Application of botrychium ternatum total extract and petroleum ether layer extract in anti-tuberculosis activity and preparation method thereof

Abstract

The invention discloses application of botrychium ternatum total extract and petroleum ether layer extract in antituberculous activity and a preparation method of the botrychium ternatum total extract and the petroleum ether layer extract, and tests prove that the botrychium ternatum total extract and the petroleum ether layer extract show remarkable antituberculous activity and particularly have a specific inhibition effect on multidrug-resistant strains (such as Mtb706). It is prompted that the compound can possibly become a potential alternative drug source for multi-drug resistant tuberculosis (MDR-TB). Meanwhile, active substances of the petroleum ether layer extract provide a clear direction for subsequent separation and identification, and the screening range of the antituberculous active ingredients in the botrychium ternatum is effectively narrowed. And a methodological foundation is laid for extraction process optimization and active component targeting research of the botrychium ternatum antituberculous drug.

Description

TECHNICAL FIELD

[0001] The application relates to application of total extract of Botrychium ternatum and petroleum ether layer extract in anti-tuberculosis activity and a preparation method thereof, and belongs to the technical field of plant extract application. BACKGROUND

[0002] It is reported that after two years of follow-up observation on patients with initial treatment sputum culture positive pulmonary tuberculosis using a standardized treatment scheme, it is found that the sputum culture non-negative conversion or positive conversion rate in the MDR-TB group is 51.7%, and that in the non-drug-resistant group is only 4.1%. The long transmission time, complex treatment scheme, long treatment course, high cost, low treatment compliance and low cure rate of drug-resistant tuberculosis have become a severe challenge for tuberculosis prevention and treatment.

[0003] At present, clinical anti-tuberculosis drugs mainly include first-line drugs such as isoniazid, rifampicin, ethambutol and pyrazinamide, and second-line oral drugs such as levofloxacin, moxifloxacin, terizidone, ethionamide, cycloserine and p-aminosalicylic acid. In recent years, new anti-tuberculosis drugs such as bedaquiline, delamanid and pretomanid and the drug clofazimine and linezolid which is reused are gradually used for drug-resistant tuberculosis treatment. Although the above chemical drugs play an important role in anti-tuberculosis treatment, the emergence of drug resistance and many side effects such as hepatotoxicity, neurotoxicity, cardiotoxicity and gastrointestinal discomfort seriously affect the treatment effect.

[0004] National medicine is the theoretical basis of medicine produced and formed by a nation in a long history of practice, and is the sum of generation after generation accumulation, which gathers long oral tradition, unique national regionalism and rich traditional background. It is very urgent and necessary to excavate and inherit the anti-tuberculosis effect of local traditional medicine for tuberculosis and drug-resistant tuberculosis treatment. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an application of total extract of Botrychium ternatum and petroleum ether layer extract in anti-tuberculosis activity and a preparation method thereof, which can overcome the shortcomings of the prior art.

[0006] The technical scheme of the present application is an application of total extract of Botrychium ternatum in anti-tuberculosis activity.

[0007] The aforementioned application of petroleum ether layer extract of Botrychium ternatum in anti-tuberculosis activity.

[0008] The preparation method of the total extract of Botrychium ternatum is as follows: 2 parts of dried and pulverized whole grass of Botrychium ternatum are weighed, soaked in 3-4 parts of 95% anhydrous ethanol for 20-40 minutes, extracted by heating reflux extraction method for 3 times, 2.0 hours each time, the filtrates of the three times are collected and combined, and the combined filtrate is concentrated under reduced pressure on a rotary evaporator under the condition of vacuum degree-0.1~0.08MPa and temperature 45-55℃ to obtain the extract.

[0009] The method for preparing the petroleum ether extract of *Pteris vittata* is as follows: a. Add the extracted *Pteris vittata* extract to boiling water and stir until dissolved to obtain a suspension. The ratio of *Pteris vittata* extract to boiling water is 1:1. b. Pour the suspension obtained in step a into a separatory funnel, then pour an equal volume of petroleum ether into the separatory funnel, shake and mix repeatedly, let stand for 5-10 minutes, and collect the petroleum ether layer after the obvious separation is visible to the naked eye. c. Add an equal volume of petroleum ether to the aqueous layer from step b and extract again. Repeat this process three times. Combine the three petroleum ether extracts and extract the petroleum ether extract using a rotary evaporator.

