007] The technical scheme of the present application provides a water extract of Rabdosia lophanthoides aerial whole grass, specifically, taking the whole grass of the aerial part of Rabdosia lophanthoides, drying and pulverizing;

[0008] Further, the Rabdosia lophanthoides aerial whole grass powder is heated and extracted at 100°C to obtain an extract;

[0009] Further, the above alcohol extract is concentrated under reduced pressure, and the water extract total extract is obtained after drying at room temperature;

[0010] Further, the water extract total extract is dissolved with water, and is extracted with solvents with polarity from weak to strong in turn;

[0011] Further, the petroleum ether extract is extracted, and the petroleum ether segment is taken after repeated extraction;

[0012] Further, the ethyl acetate extract is extracted, and the ethyl acetate segment is taken after repeated extraction;

[0013] Thus, the water extract of Rabdosia lophanthoides aerial whole grass petroleum ether segment and ethyl acetate segment is obtained, and finally the paste is obtained by evaporation and concentration at room temperature.

[0014] The beneficial effects of the technical scheme of the present application are:

[0015] (1) Through in vitro bacteriostatic test, the present application proves that the water extract of Rabdosia lophanthoides aerial whole grass provided has a significant inhibitory effect on BCG, and shows a dose-dependent effect.

[0016] (2) Through in vitro bacteriostatic half-inhibitory concentration (IC50) test, the present application proves that the different water extracts of Rabdosia lophanthoides aerial whole grass have BCG bacteriostatic effect, showing that they may contain different BCG bacteriostatic components. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 For the bacteriostatic experiment of the water extract of Rabdosia lophanthoides aerial whole grass on BCG,

[0018] Figure 2The half inhibitory concentration (IC50) of the water extract of the aboveground whole plant of Ziziphus ju Jubiana Pall. to BCG was tested. DETAILED DESCRIPTION

[0019] The application is further described in detail below, the embodiments of the application are given for the convenience of example and description, and are not exhaustive or limit the application to the disclosed forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments are chosen and described in order to better illustrate the principles and practical application of the application, and to enable those skilled in the art to understand the application in order to design various embodiments with various modifications suitable for specific purposes.

[0020] Example 1, extraction and preparation of the water extract of the aboveground whole plant of Ziziphus jujubiana Pall.

[0021] (1) Extraction of the aboveground whole plant of Ziziphus jujubiana Pall.: dry the aboveground whole plant of Ziziphus jujubiana Pall. into powder, add appropriate amount of water, heat at 100°C for 1.5h, repeat extraction three times, filter the extract, evaporate and concentrate at 40°C, dry in a water bath at 45°C for about one week to obtain the extract, which is the water extract of the aboveground whole plant of Ziziphus jujubiana Pall., about 8g of extract is obtained from 100g of whole plant.

[0022] (2) Extraction: dissolve about 500g of the water extract of the aboveground whole plant of Ziziphus jujubiana Pall. with water, extract with petroleum ether and ethyl acetate in sequence. First, extract with petroleum ether, extract three times, concentrate the extract at 40°C to obtain the petroleum ether extract, dry the petroleum ether extract in a water bath at 45°C for about one week to obtain the petroleum ether extract of the aboveground whole plant of Ziziphus jujubiana Pall.; then extract with ethyl acetate, extract three times, concentrate the extract at 40°C to obtain the ethyl acetate extract, dry the ethyl acetate extract in a water bath at 45°C for about one week to obtain the ethyl acetate extract of the aboveground whole plant of Ziziphus jujubiana Pall. Finally, obtain the water extract of the petroleum ether extract (8.084g) and the water extract of the ethyl acetate extract (20.0g).

[0023] Example 2, antibacterial experiment of the water extract of the aboveground whole plant of Ziziphus jujubiana Pall. to BCG.

[0024] (1) Experimental reagents and materials

[0025] Wangjuzi extract, sterile water, DMSO, 7H9 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).

[0026] (2) Experimental procedure

[0027] The water extract of the whole plant of Wangjuzi was dissolved in dimethyl sulfoxide (DMSO) to prepare a 200 mg / ml stock solution. The Wangjuzi extract stock solution was serially diluted by two-fold concentration gradient, 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.

[0028] 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 medium, and finally resuspended with 2 ml of 7H9 medium, and allowed to stand for 20 min.

[0029] The OD600 of the supernatant was measured, and then diluted with 7H9 medium to OD600=0.36, and further diluted by 1000-fold.

[0030] The diluted bacterial solution was added to the corresponding positions of the above-mentioned 96-well plates at 99 μl / well. The blank control wells contained 7H9 medium without bacterial solution.

[0031] After incubation at 37°C for one week, the fluorescent indicator Alamar Blue for cell metabolic activity detection was added at 10 μl / well.

[0032] After incubation at 37°C for 24 h, 75% ethanol was added to inactivate the cells, and the data was recorded and statistically analyzed by using a microplate reader at an excitation wavelength of 540 nm and an emission wavelength of 590 nm.

[0033] (3) Experimental results

[0034] The fluorescence value of the blank control wells without cells was taken as the background, and the fluorescence value of the normal cell wells without extract was taken as 100% survival rate control. The fluorescence value data of the experimental groups (three groups of cells treated differently) were converted to the relative cell viability percentage according to the following formula:

[0035] Cell viability (%) = (experimental group fluorescence value - blank control fluorescence value) / (untreated group fluorescence value - blank control fluorescence value) x 100

[0036] The average and standard deviation (SD) of 3 groups of experimental data were calculated, and a bar graph was drawn Figure 1 ).

[0037] As shown in Figure 1 , all the water extract of the whole plant of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu had a significant inhibitory effect on the BCG viability. The petroleum ether fraction and ethyl acetate fraction of the water extract of the whole plant almost completely inhibited the metabolic activity of the bacteria at a concentration of ≥0.25 mg / ml.

[0038] The water extract of the whole plant of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. Fu described above was selected to test the half-inhibitory concentration (IC50) against BCG, and the results are shown in Figure 2 . The calculation results showed that the half-inhibitory concentration (IC50) of the petroleum ether fraction and the ethyl acetate fraction of the water extract of the whole plant were 36.4 μg / ml and 11.4 μg / ml, respectively

[0039] 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 of ordinary skill in the art and related fields without creative labor should fall within the scope of protection of the present application. The structures, devices and operation methods not specifically described and explained in the present application, if not specifically described and limited, are implemented according to the conventional means in the art.

Claims

1. A water extract of the whole aerial parts of Crataegus pinnatifida Bge. characterized in that: The water extraction of the whole plant of Vitis amurensis Rupr. is a boiling water extraction.

2. The extract of Wangjuzi according to any one of claim 1, characterized in that: The alcohol extraction of the whole plant of Vitis amurensis Rupr. is obtained by extracting the water extraction of the whole plant of Vitis amurensis Rupr. with petroleum ether.

3. The extract of Wangjuzi according to any one of claim 1, characterized in that: The alcohol extraction of the whole plant of Vitis amurensis Rupr. is obtained by extracting the water extraction of the whole plant of Vitis amurensis Rupr. with ethyl acetate.

4. The extract of Wangjuzi according to any one of claims 1-3, characterized in that: The water extraction of the whole plant of Vitis amurensis Rupr. and the subsequent extraction products thereof are used for preparing anti-tuberculosis Mycobacterium products.

5. The extract of Wangjuzi according to claim 4, characterized in that: The anti-tuberculosis Mycobacterium products include anti-tuberculosis Mycobacterium drugs and other products.

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