Isodon amethystoides rhizome extract and application thereof

Abstract

According to the isodon amethystoides rhizome extract and the application thereof, underground isodon amethystoides rhizome parts are taken, the obtained extract is characterized by being alcohol extraction total extractum of rhizomes, the alcohol extraction total extractum is sequentially extracted through petroleum ether, ethyl acetate and n-butyl alcohol respectively, and the parts obtained through extraction and the remaining water-soluble part are separated. The isodon amethystoides rhizome extract disclosed by the invention has an effect of resisting mycobacteria, and the effect shows that the growth activity of mycobacteria including mycobacterium tuberculosis is inhibited. The isodon amethystoides rhizome extract disclosed by the invention can be used for preparing a medicine for resisting mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and more specifically, to an extract of the rhizome of jujube and its application. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Drug resistance in Mtb includes genetic resistance and phenotypic resistance (i.e., persistent bacteria). Mtb naturally resists most antibiotics, often requiring long-term treatment with multiple anti-tuberculosis drugs. Patients suffer greatly from the side effects of these drugs, experiencing significant psychological stress. This leads to poor patient adherence, treatment interruptions, and antibiotic overuse, ultimately driving the rapid evolution of Mtb. The accumulation of drug-resistant mutations has led to the emergence of extensively drug-resistant (XDR) and totally drug-resistant (TDR) strains. With the increasing prevalence of drug-resistant TB, treatment durations for resistant cases are longer, posing significant challenges to traditional treatment regimens. Therefore, the clinical treatment of TB urgently requires highly effective treatment regimens with shorter treatment courses, fewer drug types, and fewer side effects.

[0003] In Mycobacterium tuberculosis research, BCG (Bacillus Calmette-Guérin) and Mycolicibacterium smegmatis are the main models. Compared to Mycobacterium tuberculosis, which doubles in approximately 18-24 hours, Mycolicibacterium smegmatis doubles in only 3-5 hours, facilitating rapid data acquisition in laboratories. Therefore, Mycolicibacterium smegmatis is an ideal alternative model for tuberculosis research and is commonly used in anti-tuberculosis drug development, gene function analysis, and bacterial physiological mechanism research. BCG is a live attenuated vaccine of Mycobacterium bovis, used for tuberculosis prevention. Due to its safety and genetic similarity to Mycobacterium tuberculosis, BCG is often used as a substitute strain to simulate in vitro antibacterial experiments of human Mycobacterium tuberculosis. BCG can be used to rapidly screen compounds with in vitro antibacterial activity, providing experimental evidence for the development of new anti-tuberculosis drugs.

[0004] While Western medicine treatment for tuberculosis is relatively routine, its components have significant side effects and are prone to producing drug-resistant strains, leading to high treatment costs for drug-resistant tuberculosis. Traditional Chinese medicine (TCM) has a long history of treating tuberculosis, regulating immune function and improving the patient's constitution through methods such as tonifying Qi (e.g., Astragalus membranaceus, Ginseng) and nourishing Yin (e.g., Ophiopogon japonicus, Asparagus cochinchinensis). Simultaneously, Western anti-tuberculosis drugs can easily cause liver damage and gastrointestinal discomfort; TCM alleviates these adverse reactions and improves treatment adherence by soothing the liver and regulating Qi (Bupleurum chinense) and strengthening the spleen and stomach (Poria cocos). A current limitation of TCM in treating tuberculosis is that it must be used in combination with Western anti-tuberculosis drugs (e.g., rifampin, isoniazid) and cannot be used alone to replace conventional treatment. However, given the long history and wide availability of TCM, it is possible to screen for effective anti-tuberculosis components and develop new anti-tuberculosis preparations, with significant potential for application.

[0005] Wangzaozi, scientifically known as *Corydalis yanhusuo*, is a plant belonging to the genus *Corydalis* in the Lamiaceae family. It is a commonly used traditional Chinese medicine in Suzhou, Anhui Province, where the whole plant is often used clinically. Local people have long used it primarily to treat lung diseases such as lung abscess and chronic bronchitis. Over the years, the properties of Wangzaozi have gradually gained attention, and some unique tetracyclic diterpenoid components have been discovered and studied. However, there are currently no research reports on the use of Wangzaozi for anti-tuberculosis purposes. Summary of the Invention

[0006] The purpose of this invention is to provide a jujube rhizome extract and its application, in order to solve the technical problems existing in the background art.

[0007] This invention provides a *Ziziphus jujuba* root and stem alcohol extract, which is prepared using an ethanol solvent impregnation method. Specifically, the underground part of the *Ziziphus jujuba* root and stem is taken, dried, and then pulverized.

