Method for extracting neonorsesquiterpenes from stems and leaves of rhizoma arisaematis and application of neonorsesquiterpenes

Abstract

The invention belongs to the field of medicinal chemistry, and relates to comprehensive utilization of rhizoma arisaematis. The invention provides a method for extracting norsesquiterpenes from stems and leaves of rhizoma arisaematis and application of the norsesquiterpenes. According to the invention, 95% ethanol is adopted for heating reflux for extraction. Two new norsesquiterpene compounds are separated and identified from ethyl acetate parts of stems and leaves of rhizoma arisaematis, namely, the experimental results of the Pedatisectaene C (1) and the Pedatisectaene D (2) show that the two compounds can be used for remarkably improving PC-12 cell injury induced by A beta 25-35.

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry and relates to the comprehensive utilization of Cynanchum yunnanensis. Background Art

[0002] Yunanxing is a plant of the genus Pinellia in the family Araceae. Pinellia pedatisecta Schott), also known as tiger palm and palm leaf pinellia, is warm in nature, bitter and pungent in taste, slightly toxic, and has the effects of drying dampness and resolving phlegm, dispelling wind and stopping spasm, dispersing nodules and reducing swelling. Yunanxing is mainly produced in Yuzhou City, Henan Province. It is known as the "Four Major Yu Medicines" together with Yu Baizhi, Yu Baifu and Yu Banxia. It is sold well overseas due to its large output and good quality. However, during the large-scale use of Yunanxing, its stems and leaves are discarded, resulting in serious waste of resources. The state has clearly proposed to strengthen the comprehensive utilization research of the "non-medicinal parts" of Chinese medicinal materials, and should consider paying attention to the resource utilization rate of stems and leaves. In order to clarify the material basis of the efficacy of the stems and leaves of Yunanxing and improve its utilization rate.

[0003] Research on sesquiterpenes is mostly focused on the study of Chinese medicinal plants; the application with publication number CN112920038A discloses sesquiterpenoid compounds with antibacterial activity found in Chimonanthus salicifolius. Some studies have found a series of anti-liver cancer active ingredients in Artemisia scoparia; the complex ring system of sesquiterpenes and sesquiterpenoid derivatives, the low content of sesquiterpenes in Chinese medicinal plants, and the difficulty of separation have brought great challenges to the performance research of new sesquiterpenes; and the existing data show that new sesquiterpenoid compounds have not yet been isolated in Yunanxing. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method and application of extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa.

[0005] The technical solution of the present invention is achieved in this way: A method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa, comprising the following steps: (1) After crushing the yunnanxing, the ethanol is refluxed, filtered, and the filtrate is collected. The obtained filtrate is concentrated under reduced pressure to obtain an extract. The obtained extract is dissolved in water and extracted with petroleum ether, ethyl acetate and n-butanol in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, an n-butanol fraction and a water fraction, respectively; (2) After the ethyl acetate fraction is concentrated and dried, it is gradient eluted with a 10%-100% methanol-water solution by volume. After elution, the 20% methanol-water solution fraction is combined as component E2. In this step, a macroporous resin filler is used to perform a preliminary separation of the large-weight ethyl acetate fraction, and the 20% methanol-water solution fraction with appropriate polarity is selected.

[0006] (3) After component E2 is loaded onto a silica gel column, gradient elution II is carried out successively with an eluent composed of petroleum ether and ethyl acetate in a volume ratio of 0 - 10:1, and the fractions with a volume ratio of petroleum ether to ethyl acetate of 1:1 are combined to obtain component E2-10; silica gel packing is used in this step. According to the different polarities of the eluent, the compounds elute successively from small to large polarity, and the compounds with a polarity around petroleum ether:ethyl acetate = 1:1 are collected.

[0007] (4) Component E2-10 is continuously loaded onto a silica gel column, and gradient elution III is carried out successively with an eluent composed of petroleum ether and ethyl acetate in a volume ratio of 0 - 5:1, and the fractions with a volume ratio of petroleum ether to ethyl acetate of 2:1 are combined to obtain component E2-10-4; silica gel packing is used in this step to subdivide the small-weight component E2-10, and the compounds with a polarity of petroleum ether:ethyl acetate = 2:1 are collected.

[0008] (5) Component E2-10-4 is separated by semi-preparative HPLC, and the fractions with retention times t R = 16.9 - 18.0 min and t R = 20.0 - 22.5 min are collected, concentrated and dried to obtain new sesquiterpenoid compound 1 and new sesquiterpenoid compound 2.

