Method for extracting sesquiterpenes from leaves and stems of podophyllum hexandrum and applications thereof

Abstract

A method for extracting sesquiterpenoids from the stems and leaves of Arisaema heterophyllum and its application are disclosed. This method can effectively extract sesquiterpenoids G and I from the stems and leaves of Arisaema heterophyllum, effectively solving the problem of preparing drugs for the treatment of Alzheimer's disease. The preparation method is as follows: the dried stems and leaves of Arisaema heterophyllum are pulverized, heated under reflux, filtered, concentrated under reduced pressure, dissolved in water, and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol, respectively, and then concentrated and dried. The concentrated and dried ethyl acetate fraction is eluted, the fraction retained for the retention time is collected, concentrated and dried to obtain the final product. This method is easy to operate, scientifically sound, and can effectively extract sesquiterpenoids G and I from the stems and leaves of Arisaema heterophyllum. These compounds can significantly improve Aβ25-35-induced PC-12 cell damage, opening up new uses for the active ingredients in the stems and leaves of Arisaema heterophyllum, improving the commercial and industrial value of Arisaema heterophyllum, and simultaneously opening up new avenues for the treatment of Alzheimer's disease, resulting in significant economic and social benefits.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a method for extracting sesquiterpenoid compounds from the stems and leaves of Arisaema heterophyllum and its application. Background Technology

[0002] Yu Nanxing, the tuber of *Pinellia pedatisecta* Schott, a plant in the Araceae family, is also known as Tiger Paw or Palm-leaved Pinellia. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it possesses properties such as drying dampness and resolving phlegm, dispelling wind and relieving spasms, and dispersing nodules and reducing swelling. Since its inception in the *Shennong Bencao Jing*, Yu Nanxing has been a traditional high-quality Chinese medicinal herb. Yu Nanxing from Yuzhou City, Henan Province, its authentic producing area, is particularly popular both domestically and internationally, showing promising development prospects. While the tuber of Yu Nanxing is widely used as the medicinal part, its above-ground parts are discarded, resulting in resource waste and significantly limiting the development of its non-medicinal value. With the modernization of traditional Chinese medicine, strengthening the comprehensive development and utilization of the non-medicinal parts of Yu Nanxing and enhancing its economic value is a crucial and urgent task.

[0003] Alzheimer's disease (AD) is a degenerative disease of the central nervous system, primarily occurring in the elderly or pre-elderly. Its main characteristics include progressive cognitive impairment, behavioral disturbances, aphasia, apraxia, agnosia, and visuospatial impairment. It is accompanied by personality and behavioral changes, impairing abstract thinking and calculation abilities. It is the most common form of dementia, accounting for approximately 60-70% of cases. Alzheimer's disease is the most common type of dementia in old age, and the risk of developing it increases with age.

[0004] To further enrich the pharmacodynamic material basis of Arisaema heterophyllum stems and leaves and expand the source of medicinal materials, this invention isolated two sesquiterpenoid compounds, Pedatisectaene G and Pedatisectaene I, from the 95% ethanol reflux extract of Arisaema heterophyllum stems and leaves. Activity experiments showed that the compounds significantly improved Aβ25-35-induced PC-12 cell damage, providing a basis for further development of anti-Alzheimer's drugs. However, no related research has been publicly reported to date. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting sesquiterpenoid compounds from the stems and leaves of Arisaema heterophyllum and its application. This method can effectively extract sesquiterpenoid compounds Pedatisectaene G and Pedatisectaene I from the stems and leaves of Arisaema heterophyllum, and can effectively solve the problem of preparing drugs for the treatment of Alzheimer's disease.

[0006] The technical solution provided by this invention is a method for extracting sesquiterpene compound G (Pedatisectaene G)(1) and sesquiterpene compound I (Pedatisectaene I)(2) from the stems and leaves of Arisaema heterophyllum, with the following chemical molecular structures:

[0007]

[0008] The preparation method is as follows:

[0009] The dried stems and leaves of Arisaema heterophyllum were pulverized, heated under reflux with 95% ethanol, filtered, concentrated under reduced pressure to obtain an extract, dissolved in water, and extracted successively with petroleum ether, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, which were then concentrated and dried.