[0010] Compared with existing technologies, this invention targets tuberculosis by identifying *Dryopteris oryzae* through the analysis of traditional Chinese medicinal herbs. The earliest record of *Dryopteris oryzae*'s medicinal application is found in the *Illustrated Materia Medica*. It is sweet and bitter in taste, slightly cold in nature, and enters the lung and liver meridians. It is a perennial herb belonging to the genus *Dryopteris* of the family Dryopterisceae in the order Diplophyceales. Later, it was also recorded in minority ethnic works such as *A Compendium of Commonly Used Folk Herbs* and *Miao Medicine*, indicating its long history of medicinal use. Modern research has found that *Dryopteris oryzae* is rich in flavonoids, polysaccharides, organic acids, coumarins, phenols, saponins, and other compounds. Among these, flavonoids, terpenes, and steroids possess multiple biological activities, including antioxidant, anti-inflammatory, antibacterial, and antitumor effects.

[0011] *Dryopteris shadiola*, also known by various folk names such as "One Cloud," "Snake-like Back," "Blood-Scattering," "Sky-Breaking Cloud," "Little Spring Flower," "Snake Unseen," "Standing Golden Rooster," "Single-Horned Artemisia," "Winter Grass," and "Huanglianqi," has a short, erect rhizome, short petioles, and slender petioles for the vegetative leaves. The leaves are broadly triangular and tripinnately divided. The sporophytes have long petioles, and the sporangia are conical with loose, slightly spreading, and hairless spikelets. It mainly grows in temperate regions, typically in shady shrub forests, ravines, damp places in valleys, or grassy slopes at altitudes of 800-2500 meters. In my country, it is widely distributed throughout the country, thus possessing the advantages of abundant resources and low cost.

[0012] Meanwhile, the present invention has yielded the following results through a series of experiments: 1. Based on the observation of Mycobacterium smegma ( Mycobacterium smegmatis, M.sm Preliminary verification showed that the total extract and petroleum ether extract of *Dryopteris smegmatis* exhibited significant antibacterial activity against *Mycobacterium smegmatis*. Both the total extract (37.5 mg / mL) and the petroleum ether extract (300 mg / mL) showed high sensitivity to *Mycobacterium smegmatis* (average inhibition zones of 18.33 mm and 28.67 mm, respectively), with a MIC of 1.875 mg / mL, demonstrating their potent antibacterial potential.

[0013] 2. Based on the analysis of Mycobacterium tuberculosis ( Mycobacterium tuberculosisPreliminary validation of *Mycobacterium tuberculosis* (Mtb) H37Rv showed that the minimum inhibitory concentration (MIC) of the total extract of *Pteris vittata* against the standard strain H37Rv (ATCC27294) was 937 µg / mL, indicating that it has certain antibacterial activity. The MIC of the extract after petroleum ether extraction significantly decreased to 234 µg / mL, indicating that the lipid-soluble active ingredients in *Pteris vittata* have a stronger antibacterial effect against *Mycobacterium tuberculosis* H37Rv, approximately four times that of the total extract.

[0014] 3. Based on preliminary verification of the clinical multidrug-resistant tuberculosis strain Mtb706, the minimum inhibitory concentration (MIC) of the total extract of *Pteris vittata* against the clinical multidrug-resistant strain Mtb706 was 117 µg / mL, and the MIC of the petroleum ether extract of *Pteris vittata* against the clinical multidrug-resistant strain Mtb706 was ≤7.3 µg / mL, indicating that it has a strong antibacterial effect.

