Striga aegyptiaca glycoside A, extraction method and application thereof

Abstract

The application discloses strigasiaticaside A and an extraction method and application thereof, and finds, through chemical component research on the whole grass of striga asiatica, a new synbinaphthyl tetrahydrofuran lignan glycoside, that is, strigasiaticaside A, identifies the chemical structure of the new compound through nuclear magnetic resonance, mass spectrometry, ultraviolet, infrared and circular dichroism spectrum technology.The structure of the new compound is identified as (-) sesamin-5-O-beta-D-glucopyranoside.The anti-hepatitis activity of the new natural product is evaluated by using LPS stimulated HepG2 cells.The results show that when the concentration is 100 muM, the compound has a significant inhibitory effect on the production of inflammatory factors IL-10 and NF-kappa B protein, and has no cytotoxicity, indicating that the compound may have potential anti-hepatitis activity.

Description

Technical Field

[0001] This invention belongs to the technical field of plant active ingredients, specifically relating to strigoglycoside A, its extraction method, and its application. Background Technology

[0002] One-legged gold Striga asiatica (L.) O. Kuntze is a species of strigometal (L.) belonging to the family Scrophulariaceae. Striga This plant, also known as *Gnaphalium affine* or *Gnaphalium affine*, is widely distributed in Guangdong, Guangxi, Jiangxi, Fujian, Hainan, and other regions of China. Historical medical texts such as *Zhonghua Bencao* and *Shengcao Yaoxing Beiyao* contain detailed records and praise for its effects in relieving indigestion, strengthening the spleen, clearing heat, eliminating parasites, and treating infantile malnutrition and jaundice.

[0003] Numerous modern phytochemical studies have found that *Striga asiatica* mainly contains lignans, flavonoids, glycosides, alkaloids, and organic acids. The pharmacological effects of *Striga asiatica* are studied as follows:

[0004] Related studies have reported that the chemical components of *Striga asiatica* have many beneficial biological activities, including antibacterial, antioxidant, anti-inflammatory, antifertility, and antitumor activities, and have good therapeutic effects on digestive system diseases such as diarrhea and malnutrition in children.

[0005] Research on the effective components of *Striga asiatica*:

[0006] Strigol contains flavonoids, such as hyperoside and apigenin, phenolic acids such as caffeic acid and chlorogenic acid, and organic acids such as malic acid and citric acid. These components work together to exert the medicinal effect.

[0007] Research on the extraction technology of strigolactone:

[0008] Extraction methods mainly include modern technologies such as water extraction, alcohol extraction, ultrasonic extraction, and microwave extraction. These methods can improve extraction efficiency and ensure the quality and activity of the extract.

[0009] Research on the application areas of *Striga asiatica*:

[0010] In the agricultural sector: the market price of *Striga asiatica* is high. According to literature reports, the price of dried *Striga asiatica* medicinal material can reach 2,000 to 2,600 yuan / kg, and its cultivation value is high.

[0011] In the field of medicine: the extract of *Striga asiatica* has broad prospects in medicine and can be used to develop drugs for the treatment of diseases such as diarrhea, malnutrition, and jaundice in children.

[0012] In clinical practice, *Striga asiatica* is widely used in traditional Chinese medicine to treat indigestion and malnutrition in children. In modern clinical practice, it is also frequently used to treat jaundice-type hepatitis and anorexia, all of which have shown significant efficacy.

[0013] However, some problems still exist in the research on *Striga asiatica*:

[0014] Resource scarcity: Strigolium moniliforme mainly relies on wild resources for collection. In recent years, due to changes in the ecological environment, its distribution area has shrunk, resources have been depleted or are on the verge of depletion, and market prices have continued to rise.

[0015] Research gaps: Current research on strigolactone by scholars at home and abroad is not comprehensive enough, mainly focusing on seed germination and interaction with the host, while research on component analysis, medicinal efficacy evaluation and application is relatively limited, which further restricts its in-depth development. Summary of the Invention

[0016] To address the problem that existing technologies cannot obtain the natural product strigoglycoside A through purification and that there are currently no related applications for this compound, this invention provides strigoglycoside A, its extraction method, and its applications. The inventors studied the methanol extract of strigofera and discovered a novel bis(tetrahydrofuran) lignan glycoside. This invention reports a novel method for the extraction, separation, and preparation of a natural product, its structural identification, and its evaluation of anti-hepatitis activity.