[0010] The concentrated and dried ethyl acetate fraction was loaded onto an ODS medium-pressure column and eluted sequentially with methanol at volume concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. The fractions containing 30% methanol were combined to obtain fraction E.

[0011] Component E was dissolved in methanol, mixed with silica gel, and eluted using a gradient of petroleum ether:ethyl acetate ratios of 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 (v / v). The fractions of petroleum ether:ethyl acetate ratio of 3:1 were combined to obtain component E1.

[0012] Component E1 was dissolved in methanol and further separated. It was eluted with 70% methanol at a flow rate of 1 ml / min and a volume of 500 ml. The fractions of 100-200 ml and 200-300 ml were combined to obtain components E1-1 and E1-2, respectively.

[0013] Component E1-1 was separated by semi-preparative HPLC, loaded onto a chromatographic column, and the retention time t was collected. R The fraction with a flow rate of 20.1–22.0 min was concentrated and dried to obtain sesquiterpene compound G (Pedatisectaene G);

[0014] Component E1-2 was separated by semi-preparative HPLC, loaded onto a chromatographic column, and the retention time t was collected. R The fraction with a flow rate of 15.6–18.0 min was concentrated and dried to obtain sesquiterpene compound I.

[0015] The application of sesquiterpene compound G and sesquiterpene compound I prepared by the above method in the preparation of drugs for treating Alzheimer's disease.

[0016] The preparation method of this invention is easy to operate, scientific and reasonable, and can effectively extract sesquiterpene compound G and sesquiterpene compound I from the stems and leaves of Arisaema heterophyllum. These compounds can significantly improve Aβ25-35-induced PC-12 cell damage, opening up new uses for the active ingredients in the stems and leaves of Arisaema heterophyllum, saving resources, and improving the commercial and practical value of Arisaema heterophyllum. At the same time, it opens up a new avenue for the treatment of Alzheimer's disease, with huge economic and social benefits. Attached Figure Description

[0017] Figure 1 The chemical molecular structure diagrams of sesquiterpene compound G (Pedatisectaene G)(1) and sesquiterpene compound I (Pedatisectaene I)(2) of this invention are shown.

[0018] Figure 2 The compound Pedatisectaene G of this invention 1 1H NMR spectrum (500MHz, Acetone-d6);

[0019] Figure 3 The compound Pedatisectaene G of this invention 13 C10 NMR spectrum (125MHz, Acetone-d6);

[0020] Figure 4 The compound Pedatisectaene I of this invention 1 1H NMR spectrum (500MHz, Acetone-d6);

[0021] Figure 5 The compound Pedatisectaene I of this invention 13 C10 NMR spectrum (125MHz, Acetone-d6). Detailed Implementation

[0022] The specific embodiments of the present invention will be described in detail below with reference to specific circumstances.

[0023] In specific implementation, the present invention can be given by the following embodiments.

[0024] A method for extracting sesquiterpene compound G and sesquiterpene compound I from the stems and leaves of Arisaema heterophyllum, comprising the following steps:

[0025] Take 20 kg of dried stems and leaves of Arisaema heterophyllum, pulverize them, and heat them under reflux twice with 95% ethanol. Each time, add 200 L of 95% ethanol and reflux for 2 hours. Filter the mixture, combine the two filtrates, and concentrate under reduced pressure to obtain 1.8 kg of extract. Dissolve the extract in 3 L of water, and extract it 20 times each with petroleum ether, ethyl acetate, and n-butanol, using 4 L each time, to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. Concentrate and dry each fraction.

[0026] The concentrated and dried ethyl acetate fraction was loaded onto an ODS medium-pressure column and eluted sequentially with methanol gradients of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v / v), 1 L per gradient, at a flow rate of 8 ml / min. The fraction was checked every 250 ml using anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed in 5 days. The fractions containing 30% methanol were combined to obtain fraction E.

[0027] Component E was dissolved in methanol and mixed with 100-200 mesh silica gel at a 1:1 ratio. The sample was loaded onto a 200-300 mesh silica gel column and eluted using a gradient of petroleum ether:ethyl acetate ratios of 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 (v / v). Each gradient mobile phase consisted of 3 times the column volume, and the flow rate was 6 ml / min. The sample was checked every 200 ml using anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed in 3 days. The fractions with a petroleum ether:ethyl acetate ratio of 3:1 were combined to obtain component E1.

[0028] Component E1 was dissolved in methanol and further separated using Sephadex LH-20. It was eluted with 70% methanol at a flow rate of 1 ml / min and a volume of 500 ml. It was identified by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. The fractions of 100-200 ml and 200-300 ml were combined to obtain components E1-1 and E1-2, respectively.

[0029] Component E1-1 was separated by semi-preparative HPLC using a YMC-Pack ODS-AA column (250×10mm, 5μm particle size, 12nm pore size). The mobile phase was methanol:trifluoroacetic acid aqueous solution at a volume ratio of 35:65 (3 / 10,000), with a trifluoroacetic acid concentration of 0.03%. The flow rate was 2 mL / min, and the retention time was t. R The fraction with a flow rate of 20.1–22.0 min was concentrated and dried to obtain sesquiterpene compound G (Pedatisectaene G);

[0030] Component E1-2 was separated by semi-preparative HPLC using a YMC-Pack ODS-AA column (250×10mm, 5μm particle size, 12nm pore size). The mobile phase was methanol:trifluoroacetic acid aqueous solution at a volume ratio of 35:65 (3 / 10,000), with a trifluoroacetic acid concentration of 0.03%. The flow rate was 2 mL / min, and the retention time was t. R The fraction with a flow rate of 15.6–18.0 min was concentrated and dried to obtain sesquiterpene compound I (Pedatisectaene I).

[0031] The application of sesquiterpene compound G and sesquiterpene compound I prepared by the above method in the preparation of drugs for treating Alzheimer's disease.

[0032] The preparation method of this invention is easy to operate, scientifically sound, and can effectively extract sesquiterpene compound G (Pedatisectaene G) and sesquiterpene compound I (Pedatisectaene I) from the stems and leaves of Arisaema heterophyllum. These compounds can significantly improve Aβ. 25-35 Inducing PC-12 cell damage and determining the characteristic structure and activity of the active ingredients in the extract yielded very good technical results. The relevant experimental data are as follows:

[0033] 1. Instruments and reagents

[0034] Nuclear magnetic resonance (NMR) was performed using a Bruker AVANCE III 500 NMR spectrometer (TMS internal standard) (Bruker); infrared spectroscopy was performed using a Nicolet IS 10 Microscope Spectrometer (Thermo Scientific, USA); high-resolution mass spectrometry was performed using a Bruker Maxis HD mass spectrometer; and ultraviolet spectroscopy was performed using a Shimadzu UV-2401PC. Apparatus, LC50 high-performance liquid chromatograph, UV200 ultraviolet detector [Saipu Ruisi (Beijing) Technology Co., Ltd.], YMC-PackODS-A column (250×10mm, DS-5mm, 12mm) (YMC Ltd.), others include N-1100 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), A-1000S water pump (Shanghai Ailang Instrument Co., Ltd.), N-1111 chilled water circulation system (Shanghai Ailang Instrument Co., Ltd.), FDU-2110 freeze dryer (Shanghai Ailang Instrument Co., Ltd.), DFZ-60508 vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.), AB204-N 0.01% precision analytical balance (METTLER TOLEDO), CO2 3111 incubator (Thermo), ECLPSE TS100 inverted microscope (Nikon), Centrifuge-5804R high-speed centrifuge (Eppendorf), Multiskan MK3 microplate reader (Thermo Fisher), Advantage A10 ultrapure water system (Sartorius), BCD-206TAS ultra-low temperature freezer (Haier), DZF-6050B vacuum drying oven (Beijing Hengtaifengke Test Equipment Co., Ltd.), HVA-85 autoclave (Hirayama).

[0035] Highly differentiated rat adrenal medullary pheochromocytoma cell line (PC-12), Aβ 25-35 Lyophilized powder (Sangon Biotech Co., Ltd.), fetal bovine serum (Hangzhou Sijiqing Company), DMEM medium (Gibco Invitrogen), MTT (Beijing Solarbio Science & Technology Co., Ltd.), DMSO (Shanghai Maclean Biotechnology Co., Ltd.).

[0036] The stems and leaves of Pinellia pedatisecta Schott were collected in Yuzhou, Henan Province in November 2023 and identified by Professor Chen Suiqing and Professor Dong Chengming of Henan University of Traditional Chinese Medicine as dried stems and leaves of Pinellia pedatisecta Schott.

[0037] 2. Structural Feature Identification

[0038] Pedatisectaene G(1): Yellow oil. HR-ESI-MS shows a quasi-molecular ion peak at m / z 263.1257 [M+H]. + ,(calcd for C 13 H 21 O4,263.1254), its molecular formula was determined to be C 13 H 20 O4;UV(MeOH)λ max :203,227,262nm; IR(iTR)ν max 3322, 1769, 1031cm -1 See the diagram. Figure 2-3 The NMR data are shown in Table 1;

[0039] Pedatisectaene I(2): Yellow oil. HR-ESI-MS showed a quasi-molecular ion peak at m / z 247.0943 [M+Na]. + ,(calcd for C 12 H 15 O4Na, 247.0941), its molecular formula was determined to be C 12 H 15 O4;UV(MeOH)λ max :204,231nm; IR(iTR)ν max :3411,2933,1720,1651cm -1 See the diagram. Figure 4-5 The NMR data are shown in Table 1;

[0040] The chemical molecular structures of Pedatisectaene G(1) and Pedatisectaene I(2) are as follows:

[0041]

[0042] Table 1. NMR data of the compounds in Acetone-d6

[0043]

[0044]

[0045] 3. Activity Experiment

[0046] 3.1Aβ 25-35 Freeze-dried powder preparation

[0047] Aβ 25-35 The freeze-dried powder was removed from the -20°C freezer and left at room temperature for half an hour. Then, in a laminar flow hood, Aβ was dissolved in sterile double-distilled water. 25-35The powder was prepared into a 1M stock solution, aged in a 37℃, 5% CO2 incubator for 7 days, sealed and labeled, and stored in a -20℃ refrigerator for later use.

[0048] 3.2 Cell Culture

[0049] PC-12 cells were cultured in DMEM medium containing 10% FBS in a 37°C incubator with 5% CO2. When the cells reached 80% confluence, they were passaged every 2 days.

[0050] 3.3 MTT assay for the effect of compounds in *Yu Nanxing* on Aβ 25-35 Experiment on the effect of inducing PC-12 cell viability

[0051] PC-12 cells were cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase, and then cultured at a cell density of 5 × 10⁶ cells / year. 4 Inoculated at 1 / mL into 96-well plates, and after 24 hours, the cells were divided into a normal group (CON) and a model group (M, Aβ). 25-35 Cells were cultured in two groups: one receiving 0.1 μM of MTT solution and the other receiving 0.1 μM of Aβ25-35. After culturing for 24 h, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for 4 h. The liquid in the wells was discarded, and 150 μL of DMSO was added to each well. The mixture was shaken for 10 min to completely dissolve the cells, and the OD value of each well was measured at 490 nm using a microplate reader. The experiment was repeated three times, and cell viability was calculated as: cell viability = OD value of each group / OD value of the normal group.

[0052] The results showed that the two compounds significantly improved PC-12 cell viability compared with the model group (P<0.01), as shown in Table 2, suggesting that both compounds could improve Aβ. 25-35 Induces damage to PC-12 cells.

[0053] Table 2. Effects of compounds on Aβ 25-35 Effects of inducing PC-12 cell damage n=4)

[0054]

[0055]

[0056] In summary, this invention uses a reflux method with 95% ethanol to extract the stems and leaves of Arisaema heterophyllum. The sesquiterpenoid compounds Pedatisectaene G and Pedatisectaene I were isolated and identified from the ethyl acetate fraction of the extract. (The last sentence appears to be incomplete and requires further context.) 25-35 An in vitro model was established by inducing PC-12 cells, and the activity of two compounds was screened. The results showed that both compounds 1 and 2 could significantly improve Aβ. 25-35Inducing PC-12 cell damage further enriches the pharmacodynamic material basis of *Yunanxing*, enabling its further application in the preparation of drugs for treating Alzheimer's disease. This opens up new avenues for the medicinal value of *Yunanxing* and the treatment of Alzheimer's disease, with significant social and economic benefits.

Claims

1. A method for extracting sesquiterpenoid compounds from the stems and leaves of Arisaema heterophyllum, characterized in that, The sesquiterpenoids mentioned are sesquiterpenoid compound G (Pedatisectaene G) (1) and sesquiterpenoid compound I (Pedatisectaene I) (2), with the following chemical molecular structures: ； Its preparation method is as follows: Take 20 kg of dried stems and leaves of Arisaema heterophyllum, pulverize them, and heat them under reflux twice with 95% ethanol. Each time, add 200 L of 95% ethanol and reflux for 2 h. Filter, combine the two filtrates, concentrate under reduced pressure to obtain 1.8 kg of extract, dissolve it in 3 L of water, and extract it 20 times each with petroleum ether, ethyl acetate, and n-butanol, with a volume of 4 L each time, to obtain petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. Concentrate and dry each fraction. The ethyl acetate fraction was loaded onto an ODS medium-pressure column and eluted sequentially with methanol gradients of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v / v), 1 L per gradient, at a flow rate of 8 ml / min. The fraction was checked every 250 ml using anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed in 5 days. The fractions containing 30% methanol were combined to obtain fraction E. Component E was dissolved in methanol and mixed with 100-200 mesh silica gel at a 1:1 ratio. The sample was loaded onto a 200-300 mesh silica gel column and eluted using gradients of petroleum ether:ethyl acetate at ratios of 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 (v / v). Each gradient mobile phase consisted of 3 times the column volume, and the flow rate was 6 ml / min. The sample was checked every 200 ml using anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed in 3 days. The fractions of petroleum ether:ethyl acetate at a ratio of 3:1 were combined to obtain component E1. Component E1 was dissolved in methanol and further separated using Sephadex LH-20. It was eluted with 70% methanol at a flow rate of 1 ml / min and a volume of 500 ml. It was identified by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. The fractions of 100-200 ml and 200-300 ml were combined to obtain components E1-1 and E1-2, respectively. Component E1-1 was separated by semi-preparative HPLC. The particle size was 250×10 mm, and the particle size was 5. μ A YMC-Pack ODS-AA column with a pore size of 12 nm was used. The mobile phase was methanol:trifluoroacetic acid aqueous solution at a volume ratio of 35:65 (3 / 10,000), with a trifluoroacetic acid aqueous solution concentration of 0.03%. The flow rate was 2 ml / min, and the retention time was t. R The fraction was concentrated and dried over a period of 20.1 to 22.0 min to obtain compound Pedatisectaene G; Component E1-2 was separated by semi-preparative HPLC. The particle size was 250×10 mm, and the particle size was 5. μ A YMC-Pack ODS-AA column with a pore size of 12 nm was used. The mobile phase was methanol:trifluoroacetic acid aqueous solution at a volume ratio of 35:65 (3 / 10,000), with a trifluoroacetic acid aqueous solution concentration of 0.03%. The flow rate was 2 ml / min, and the retention time was t. R The fraction was concentrated and dried over a period of 15.6 to 18.0 minutes to obtain compound Pedatisectaene I.

2. The use of the sesquiterpenoid compounds Pedatisectaene G and Pedatisectaene I prepared by the method of claim 1 in the preparation of drugs for treating Alzheimer's disease.

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