A sesquiterpene lactone compound in isodon japonicus and extraction method and application thereof

Abstract

The application relates to the technical field of medicines, in particular to a sesquiterpene lactone compound in the fruit of Styrax pubescens, an extraction method and application thereof. A sesquiterpene lactone compound CA50 is extracted from the fruit of Styrax pubescens, and the structure is shown as formula I. The preliminary experiment verifies that the CA50 can reduce the levels of HBV core DNA and HBsAg in HepAD38 cells, and has the potential to prepare anti-hepatitis B virus drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a sesquiterpene lactone compound from Tianmingjing, its extraction method, and its application. Background Technology

[0002] Hepatitis B (HBV) is a global public health problem caused by the hepatitis B virus. According to the World Health Organization, there are approximately 296 million HBV-infected individuals worldwide, with about 70-90 million currently infected in my country, accounting for one-quarter of global cases and ranking first in disease burden. After infection, persistent viral replication can lead to abnormal liver function, liver fibrosis, and cirrhosis. Approximately 15%-25% of chronically infected individuals eventually progress to liver cancer or end-stage liver disease. HBV transmission is primarily through mother-to-child transmission, blood exposure, and iatrogenic infection. Although widespread vaccination has significantly reduced the incidence of new infections, the risk of reinfection due to lack of vaccination, immune escape, or antibody decay still exists. Patients in the immune tolerance phase, in particular, lack effective treatment options and become a potential source of continued viral transmission. Epidemiological data shows that the younger the age of HBV infection, the higher the risk of chronicity, further exacerbating the pressure on public health prevention and control.

[0003] Currently, first-line anti-HBV drugs in clinical practice mainly consist of nucleoside (acid) analogs (NAs) and interferon-alpha (PEG-IFNα). NAs reduce HBV DNA load by inhibiting viral reverse transcriptase, but they cannot eliminate viral covalently closed circular DNA (cccDNA), and long-term use easily induces drug resistance mutations (such as YMDD mutations), leading to virological breakthrough. Although interferon can activate the host immune response, the response rate is less than 30%, and it easily causes serious adverse reactions such as liver damage and thyroid dysfunction. Neither class of drugs can effectively reduce hepatitis B surface antigen (HBsAg) levels, and the viral rebound rate is high after NAs are discontinued, requiring lifelong medication. In addition, the average annual treatment cost for patients exceeds 6,600 yuan, creating a vicious cycle of economic burden and poor long-term medication adherence, resulting in a clinical dilemma. Therefore, developing new drugs that combine high efficacy, safety, and cost-effectiveness has become an important need for overcoming the bottleneck in hepatitis B treatment in my country.

[0004] Compared to chemically synthesized drugs, natural products have greater structural diversity, lower risk of drug resistance, and their drug properties can be optimized through structural modification. For example, CN113735920A discloses a cyanogenic glycoside compound, Menisdaurin F, extracted from fresh Rhizophora stylosa hypocotyl slices, which has anti-hepatitis B virus activity; CN108096307B discloses an extract from Prunus mume with anti-hepatitis B virus activity.

[0005] The Chinese medicinal herb *Tianmingjing* is *Tianmingjing*, a plant belonging to the genus *Tianmingjing* in the family Asteraceae. Carpesium abrotanoidesThe whole herb of *L.*. It enters the liver and lung meridians, and is pungent and cold in nature. The *Dictionary of Traditional Chinese Medicine* records its use for tonsillitis, sore throat, malaria, acute hepatitis, acute and chronic infantile convulsions, intestinal parasites, blood stasis, epistaxis, hematuria, boils and carbuncles, and itchy skin rashes. Clinically, a decoction with ginger, taken orally, can treat acute icteric infectious hepatitis. Despite records and clinical applications in treating hepatitis, the underlying material basis remains unclear.

[0006] In light of the above background, this application is hereby submitted. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a sesquiterpene lactone compound from Tianmingjing, its extraction method and application.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] Technical Topic 1

[0010] A sesquiterpene lactone compound, the structure of which is shown in Formula I:

[0011] .

[0012] Technical Theme Two

[0013] A method for extracting sesquiterpene lactones as described in Technical Subject 1, comprising the following steps:

[0014] S1: Extract the dried, mature fruit of *Symplocos tianmingjing* with an aqueous ethanol solution, recover the solvent, and obtain the extract;

[0015] S2: Disperse the extract in saturated brine and extract it sequentially with petroleum ether and dichloromethane to obtain two extracts;

[0016] S3: The dichloromethane fraction was separated by silica gel column chromatography with gradient elution of petroleum ether-ethyl acetate at a volume ratio of 4:1 to 0:1 to obtain fractions B1.1-B1.66.

[0017] S4: Combine fractions B1.44-B1.46 and separate them by silica gel column chromatography with gradient elution of dichloromethane-acetone at a volume ratio of 40:1 to 10:1 to obtain fractions B2.1-B2.65.

[0018] S5: Combine fractions B2.44-B2.46 and separate them by LH20 gel column chromatography, eluting with methanol isocratic to obtain fractions B3.1-B3.61;

[0019] S6: Combine fractions B3.44-B3.53 and separate and purify them using semi-preparative high-performance liquid chromatography to obtain compound CA50.

[0020] As a further improvement of the present invention, the mass-to-volume ratio of dried mature fruit of S1 Tianmingjing to ethanol aqueous solution is 1Kg:2-3L, the ethanol aqueous solution is 95% ethanol, and the extraction is a soaking extraction, which is performed 3-5 times, each time for 2-4 days.

[0021] As a further improvement of the present invention, the mass-to-volume ratio of extract to saturated brine in S2 is 1 kg: 1.5-2 L, and the volume ratio of saturated brine to petroleum ether and dichloromethane is 1:1-1.2:1-1.2.

[0022] As a further improvement of the present invention, the mass-to-volume ratio of dried mature fruit of S1 Tianmingjing to ethanol aqueous solution is 1Kg:2.5L, and the extraction is a soaking extraction, which is performed 3 times, each time for 3 days.

[0023] As a further improvement of the present invention, the mass-to-volume ratio of extract to saturated brine in S2 is 1 kg: 1.75 L, and the volume ratio of saturated brine to petroleum ether and dichloromethane is 1:1:1.

[0024] As a further improvement of the present invention, the elution gradient of petroleum ether-ethyl acetate in S3 is 4:1, 1:1, 0:1;

[0025] 4:1, 1:1, and 0:1 represent the volume ratios of petroleum ether to ethyl acetate; the elution gradient of dichloromethane-acetone in S4 is 40:1, 20:1, and 10:1.

[0026] 40:1, 20:1, and 10:1 are the volume ratios of dichloromethane to acetone.

[0027] As a further improvement of the present invention, in S6, the separation and purification is carried out by gradient elution of acetonitrile-water with a volume ratio of 45:55→100:0.

[0028] Technical Theme 3

[0029] The use of a sesquiterpene lactone compound as described in Technical Subject 1 in the preparation of an anti-hepatitis B virus drug.

[0030] As a further improvement of the present invention, the drug can reduce HBV core DNA and HBsAg levels.

[0031] This application yielded a previously unreported sesquiterpene lactone compound (CA50) extracted from the fruit of *Heliotropium indicum*. Preliminary experimental verification showed that CA50 can reduce HBV core DNA and HBsAg levels in HepAD38 cells, demonstrating its potential for the preparation of anti-hepatitis B virus drugs. Attached Figure Description

[0032] Figure 1This is the HR-ESI-MS spectrum of compound CA50;

[0033] Figure 2 It is compound CA50. 1 H NMR spectrum;

[0034] Figure 3 It is compound CA50. 13 C NMR spectrum;

[0035] Figure 4 It is compound CA50. 1 H- 1 H COSY spectrum;

[0036] Figure 5 This is the HSQC spectrum of compound CA50;

[0037] Figure 6 This is the HMBC spectrum of compound CA50;

[0038] Figure 7 This is the NOESY spectrum of compound CA50;

[0039] Figure 8 This is a graph showing the inhibition results of compound CA50 on intracellular HBV core DNA and HBsAg. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0041] Example 1

[0042] S1: Take 8.0 kg of dried mature fruit of *Epiphyllum indicum* and extract it by soaking it in 95% ethanol (20 L) at room temperature three times for three days each time. The solvent was recovered to obtain 229.0 g of extract.

[0043] S2: Disperse the extract in 400ml of saturated saline solution, and extract it sequentially with 400ml of petroleum ether and 400ml of dichloromethane. After distilling the solvent, obtain the petroleum ether extract and the dichloromethane extract, respectively.

[0044] S3: The dichloromethane extract (B, 73.1 g) was separated by silica gel column chromatography at atmospheric pressure, and eluted with a petroleum ether-ethyl acetate gradient of 4:1 to 0:1 (v / v), yielding fractions B1.1-B1.66.

[0045] S4: Combine fractions B1.44-B1.46 and separate them by silica gel column chromatography with a dichloromethane-acetone gradient elution of 40:1 to 10:1 (v / v).

[0046] S5: Combine fractions B2.44-B2.46 and separate them by LH20 gel column chromatography, eluting with methanol isocratic to obtain fractions B3.1-B3.61.

[0047] S6: Fractions B3.44-B3.53 were combined and separated by semi-preparative high-performance liquid chromatography (HPLC). The chromatographic conditions were: GRACEAllsphere ODS-2 (22×250 mm, 5 μm), flow rate 3.0 mL / min, with acetonitrile-water gradient elution from 0 to 35 min at a volume ratio of 45:55 → 100:0. R CA50 was obtained at 9.5 min.

[0048] CA50 is a colorless needle-like crystal. α ]20 D+48.0( c 0.10, MeOH). Positive ion HR-ESI-MS ( Figure 1 (Given quasi-molecular ion peaks) m / z The value is 263.1286 [M+H]. + (calcd for C 15 H 19 O4 (263.1278), the molecular formula is deduced to be C 15 H 18 O4, the calculated degree of unsaturation is 7.

[0049] That 1 H-NMR ( Figure 2 )and 13 C-NMR ( Figure 3 The data is shown in Table 1. 1 H- 1 H COSY spectrum as follows Figure 4 As shown, the HSQC spectrum is as follows Figure 5 As shown, the HMBC spectrum is as follows Figure 6 As shown, the NOESY spectrum is as follows Figure 7 As shown.

[0050] Table 1

[0051]

[0052] The above data confirms that the structure of compound CA50 is as shown in Formula I.

[0053]

[0054] Example 1

[0055] This application uses the HBV stable replication cell line HepAD38 (source: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences) to investigate the inhibitory effect of CA50 on intracellular HBV core DNA and HBsAg at non-toxic doses.

[0056] Experimental methods:

[0057] 1. Sample preparation: Dissolve CA50 in DMSO to prepare a 100mM stock solution and store at -20℃.

[0058] 2. Cytotoxicity assay: The CA50 stock solution was diluted to 500 μM using complete culture medium. Starting from 500 μM, a three-fold serial dilution was performed, resulting in eight concentration points. HepAD38 cells were added to 48-well plates, with 2 × 10⁻⁶ cells added to each well. 4 Each sample was cultured in 500 μl of the above-mentioned complete medium containing CA50 at 37°C. After 4 days of culture, cytopathic effect (CPE) was observed and cytotoxicity was determined. The maximum non-toxic concentration TC0 was determined to be 6.2 μM, and the half-maximal toxic concentration TC was calculated using the Reed-Muench method. 50 =18.52μM.

[0059] 3. Anti-HBV efficacy determination: The test sample was applied to cells at the maximum non-toxic concentration TC0 = 6.2 μM.

[0060] Add HepAD38 to the 48-well plate, adding 2×10 to each well. 4 Cells were cultured in 500 μl of complete culture medium containing 6.2 μM CA50 at 37°C for 4 days. Cells and supernatant were then collected for efficacy evaluation. HBsAg was detected using an ELISA kit (purchased from Komei Diagnostics Technology Co., Ltd.). Intracellular HBV core DNA was extracted and its content was detected using real-time quantitative PCR (qPCR), and the relative content was calculated.

[0061] 4. Measurement results: such as Figure 8 As shown, CA50 has a certain inhibitory effect on the secretion of HBV core DNA and HBsAg in HepAD38 cells at the maximum non-toxic concentration.

[0062] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a sesquiterpene lactone compound with the structure shown in Formula I in the preparation of an anti-hepatitis B virus drug; .

2. The application according to claim 1, characterized in that, The drug can reduce HBV core DNA and HBsAg levels.

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