[0017] The technical solution of the present invention is as follows:

[0018] This invention investigated the chemical constituents of the whole herb *Striga asiatica*, discovering a novel bis(tetrahydrofuran) lignan glycoside, strigasiaticaside A. Its chemical structure was identified using nuclear magnetic resonance (NMR), mass spectrometry (MS), ultraviolet (UV), infrared (IR), and circular dichroism (CD). The structure of this new compound was identified as (-)-sesamin-5-O-β-D-glucopyranoside. The anti-hepatitis activity of this new natural product was evaluated using LPS-stimulated HepG2 cells. The results showed that at a concentration of 100 μM, this compound significantly inhibited the production of inflammatory factors IL-10 and NF-κB protein without cytotoxicity, indicating that this compound possesses potential anti-hepatitis activity.

[0019] The objective of this invention is achieved through the following technical solution:

[0020] Strigasiaticaside A, the structure of which was identified as (-)-sesamin-5-O-β-D-glucopyranoside, was named Strigasiaticaside A.

[0021] This invention also relates to a method for extracting the above-mentioned strigoglycoside A, comprising the following steps:

[0022] S1. The dried whole herb of *Striga asiatica* was crushed into small pieces and extracted three times by heating and reflux with 75% methanol at a material-to-liquid ratio of 1:20, each time for 3 hours.

[0023] S2. After filtering with gauze, the methanol extracts were combined, and the solvent was recovered by rotary evaporator to obtain the methanol extract of *Striga asiatica*.

[0024] S3. The methanol extract of *Striga asiatica* obtained in S2 was dispersed and dissolved in distilled water. According to the ratio of methanol extract of *Striga asiatica* to distilled water = 100g: 300mL, petroleum ether, ethyl acetate, and n-butanol with boiling ranges of 60℃~90℃ were used for extraction in sequence, according to the polarity of the solvent. Each extraction was performed 5 times to obtain different extract fractions. After vacuum concentration and recovery, petroleum ether extract, ethyl acetate extract, and n-butanol extract were obtained.

[0025] S4. Take 21.5g of ethyl acetate (EtOAc) extract, dissolve it in 20mL-50mL of methanol, add the sample to an equal volume of silica gel in small amounts several times, heat in a water bath, and grind continuously to obtain a uniform particle size and dry sample-silica gel mixture. Separate by normal silica gel column chromatography, using dichloromethane-methanol gradient elution at a gradient of 50:1→1:3. Detect and combine identical fractions by TLC to obtain 12 components, named E1~E12.

[0026] Component E8 was prepared and separated by RP-HPLC with a mobile phase of methanol-water and a gradient of 30:70→80:20, v / v; flow rate: 3 mL / min. Gradient elution yielded compound 1. R =34.0 min, compound 1 was finally identified as (-)-sesamin-5-O-β-D-glucopyranoside [(-)-sesamin-5-O-β-D-glucopyranoside], and named strigasiaticaside A.

[0027] Furthermore, the material-to-liquid ratio mentioned in S1 is based on the ratio of medicinal material mass (g) to extraction solvent volume (mL).

[0028] Furthermore, as described in S4, an equal amount of silicone is added, wherein the silicone is 100 mesh to 200 mesh.

[0029] Furthermore, the separation described in S4 is performed by forward silica gel column chromatography, wherein the silica gel is 200-300 mesh.

[0030] This invention also relates to the application of the above-mentioned strigoglycoside A in the preparation of anti-hepatitis active drugs.

[0031] Furthermore, strigoglycoside A significantly inhibits the production of inflammatory factors IL-10 and NF-κB protein without cytotoxicity, indicating that the compound has potential anti-hepatitis activity, specifically for the preparation of an anti-hepatitis drug that inhibits the production of inflammatory factors IL-10 and NF-κB protein.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The extraction method of strigoglycoside A described in this invention adopts heating reflux extraction, which is simple to operate; pure product can be prepared by one-step silica column chromatography followed by one-step reversed-phase HPLC, which is convenient and fast, and has strong reproducibility.

[0034] 2. The strigoglycoside A described in this invention is a novel compound (natural product), and its chemical structure and spectroscopic data are reported for the first time. Pharmacological and pharmacodynamic studies on this compound are currently lacking.

[0035] 3. The application of strigoglycoside A in the preparation of anti-hepatitis active drugs described in this invention fills the research gap of this compound in this field; based on the traditional use of strigolactone, the application of strigoglycoside A in the research of anti-hepatitis active drugs has historical traceability and rationality; in addition, natural products have the characteristics of high efficiency, low toxicity and few side effects. Attached Figure Description

[0036] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0037] Figure 1 The chemical structure diagram of strigoglycoside A described in Example 1 of this invention is shown.

[0038] Figure 2 This is a graph showing the effect of strigoglycoside A on LPS-induced HepG2 cell survival rate as described in the experimental examples of this invention.

[0039] Figure 3 The effect of strigoglycoside A on the expression levels of IL-10 and NF-κB proteins in LPS-induced HepG2 cells is shown in the experimental example of this invention (n=3).

[0040] Figure 4 The strigoglycoside A described in Example 3 of this invention 1 H NMR spectrum (CD3OD);

[0041] Figure 5 The strigoglycoside A described in Example 3 of this invention 1 A magnified view of a portion of the H NMR spectrum (CD3OD);

[0042] Figure 6 The strigoglycoside A described in Example 3 of this invention 13 C10 NMR spectrum (CD3OD);

[0043] Figure 7 The strigoglycoside A described in Example 3 of this invention 13 A magnified view of a partial 3C NMR spectrum (CD3OD);

[0044] Figure 8 This is the DEPT spectrum (CD3OD) of strigoglycoside A as described in Example 3 of the present invention.

[0045] Figure 9 This is a partially enlarged DEPT spectrum (CD3OD) of strigoglycoside A as described in Example 3 of the present invention.

[0046] Figure 10 The HSQC spectrum (CD3OD) of strigoglycoside A described in Example 3 of this invention is shown.

[0047] Figure 11 The HMBC spectrum (CD3OD) of strigoglycoside A described in Example 3 of this invention is shown.

[0048] Figure 12 The strigoglycoside A described in Example 3 of this invention 1 H- 1 H COSY spectrum (CD3OD);

[0049] Figure 13 This is the NOESY spectrum (CD3OD) of strigoglycoside A as described in Example 3 of the present invention.

[0050] Figure 14 The HRESIMS spectrum of strigoglycoside A described in Example 3 of this invention;

[0051] Figure 15 This is the CD spectrum of strigoglycoside A as described in Example 3 of the present invention. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0053] Example 1:

[0054] The extraction method of strigoglycoside A includes the following steps:

[0055] Extraction and separation methods:

[0056] Two kg of dried whole herb of *Striga asiatica* was crushed into small pieces and extracted three times with 75% methanol under reflux at a material-to-liquid ratio of 1:20 (g of herb mass / mL of extraction solvent), each time for 3 hours. After filtering with gauze, the methanol extracts were combined and the solvent was recovered using a rotary evaporator to obtain 360.0 g of *Striga asiatica* methanol extract.

[0057] The total extract was dispersed and dissolved in appropriate amount of distilled water. Depending on the polarity of the solvent, it was extracted with petroleum ether (60-90), ethyl acetate, and n-butanol in sequence, five times each, to obtain different extract fractions. After vacuum concentration and recovery, 11.7 g of petroleum ether extract, 21.5 g of ethyl acetate extract, and 69.5 g of n-butanol extract were obtained.

