A saponin compound, a preparation method and application thereof

Abstract

This application relates to the pharmaceutical field, specifically to a saponin compound, its preparation method, and its applications. This application provides a new class of compounds, including compound 1 and compound 2, and also isolates existing compounds 3-5. These compounds exhibit significant neuroprotective effects. Specifically, when compound 1 (12-dehydroxy-Gynostemma pentaphyllum saponin III) is at a concentration of 50 μM, compound 1 exhibits weak AChE inhibitory activity. Compounds 1-5 show significant protective activity against 6-OHDA-induced SH-SY5Y cell damage. Furthermore, compounds 2 and 3 effectively alleviate 6-OHDA-induced oxidative stress damage by activating the Nrf2 / HO-1 / NQO1 antioxidant signaling pathway and upregulating the expression of endogenous antioxidant enzymes, thereby exerting neuroprotective effects. Compound 1 also shows good efficacy in relieving paralysis in *A. adenophora* nematodes, while compounds 3 and 4 have some paralysis-relieving effects on *A. adenophora* nematodes.

Description

Technical Field

[0001] This application relates to the pharmaceutical field, and in particular to a saponin compound, its preparation method, and its application. Background Technology

[0002] Gynostemma pentaphyllum (Thunb.) Makino is a perennial herbaceous climbing plant belonging to the Cucurbitaceae family, widely distributed in East and South Asia. It has a long history of medicinal and edible use in traditional Chinese medicine. Gynostemma pentaphyllum is rich in various bioactive components such as saponins, polysaccharides, and flavonoids, with gypenosides being its most important active ingredient. Extracts of Gynostemma pentaphyllum and its saponins possess various biological activities, including lowering blood sugar and lipids, protecting the liver, anti-tumor effects, and regulating intestinal flora. It also has significant protective effects on the nervous system, showing therapeutic potential in various neuropsychiatric disease models such as depression, Alzheimer's disease, Parkinson's disease, secondary dementia, stroke, and optic neuritis.

[0003] Although modern medicine has gained a deeper understanding of the pathological mechanisms of these neurodegenerative diseases, most current treatments can only alleviate symptoms and are often accompanied by dose-limiting adverse reactions, making it difficult to effectively curb the progression of the disease. Therefore, exploring neuroprotective agents with multi-target regulatory functions and high safety profiles from natural products has become an important direction in drug development. However, Gynostemma pentaphyllum from different geographical sources exhibits significant differences in saponin types, flavonoid content, and fingerprint characteristics. These differences in chemical composition lead to significant regional characteristics in the antioxidant, anti-inflammatory, and anti-tumor activities of Gynostemma pentaphyllum from different origins.

[0004] Therefore, there is an urgent need to discover new gypenosides with excellent neuroprotective activity, and to provide a reference for the in-depth development of gypenosides and the research and development of drugs for the treatment of nervous system diseases. Summary of the Invention

[0005] To address or partially address the problems existing in the related technologies, this application provides a saponin compound comprising compound 1 and compound 2; compound 1 is 12-deoxygynosaponin III and compound 2 is 12-deoxygynosaponin IV.

[0006] Furthermore, the structural formula of compound 1 is as shown in Formula 1, and the structural formula of compound 2 is as shown in Formula 2; Formula 1, Equation 2.

[0007] On the other hand, this application also provides a method for preparing the above-mentioned saponin compounds, the preparation method comprising the following steps: S1, collect the aerial parts of Gynostemma pentaphyllum from Zhaotong, dry them, extract them by reflux with ethanol, concentrate them under reduced pressure using a rotary evaporator and dry them to obtain the extract, denoted as GPZJ; S2, after column chromatography separation and fragmentation of GPZJ, elution is performed to obtain GPZJ-1 to GPZJ-8; S3, after dissolving GPZJ-6 in pyridine, the mixture is separated, fragmented, and eluted to obtain GPZJ-6A-GPZJ-6L; S4, GPZJ-6J was dissolved in methanol and mixed with silica gel. After separation and elution by normal phase silica gel column chromatography, the samples were detected and combined to obtain GPZJ-6J1 to GPZJ-6J13. S5, of which GPZJ-6J7 is pure compound 1 after separation and purification; S6, wherein GPZJ-6J9 was dissolved in methanol and mixed with silica gel, separated and fragmented, eluted, and then combined after TLC detection to obtain GPZJ-6J9A to GPZJ-6J9G, wherein GPZJ-6J9A was separated and purified to obtain compound 2.