[0008] Furthermore, at room temperature, the root and stem powder of *Ziziphus jujuba* was extracted with fresh 95% ethanol to obtain an extract.

[0009] Furthermore, the above alcohol extract was concentrated under reduced pressure and dried at room temperature to obtain the total alcohol extract of jujube rhizome.

[0010] Further, the total extract was dissolved in water and then extracted sequentially with solvents of increasing polarity.

[0011] Further, the sample was extracted with petroleum ether, and the petroleum ether fraction was obtained after repeated extraction.

[0012] Further, the sample was extracted with ethyl acetate, and the ethyl acetate fraction was obtained after repeated extraction.

[0013] Further, the sample was extracted with n-butanol, and the n-butanol fraction was obtained after repeated extraction.

[0014] Further, after extraction, the water is collected.

[0015] This yielded the total alcohol extract of the underground rhizome of *Ziziphus jujuba*, including petroleum ether, ethyl acetate, n-butanol, and water fractions. Each extraction fraction was obtained sequentially according to the polarity of the extraction solvent, from weakest to strongest. Therefore, components similar to those soluble in each extraction solvent were separated from the total alcohol extract, resulting in a more refined composition. The total alcohol extract of *Ziziphus jujuba* rhizome and its various extraction fractions were finally evaporated and concentrated at room temperature to obtain a paste.

[0016] The beneficial effects of the technical solution of this invention are:

[0017] (1) Through in vitro antibacterial tests, the present invention confirms that the provided jujube rhizome alcohol extract has a significant inhibitory effect on Mycobacterium smegmatis and BCG, and the effect is dose-dependent. Its subsequent extraction fractions, including petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction, also have different degrees of inhibitory effect on Mycobacterium smegmatis and BCG.

[0018] (2) Through in vitro antibacterial half-inhibitory concentration (IC50) test, the present invention confirms that the provided ethanol extract of the rhizome of the Chinese jujube and its respective extract fractions have different antibacterial effects against Mycobacterium smegmatis and BCG, indicating that they may contain different antibacterial components of Mycobacterium smegmatis and BCG. Attached Figure Description

[0019] Figure 1 This invention provides an antibacterial experiment on the ethanol extract of jujube rhizome against Mycobacterium smegmae.

[0020] Figure 2 The half-maximal inhibitory concentration (IC50) of the ethanol extract of *Ziziphus jujuba* rhizome against *Mycobacterium smegmatis* was tested according to the present invention.

[0021] Figure 3 This invention provides an antibacterial experiment on BCG using the alcoholic extract of jujube rhizome.

[0022] Figure 4 This invention relates to the test of the half-maximal inhibitory concentration (IC50) of the ethanol extract of jujube rhizome against BCG. Detailed Implementation

[0023] The present invention will now be described in further detail. The embodiments of the invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose.

[0024] Example 1: Extraction and preparation of *Ziziphus jujuba* rhizome samples.

[0025] (1) Extraction of *Ziziphus jujuba* rhizome: The dried *Ziziphus jujuba* rhizome was ground into powder. 100 grams of powder was added to 500 ml of 95% ethanol (volume percentage) and repeatedly extracted at room temperature. After filtering the extract, it was evaporated and concentrated at 40°C, and then dried in a water bath at 45°C for about one week to obtain the extract, which is the total extract of *Ziziphus jujuba* rhizome. Approximately 26 grams of total extract were obtained from every 100 grams of rhizome.

[0026] (2) Extraction: 26g of the total extract of *Ziziphus jujuba* rhizome was added to a 500ml separatory funnel, and 200ml of water was added and stirred to form a suspension. First, 200ml of petroleum ether was added for extraction, and the extraction was repeated 3 times. The extract was concentrated at 40°C to obtain the petroleum ether fraction extract. The petroleum ether fraction extract was dried in a 45°C water bath for one week to obtain the petroleum ether fraction extract of *Ziziphus jujuba* rhizome, yielding approximately 6.8g. Then, 200ml of ethyl acetate was added for extraction, and the extraction was repeated 3 times. The extract was concentrated at 40°C to obtain the ethyl acetate fraction extract. The ethyl acetate fraction extract was dried in a 45°C water bath for one week to obtain the ethyl acetate fraction extract of *Ziziphus jujuba* rhizome. The ethyl acetate fraction extract yielded approximately 8.0 g; then, 200 mL of n-butanol was added for extraction, and the extraction was repeated three times. The extract was concentrated at 40°C to obtain a n-butanol fraction extract. The n-butanol fraction extract was dried in a water bath at 45°C for one week to obtain a n-butanol fraction extract of *Ziziphus jujuba* rhizome, yielding approximately 4.5 g; the remaining extract was concentrated at 40°C to obtain an aqueous fraction extract. The aqueous fraction extract was dried in a water bath at 45°C for one week to obtain an aqueous fraction extract of *Ziziphus jujuba* rhizome, yielding approximately 3.0 g.