[0058] 21.5 g of ethyl acetate (EtOAc) extract was dissolved in an appropriate amount of methanol. The sample was added in small, repeated amounts to an equal volume of silica gel (100-200 mesh). The mixture was heated in a water bath while continuously grinding to obtain a uniformly sized, dry sample-silica gel mixture. This mixture was then separated by normal silica gel column chromatography (silica gel: 200-300 mesh) using a gradient elution of dichloromethane-methanol (50:1→1:3). TLC analysis combined identical fractions yielded 12 fractions, E1-E12. Fraction E8 (100.0 mg) was preparatively separated by RP-HPLC using methanol-water (30:70→80:20, v / v; flow rate: 3 mL / min) as the mobile phase, with gradient elution to obtain compound 1 (5.3 mg, t...). R =34.0 min).

[0059] Example 2:

[0060] The extraction method of strigoglycoside A includes the following steps:

[0061] S1. The dried whole herb of *Striga asiatica* was crushed into small pieces and extracted three times by heating and reflux with 75% methanol at a material-to-liquid ratio of 1:20, each time for 3 hours.

[0062] The material-to-liquid ratio is based on the ratio of medicinal material mass (g) to extraction solvent volume (mL).

[0063] S2. After filtering with gauze, the methanol extracts were combined, and the solvent was recovered by rotary evaporator to obtain the methanol extract of *Striga asiatica*.

[0064] S3. The methanol extract of *Striga asiatica* obtained in S2 was dispersed and dissolved in distilled water. According to the ratio of methanol extract of *Striga asiatica* to distilled water = 100g: 300mL, petroleum ether, ethyl acetate, and n-butanol with boiling ranges of 60℃~90℃ were used for extraction in sequence, according to the polarity of the solvent. Each extraction was performed 5 times to obtain different extract fractions. After vacuum concentration and recovery, petroleum ether extract, ethyl acetate extract, and n-butanol extract were obtained.

[0065] S4. Take 21.5g of ethyl acetate (EtOAc) extract, dissolve it in 20mL-50mL of methanol, add the sample to an equal volume of silica gel (100-200 mesh) in small amounts several times, heat in a water bath, and grind continuously to obtain a uniform particle size and dry sample-silica gel mixture. Separate by normal silica gel column chromatography (200-300 mesh), elute with dichloromethane-methanol gradient (50:1 → 1:3), and combine identical fractions by TLC to obtain 12 components, named E1~E12.

[0066] Component E8 was prepared and separated by RP-HPLC with a mobile phase of methanol-water and a gradient of 30:70→80:20, v / v; flow rate: 3 mL / min. Gradient elution yielded compound 1. R =34.0 min, compound 1 was finally identified as (-)-sesamin-5-O-β-D-glucopyranoside [(-)-sesamin-5-O-β-D-glucopyranoside], and named strigasiaticaside A.

[0067] Example 3:

[0068] Structural identification of strigoglycoside A:

[0069] Compound 1: 1 H and 13 C NMR data (Table 1); [α] -6.02 ( c 0.10, MeOH); UV (MeOH)λ max (log ε) 204 (1.04) nm; ECD ( c 1.0 mg / mL, MeOH) λ max (Δε) 203 (-4.88) nm; HR-ESI-MS m / z 555.1456 [M + Na] + (calcd. for C 26 H 28 O 12Na, 555.1478); IR (microscope)ν max 3373, 2923, 2853, 1633, 1503, 1489, 1440, 1246, 1071, 1035, 930, 734 cm -1 White amorphous powder.

[0070] Compound 1 is a white amorphous powder, as determined by high-resolution mass spectrometry data (HR-ESI-MS). m / z 555.1456 [M + Na] + (Theoretical value is C) 26 H 28 O 12 Analysis of Na (555.1478) yielded its molecular formula as C. 26 H 28 O 12 This leads to the conclusion that the structure contains 13 degrees of unsaturation. Figure 14 );

[0071] Figure 1 The chemical structure diagram of strigoglycoside A described in Example 3 is shown below;

[0072] Figure 14 The HRESIMS spectrum of strigoglycoside A described in Example 3;

[0073] exist 1 In the H-NMR (CD3OD, 400 MHz) spectrum, δ 6.87 (1H, d, J = 1.68 Hz), 6.83 (1H, dd, J = 7.96, 1.68 Hz), 6.77 (1H, d, J = 7.96 Hz) and 6.60 (1H, d, J = 1.52 Hz), 6.74 (1H, d, J = 1.52 Hz) represent five proton signals in the ABX and AX benzene ring systems; δ 4.69 (1H, d, J = 4.60 Hz) and δ 4.66 (1H, d, J = 5.08 Hz) represent two sets of methoxy proton signals; δ 4.22 (2H, dd, J = 9.0, 4.16 Hz) and δ 3.84 (2H, δ(m) represents two groups of methylene proton signals; δ 5.93 (1H, d, J = 1.20), 5.91 (1H, d, J = 1.20), and 5.91 (2H, s) represent two groups of methylenedioxy proton signals; δ 3.07 (2H, m) represents two groups of methylene proton signals. Furthermore, 1The presence of a terminal proton signal δ 5.05 (1H, d, J = 7.44 Hz) in the 1H-NMR spectrum indicates the presence of a single monosaccharide in the structure. Figure 4 );

[0074] Figure 4 The strigoglycoside A described in Example 3 1 H NMR spectrum;

[0075] Figure 5 The strigoglycoside A described in Example 3 1 A magnified view of a portion of the H NMR spectrum;

[0076] exist 13 In the C-NMR and DEPT (CD3OD, 100 MHz) spectra, there were a total of 26 carbon signals, of which 18 signals were from the lignan core of a two-molecule C6-C3 skeleton, the two carbon signals at δ 102.8 (C-10) and 102.39 (C-10′) were from two -OCH2O- groups on the lignan core, and the other 6 carbon signals were from a single hexavalent pyranose group (…). Figure 6 , Figure 8 );

[0077] Figure 6 The strigoglycoside A described in Example 3 13 C NMR spectrum;

[0078] Figure 7 The strigoglycoside A described in Example 3 13 A magnified view of a portion of the C NMR spectrum;

[0079] Figure 8 The DEPT spectrum of strigoglycoside A described in Example 3;

[0080] Figure 9 This is a partially enlarged view of the DEPT spectrum of strigoglycoside A described in Example 3;

[0081] Comprehensive analysis 1 H-NMR and 13C-NMR data and classic olefin signals δ 137.34 (C-1), 102.44 (C-2), 136.42 (C-3), 150.86 (C-4), 142.01 (C-5), 110.7 (C-6) and 136.44 (C-1′), 107.56 (C-2′), 149.39 (C-3′), 148.61 (C-4′), 108.98 (C-5′), 120.68 (C-6′); two biphenyl methine group signals δ 87.06 (C-7) and 87.35 (C-7′); two methine group signals δ 55.51 (C-8) and 55.54 (C-8′); two methylene group signals δ 72.87 (C-9). The presence of 72.64 (C-9′) indicates that this compound is a furofuran-type lignan. The compound's... 1 H-NMR and 13 The C-NMR data are very similar to those of the known compound (-)-sesamin, except that compound 1 lacks one aromatic hydrogen atom in the benzene ring and has an additional C-H signal from glucose. Comparison of compound 1 with termitomenin D also reveals similar NMR data, differing only in the sugar substitution position. The coupling constant of the terminal proton δ 5.05 (1H, d, J = 7.44 Hz) reveals that pyranose is in the β configuration (…). Figure 4 Further analysis of the HMBC spectrum revealed that the hydrogen at position 1′′ of pyranose [δ 5.05 (1H, d, J = 7.44 Hz)] is connected to the carbon at position 5 of the parent nucleus (δ 142.01). Figure 11 );

[0082] Figure 11 The HMBC spectrum of strigoglycoside A described in Example 3;

[0083] Analysis of the NOESY spectrum revealed a correlation between H-1′′ and H-6, further confirming that glucose is attached at the C-5 position; H-9a was correlated with H-8, and H-8 was correlated with H-2 and H-6 respectively; H-9′a was correlated with H-8′, and H-8′ was correlated with H-2′ and H-6′ respectively, indicating that H-8 and H-8′ are in cis-form, H-7 and H-7′ are also in cis-form, and H-7 / H-7′ and H-8 / H-8′ are in trans-form. Figure 13 );

[0084] Figure 13 The NOESY spectrum of strigoglycoside A described in Example 3;

[0085] The above evidence indicates that the relative configuration of compound 1 is (7R,8S,7′R,8′S)-1 or (7S,8R,7′S,8′R)-1. Finally, by combining CD spectrum with ECD calculations, the absolute configuration of the chiral carbons at positions 7, 8, 7', and 8' is determined to be (7R,8S,7′R,8′S)-1. Figure 15 ).