[0008] Furthermore, in S1, the reflux extraction is performed by refluxing with 70% ethanol at 50°C three times, for 4 hours each time.

[0009] Furthermore, in S4, the mass ratio of GPZJ-6J to silicone is 3.18:9.

[0010] Furthermore, the mass ratio of GPZJ-6J9 to silicone in S6 is 0.46:1.

[0011] Furthermore, in S3, the ratio of GPZJ-6 to pyridine is 1:5 (mg / ml).

[0012] Furthermore, the separation and purification methods in S5 and S6 are semi-preparative high-performance liquid chromatography.

[0013] On the other hand, this application also provides the use of the above-mentioned saponin compounds in the preparation of drugs for neuroprotective diseases.

[0014] Furthermore, the neuroprotective diseases include Alzheimer's disease and Parkinson's disease.

[0015] Beneficial effects 1. Compounds 1-2 provided in this application are all novel compounds. Compound 1 (12-dehydroxy-Gynostemma pentaphyllum saponin III) exhibits weak AChE inhibitory activity at a concentration of 50 μM, with an inhibition rate of 25.84% ± 1.38%. At a concentration of 2.5 μM, it shows significant protective activity against 6-LHDA-induced damage to SH-SY5Y cells, with a cell survival rate of 73.60%, compared to 50.87% in the blank control group. It also shows good efficacy in alleviating paralysis in AD nematodes. Compound 2 is also a novel compound. At a concentration of 2.5 μM, it shows significant protective activity against 6-LHDA-induced damage to SH-SY5Y cells, with a cell survival rate of 85.69%, compared to 52.64% in the blank control group. It can also effectively reduce 6-OHDA-induced oxidative stress damage by activating the Nrf2 / HO-1 / NQO1 antioxidant signaling pathway and upregulating the expression of endogenous antioxidant enzymes.

[0016] 2. Although compounds 3-5 are previously reported compounds, they also have neuroprotective effects.

[0017] Compound 3, at a concentration of 2.5 μM, showed significant protective activity against 6-LHDA-induced damage to SH-SY5Y cells, with a cell survival rate of 84.96%, compared to 53.88% in the blank control group. Compound 4, at a concentration of 2.5 μM, also showed significant protective activity against 6-LHDA-induced damage to SH-SY5Y cells, with a cell survival rate of 82.31%, compared to 80.85% in the blank control group. Compound 5, at a concentration of 2.5 μM, showed significant protective activity against 6-LHDA-induced damage to SH-SY5Y cells, with a cell survival rate of 74.27%, compared to 49.42% in the blank control group. Furthermore, compounds 3 and 4 showed some alleviating effect on paralysis in *A. adenocarcinoma*.

[0018] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0019] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0020] Figure 1 This is a flowchart of the extraction and separation process of chemical components from Gynostemma pentaphyllum; Figure 2 It is the chemical structure of compound 1 (12-deoxygynosaponin Ⅲ).

[0021] Figure 3It is the chemical structure of compound 2 (12-deoxygynosaponin Ⅳ).

[0022] Figure 4 The chemical structure of compound 3 (gynosaponin Ⅲ) Figure 5 It is the chemical structure of compound 4 (gynosaponin Ⅳ).

[0023] Figure 6 This is the chemical structure of compound 5 (gynosaponin V).

[0024] Figure 7 This shows the effect of compounds on the AD model of nematodes; the left figure shows the non-paralytic effect of compound 4 (WGZ7) over 24-36 hours. Rates; the right figure shows the non-paralyzing rates of compounds 1 (WGZ13), 2 (WGZ10), 3 (WGZ9), and 5 (WGZ11) over 24-36 hours. Other data are irrelevant to this application and are not explained.