[0009] In the above step (1), the ethanol used for ethanol reflux is 95% ethanol; the number of extractions is 20 - 30 times.

[0010] In the above step (2), the flow rate of gradient elution I is 8 - 10 ml / min and the time is 5 - 7 days.

[0011] In the above step (3), the flow rate of gradient elution II is 5 - 7 ml / min and the time is 2 - 3 days.

[0012] In the above step (4), the flow rate of gradient elution III is 2 - 4 ml / min and the time is 2 - 3 days.

[0013] In the above step (5), the mobile phase for semi-preparative HPLC separation is an aqueous methanol solution with a volume fraction of 25%, and the water in the solvent of the aqueous methanol solution contains three ten-thousandths of trifluoroacetic acid; the flow rate of semi-preparative HPLC separation is 2 ml / min.

[0014] The structural formula of the above new sesquiterpenoid compound 1 is: ; the structural formula of new sesquiterpenoid compound 2 is: .

[0015] In a second aspect, there is also provided a pharmaceutical composition, which contains any one of the following compounds: or .

[0016] The third aspect provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating diseases caused by A β 25-35 -induced PC-12 cell damage.

[0017] Furthermore, the above-mentioned disease is Alzheimer's disease.

[0018] The present invention has the following beneficial effects: 1. In this study, 95% ethanol was used for heating under reflux for extraction. Two new norsesquiterpene compounds, namely Pedatisectaene C (1) and Pedatisectaene D (2), were isolated and identified from the ethyl acetate fraction of the stems and leaves of Arisaema heterophyllum Blume. The experimental results showed that both compounds could significantly improve A β 25-35 -induced PC-12 cell damage, and solved the problem of its application in the preparation of medicaments for treating Alzheimer's disease.

[0019] 2. No scholar has ever systematically separated the chemical components in the stems and leaves of Arisaema heterophyllum Blume. This study is the first to isolate and purify the norsesquiterpene compounds therein, which has explored the medicinal value and commercial value of the stems and leaves of Arisaema heterophyllum Blume, and has remarkable economic and social benefits.

[0020] 3. In this study, a method of cross-using various chromatographic packings was adopted to obtain new norsesquiterpene compounds more quickly and efficiently and meet the implementation requirements in the industry. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 is the structure of the compound.

[0023] Figure 2 is for compound 1 1 H NMR spectrum (500 MHz, CD 3 OD).

[0024] Figure 3 is for compound 1 13 C NMR spectrum (500 MHz, CD 3 OD).

[0025] Figure 4 is for compound 2 11H NMR spectrum (500 MHz, CD 3 OD).

[0026] Figure 5 For compound 2 13 13C NMR spectrum (500 MHz, CD 3 OD). Detailed implementation manners

[0027] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0028] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0029] Instruments and reagents Bruker AVANCE Ⅲ 500 nuclear magnetic resonance spectrometer for nuclear magnetic resonance (TMS internal standard) (Bruker); Nicolet is 10 Microscope Spectrometer for infrared spectrum (Thermo Scientific, USA); Bruker maxis HD mass spectrometer for high-resolution mass spectrometry; Shimadzu UV-2401PC apparatus for ultraviolet spectrum; LC50 type high-pressure preparative liquid chromatograph; UV200 type ultraviolet detector [Sepurest (Beijing) Technology Co., Ltd.]; YMC-Pack ODS-A chromatographic column (250×10 mm. D. S-5 mm, 12 mm) (YMC Co., Ltd.); The rest includes N-1100 type rotary evaporator (Shanghai Ailang Instrument Co., Ltd.); A-1000S type water aspirator (Shanghai Ailang Instrument Co., Ltd.); N-1111 type chilled water circulation device (Shanghai Ailang Instrument Co., Ltd.); FDU-2110 type freeze dryer (Shanghai Ailang Instrument Co., Ltd.); DFZ-60508 type vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); AB204-N ten-thousandth precision analytical balance (METTLER TOLEDO); Carbon dioxide 3111 type incubator (Thermo); ECLPSE TS100 inverted microscope (Nikon); Centrifuge-5804R high-speed centrifuge (Eppendorf); Multiskan MK3 microplate reader (Thermo Fisher); Advantage A10 ultrapure water instrument (Sartorius); BCD-206TAS ultra-low temperature refrigerator (Haier Company); DZF-6050B vacuum drying oven (Beijing Hengtaifengke Test Equipment Co., Ltd.); HVA-85 autoclave (Hirayama).

[0030] Highly differentiated rat adrenal medulla pheochromocytoma cell line (PC-12); A β 25-35 Lyophilized powder (Sangon Biotech Co., Ltd.); Fetal bovine serum (Hangzhou Sijiqing Company); DMEM medium (Gibco Invitrogen Company); MTT (Beijing Solarbio Science & Technology Co., Ltd.); DMSO (Shanghai Macklin Biochemical Co., Ltd.).

[0031] The stems and leaves of Arisaema heterophyllum Blume were collected from Yuzhou, Henan in November 2023, and identified as the dried stems and leaves of Arisaema heterophyllum Blume ( Pinellia pedatisecta Schott) by Professor Chen Suiqing and Professor Dong Chengming of Henan University of Chinese Medicine.

[0032] Example 1 A method for extracting new norsesquiterpenoids from the stems and leaves of Arisaema heterophyllum Blume, the steps are as follows: (1) Take 20 kg of dried stems and leaves of Arisaema heterophyllum Blume, crush them, reflux with 95% ethanol twice, 2 h each time, filter, combine the filtrates and concentrate under reduced pressure to obtain 1.8 kg of extract, dissolve it in 3 L of water, and extract it 20 times with 4 L of petroleum ether, ethyl acetate, and n-butanol in turn to obtain petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction.

[0033] (2) After concentrating and drying each part, 60 g of ethyl acetate fraction E was loaded onto an ODS medium-pressure column and eluted successively with a gradient of 10%-100% methanol containing water at a flow rate of 8 ml / min. Detection was carried out every 250 ml, and the amount of each gradient mobile phase was judged by anisaldehyde-sulfuric acid thin-layer chromatography. Elution was completed in 5 days, and the fractions eluted with 20% methanol were combined and labeled as fraction E2.

[0034] (3) After dissolving fraction E2 in methanol, it was loaded onto a silica gel column. The sample was mixed with silica gel of 100-200 mesh at a ratio of 1:1, and the column was packed with silica gel of 200-300 mesh. It was eluted with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 6 ml / min. The ratios used were successively petroleum ether:ethyl acetate = 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. The amount of each gradient mobile phase was judged by anisaldehyde-sulfuric acid thin-layer chromatography. Elution was completed in 2 days, and the fractions eluted with petroleum ether:ethyl acetate = 1:1 were combined and labeled as fraction E2-10.

[0035] (4) Fraction E2-10 was continuously loaded onto a silica gel column. The sample was mixed with silica gel of 100-200 mesh at a ratio of 1:1, and the column was packed with silica gel of 200-300 mesh. It was eluted with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 3 ml / min. The ratios used were successively petroleum ether:ethyl acetate = 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. Detection was carried out by anisaldehyde-sulfuric acid thin-layer chromatography. Elution was completed in 2 days, and the fractions eluted with petroleum ether:ethyl acetate = 2:1 were combined and labeled as fraction E2-10-4.

[0036] (5) Fraction E2-10-4 was separated by semi-preparative HPLC and loaded onto a YMC-Pack ODS-AA chromatographic column with a specification model of 250×10 mm, particle size 5 μ μm, pore size 12 nm. The mobile phase was methanol:water (the content of trifluoroacetic acid was three ten-thousandths) 25:75, and the flow rate was 2 ml / min. The fractions with retention times of t R = 16.9~18.0 min and t R = 20.0~22.5 min were collected, concentrated and dried to obtain compound 1 (Pedatisectaene C (1)) and compound 2 (Pedatisectaene D (2)).

[0037] Structure Identification Pedatisectaene C (1): Yellow oil. HR-ESI-MS gave the quasi-molecular ion peak m / z 229.1065[M + H] + , (calcd for C 11 H17 O 5 , 229.1070), and its molecular formula was determined to be C 11 H 16 O 5 ; UV (MeOH) λ max : 212 nm; IR (iTR) ν max : 3377, 1744, 1680, 1209, 1143 cm -1 ; Its hydrogen spectrum and carbon spectrum are as shown in Figure 2 and Figure 3 .

[0038] Pedatisectaene D (2): Yellow oil. HR-ESI-MS gave the quasi-molecular ion peak m / z 213.1116 [M+H] + , (calcd for C 11 H 17 O 4 , 213.1121), and its molecular formula was determined to be C 11 H 16 O 4 ; UV (MeOH) λ max : 212 nm; IR (iTR) ν max : 3426, 2923, 1731, 1678, 1202, 1141 cm -1 ; Its hydrogen spectrum and carbon spectrum are as shown in Figure 4 and Figure 5 .

[0039] The NMR data are shown in Table 1: Table 1 NMR data of the compounds in CD 3 OD .

[0040] Example 2 A method for extracting new norsesquiterpenes from the stems and leaves of Arisaema heterophyllum Blume, the steps are as follows: (1) Take 20 kg of dried stems and leaves of Arisaema heterophyllum Blume, crush them, heat and reflux with 95% ethanol twice, 2 h each time, filter, and concentrate the combined filtrate under reduced pressure to obtain 1.8 kg of extract, dissolve it in 3 L of water, and extract it 20 times with 4 L of petroleum ether, ethyl acetate, and n-butanol in turn to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction.

[0041] (2) After concentrating and drying each part, 60 g of the ethyl acetate fraction E was loaded onto an ODS medium-pressure column and eluted successively with a gradient of 10%-100% methanol containing water at a flow rate of 9 ml / min. Detection was carried out every 250 ml, and the amount of each gradient mobile phase was judged by anisaldehyde-sulfuric acid thin-layer chromatography. The elution was completed in 6 days. The fractions eluted with 20% methanol were combined and labeled as fraction E2.

[0042] (3) After dissolving fraction E2 in methanol, it was loaded onto a silica gel column. The sample was mixed with silica gel of 100-200 mesh at a ratio of 1:1, and the column was packed with silica gel of 200-300 mesh. It was eluted with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 5 ml / min. The ratios used were successively petroleum ether:ethyl acetate = 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. The amount of each gradient mobile phase was judged by anisaldehyde-sulfuric acid thin-layer chromatography. The elution was completed in 3 days. The fractions eluted with petroleum ether:ethyl acetate = 1:1 were combined and labeled as fraction E2-10.

[0043] (4) Fraction E2-10 was continuously loaded onto a silica gel column. The sample was mixed with silica gel of 100-200 mesh at a ratio of 1:1, and the column was packed with silica gel of 200-300 mesh. It was eluted with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 4 ml / min. The ratios used were successively petroleum ether:ethyl acetate = 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. Detection was carried out by anisaldehyde-sulfuric acid thin-layer chromatography. The elution was completed in 3 days. The fractions eluted with petroleum ether:ethyl acetate = 2:1 were combined and labeled as fraction E2-10-4.

[0044] (5) Fraction E2-10-4 was separated by semi-preparative HPLC and loaded onto a YMC-Pack ODS-AA chromatographic column with a specification model of 250×10 mm, particle size 5 μ μm, pore size 12 nm. The mobile phase was methanol:water (the content of trifluoroacetic acid was three ten-thousandths) 25:75, and the flow rate was 2 ml / min. The fractions with retention times t R = 16.9~18.0 min and t R = 20.0~22.5 min were collected, concentrated and dried to obtain compound 1 (Pedatisectaene C (1)) and compound 2 (Pedatisectaene D (2)).

[0045] Pedatisectaene C (1): Yellow oil. Its characterization data are the same as those in Example 1.

[0046] Pedatisectaene D (2): Yellow oil. Its characterization data are the same as those in Example 1.

[0047] Example 3 A method for extracting new norsesquiterpenoids from the stems and leaves of Arisaema heterophyllum Blume var. pekingense (Engl.) Hsiao & K. C. Hsia is as follows: (1) Take 20 kg of dried stems and leaves of Arisaema heterophyllum Blume var. pekingense (Engl.) Hsiao & K. C. Hsia, crush them, heat and reflux with 95% ethanol twice, 2 h each time, filter, and after combining the 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 4 L of petroleum ether, ethyl acetate, and n-butanol to obtain a petroleum ether fraction, an ethyl acetate fraction, an n-butanol fraction, and an aqueous fraction.

[0048] (2) After concentrating and drying each fraction, take the ethyl acetate fraction E (60 g) and load it onto an ODS medium-pressure column. Elute it successively with a gradient of 10%-100% methanol containing water at a flow rate of 8.5 ml / min, and detect it once every 250 ml. The amount of the mobile phase for each gradient is judged by thin-layer chromatography with anisaldehyde-sulfuric acid. The elution is completed in 7 d. Combine the fractions eluted with 20% methanol and label them as component E2.

[0049] (3) After dissolving component E2 in methanol, load it onto a silica gel column. Mix the sample with silica gel of 100-200 mesh at a ratio of 1:1, pack the column with silica gel of 200-300 mesh, and elute it with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 7 ml / min. The ratios used are successively petroleum ether:ethyl acetate = 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. The amount of the mobile phase for each gradient is judged by thin-layer chromatography with anisaldehyde-sulfuric acid. The elution is completed in 2 d. Combine the fractions with petroleum ether:ethyl acetate = 1:1 and label them as component E2-10.

[0050] (4) Component E2-10 is continuously loaded onto a silica gel column. Mix the sample with silica gel of 100-200 mesh at a ratio of 1:1, pack the column with silica gel of 200-300 mesh, and elute it with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 4 ml / min. The ratios used are successively petroleum ether:ethyl acetate = 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. Detect it by thin-layer chromatography with anisaldehyde-sulfuric acid. The elution is completed in 2.5 d. Combine the fractions with petroleum ether:ethyl acetate = 2:1 and label them as component E2-10-4.

[0051] (5) Component E2-10-4 is separated by semi-preparative HPLC. Load it onto a YMC-Pack ODS-AA chromatographic column with a specification model of 250×10 mm, a particle size of 5 μ μm, and a pore size of 12 nm. The mobile phase is methanol:water (the content of trifluoroacetic acid is three ten-thousandths) 25:75, and the flow rate is 2 ml / min. Collect the fractions with retention times t R = 16.9~18.0 min and t RFractions with a retention time of 20.0 - 22.5 min were concentrated and dried to obtain Compound 1 (Pedatisectaene C (1)) and Compound 2 (Pedatisectaene D (2)).

[0052] Pedatisectaene C (1): Yellow oil. Its characterization data is the same as that in Example 1.

[0053] Pedatisectaene D (2): Yellow oil. Its characterization data is the same as that in Example 1.

[0054] Example 4 A method for extracting new norsesquiterpenes from the stems and leaves of Arisaema heterophyllum Blume var. pekingense (Engl.) Hsiao & K. C. Hsia is as follows: (1) Take 20 kg of dried stems and leaves of Arisaema heterophyllum Blume var. pekingense (Engl.) Hsiao & K. C. Hsia, crush them, reflux with 95% ethanol twice, 2 h each time, filter, combine the filtrates and concentrate under reduced pressure to obtain 1.8 kg of extract. Dissolve it in 3 L of water and extract it 20 times with 4 L of petroleum ether, ethyl acetate, and n-butanol respectively to obtain petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction.

[0055] (2) After concentrating and drying each fraction, take the ethyl acetate fraction E (60 g) and load it onto an ODS medium-pressure column. Elute it successively with a gradient of 10% - 100% methanol containing water at a flow rate of 10 ml / min. Detect once every 250 ml. The amount of mobile phase used for each gradient is judged by thin-layer chromatography with anisaldehyde-sulfuric acid. The elution is completed in 5 days. Combine the fractions eluted with 20% methanol and label them as Component E2.

[0056] (3) Dissolve Component E2 in methanol and load it onto a silica gel column. Mix the sample with silica gel (100 - 200 mesh) at a ratio of 1:1. Pack the column with silica gel (200 - 300 mesh). Elute it with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 5 ml / min. The ratios used are successively petroleum ether:ethyl acetate = 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. The amount of mobile phase used for each gradient is judged by thin-layer chromatography with anisaldehyde-sulfuric acid. The elution is completed in 3 days. Combine the fractions with a ratio of petroleum ether:ethyl acetate = 1:1 and label them as Component E2-10.

[0057] (4) Component E2-10 is continuously loaded onto a silica gel column. Mix the sample with silica gel (100 - 200 mesh) at a ratio of 1:1. Pack the column with silica gel (200 - 300 mesh). Elute it with a gradient of petroleum ether:ethyl acetate as the mobile phase at a flow rate of 2 ml / min. The ratios used are successively petroleum ether:ethyl acetate = 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. Detect by thin-layer chromatography with anisaldehyde-sulfuric acid. The elution is completed in 3 days. Combine the fractions with a ratio of petroleum ether:ethyl acetate = 2:1 and label them as Component E2-10-4.

[0058] (5) Component E2-10-4 was separated by semi-preparative HPLC using a YMC-Pack ODS-AA column with the specifications of 250×10 mm, particle size 5 μ μm, pore size 12 nm. The mobile phase was methanol:water (containing 0.03% trifluoroacetic acid) at a ratio of 25:75, and the flow rate was 2 ml / min. The fractions with retention times t R = 16.9 - 18.0 min and t R = 20.0 - 22.5 min were collected, concentrated and dried to obtain Compound 1 (Pedatisectaene C (1)) and Compound 2 (Pedatisectaene D (2)).

[0059] Pedatisectaene C (1): Yellow oil. Its characterization data is the same as that in Example 1.

[0060] