Schisandracean triterpenoids, preparation method and application thereof

Abstract

The application belongs to the technical field of natural product chemistry, and discloses a schisandrin triterpenoid compound, a preparation method and application thereof. The schisandrin triterpenoid compound is prepared from dried fruits of Schisandra sphenanthera as raw materials, through organic solvent extraction, combined with silica gel normal phase column chromatography, ODS reverse phase column chromatography and preparative high performance liquid chromatography multistage chromatography separation and purification, and finally the target compound is obtained. The biological activity test results prove that the compound has excellent antioxidant stress, anti-ultraviolet radiation stress and in-vitro anti-inflammatory biological activity, can improve the body tolerance of Caenorhabditis elegans to oxidative and ultraviolet stress damage, and can inhibit the release of NO of macrophages induced by lipopolysaccharide. Therefore, the compound has potential application value in preparation of antioxidant, anti-light damage and anti-inflammatory related drugs, functional health care food and daily chemical functional products.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, and discloses a triterpenoid compound of Schisandra chinensis, its preparation method and application. Background Technology

[0002] Inflammatory response and oxidative stress are two key physiological and pathological response mechanisms that the body generates in response to exogenous stimuli and endogenous pathological damage. They are closely related to the occurrence and development of various diseases such as aging, cardiovascular disease, neurodegenerative disease, metabolic disease, and photodamage to the skin.

[0003] Excessive accumulation of reactive oxygen species (ROS) can disrupt intracellular redox homeostasis, leading to lipid peroxidation, protein damage, and DNA damage, thereby accelerating aging and promoting inflammatory responses. Furthermore, ultraviolet radiation is a significant environmental factor causing photoaging and oxidative damage to the skin, inducing the production of large amounts of ROS in the epidermis and dermis, resulting in cell damage and inflammatory responses. Therefore, discovering natural active ingredients with multiple activities including antioxidant, anti-UV damage, and anti-inflammatory properties from natural resources has become an important direction in new drug development and functional product development.

[0004] Schisandra grandiflora ( Schisandra grandiflora Schisandra chinensis is a perennial deciduous vine belonging to the genus Schisandra in the family Schisandraceae. It typically grows in mountain forest edges and shrublands at altitudes of 1800–3100 m. Schisandra species have a long history of use in folk medicine and traditional medicine. The dried fruit of *Schisandra chinensis* is a commonly used medicinal material, often used to astringe the lungs and stop sweating, replenish qi and promote body fluid production, and tonify the kidneys and calm the mind. It has high medicinal value in treating chronic cough and asthma, thirst due to fluid depletion, palpitations, and insomnia. In recent years, Schisandra species have received widespread attention in the field of natural product chemistry due to their rich content of structurally diverse lignans and triterpenoid secondary metabolites. However, there are few reports on the systematic isolation and multi-effect pharmacological evaluation of the triterpenoid active components in *Schisandra chinensis*, and its active material basis needs further exploration. Summary of the Invention

[0005] Therefore, the object of the present invention is to provide a triterpenoid compound of Schisandra chinensis having the structure shown in formula (I): Formula (I) .

[0006] Another object of the present invention is to provide a method for preparing triterpenoid compounds from Schisandra chinensis, comprising the following steps: (1) Extraction: Using dried fruit of Schisandra chinensis as raw material, ethanol was used for cold soaking at room temperature. After solid-liquid separation, the extract was collected and concentrated under reduced pressure to obtain ethanol extract. The ethanol extract was then dispersed in water to prepare a suspension. (2) Fractional extraction: First, add petroleum ether of the same volume as the suspension to the suspension obtained in step (1) to extract and separate the layers, collect the lower aqueous phase extract, repeat the extraction 3 to 4 times, and combine all the aqueous phase extracts; then add ethyl acetate of the same volume as the aqueous phase extract to the combined aqueous phase extract to extract and separate the layers, collect the upper ethyl acetate extract, and repeat the extraction 3 to 4 times. (3) First silica gel column chromatography separation: The ethyl acetate fraction obtained in step (2) was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the elution system, and the fraction Fr.2 was collected; (4) Polyamide column chromatography separation: The fraction Fr.2 obtained in step (3) was subjected to polyamide column chromatography and eluted with an ethanol-water gradient to collect fraction Fr.2-1; (5) Second silica gel column chromatography separation: The fraction Fr.2-1 obtained in step (4) was subjected to silica gel column chromatography with gradient elution using dichloromethane-methanol as the elution system, and fraction Fr.2-1-3 was collected; (6) ODS reversed-phase column chromatography separation: The fraction Fr.2-1-3 obtained in step (5) was subjected to ODS reversed-phase column chromatography with gradient elution using methanol-water as the elution system, and the fraction Fr.2-1-3-17 was collected. (7) Semi-preparative high performance liquid chromatography purification: The component Fr.2-1-3-17 obtained in step (6) was subjected to semi-preparative high performance liquid chromatography, and was eluted with methanol-water as the mobile phase. The eluent of the target peak was collected and concentrated to obtain the crude product. The crude product was subjected to semi-preparative high performance liquid chromatography, and was eluted with acetonitrile-water as the mobile phase. The crude product was concentrated to obtain the triterpenoid compounds of Schisandra chinensis.

[0007] Preferably, in step (1), the volume fraction of ethanol is 95%; the mass-to-volume ratio of the raw material to ethanol in the cold maceration extraction is 1 kg:(2-4) L; and the cold maceration extraction time is 60-84 h.

[0008] Preferably, in step (3), the gradient elution volume ratio range of the petroleum ether-ethyl acetate elution system is 20:1 to 0:1.

[0009] Preferably, in step (3), the component Fr.2 is the elution component when the volume ratio of petroleum ether to ethyl acetate is 2:1.

[0010] Preferably, in step (4), the gradient elution volume fraction of the ethanol aqueous solution is in the range of 30% to 90%.

[0011] Preferably, in step (4), the component Fr.2-1 is the elution component with an ethanol volume fraction of 30%.

[0012] Preferably, in step (5), the gradient elution volume ratio range of the dichloromethane-methanol elution system is 100:1 to 0:1.

[0013] Preferably, in step (5), the component Fr.2-1-3 is the elution component when the volume ratio of dichloromethane to methanol is 50:1.

[0014] Preferably, in step (6), the gradient elution volume ratio range of the methanol-water elution system is 10:90 to 95:5.

[0015] Preferably, in step (6), the elution flow rate of the ODS reversed-phase column chromatography is 15 mL / min; the component Fr.2-1-3-17 is the elution component when the volume ratio of methanol to water is 50:50.

[0016] Preferably, in step (7), the volume ratio of methanol to water in the methanol-water mobile phase is 43:57, the elution flow rate of the semi-preparative high performance liquid chromatography is 8 mL / min, and the retention time of the target peak is 14 min; the volume ratio of acetonitrile to water in the acetonitrile-water mobile phase is 28:72.

[0017] Another object of the present invention is to provide the application of Schisandra chinensis triterpenoids in the preparation of anti-inflammatory products.

[0018] Preferably, the anti-inflammatory product is a drug, health product, or functional product used to treat or prevent inflammatory diseases mediated by nitric oxide.

[0019] Another objective of this invention is to provide the application of Schisandra chinensis triterpenoids in the preparation of anti-stress products.

[0020] Preferably, the anti-stress product is a drug, health product, or functional product that enhances the body's tolerance to oxidative stress and / or ultraviolet radiation stress.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention isolates a novel compound, Arisandilactone F, from the dried fruit of Schisandra chinensis. After in vitro cell and nematode in vivo activity verification, the compound has antioxidant stress, anti-ultraviolet light damage and anti-inflammatory activities. It can inhibit the release of nitric oxide (NO), a key inflammatory mediator, and prolong the survival time of nematodes under stress. It can be developed into anti-inflammatory, anti-aging, and anti-photodamage drugs, health foods or skin functional preparations with a wide range of applications.

[0022] (2) The present invention adopts a petroleum ether-ethyl acetate fractional extraction system. Petroleum ether removes low-polarity impurities such as oils and pigments in advance, and then enriches the target compound in the ethyl acetate phase, which greatly reduces the load of subsequent chromatographic separation and effectively solves the problem of complex impurities and difficult separation of Schisandra chinensis extract.

[0023] (3) The present invention uses general commercial chromatographic packing materials such as silica gel, polyamide, and ODS throughout the process. The raw materials are readily available and the procurement cost is low. It is equipped with conventional organic solvents such as petroleum ether, ethyl acetate, methanol, and ethanol. The solvent boiling points are moderate and easy to distill and recover. The whole process is mild and does not require high temperature, high pressure or special reaction equipment. It is easy to operate, safe and controllable, and has strong process stability, and has the potential for industrial production.

[0024] (4) This invention uses multi-stage column chromatography gradient elution combined with two-and-a-half-step preparative HPLC fine purification to remove homologous impurities with similar polarity step by step, and finally obtains high-purity monomer compounds without the need for complex derivatization treatment. The product structure is stable, which is convenient for subsequent active formulation development and in-depth pharmacological research. Attached Figure Description

[0025] Figure 1 The ultraviolet spectrum of the target compound prepared in Example 1 of this invention; Figure 2 The hydrogen nuclear magnetic resonance spectrum of the compound Arisandilactone F prepared in Example 1 of this invention in deuterated methanol (MeOD); Figure 3 The image shows the carbon NMR spectrum of the compound Arisandilactone F prepared in Example 1 of this invention in deuterated methanol (MeOD). Figure 4 The image shows the heteronuclear single quantum correlation (HSQC) spectrum of the compound Arisandilactone F prepared in Example 1 of this invention in deuterated methanol (MeOD); Figure 5 The image shows the heteronuclear multibond correlation (HMBC) spectrum of the compound Arisandilactone F prepared in Example 1 of this invention in deuterated methanol (MeOD); Figure 6 The hydrogen-hydrogen correlation spectrum (HH COSY) of the compound Arisandilactone F prepared in Example 1 of this invention in deuterated methanol (MeOD); Figure 7 The image shows the nuclear Overhausen effect (NOESY) spectrum of the compound Arisandilactone F prepared in Example 1 of this invention in deuterated methanol (MeOD); Figure 8(A) is a graph showing the survival rate under ultraviolet stress in Application Example 1 of the present invention; (B) is a graph showing the survival rate under oxidative stress in Application Example 1 of the present invention. Figure 9 The NO concentration-absorbance standard curve is shown in Example 2 of this invention. Figure 10 This is a bar chart showing the NO concentration in the cell supernatant of each group in Application Example 2 of the present invention. Detailed Implementation

[0026] Example 1 The raw material used in this embodiment, dried fruit of Schisandra chinensis, is a commercially available Chinese medicinal material. The original collection site is a mountainous area at an altitude of 1900 m in southwestern Yunnan. It has been identified by botanical taxonomy as the dried mature fruit of Schisandra chinensis in the Schisandraceae family.

[0027] In this embodiment, the silica gel column chromatography packing material is 200-300 mesh silica gel with a particle size of 54-70 μm. The mass ratio of the sample component to the silica gel packing material is 1:40, and gravity elution is performed at room temperature and pressure. In this embodiment, the polyamide column chromatography packing material is 80-100 mesh polyamide with a mass ratio of the sample component to the polyamide packing material of 1:30, and elution is performed at room temperature and pressure. In this embodiment, the ODS reversed-phase column chromatography uses octadecylsilane-bonded silica gel with a particle size of 50 μm, a carbon loading of 17%, and a pore size of 120 Å. The operation is at room temperature, with constant flow pump speed control and a fixed elution flow rate of 15 mL / min. The mass ratio of the sample component to the ODS packing material is 1:20. In this embodiment, the semi-preparative high-performance liquid chromatography uses a 250 × 20 mm column with a particle size of 5 μm. The column temperature is kept constant at 25℃, and constant flow elution is performed. The flow rates of both the methanol-water mobile phase and the acetonitrile-water mobile phase are 8 mL / min, and the detection wavelength is 248 nm.

[0028] This embodiment provides a method for preparing triterpenoid compounds from Schisandra chinensis, the specific steps of which are as follows: (1) Weigh 4.0 kg of dried Schisandra chinensis fruit as raw material, use 95% ethanol aqueous solution as extraction solvent, add extraction solvent at a mass-volume ratio of 1 kg: 3 L of raw material to extraction solvent, and cold soak extraction at room temperature for 72 h; after extraction, perform solid-liquid separation, collect the extract, use rotary evaporator to concentrate under reduced pressure to remove solvent, and obtain ethanol extract. Disperse the obtained ethanol extract in an appropriate amount of water to prepare a suspension.

[0029] (2) First, add petroleum ether of the same volume as the suspension to the suspension obtained in step (1), shake thoroughly to extract, let stand to separate the layers, discard the upper petroleum ether organic phase, collect the lower aqueous phase extract, repeat the extraction 4 times, and combine all the aqueous phase extracts; then add ethyl acetate of the same volume as the aqueous phase extract, shake thoroughly to extract, let stand to separate the layers, collect the upper ethyl acetate extract, discard the lower aqueous phase, repeat the extraction 4 times, combine all the ethyl acetate extracts, and concentrate under reduced pressure using a rotary evaporator to obtain the ethyl acetate fraction extract.

[0030] (3) The ethyl acetate extract obtained in step (2) was separated by silica gel column chromatography. Gradient elution was performed using a petroleum ether-ethyl acetate (v / v) system with elution ratios of 20:1, 15:1, 10:1, 5:1, 1:1 and 0:1 respectively. The eluted components were combined according to the thin-layer chromatography (TLC) detection results, and a total of 5 components were obtained, which were denoted as Fr.1 to Fr.5 respectively. Among them, Fr.2 was the component obtained by elution with petroleum ether-ethyl acetate (2:1, v / v).

[0031] (4) Take the Fr.2 component obtained in step (3) and further separate it by polyamide column chromatography. Use ethanol aqueous solution with volume fractions of 30%, 50%, 70% and 90% for gradient elution. Combine the eluted components according to the TLC detection results to obtain a total of 4 components, which are denoted as Fr.2-1 to Fr.2-4 respectively. Among them, Fr.2-1 is the component obtained by elution with 30% ethanol aqueous solution.

[0032] (5) Take the Fr.2-1 fraction obtained in step (4) and further separate it by silica gel column chromatography. Use a dichloromethane-methanol (v / v) system for gradient elution with elution ratios of 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 1:1 and 0:1 respectively. Combine the eluted fractions according to the TLC detection results to obtain a total of 8 fractions, which are denoted as Fr.2-1-1 to Fr.2-1-8 respectively. Among them, Fr.2-1-3 is the fraction obtained by dichloromethane-methanol (50:1, v / v) elution.

[0033] (6) Take the Fr.2-1-3 fraction obtained in step (5) and further separate it by ODS reversed-phase column chromatography. Use a methanol-water (v / v) system for gradient elution with elution ratios of 10:90, 25:75, 40:60, 50:50, 60:40, 70:30, 85:15, 90:10 and 95:5 respectively, and the flow rate is 15 mL / min. Combine the eluted fractions according to the TLC detection results to obtain a total of 21 fractions, which are denoted as Fr.2-1-3-1 to Fr.2-1-3-21 respectively. Among them, Fr.2-1-3-17 is the fraction obtained by methanol-water (50:50, v / v) elution.

[0034] (7) Take the Fr.2-1-3-17 fraction obtained in step (6), use methanol-water (43:57, v / v) as the mobile phase, and perform semi-preparative high performance liquid chromatography isocratic elution at a flow rate of 8 mL / min. Collect the target peak with a retention time of 14 min, and after concentration to remove the solvent, obtain the crude product. The crude product is further subjected to semi-preparative high performance liquid chromatography isocratic elution using acetonitrile-water (28:72, v / v) as the mobile phase. Collect the target chromatographic peak, concentrate and dry to obtain the target compound (Arisandilactone F).

[0035] The compound Arisandilactone F prepared in this example is a white powder. The characterization results of this compound are as follows: Specific rotation: [ α ]20 D +27.8 ( c 0.1, CD3OD).

[0036] Ultraviolet spectrum such as Figure 1 As shown, this compound exhibits maximum ultraviolet absorption at a wavelength of 261 nm in methanol solution, with a logarithmic molar absorptivity of 2.52.

[0037] High-resolution mass spectrometry: HRESIMS m / z 551.2255 [M + Na] + (calcd. for C 29 H 36 O9Na, 551.2252).

[0038] 1H NMR spectrum: 1 H NMR (CD3OD, 600 MHz), see Table 1.

[0039] Carbon NMR spectrum: 13 C NMR (CD3OD, 150 MHz), see Table 1.

[0040] Table 1. NMR data of compounds Arisandilactone A and Arisandilactone F in MeOD. The structure of compound Arisandilactone A is as follows: Table 1 shows that the target compound contains 5 methyl proton signals in its 1H NMR spectrum: δH 1.20 (3H, s, Me-18), δH 1.13 (3H, d, J= 6.4 Hz, Me-21), 1.88 (3H, s, Me-27), 1.24 (3H, s, Me-29), 1.33 (3H, s, Me-30); 10 methylene proton signals δH 3.50 (H, m, H-2α), 2.48 (1H, d, J = 18.4 Hz, H-2β), 1.99 (1H, m, H-6α), 1.62 (1H, m, H-6β), 2.11 (1H, m, H-7α), 1.60 (1H, m, H-7β), 1.43 (1H, m, H-11α), 2.04 (1H, m, H-11β), 1.10 (1H, overlap, H-12α), 2.33 (1H, m, H-12β); δH signals for four methine protons: 2.25 (1H, m, H-5), 2.36 (1H, m, H-8), 2.29 (1H, m, H-17), 3.49 (1H, m, H-20); δH signals for four oxymethine protons: 4.27 (1H, d, J = 5.7 Hz, H-1), 3.95 (1H, s, H-19), 3.74 (1H, d, J = 9.4Hz, H-22), 4.98 (1H, d, J = 1.3 Hz, H-23); 2 olefin proton signals δH 5.77 (1H, d, J =8.7 Hz, H-16), 7.17 (H, s, H-24). The carbon NMR spectrum showed 29 carbon signals, including 3 carbonyl carbon signals δC 178.3 (C-3), 201.5 (C-14), 183.3 (C-26); 2 groups of olefin carbon signals δC 154.9 (C-15), 113.2 (C-16), 149.6 (C-24), 130.7 (C-25); and 8 oxygen-containing carbon signals δC 80.2 (C-1), 85.5 (C-4), 99.5 (C-10), 89.1 (C-9), 86.2 (C-13), 69.4 (C-19), 88.6 (C-22), 77.0 (C-23).

[0041] according to Figure 5The HMBC spectrum shown, along with the related signals H-8 / C-14, H-9 / C-13, H-11 / C-19, C-13, H-12 / C-9, C-13, H-16 / C-14, C-17, H-17 / C-15, H-21 / C-17, C-22, and C-23, combined with... Figure 6 The 1H-1H COSY correlation signals H-12 / H-13, H-17 / H-18, H-17 / H-21 and the carbon signals δC 89.1 (C-9), 201.5 (C-14), 154.9 (C-15) shown can be used to deduce the D, E and F rings.

[0042] A comparison of the spectra of compound Arisandilactone F with those of the known compound Arisandilactone A revealed that in Arisandilactone A, the carbon signals at positions C-24 and C-25 are located in the low-field region, indicating saturated carbons (δC 34.2 and 35.3). In Arisandilactone F, the carbon signals at positions C-24 and C-25 were not observed in the low-field region of Arisandilactone A. Instead, two olefinic carbon signals (δC 149.6 and 130.7) were observed in the high-field region. It is speculated that the C-24 and C-25 positions of compound Arisandilactone F are double bonds.

[0043] exist Figure 4 In the HSQC spectrum shown, only one H (δH 7.17) is directly connected to C-24 (δC 149.6), and no H was observed connected to C-25, indicating that C-25 is a quaternary olefin. In the HMBC spectrum, H-24 is correlated with C-23 / C-25 / C-26, H-27 is correlated with C-24 / C-25 / C-26, and H-23 is correlated with C-24 / C-25 / C-27. Combined with the 1H-1H COSY correlation signals H-23 / H-24 / H-22 and H-24 / H3-27, the existence of a double bond between C-24 and C-25 can be confirmed. Meanwhile, high-resolution mass spectrometry shows that the molecular formula of compound Arisandilactone F has one more degree of unsaturation than that of Arisandilactone A. Furthermore, the HSQC and HMBC spectra mentioned above prove that C-24 and C-25 are sp² hybridized and there are no other unsaturated functional groups present. Therefore, this degree of unsaturation must be attributed to the double bond formed between C-24 and C-25.

[0044] Furthermore, the NOESY related signals H-23 / H-24 / H-22 / Me-27, Me-29 / H-1, H-17 / Me-21, and Me-30 / H-5 are consistent with those of compound Arisandilactone A. Therefore, the structure of compound Arisandilactone F can be determined, as shown in formula (I): Formula (I) .

[0045] Application Example 1 This application example uses the N2 strain of wild-type Caenorhabditis elegans to conduct oxidative stress and ultraviolet stress tests. By measuring the survival rate of the nematodes, the antioxidant and anti-ultraviolet stress activating properties of the compound Arisandilactone F are evaluated. The specific steps are as follows: First, wild-type Caenorhabditis elegans of strain N2 was cultured synchronously. After growing to the L4 larval stage, the nematodes were divided into groups: the experimental group was inoculated with NGM medium containing the compound Arisandilactone F (concentration 50 μM), and the blank control group was treated with an equal volume of ordinary NGM medium with M9 buffer. Both groups of nematodes were placed in a constant temperature environment of 20 ℃ for 5 days to complete the drug pre-incubation.

[0046] Oxidative stress survival rate determination: From the two groups of pre-incubated nematodes, 20 individuals were randomly selected from each group and transferred to a drug-free NGM plate containing 30 mmol / L H2O2 to construct an oxidative stress model. The survival of nematodes was observed and recorded every 0.5–1 h until all nematodes on the plate died. The criterion for determining nematode death was that there was no stress withdrawal response when the nematode's body was gently touched with a platinum wire, and the physiological activity of pharyngeal pumping ceased and spontaneous movement disappeared.

[0047] UV stress survival rate determination: Twenty nematodes were randomly selected from each of the two pre-incubated samples and transferred to blank E. coli plates without OP50 coating. After opening the petri dish, UV irradiation was performed to establish a UV damage model. The survival of nematodes was observed and recorded every 0.5–1 h until all nematodes in the plate died. The criteria for determining nematode mortality were consistent with those used in the oxidative stress survival rate determination experiment.

[0048] To eliminate random error, both the oxidative stress survival experiment and the UV stress survival experiment were independently repeated three times. Results are expressed as mean ± standard deviation (Mean ± SD), and the differences in survival curves were statistically analyzed using the Log-rank test. The results showed that under oxidative stress conditions, compared with the blank control group, the experimental group significantly prolonged the average survival time of *C. elegans*, with an extension rate of 52.33% (calculated based on the median survival time of each group). The difference was statistically significant (P < 0.001), indicating that this compound has good antioxidant activity. Survival curves are shown below. Figure 8 (B); Under ultraviolet stress, compared with the blank control group, the experimental group also significantly improved the survival rate of *C. elegans*, with an average survival time extension rate of 22.89%. The survival curve is shown in [Figure number missing]. Figure 8 (A)

[0049] Application Example 2 This application example uses lipopolysaccharide (LPS) to induce RAW 264.7 (mouse mononuclear macrophage leukemia cells), and evaluates the in vitro anti-inflammatory activity of compound Arisandilactone F by detecting the nitric oxide (NO) release level in the cell supernatant. The specific steps are as follows: RAW 264.7 cells were fed at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 100 μL of cell suspension per well in 96-well cell culture plates. The plates were then incubated until cells adhered and reached a suitable confluence. The original culture supernatant was discarded, and 100 μL of the corresponding treatment solution was added to each well. Four parallel treatment groups were set up, with three replicates per group. The experiment was independently repeated three times. Blank control group: Only an equal volume of basal culture medium was added, without LPS or drug stimulation; Model control group: LPS (1 μg / mL) was added only; Positive control group: LPS (1 μg / mL) and dexamethasone (100 μM) were added simultaneously. Test compound group: LPS (1 μg / mL) and compound Arisandilactone F (100 μM) were added simultaneously.

[0050] The four parallel treatment groups were cultured at 37 ℃ and 5% CO2 for 24 h. After culture, 50 μL of cell culture supernatant was transferred to a new 96-well plate. After equilibration in the dark, 50 μL of Griess reagent I and 50 μL of Griess reagent II were added sequentially, and the color reaction was carried out at room temperature in the dark for 10–15 min. Subsequently, the absorbance (A) of each well was measured at 560 nm using a microplate reader. Simultaneously, 0–100 μM NaNO2 standard solutions were prepared, and absorbance was measured using the same color development and detection procedure. A NO concentration-absorbance standard curve was plotted (see [link to standard curve]). Figure 9 The NO concentration in each group of samples was calculated by combining the sample absorbance values.

[0051] Depend on Figure 10 It was found that compound Arisandilactone F exhibited certain inhibitory activity against LPS-induced nitric oxide production in RAW264.7 cells. Compared with the control group, the NO content was significantly increased after LPS treatment, while the NO content decreased after treatment with compound Arisandilactone F. The inhibition rate of NO production in LPS-induced RAW264.7 macrophages by compound Arisandilactone F was 36.93 ± 3.57%, while that in the dexamethasone positive control group was 77.50 ± 1.45%, both showing significant inhibitory effects compared to the model group. This example demonstrates, through a classic LPS-induced macrophage in vitro inflammation model, that compound Arisandilactone F can effectively inhibit the production of NO, a key mediator in the inflammatory response, exhibiting clear in vitro anti-inflammatory activity. This provides important preliminary in vitro cell experiments to support the development of anti-inflammatory drugs and functional formulations based on this compound.