[0025] Figure 8 The effects of treatment with five compounds (5 μM, 24 h) on the protein expression of Nrf2, HO-1, and NQO1 in nerve cells. Detailed Implementation Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0026] Studies have confirmed that Gynostemma pentaphyllum extract and its saponins can exert neuroprotective effects by alleviating oxidative stress damage, inhibiting neuroinflammation, and regulating autophagy and apoptosis. Specifically, total saponins from Gynostemma pentaphyllum can reduce abnormal overexpression of tau protein and improve neuronal function by regulating glycerophospholipid metabolism pathways. Ethanol extracts of Gynostemma pentaphyllum can regulate the expression of mitogen-activated protein kinase (MAPK) family and mitochondrial apoptosis-related proteins, protecting dopaminergic neurons and improving motor dysfunction in mice. For neurological injury following cerebral hemorrhage, Gynostemma pentaphyllum saponin XVII can activate the phosphatidylinositol 3-kinase (PI3K) / Akt signaling pathway, alleviating oxidative stress and inflammatory responses, and promoting neurological function recovery. Furthermore, Gynostemma pentaphyllum saponins can regulate the gut microbiota and its metabolite trimethylamine-N-oxide (TMAO) cycle, indirectly affecting the central nervous system microenvironment, providing a new perspective for intervening in neurodegenerative diseases. The study also found that Gynostemma pentaphyllum saponins can antagonize glutamate-induced oxidative neurological damage, with mechanisms involving maintaining mitochondrial membrane potential stability, increasing intracellular glutathione levels, and inhibiting excessive nitric oxide release. These studies collectively reveal the multi-target, multi-pathway synergistic regulatory characteristics of Gynostemma pentaphyllum's active components on neurological function. Parkinson's disease (PD) is the second leading cause of neurodegenerative disease worldwide, characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Numerous studies have shown that oxidative stress is a core component of PD pathogenesis. 6-hydroxydopamine (6-OHDA), a classic dopaminergic neurotoxin, can induce reactive oxygen species (ROS) bursts, deplete endogenous antioxidant enzymes (such as superoxide dismutase SOD), and trigger lipid peroxidation (increased malondialdehyde (MDA), ultimately leading to dopaminergic neuronal apoptosis. Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor regulating the endogenous antioxidant system. It can initiate the expression of downstream phase II detoxification enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), forming the first line of defense for cells against oxidative damage.

[0027] Located in the Wumeng Mountains, Zhaotong Prefecture in Yunnan Province boasts high altitude, large diurnal temperature variations, and soil rich in trace elements, fostering unique medicinal herb resources. Zhaotong Gynostemma pentaphyllum, a commonly used local herb, has long been used for its effects of "clearing heat and dampness, calming the mind and aiding sleep." However, to date, the chemical composition characteristics of Zhaotong Gynostemma pentaphyllum and its neuroprotective effects in Parkinson's disease models have not been systematically reported. Based on this research background, this paper focuses on studying the neuroprotective activity of Gynostemma pentaphyllum collected from Zhaotong City, Yunnan Province. This research fills a gap in the development of the medicinal value of Zhaotong Gynostemma pentaphyllum and also promotes the discovery of anti-PD lead compounds based on this resource.

[0028] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0029] Example 1 1. Extraction and separation of saponin compounds Two kg of dried powder from the aerial parts of Gynostemma pentaphyllum collected in Zhaotong City, Yunnan Province, was extracted three times with 70% ethanol under reflux at 50°C for four hours each time. The combined extracts were concentrated under reduced pressure using a rotary evaporator and dried to obtain 180 g of extract, denoted as GPZJ (180 g). 160 g of GPZJ was dissolved in 300 mL of distilled water and separated by column chromatography using a D101 macroporous resin (6 L) column with gradient elution using an ethanol-water solvent system (0%, 30%, 50%, 70%, 90% M / W). The solvent in the eluent was recovered using a rotary evaporator, and the eluent was analyzed by thin-layer chromatography (TLC) to obtain 8 fractions: GPZJ-1 (4.1 g), GPZJ-2 (56.9 g), GPZJ-3 (8.0 g), GPZJ-4 (10.5 g), GPZJ-5 (24.0 g), GPZJ-6 (11.9 g), GPZJ-7 (4.2 g), and GPZJ-8 (11.9 g).

[0030] GPZJ-6 (11.9g) was taken in batches (11g) and dissolved in pyridine (solid-to-material ratio 1:5, mg / mL). Separation was performed using medium-pressure column chromatography with fractional separation. A gradient elution system of methanol-water solvent (0%, 30%, 50%, 70%, 90% M / W) was used as the eluent. After solvent recovery under low-temperature reduced pressure using a rotary evaporator, the fractions were analyzed by TLC and combined to obtain 12 fractions: GPZJ-6A (200mg), GPZJ-6B (300mg), GPZJ-6C (120mg), GPZJ-6D (80mg), GPZJ-6E (250mg), GPZJ-6F (1.1g), GPZJ-6G (980mg), GPZJ-6H (740mg), GPZJ-6I (620mg), GPZJ-6J (3.18g), GPZJ-6K (1.2g), and GPZJ-6L (1.3g).

[0031] GPZJ-6I (620 mg) was dissolved in methanol (solid-to-solution ratio 1:5, mg / mL), mixed with 1.2 g silica gel (80-100 mesh), and separated by normal-phase silica gel column chromatography. Gradient elution was performed using a dichloromethane:methanol solvent system as the eluent (9:1, 4:1, 3:1, 1:1). After TLC detection, four fractions were obtained: GPZJ-6I1~GPZJ-6I4. Among them, GPZJ-6I2 (90 mg) was purified into pure compound 5 (50 mg) by semi-preparative high-performance liquid chromatography. The chromatographic column was a Cosmosil Packed Column 250×10 mm, the mobile phase was 35% acetonitrile, and the retention time was 9.45 min.

[0032] GPZJ-6J (3.18 g) was dissolved in methanol (solid-to-liquid ratio 1:5, mg / mL) and mixed with 9 g of silica gel (80-100 mesh). The mixture was then separated by normal-phase silica gel column chromatography, using a dichloromethane:methanol solvent system as the eluent (9:1, 8:1, 6:1, 4:1, 1:1) for gradient elution. After TLC analysis, 13 fractions were obtained: GPZJ-6J1 (190 mg), GPZJ-6J2 (86 mg), GPZJ-6J3 (20 mg / mL), GPZJ-6J4 (20 mg / mL), GPZJ-6J5 (20 mg / mL), GPZJ-6J6 ... mg), GPZJ-6J4 (150mg), GPZJ-6J5 (420mg), GPZJ-6J6 (140mg), GPZJ-6J7 (120mg), GPZJ-6J8 (450mg), GPZJ-6J9 (920mg), GPZJ-6J10 (520mg), GPZJ-6J11 (240mg), GPZJ-6J12 (500mg), GPZJ-6J13 (190mg).

[0033] GPZJ-6J7 (120 mg) was purified by semi-preparative high performance liquid chromatography (HPLC) to obtain pure compound 1 (25 mg). The chromatographic column was a YMC ODS-AQ Column 250×10 mm, the mobile phase was 39% acetonitrile, the flow rate was 3 mL / min, and the retention time was 24.6 min.

[0034] GPZJ-6J9 (920 mg) was dissolved in methanol and mixed with 2 g of silica gel (80-100 mesh). The mixture was then separated by normal-phase silica gel column chromatography, using dichloromethane:methanol (6 / 1) as the eluent. After TLC analysis, seven fractions were obtained: GPZJ-6J9A to GPZJ-6J9G. GPZJ-6J9A (310 mg) was purified by semi-preparative high-performance liquid chromatography to obtain pure compound 3 (130 mg). t R =20.51min) and 2 (18mg, t R =23.34 min), the chromatographic column was a YMC ODS-AQ Column 250×10 mm, and the mobile phase was 38% acetonitrile.

[0035] GPZJ-6J12 (500mg) was dissolved in methanol and mixed with 1g of silica gel (80-100 mesh). The mixture was separated by normal phase silica gel column chromatography and eluented with dichloromethane:methanol solvent 6 / 1. After TLC detection, the fractions were combined to obtain 7 fractions: GPZJ-6J12A~GPZJ-6J12E.

[0036] GPZJ-6J12B (86 mg) was purified by semi-preparative high performance liquid chromatography (HPLC) to obtain pure compound 4 (38 mg). The chromatographic column was a YMC ODS-AQ Column 250×10 mm, the mobile phase was 37% acetonitrile, the flow rate was 3 mL / min, and the retention time was 34.01 min.

[0037] 2. Physicochemical properties of compounds Compound 1, white powder, [α]25 D -8.89 (c = 0.09, methanol); UV (MeOH)λ max (nm) (logε): 203.60 (3.42); IR (KBr): 3412, 2942, 2875, 1632, 1450, 1384, 1 308, 1261, 1218, 1159, 1074, 1043, 910, 868, 839, 812, 675, 640, 590, 573, 557 cm -1 ESI-MS m / z 938 [M+Na] + HRESIMS m / z937.5492 [MH] - (calcd for C) 53 H 89 O 24 Na, 937.5603). 1 H-NMR (500MHz, C5D5N) and 13 C-NMR (125MHz, C5D5N) data are shown in Table 1.

[0038] Compound 2, white powder, [α]25 D -20.20 (c = 0.10, methanol); UV (MeOH)λ max (nm) (logε): 203.20 (3.58); IR (KBr): 3412, 2939, 2875, 1633, 1451, 1384 , 1311, 1265, 1217, 1073, 1040, 914, 867, 837, 811, 686, 660, 636, 591, 420 cm -1 ESI-MS m / z 1084 [M+Na] + HRESIMS m / z 1083.6074 [MH] - (calcd for C) 53 H 89 O 24 Na, 1083.6182). 1H-NMR (500MHz, C5D5N) and 13 C-NMR (125 MHz, C5D5N) data are shown in Table 1.

[0039] Table 1. Compounds 1 and 2 1 H and 13 C NMR data Example 2: Methods and Results for Evaluating Acetylcholinesterase Activity 110 μL of PBS solution, 40 μL of 0.1 U / mL AChE (acetylcholinesterase) solution, and 10 μL of each compound solution (2 mg / mL) were added sequentially to the wells of a 96-well plate, and the plate was pre-incubated at 37 °C for 20 min. Then, 40 μL of 6.25 mmol / L LTNB solution and 40 μL of 6.25 mM thioacetylcholine iodide (AtCHI) solution were added, mixed, and incubated at 37 °C for 20 min. After incubation, the 96-well plate was immediately placed in a -20 °C refrigerator for 10 min to terminate the reaction. The absorbance was then measured at 405 nm using a microplate reader. Triple-well experiments were performed, and the average value was taken. A control group without enzyme and a blank control group without enzyme and sample were also included, with tacrine as a positive control. All reactions were repeated three times. The inhibition rate was calculated according to formula (1).

[0040] Formula (1): A: Sample group (ODsample) B: Sample control group, without enzyme, replaced with the same volume of PBS (OD200). sample blank ) C: Control group, no sample added, replaced with the same volume of PBS (OD2000). control ) D: Blank control group, no enzyme or sample added (OD) control blank ) The results are shown in Table 2. At a concentration of 50 μM, the new compound 1 (12-dehydroxy-Gynostemma pentaphyllum saponin III) exhibited AChE inhibitory activity with an inhibition rate of 25.84% ± 1.38%.

[0041] Table 2. Inhibitory activity of the compounds against acetylcholinesterase Example 3: Methods and results of the protective effect against nerve cell damage. 1. The neuroprotective effects of the compound were evaluated using a 6-OHDA-induced SH-SY5Y cell injury model.

[0042] 1.1 Establishment of a 6-OHDA-induced SH-SY5Y cell damage model Frozen SH-SY5Y cells were revived, centrifuged, resuspended, and seeded into DMEM medium containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were then cultured in a 37°C, 5% CO2 incubator. When the cells grew to about 80% of the bottom of the culture dish, a 6-OHDA-induced SH-SY5Y cell damage model was established.

[0043] 1.2. CCK-8 method analysis and detection 6-OHDA stock solution (20mM): Accurately weigh 10mg of 6-OHDA and dissolve it in 2mL of physiological saline to prepare a 100mM 6-OHDA solution.

[0044] This embodiment uses the CCK-8 cytotoxicity assay kit to detect the protective effect of the compound against 6-OHDA-induced SH-SY5Y cell toxicity. The specific steps are as follows: SH-SY5Y cells in logarithmic growth phase were taken, the supernatant was discarded, and the cells were washed with sterile PBS. After removing the PBS, 1 mL of trypsin was added for digestion. The cells were incubated at 37°C with 5% CO2 for 2 min. Digestion was terminated with fresh DMEM medium to form a cell suspension, which was then transferred to 15 mL centrifuge tubes. The cells were then centrifuged at a rate of 1×10⁻⁶ cells / mL. 4 Seed each cell type 1 / well in a 96-well plate. Add 100 μL of DMEM medium containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin to each well and incubate overnight. Add solutions of compounds 1-5 (concentration gradient of 2.5, 5, 10, 20, 50, 100, 150, 200 μM) and incubate for 24 h. Add 100 μM 6-OHDA and incubate for 24 h. Discard the supernatant, wash with PBS, aspirate, add 100 μL of medium and 10 μL of CCK-8 to each well, return to the incubator, and incubate for 30 min. Measure the absorbance at 450 nm using a microplate reader. Calculate cell viability.

[0045] To further elucidate the molecular mechanism of the neuroprotective effects of the candidate compounds, the protein expression levels of Nrf2, HO-1, and NQO1 in the blank control group and the five compound treatment groups (5 μM, 24 h) were detected by Western blotting. β-Actin was used as an internal reference for gray value normalization analysis.

[0046] 1.3 Results and Analysis As shown in Table 3, the molar concentrations of the five compounds in the activity test system were 0.3125, 0.625, 1.25, 2.5, 5, 10, 25, and 50 μM. All compounds exhibited good safety within the tested concentration range, with cell viability generally exceeding 80%. Compound 3 showed a viability of 95.38% at 5 μM, and compound 2 showed a viability as high as 98.44% at 5 μM. Compound 4 exhibited some cytotoxicity at concentrations of 1.25–25 μM, with a viability of 46.85%, indicating significant cytotoxicity. The neuroprotective effects of each compound were evaluated in a 100 μM 6-OHDA-induced cell injury model (Table 4). Cell viability in the model group without added compounds ranged from 49.42% to 53.88%. Compounds 2 and 3 showed the strongest protective activity, with compound 3 achieving cell viability of 84.96% and 86.14% at concentrations of 2.5 μM and 5 μM, respectively. Compound 2 showed protective survival rates of 85.69% and 85.73% at the same concentrations. Both compounds exhibited protective effects at low concentrations of 0.3125–1.25 μM. Although compound 4 achieved protective survival rates of 80.52% and 82.31% at 2.5–5 μM, its own cell survival rates at the same concentrations were only 51.65% and 51.63%, respectively. This protective effect may stem from the complex effects of its own toxicity on cell viability rather than true neuroprotection. Compound 1 showed moderate protective activity at concentrations of 1.25–5 μM, with a cell survival rate of approximately 70%.

[0047] Western blot results showed that ( Figure 8 Compared with the normal control group, all tested compounds upregulated the expression levels of Nrf2 and its downstream target proteins HO-1 and NQO1 to varying degrees.

[0048] Compounds 2 and 3 exert neuroprotective effects by activating the Nrf2 / HO-1 / NQO1 antioxidant signaling pathway, upregulating the expression of endogenous antioxidant enzymes, and effectively alleviating 6-OHDA-induced oxidative stress damage.

[0049] Table 3. Toxicity of five compounds to SH-SY5Y cells (cell viability %) Note: The cell viability of SH-SY5Y cells treated with compounds 1, 2, and 3 was significantly higher than that of cells treated with 6-OHDA (P < 0.0001). The cell viability of SH-SY5Y cells treated with compound 4 (concentrations of 1.25-50 μM) was significantly lower than that of cells treated with 6-OHDA (P < 0.0001), indicating that compound 4 was more toxic to SH-SY5Y cells than 6-OHDA (100 μM) within this concentration range. There was no significant difference in cell viability between the 0.3125 and 0.625 μM treatment groups and the 6-OHDA treatment group. The cell viability of SH-SY5Y cells treated with compound 5 (concentrations of 5-50 μM) was significantly lower than that of cells treated with 6-OHDA (P < 0.0001), indicating that compound 5 was more toxic to SH-SY5Y cells than 6-OHDA (100 μM).

[0050] Table 4. Protective activity of five compounds against 6-OHDA-induced damage in SH-SY5Y cells (cell viability %) Note: P < 0.001, and for the rest P < 0.0001 Example 4: Neuroprotective activity against CL4176 (AD nematode), a model of Alzheimer's disease. 1.1 Experimental Materials Nematode strain: CL4176 dvls27 [myo-3p::A-Beta(1-42)::let-8513'UTR+rol-6(su1006)]; cultured on NGM plates with Escherichia coli OP50 as food.

[0051] 1.2 Medicines and Reagents Dimethyl sulfoxide, sodium hydroxide, sodium hypochlorite, and ethanol were all analytical grade and purchased from Yunnan Liyan Technology Co., Ltd.; tryptone, yeast extract, sodium chloride, agar, peptone, magnesium sulfate, calcium chloride, cholesterol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Sangon Biotech (Shanghai).

[0052] 1.3 Experimental Methods Synchronized L1-stage transgenic CL4176 nematodes were inoculated onto culture dishes of the control group (100 μg / mL DMSO) and the experimental group (100 μg / mL DH compound). After incubation at 16℃ to L3 stage, the nematodes were transferred to 25℃ for 24 h. Paralysis was then observed, with counts every 2 h until all nematodes were paralyzed. The non-paralysis rate was calculated according to Zhao P's method; paralysis was defined as the absence of complete body movement or movement only in the head.

[0053] 1.4 Results and Analysis To evaluate the effects of different WGZ compounds on Aβ-induced toxicity, CL4176, which expresses human Aβ protein, was selected for the experiment. Figure 7 It can be seen that during the 24-36 hours of heating, compounds 2 and 5 showed no significant difference in the non-paralyzing rate of AD nematodes compared to the control group. When compounds 3 and 4 were transferred to 25℃ for 36 hours, 32.76% and 32.14% of the nematodes remained unparalyzed, respectively, compared to only 15.38% in the control group, indicating that compounds 3 and 4 had a certain effect in alleviating paralysis in AD nematodes. When compound 1 was transferred to 25℃ for 36 hours, 55.1% of the nematodes remained unparalyzed, compared to only 15.38% in the control group, indicating that compound 1 had a better effect in alleviating paralysis in AD nematodes.

[0054] In summary, compounds 1, 3, and 4 exerted neuroprotective effects, alleviated the paralytic effect of *A. adenophora*, significantly reduced Aβ-induced toxicity, and effectively inhibited Aβ accumulation in nerves.

[0055] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A saponin compound, characterized in that, The saponin compounds include compound 1 and compound 2; compound 1 is 12-deoxygynosaponin III and compound 2 is 12-deoxygynosaponin IV.

2. The saponin compound according to claim 1, characterized in that, The structural formula of compound 1 is as shown in Formula 1, and the structural formula of compound 2 is as shown in Formula 2; Formula 1, Formula 2.

3. A method for preparing a saponin compound as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: S1, collect the aerial parts of Gynostemma pentaphyllum from Zhaotong, dry them, extract them by reflux with ethanol, concentrate them under reduced pressure using a rotary evaporator and dry them to obtain the extract, denoted as GPZJ; S2, after column chromatography separation and fragmentation of GPZJ, elution is performed to obtain GPZJ-1 to GPZJ-8; S3, after dissolving GPZJ-6 in pyridine, the mixture is separated, fragmented, and eluted to obtain GPZJ-6A-GPZJ-6L; S4, GPZJ-6J was dissolved in methanol and mixed with silica gel. After separation and elution by normal phase silica gel column chromatography, the samples were detected and combined to obtain GPZJ-6J1 to GPZJ-6J13. S5, of which GPZJ-6J7 is pure compound 1 after separation and purification; S6, wherein GPZJ-6J9 was dissolved in methanol and mixed with silica gel, separated and fragmented, eluted, and then combined by TLC to obtain GPZJ-6J9A to GPZJ-6J9G, wherein GPZJ-6J9A was separated and purified to obtain compound 2.

4. The preparation method according to claim 3, characterized in that, The reflux extraction in S1 involves refluxing with 70% ethanol at 50°C three times, for 4 hours each time.

5. The preparation method according to claim 3, characterized in that, The mass ratio of GPZJ-6J to silicone in S4 is 3.18:

9.

6. The preparation method according to claim 3, characterized in that, The mass ratio of GPZJ-6J9 to silicone in S6 is 0.46:

1.

7. The preparation method according to claim 3, characterized in that, In S3, the ratio of GPZJ-6 to pyridine is 1:5 (mg / ml).

8. The preparation method according to claim 3, characterized in that, The separation and purification methods in S5 and S6 are semi-preparative high-performance liquid chromatography.

9. The use of a saponin compound as described in claim 1 in the preparation of a neuroprotective drug.

10. The application according to claim 8, characterized in that, The neuroprotective diseases mentioned include Alzheimer's disease and Parkinson's disease.

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