[0015] This demonstrates that the total extract and petroleum ether extract of *Fernonia yinyuanensis* exhibit significant anti-tuberculosis activity, particularly showing specific inhibitory effects against multidrug-resistant tuberculosis (Mtb706), suggesting it may be a potential alternative drug source for MDR-TB. Furthermore, the active substances in the petroleum ether extract provide a clear direction for subsequent isolation and identification, effectively narrowing the screening range of anti-tuberculosis active components in *Fernonia yinyuanensis*. This lays a methodological foundation for optimizing the extraction process of anti-tuberculosis drugs from *Fernonia yinyuanensis* and for targeted research on active components. Attached Figure Description

[0016] Figure 1 Antibacterial activity of total extract and petroleum ether extract of *Dryopteris smegmatis* against *Mycobacterium smegmatis* (solid agar disc diffusion method); Figure 1-total extract of *Dryopteris smegmatis* 37.5 mg / mL; 2-petroleum ether extract of *Dryopteris smegmatis* 300 mg / mL; 3-kanamycin 250 μg / mL.

[0017] Figure 2 Minimum inhibitory concentrations (MICs) of total extract and petroleum ether extract of *Dryopteris smegmatis* against *Mycobacterium smegmatis* (micro-broth dilution method for drug susceptibility testing); 1-Kanamycin concentration gradient (μg / mL): 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0626; 2-Total extract of *Dryopteris smegmatis*; 3-Petroleum ether extract of *Dryopteris smegmatis*; 2- and 3-Concentration gradients (μg / mL): 15000, 7500, 3750, 1875, 937, 468, 234, 117, 58.5, 29.2; 4-DMSO control, final concentration: 10%.

[0018] Figure 3Microplate susceptibility testing results of Mycobacterium tuberculosis standard strain H37Rv; 1-total extract of *Pteris vittata*; 2-blank control of total extract of *Pteris vittata*; 3-petroleum ether extract of *Pteris vittata*; 4-blank control of petroleum ether extract of *Pteris vittata*; 5-rifampin; 6-DMSO control; Concentration gradients (µg / mL) of 1-2-3-4 in the figure: 7500, 3750, 1875, 937, 468, 234, 117, 58.5, 29.2, 14.6, 7.3; Concentration gradient (µg / mL) of 5: 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625.

[0019] Figure 4 Microplate susceptibility testing results of clinically multidrug-resistant strain Mtb706; Figure 1: Total extract of *Dryopteris crassirhizoma*; 2: Total extract of *Dryopteris crassirhizoma* (blank drug control); 3: Petroleum ether extract of *Dryopteris crassirhizoma*; 4: Petroleum ether extract of *Dryopteris crassirhizoma* (blank drug control); 5: Rifampin; 6: DMSO control. Concentration gradients 1-2-3-4 (µg / mL): 7500, 3750, 1875, 937, 468, 234, 117, 58.5, 29.2, 14.6, 7.3; Concentration gradient 5 (µg / mL): 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625. Detailed Implementation

[0020] This embodiment is illustrated through an experimental method.

[0021] Test equipment 1.1 Plant materials The *Dryopteris* used in this experiment was purchased in Bijie, Guizhou Province in October 2023. It belongs to the genus *Dryopteris* of the family Dryopteris in the order Phyllostachyales. Botrychium teratum (thunb) Sw).

[0022] Experimental reagents Petroleum ether (analytical grade), dichloromethane (analytical grade), silica gel, thin-layer chromatography silica gel plate (GF254), Middlebrook 7H9 broth, Middlebrook 7H10 agar, Tween-80, glucose, neutral Roche medium, 96-well antimicrobial susceptibility plate (blank).

[0023] Instruments and equipment Floor-standing continuous pulverizer, digital display constant temperature water bath, circulating water multi-purpose vacuum pump, rotary evaporator, electric constant temperature drying oven, pure water system, biosafety cabinet, vertical high pressure steam sterilizer, BSP-TB96 microbial susceptibility analysis system and BSJ-9612 automatic sampler, BAC spreade1100C ultrasonic disperser.

[0024] 2. Experimental Methods 2.1 Extraction of total extract from *Dryopteris crassirhizoma* and extraction of petroleum ether extract from *Dryopteris crassirhizoma* Weigh 20 kg of the whole plant of *Pteris vittata* after pulverization, soak it in 60 kg of 95% anhydrous ethanol for half an hour, and use the heating reflux extraction method to extract it 3 times, each time for 2.0 h. Filter and collect the filtrates from the three extractions and combine them. Maintain a vacuum of -0.1~0.08 MPa and a temperature of 50℃ on a rotary evaporator, and concentrate under reduced pressure to obtain 3.0 kg of total extract of *Pteris vittata*. The extracted *Pteris vittata* extract was added to an equal mass of boiling water and stirred until dissolved to form a suspension. The suspension was then used for extraction experiments. The extraction process involved pouring the suspension into a separatory funnel, adding an equal volume of petroleum ether, repeatedly shaking to mix, and allowing it to stand for 5-10 minutes. Once clear separation was visible to the naked eye, the petroleum ether layer was collected. An equal volume of petroleum ether was added to the aqueous layer for further extraction. This process was repeated three times. The three petroleum ether extracts were combined and extracted using a rotary evaporator to obtain the *Pteris vittata* petroleum ether extract.

[0025] The obtained total extract and petroleum ether extract of *Dryopteris crassirhizoma* were sealed and stored for later use.

[0026] Preparation of culture medium 1) Middle brook 7H9 broth: Weigh the appropriate amount of culture medium according to the instructions, add 0.2% glycerol to a final concentration, and autoclave at 121 °C for 10 min. When culturing Mycobacterium smegma, add glucose and Tween 80 to a final concentration of 0.5% and 0.05% respectively just before use; when culturing Mycobacterium tuberculosis, add OADC and Tween 80 to a final concentration of 10% and 0.05% respectively just before use.

[0027] 2) Middle Brook 7H10 agar plates: Weigh the corresponding culture medium according to the instructions, add glycerol to a final concentration of 0.5%, boil to completely dissolve the culture medium, then autoclave at 121 ℃ for 10 min, cool appropriately, add glucose to a final concentration of 0.5%, and pour to prepare 7H10 agar plates.

[0028] Strains and preparation: 1) Experimental strain: Primary culture: 100 μL of *Mycobacterium smegmatis* frozen at -80℃ was inoculated into 30 mL Middle brook 7H9 broth and cultured at 37℃ with shaking at 110 rpm until turbidity was reached. Secondary culture: 1% of the primary culture was inoculated into 30 mL Middle brook 7H9 secondary culture and cultured at 37℃ with shaking at 110 rpm until OD600 = 0.5–1.

[0029] 2) Experimental strains (both provided by Beijing Center for Disease Control and Prevention): Mycobacterium tuberculosis standard strain H37Rv frozen at -80℃, and clinically multidrug-resistant strain Mtb706. After thawing, they were inoculated into neutral Löwenstein medium for resuscitation culture.

[0030] Drug susceptibility testing for Mycobacterium smegma: (1) Antimicrobial susceptibility testing by solid agar paper disc diffusion method: Mycobacterium smegmatis was cultured in Middle brook 7H9 to the logarithmic phase (OD600=0.5-1), diluted to the theoretical OD600=0.01, and 100 μL of bacterial suspension was spread on Middle brook 7H10 agar plate (9 cm in diameter). Sterile filter paper discs (6 mm in diameter) were placed on the agar plate with sterile forceps, and 3 μL of the sample was spotted on the filter paper disc. At the same time, kanamycin was used as an anti-tuberculosis drug control. The plate was incubated at 37℃ for 3 days, and the diameter of the inhibition zone was observed, recorded and measured.

[0031] (2) Micro-broth dilution method for drug sensitivity testing (resazil color development) 1) Culture Mycobacterium smegmatis in Middle brook 7H9 to the logarithmic phase (OD600=0.5-1), dilute to OD600=0.5, and then dilute 100 times for later use.

[0032] 2) Drug dilution (taking one set as an example): Add 180 μL Middle brook 7H9 to the first well, then add 20 μL of antibiotic (the concentration can be set according to your own situation; the final concentration of antibiotic in the first well should be 1 / 20 of the stock solution concentration), and mix well; add 100 μL Middle brook 7H9 to each of wells 2-10; take 100 μL from well 1 and add it to well 2, mix well, take 100 μL from well 2 and add it to well 3, and so on, performing 2-fold dilutions. After mixing well 10, discard 100 μL.

[0033] 3) Add 100 μL of the prepared bacterial solution diluted in step 1 to each of the 1-10 wells in each row.

[0034] 4) Control settings: Add 200 μL of Middle brook 7H9 to well 11 as a negative control; add 100 μL of the bacterial culture prepared in step 1 and 100 μL of Middle brook 7H9 to well 12 as a positive control.

[0035] 5) Culture and observation records: Cultured at 100 rpm and 37℃ for 2 days.

[0036] 6) Add 10 μL of 0.1% (w / v%) sodium resazurin solution (water-soluble, filtered for sterilization) to each well, incubate at 37°C for 1 day at 100 rpm.

[0037] 7) Observation results: The growth of bacteria is judged by the change in the color of azadirachtin (during the growth of bacteria, its metabolism reduces azadirachtin, turning its blue color into pink). The lowest drug concentration that will not turn blue into pink is its MIC.

[0038] 2.5 Drug susceptibility testing for Mycobacterium tuberculosis (micro-broth dilution) 1) Prepare bacterial culture medium: Culture Mycobacterium tuberculosis in neutral Löwenstein medium to the logarithmic phase, pick colonies and put them into 0.9% physiological saline, adjust the McFarland turbidity to 1, and then inoculate them into fresh Middle brook 7H9 liquid medium at a ratio of 1:100.

[0039] 2) Add 180µL of Middle brook 7H9 liquid medium to each well in the first column of the 96-well plate, and add 100µL of Middle brook 7H9 liquid medium to each well in columns 2 to 12.

[0040] 3) Add 20 µL of total extract of *Pteris vittata* (150 mg / mL) to wells A1-C1 of the 96-well plate, and add 20 µL of petroleum ether extract of *Pteris vittata* (150 mg / mL) to wells D1-F1. Add 20 µL of rifampin (1280 µg / mL) to well G1 as a conventional drug control, and add 20 µL of DMSO to well H1 as a control.

[0041] 4) Use a pipette to mix the sample in column 1 by blowing and aspirating. Transfer 100 µL to column 2 and mix by blowing and aspirating. Repeat this process until column 11, discarding 100 µL at a time. Column 12 is a positive control with only bacterial suspension added.

[0042] 5) Add 100 µL of the diluted bacterial culture solution from 1) to each well in columns 1 to 12 (leave 2 wells in column 12 without bacterial culture solution as negative controls).

[0043] 6) After sealing with the sealing film, place in a 37℃ incubator for 10 days of static incubation. Scan the drug sensitivity plate and observe the experimental results. If no colonies grow in the negative control wells and colonies grow in the positive control wells, the results are considered valid. Provided that Mycobacterium tuberculosis grows wells wells wells wells, the drug concentration at which each drug inhibits 99% of Mycobacterium tuberculosis growth is read as the MIC. If this concentration is less than or equal to the preset breakpoint concentration, it is interpreted as sensitive (S); if it is greater than the corresponding value, it is interpreted as resistant (R).

[0044] Activity test results of total extract and petroleum ether extract of *Dryopteris crassirhizoma* 3.1 Verification of the antibacterial effects of total extract and petroleum ether extract of *Pteris vittata* on *Mycobacterium smegmatis*: Under the above conditions, the total extract and petroleum ether extract of *Pteris vittata* were subjected to in vitro inhibition experiments against *Mycobacterium smegmatis* using a combination of solid agar diffusion method and 96-well micro-detection method. Kanamycin was used as an anti-tuberculosis drug control. For statistics on the diameter of the inhibition zone, please refer to [link to relevant data]. Figure 1 and Figure 2 As shown.

[0045] The results of the antibacterial activity against Mycobacterium smegma using the total extract and petroleum ether extract of *Pteris vittata* are shown in Table 1. The average inhibition zones of the total extract and petroleum ether extract were 18.33 mm and 28.67 mm, respectively, demonstrating significant antibacterial activity. Simultaneously, the minimum inhibitory concentration (MIC) of the total extract and petroleum ether extract against Mycobacterium smegma was determined using a 96-well plate micro-detection method. Kanamycin was used as an anti-tuberculosis drug control. DMSO showed almost no toxicity against Mycobacterium smegma, while kanamycin exhibited better antibacterial activity. The MIC of both the total extract and petroleum ether extract against Mycobacterium smegma was 1875 μg / mL (as shown in Table 2). Table 1. Statistical results of antibacterial activity against Mycobacterium smegmatis from total extract and petroleum ether extract of *Fernula syringae*.

[0046] Note: The activity detection method is the paper disc diffusion method. The criteria for determining the inhibition zone are as follows: inhibition zone diameter less than 8 mm indicates low sensitivity ("none"); inhibition zone diameter between 8 and 13 mm indicates moderate sensitivity ("+"); inhibition zone diameter between 13 and 19 mm indicates moderate sensitivity ("++"); and >19 mm indicates high sensitivity ("+++"). Sample volume is 3 μL.

[0047] Table 2. Minimum inhibitory concentrations (MICs) of total extract and petroleum ether extract of *Dryopteris smegmatis* against *Mycobacterium smegmatis*.

[0048] Note: "-" indicates that the antibacterial effect is not obvious or has no effect.

[0049] 4. Inhibitory effect against standard strains of Mycobacterium tuberculosis: Using the method described in section 2.5, with rifampin as an anti-tuberculosis drug control: the minimum inhibitory concentrations (MICs) of total extract and petroleum ether extract of *Pteris vittata* against the standard strain H37Rv (ATCC27294) were 937 µg / mL and 234 µg / mL, respectively (results are shown in Figure 2.5). Figure 3 As shown in Table 3, the statistical results are presented.

[0050] Table 3. Minimum inhibitory concentrations (MICs) of total extract and petroleum ether extract of *Dryopteris crassirhizoma* against standard strains.

[0051] 5. Antibacterial effect against clinically multidrug-resistant strain Mtb706 Using the methods described in section 2.5, with rifampin as the anti-tuberculosis drug control, the minimum inhibitory concentration (MIC) of the total extract of *Pteris vittata* against the clinically multidrug-resistant strain Mtb706 was 117 µg / mL, and the MIC of the petroleum ether extract of *Pteris vittata* against the clinically multidrug-resistant strain Mtb706 was ≤7.3 µg / mL (results are shown in Figure 2.5). Figure 4 As shown in the figure, the statistical results are shown in Table 4.

[0052] Table 4. Minimum inhibitory concentrations (MICs) of total extract and petroleum ether extract of *Dryopteris crassirhizoma* against clinically resistant bacteria.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of a total extract of *Pteris vittata* in antituberculosis activity.

2. The application of the petroleum ether extract of *Pteris vittata* according to claim 1 in its anti-tuberculosis activity.

3. The application of the total extract of *Pteris vittata* according to claim 1 in its anti-tuberculosis activity, characterized in that: The method for preparing the total extract of *Pteris vittata* is as follows: weigh 2 parts of dried and pulverized whole *Pteris vittata* herb, soak them in 3-4 parts of 95% anhydrous ethanol for 20-40 minutes, and use the heating reflux extraction method to extract 3 times, 2.0 hours each time. Filter, collect and combine the filtrates from the 3 extractions, and concentrate them under reduced pressure on a rotary evaporator while maintaining a vacuum of -0.1~0.08MPa and a temperature of 45-55℃.

4. The application of the total extract of *Pteris vittata* according to claim 3 in its anti-tuberculosis activity, characterized in that, The method for preparing the petroleum ether extract of *Pteris vittata* is as follows: a. Add the extracted *Pteris vittata* extract to boiling water and stir until dissolved to obtain a suspension. The ratio of *Pteris vittata* extract to boiling water is 1:

1. b. Pour the suspension obtained in step a into a separatory funnel, then pour an equal volume of petroleum ether into the separatory funnel, shake and mix repeatedly, let stand for 5-10 minutes, and collect the petroleum ether layer after the obvious separation is visible to the naked eye. c. Add an equal volume of petroleum ether to the aqueous layer from step b and extract again. Repeat this process three times. Combine the three petroleum ether extracts and extract the petroleum ether extract using a rotary evaporator.

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