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A triterpenoid compound from Schisandra chinensis, characterized in that, It has the structure shown in equation (I): Equation (I) .

2. The method for preparing the triterpenoid compounds of Schisandra chinensis according to claim 1, characterized in that, Includes the following steps: (1) Extraction: Using dried fruit of Schisandra chinensis as raw material, ethanol was used for cold soaking at room temperature. After solid-liquid separation, the extract was collected and concentrated under reduced pressure to obtain ethanol extract. The ethanol extract was then dispersed in water to prepare a suspension. (2) Fractional extraction: First, add petroleum ether of the same volume as the suspension to the suspension obtained in step (1) to extract and separate the layers, collect the lower aqueous phase extract, repeat the extraction 3 to 4 times, and combine all the aqueous phase extracts; then add ethyl acetate of the same volume as the aqueous phase extract to the combined aqueous phase extract to extract and separate the layers, collect the upper ethyl acetate extract, and repeat the extraction 3 to 4 times. (3) First silica gel column chromatography separation: The ethyl acetate fraction obtained in step (2) was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the elution system, and the fraction Fr.2 was collected; (4) Polyamide column chromatography separation: The fraction Fr.2 obtained in step (3) was subjected to polyamide column chromatography and eluted with an ethanol-water gradient to collect fraction Fr.2-1; (5) Second silica gel column chromatography separation: The fraction Fr.2-1 obtained in step (4) was subjected to silica gel column chromatography with gradient elution using dichloromethane-methanol as the elution system, and fraction Fr.2-1-3 was collected; (6) ODS reversed-phase column chromatography separation: The fraction Fr.2-1-3 obtained in step (5) was subjected to ODS reversed-phase column chromatography with gradient elution using methanol-water as the elution system, and the fraction Fr.2-1-3-17 was collected. (7) Semi-preparative high performance liquid chromatography purification: The component Fr.2-1-3-17 obtained in step (6) was subjected to semi-preparative high performance liquid chromatography, and was eluted with methanol-water as the mobile phase. The eluent of the target peak was collected and concentrated to obtain the crude product. The crude product was subjected to semi-preparative high performance liquid chromatography, and was eluted with acetonitrile-water as the mobile phase. The crude product was concentrated to obtain the triterpenoid compounds of Schisandra chinensis.

3. The method for preparing triterpenoid compounds from Schisandra chinensis according to claim 2, characterized in that, In step (1), the volume fraction of ethanol is 95%; the mass-volume ratio of the raw material to ethanol in the cold maceration extraction is 1 kg:(2-4)L; and the cold maceration extraction time is 60-84 h.

4. The method for preparing triterpenoid compounds from Schisandra chinensis according to claim 2, characterized in that, In step (3), the component Fr.2 is the elution component when the volume ratio of petroleum ether to ethyl acetate is 2:

1.

5. The method for preparing triterpenoid compounds from Schisandra chinensis according to claim 2, characterized in that, In step (4), the component Fr.2-1 is the elution component when the volume fraction of ethanol is 30%.

6. The method for preparing triterpenoid compounds from Schisandra chinensis according to claim 2, characterized in that, In step (5), the component Fr.2-1-3 is the elution component when the volume ratio of dichloromethane to methanol is 50:

1.

7. The method for preparing triterpenoid compounds from Schisandra chinensis according to claim 2, characterized in that, In step (6), the elution flow rate of the ODS reversed-phase column chromatography is 15 mL / min; the component Fr.2-1-3-17 is the elution component when the volume ratio of methanol to water is 50:

50.

8. The method for preparing triterpenoid compounds from Schisandra chinensis according to claim 2, characterized in that, In step (7), the volume ratio of methanol to water in the methanol-water mobile phase is 43:57, the elution flow rate of the semi-preparative high performance liquid chromatography is 8 mL / min, and the retention time of the target peak is 14 min; the volume ratio of acetonitrile to water in the acetonitrile-water mobile phase is 28:

72.

9. The use of the Schisandra chinensis triterpenoid compound according to claim 1 in the preparation of anti-inflammatory products.

10. The use of the Schisandra chinensis triterpenoid compound according to claim 1 in the preparation of anti-stress products.

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