[0027] Example 2: Antibacterial experiment of the extract of jujube rhizome against Mycobacterium smegmatis.

[0028] (1) Experimental reagents and materials

[0029] Rhizoma extract of Ziziphus jujuba, sterile water, DMSO, 7H9 medium (BD DIFCO™ Middlebrook 7H9Broth, catalog number 271310), OADC enrichment broth (Tube Middlebrook OADC Enrich, catalog number 211886), tetrabutylphenol tyloxapol (Sigma-Aldrich, catalog number T8761-50G), Alamar Blue reagent (Invitrogen™, catalog number DAL1100), EP tubes, 96-well plates, and Mycobacterium smegmatis (provided by the University of Science and Technology of China).

[0030] (2) Experimental steps

[0031] Except for the aqueous extract, all extracts from the rhizome of *Ziziphus jujuba* were dissolved in dimethyl sulfoxide (DMSO). The aqueous extract was dissolved in sterile water to prepare stock solutions of 100-400 mg / mL. These stock solutions were serially diluted two-fold and added to the corresponding 96-well plates at 1 μl / well. Each extract was tested in triplicate. Wells containing DMSO (final concentration 1%) served as controls.

[0032] Mycobacterium smegmatis cultured to the logarithmic growth phase (4 ml 7H9 medium, 37℃, 200 rpm, 2 days, OD600=1) was collected and centrifuged at 12000 rpm for 1 min, then RT. The supernatant was discarded in a clean bench, the sample was washed twice with 7H9 medium, and finally resuspended thoroughly with 2 ml 7H9 medium and allowed to stand for 20 min.

[0033] Take the supernatant and measure the OD600, then dilute it with 7H9 medium to OD600=0.2, and then further dilute it 1000 times.

[0034] Add the diluted bacterial solution to the corresponding positions in the 96-well plate, 99 μl / well. The wells containing 7H9 medium without bacteria serve as blank controls.

[0035] After culturing at 37℃ for 24 hours, add 10 μl of Alamar Blue, a fluorescent indicator for detecting cell metabolic activity, to each well.

[0036] After incubation at 37℃ for 15 hours, the samples were detected using an ELISA reader with an excitation wavelength of 540nm and an emission wavelength of 590nm. The fluorescence values ​​received by the instrument at 590nm were recorded and statistically analyzed.

[0037] (3) Experimental results

[0038] 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:

[0039] Cell viability (%) = (Fluorescence value of experimental group - Fluorescence value of blank control) / (Fluorescence value of untreated group - Fluorescence value of blank control) × 100

[0040] Calculate the mean and standard deviation (SD) of the three sets of experimental data, and plot a bar chart. Figure 1 ).like Figure 1As shown, all the rhizome alcohol extracts and subsequent fractional extracts of *Ziziphus jujuba* showed significant inhibitory effects on *Mycobacterium smegmatis* activity, although the degree of inhibition varied. Specifically, the petroleum ether and n-butanol fractions of the rhizome alcohol extract significantly inhibited bacterial metabolic activity at concentrations ≥0.5 mg / ml, and almost completely inhibited it at concentrations ≥1 mg / ml and 4 mg / ml, respectively. The total alcohol extract and ethyl acetate fraction of the rhizome alcohol extract showed similar inhibitory abilities, almost completely inhibiting bacterial metabolic activity at concentrations ≥2 mg / ml. The aqueous alcohol extract showed slightly weaker inhibitory activity, but this increased with increasing concentration. The inhibitory activity of all extracts on *Mycobacterium smegmatis* activity showed a concentration-dependent effect within the range of 0.25 mg / ml to 4 mg / ml.

[0041] The total extract, ethyl acetate fraction, petroleum ether fraction, and n-butanol fraction of the above-mentioned rhizome alcohol extract were tested for their half-maximal inhibitory concentration (IC50) against Mycobacterium smegma. The results are as follows: Figure 2 The calculated half-maximal inhibitory concentrations (IC50) of the total extract, ethyl acetate fraction, petroleum ether fraction, and n-butanol fraction of the rhizome extract were 862.9 μg / ml, 441 μg / ml, 268.7 μg / ml, and 7.0 μg / ml, respectively. The n-butanol fraction of the rhizome extract showed significantly better antibacterial effect than the other extract fractions.

[0042] Example 3: Antibacterial experiment of the rhizome extract of *Ziziphus jujuba* against BCG.

[0043] (1) Experimental reagents and materials

[0044] Jujube extract, sterile water, DMSO, 7H9 culture medium (BD DIFCO™ Middlebrook 7H9 Broth, catalog number 271310), OADC enrichment broth (Tube Middlebrook OADC Enrich, catalog number 211886), tetrabutylphenol tyloxapol (Sigma-Aldrich, catalog number T8761-50G), Alamar Blue reagent (Invitrogen™, catalog number DAL1100), EP tubes, 96-well plates, and BCG (provided by the First Affiliated Hospital of the University of Science and Technology of China).

[0045] (2) Experimental steps

[0046] Collect BCG cultured to the logarithmic growth phase (8 ml of bacterial culture, incubated at 37°C with OD600 = 0.2), centrifuge at 12000 rpm for 1 min, then RT. Discard the supernatant in a clean bench, wash twice with 7H9 medium, and finally resuspend thoroughly in 2 ml of 7H9 medium and incubate for 20 min.

[0047] Take the supernatant and measure the OD600. Then dilute it with 7H9 medium to OD600=0.36, and then further dilute it 1000 times.

[0048] Add the diluted bacterial solution to the corresponding positions in the 96-well plate, 99 μl / well. The blank control is 7H9 medium without bacterial solution.

[0049] 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.

[0050] 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.

[0051] (3) Experimental results

[0052] 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:

[0053] Cell viability (%) = (Fluorescence value of experimental group - Fluorescence value of blank control) / (Fluorescence value of untreated group - Fluorescence value of blank control) × 100

[0054] Calculate the mean and standard deviation (SD) of the three sets of experimental data, and plot a bar chart. Figure 3 ).like Figure 3 As shown, all the rhizome alcohol extracts and subsequent fractional extracts of *Ziziphus jujuba* showed significant inhibitory effects on BCG activity. The total alcohol extract of *Ziziphus jujuba* rhizome, including the petroleum ether and ethyl acetate fractions, significantly inhibited the metabolic activity of the bacteria at concentrations ≥0.5 mg / ml, and almost completely inhibited BCG metabolic activity at concentrations ≥1 mg / ml, ≥0.5 mg / ml, and ≥0.25 mg / ml, respectively. The aqueous alcohol extract of the rhizome showed weaker inhibitory activity, but this increased with increasing concentration. The n-butanol extract of the rhizome showed weak inhibitory activity, and no concentration-dependent effect was observed within the range of extract concentrations used.

[0055] The total extract, ethyl acetate, and petroleum ether fractions of the rhizome ethanol extract, which exhibit strong BCG inhibition activity as described above, were tested for their half-maximal inhibitory concentration (IC50) against BCG. The results are as follows: Figure 4 The calculated half-maximal inhibitory concentrations (IC50) of the total extract of rhizome alcohol, ethyl acetate, and petroleum ether were 322.8 μg / ml, 55.6 μg / ml, and 263.6 μg / ml, respectively.

[0056] 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 rhizome extract of jujube, characterized in that: Including the alcohol extract of jujube root and stem.

2. The extract of *Ziziphus jujuba* root and rhizome according to claim 1, characterized in that: The ethanol extract of the rhizome of the jujube tree is a 95% ethanol extract.

3. The extract of *Ziziphus jujuba* rhizome according to any one of claims 1-2, characterized in that: The ethanol extract of jujube rhizome was obtained by petroleum ether extraction to obtain the petroleum ether fraction of jujube rhizome ethanol extract.

4. A rhizome extract of Ziziphus jujuba according to any one of claims 1-2, characterized in that: The ethanol extract of the rhizome of the Chinese jujube was extracted with ethyl acetate to obtain the ethyl acetate fraction of the ethanol extract of the Chinese jujube.

5. A rhizome extract of *Ziziphus jujuba* according to any one of claims 1-2, characterized in that: The ethanol extract of the rhizome of the Chinese jujube was extracted with n-butanol to obtain the n-butanol fraction of the ethanol extract of the Chinese jujube.

6. The extract of *Ziziphus jujuba* rhizome according to any one of claims 1-2, characterized in that: The water-soluble portion remaining after extraction of the jujube root and stem alcohol extract is the water fraction of the jujube alcohol extract.

7. A rhizome extract of Ziziphus jujuba according to any one of claims 1-6, characterized in that: Application of the ethanol extract of jujube rhizome and its subsequent extracts in the preparation of products for anti-tuberculosis mycobacterium.

8. The extract of *Ziziphus jujuba* root and rhizome according to claim 7, characterized in that: The anti-tuberculosis mycobacterial products include anti-tuberculosis drugs and other products.

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