[0086] Figure 15 The CD spectrum of strigoglycoside A described in Example 3 is shown.

[0087] This invention utilizes a simple and rapid acid hydrolysis and saccharide derivatization technique to determine the absolute configuration of glucopyranoside. RP-HPLC analysis of the derivatized product confirmed that the absolute configuration of glucopyranoside in compound 1 is D-type. Therefore, the new compound 1 was ultimately identified as (-)-sesamin-5-O-β-D-glucopyranoside and named strigasiaticaside A.

[0088] Figure 10 The HSQC spectrum of strigoglycoside A described in Example 3;

[0089] Figure 12 The strigoglycoside A described in Example 3 1 H- 1 H COSY spectrum;

[0090] Table 1. NMR data of new compound 1 ( 1 H / 400 and 13 (C / 100 MHz)

[0091]

[0092] Experimental example:

[0093] Activity assay:

[0094] 1. Cell Culture

[0095] HepG2 cells exhibit adherent growth characteristics. Poly-L-lysine (PLL) can enhance cell adhesion between the culture dish and the cells, promoting adherent growth. A 10 mg / mL PLL solution was prepared by diluting the PLL solution with sterile PBS buffer, added to the cell culture dish, and allowed to stand for 10 min before being aspirated. HepG2 cells were seeded in complete culture medium MEM (containing 10% FBS and 1% penicillin antibiotics) and cultured in a 37 ℃, 5% CO2 incubator. When the cell mass reached 80%–90%, the cells were passaged, and cells in the logarithmic growth phase were used for experiments.

[0096] 2. CCK8 cytotoxicity assay

[0097] Prepare a 10 mg / mL poly-L-lysine (PLL) solution by diluting with sterile PBS buffer. Add 50–100 μL to each well of a 96-well plate, let stand for 10 min, and then aspirate the liquid. Take logarithmically grown HepG2 cells and add them to each well at a concentration of 5 × 10⁶ cells / well. 4 100 μL of cell suspension was added to each pretreated 96-well plate. The seeded 96-well plates were then incubated at 37 °C with 5% CO2 for 24 hours. High-concentration drugs were diluted with complete culture medium to create concentration gradients of 400, 200, 100, 50, 25, and 12.5 μM. After 24 hours of culture, the cells from the 96-well plates were removed, the old culture medium was discarded, and a blank control group was retained. Three compounds were tested per 96-well plate, with three replicates for each drug concentration. 100 μL of drug-containing complete culture medium was added to each well. The 96-well plates were then incubated at 37 °C with 5% CO2 for 24 hours. After 24 hours, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37 °C with 5% CO2 for another hour. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0098] 3. IL-10 and NF-κB experiments

[0099] Prepare a 10 mg / mL poly-L-lysine (PLL) solution by diluting with sterile PBS buffer and add it to a 6-well plate. Let it stand for 10 min and then discard the solution. Take logarithmically growing HepG2 cells and seed them into two 6-well plates at a ratio of 2 mL cell suspension per well. Incubate the plates at 37 ℃ and 5% CO2 for 24 hours. Set up a normal group, a model group (LPS 100 ng / mL), a positive control group (silymarin 20 μM), and a drug treatment group (monomer compound 400 μM + LPS 100 ng / mL, 200 μM + LPS 100 ng / mL, 100 μM + LPS 100 ng / mL). After LPS induction for 24 hours, administer the drug and incubate at 37 ℃ and 5% CO2 for 24 hours. After 24 hours, end the cell culture, discard the culture medium, wash the cells twice with clean PBS buffer, and add 100 mg / mL of the solution to each well. Transfer μL of lysis buffer (RIPA lysis buffer: PMSF = 100:1) to an EP tube and lyse on ice for 30 min, vortexing every 10 min to ensure complete cell lysis. Centrifuge the lysed protein at 12000 rpm for 10 min at 4 ℃ and store at -20 ℃ for later use.

[0100] Prepare standard solutions: A 5 mg / mL standard protein solution was used as the initial concentration. The solution was diluted with ultrapure water to concentrations of 2.5, 1, 0.5, 0.25, 0.125, 0.063, and 0 mg / mL, resulting in eight working concentrations. Prepare BCA working solution: 10 mL of solution A:B = 50:1 was prepared and mixed thoroughly. 100 μL of BCA working solution was added to each well of a 96-well plate. For each working concentration of standard protein, two replicates were set up in the 96-well plate containing BCA working solution, with 5 μL per well. For sample protein, the supernatant was collected, and three replicates were made for each sample. After shielding from light, the plates were incubated at 37 ℃ for 30 min. After incubation, the absorbance at 562 nm was measured using a microplate reader. The average absorbance of the standard protein solution was calculated, and a scatter plot was created with the standard protein concentration as the x-axis and the average OD value as the y-axis. A standard curve was plotted, and the formula (R0) was generated. 2 ≥ 0.99); input the OD value of the sample protein, calculate the sample protein concentration according to the formula, add 6 μL of 5× protein loading buffer to each sample, and then dilute with water until the total loading volume and protein amount of each protein sample are equal; after the protein sample preparation is completed, vortex mix, centrifuge at 2000 rmp for 10 s, and store at -20 ℃ for later use.

[0101] Assemble the gel on the gel casting rack. Prepare separating and stacking gels of different concentrations according to the protein molecular weight. Add separating gel to the lower edge of the green bar, then add ultrapure water to slightly fill the gap. Let it stand for 20-30 minutes until the separating gel solidifies. Add the prepared stacking gel and insert the 10-well comb. Let it stand for 1 hour until the stacking gel solidifies. Immediately after solidification, perform electrophoresis or store the solidified gel at 4°C. Take the freshly solidified gel or the gel stored at 4°C, gently pull out the comb, and rinse the sample wells in the electrolytic buffer to remove the gel strands in the sample wells to prevent affecting the sample loading effect. Take the protein samples out of the -20°C freezer, heat them in a metal bath at 100°C for 6 minutes, and then centrifuge at 12000 rpm for 1 minute. Add 8 μL of marker to each well. Load all protein samples with fixed protein amounts into each well at 30 μL. Insert the electrodes and start electrophoresis. Maintain a constant voltage of 100 V for 30 minutes, then adjust to a constant voltage of 130 V for 1-2 minutes. h, electrophoresis until the target protein is fully separated; after the target protein is separated, cut PVDF membranes and filter paper of the same size according to the gel. The PVDF membrane is activated with methanol. From bottom to top, place the black block of the clamp → sponge pad → filter paper → gel → PVDF membrane → filter paper → sponge pad → white block of the clamp, clamp the clamp tightly, place it in the electrophoresis tank, insert the electrodes, and transfer the membrane at a constant voltage of 100 V for 1.5 h; after the protein is completely transferred into the PVDF membrane, cut the target protein band, place it in blocking buffer (1×TBST solution containing 5% skim milk powder), and block it on a shaker for 1 h; after blocking, wash the membrane with TBST solution, repeat 3 times, 10 min each time; dilute the primary antibody with antibody diluent at an appropriate ratio, place the membrane in the primary antibody and incubate overnight (8~10 h) at 4 ℃; the next day, wash the membrane with TBST solution, repeat 3 times, 10 min each time, and incubate it in the secondary antibody corresponding to the species of the primary antibody for 1 h; after the secondary antibody incubation is complete, wash the membrane with TBST solution, repeat 3 times, 10 min each time. min; Mix solution A and solution B of the enhanced chemiluminescence reagent (ECL luminescent solution) in equal proportions, absorb the liquid on the membrane, add the luminescent solution, gently shake to distribute it evenly on the membrane, use a chemiluminescence imaging system to detect and acquire protein band images, with GAPDH as the internal reference protein.

[0102] Figure 2 The figure shows the effect of strigoglycoside A on LPS-induced survival rate of HepG2 cells as described in the experimental example. Figure 2 The results showed that strigoglycoside A had little effect on the survival rate of HepG2 cells and was not cytotoxic in the concentration range of 0-100 μmol / L; however, strigoglycoside A had significant cytotoxicity on HepG2 cells at concentrations of 200 μmol / L and 400 μmol / L.

[0103] Figure 3The figure shows the effect of strigoglycoside A on the expression levels of IL-10 and NF-κB proteins in LPS-induced HepG2 cells, as described in the experimental example. Figure 3 The results showed that 100 nM LPS induction significantly increased the expression levels of IL-10 and NF-κB proteins in HepG2 cells. However, strigoglycoside A at concentrations of 100 μM, 200 μM, and 400 μM significantly reduced the expression of the above proteins, and the effect was better than that of the positive control silymarin.

[0104] Results and Discussion:

[0105] This application describes the extraction and preparation of a novel natural lignan glycoside compound (Striga asiatica glycoside A) from the traditional hepatoprotective medicinal plant *Striga asiatica*. The monomeric compound was rapidly prepared by extraction under normal pressure and reflux, followed by one-step silica gel column chromatography and one-step reversed-phase HPLC purification. Striga asiatica glycoside A exhibits significant anti-hepatitis activity in the concentration range of 0 μmol / L–100 μmol / L; at tested concentrations of 200 μmol / L and 400 μmol / L, it demonstrates significant HepG2 cytotoxicity and anti-hepatocellular carcinoma activity.

[0106] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Strigosin A, characterized in that: The structure of strigoglycoside A is: 。 2. The extraction method of strigoglycoside A according to claim 1, characterized in that: Includes the following steps: S1. The dried whole herb of *Striga asiatica* was crushed into small pieces and extracted three times by heating and reflux with 75% methanol at a material-to-liquid ratio of 1g:20mL, each time for 3 hours. S2. After filtering with gauze, the methanol extracts were combined, and the solvent was recovered by rotary evaporator to obtain the methanol extract of *Striga asiatica*. S3. The methanol extract of *Striga asiatica* obtained in S2 was dispersed and dissolved in distilled water. According to the ratio of methanol extract of *Striga asiatica* to distilled water = 100g: 300mL, petroleum ether, ethyl acetate, and n-butanol with boiling ranges of 60℃~90℃ were used for extraction in sequence, according to the polarity of the solvent. Each extraction was performed 5 times to obtain different extract fractions. After vacuum concentration and recovery, petroleum ether extract, ethyl acetate extract, and n-butanol extract were obtained. S4. Take 21.5g of ethyl acetate extract, dissolve it in 20mL-50mL of methanol, add the sample to an equal amount of silica gel in small amounts several times, heat in a water bath, and grind continuously to obtain a uniform particle size and dry sample-silica gel mixture. Separate by normal silica gel column chromatography, using dichloromethane-methanol gradient elution at a gradient of 50:1→1:

3. Detect and combine identical fractions by TLC to obtain 12 components, named E1~E12. Component E8 was prepared and separated by RP-HPLC with a mobile phase of methanol-water gradient of 30:70→80:20, v / v; flow rate: 3 mL / min. Gradient elution yielded strigoglycoside A. R =34.0 min, the structure of strigoglycoside A is: 。 3. The extraction method of strigoglycoside A according to claim 2, characterized in that: The addition of an equal amount of silica gel as described in S4, wherein the silica gel is 100 mesh to 200 mesh.

4. The extraction method of strigoglycoside A according to claim 2, characterized in that: The separation described in S4 is performed by forward silica gel column chromatography, wherein the silica gel is 200-300 mesh.

5. The use of strigoglycoside A according to claim 1 in the preparation of an anti-hepatitis active drug.

6. The application of strigoglycoside A according to claim 5 in the preparation of an anti-hepatitis active drug, characterized in that: This is used to prepare an anti-hepatitis active drug that inhibits the production of inflammatory factors IL-10 and NF-κB protein.

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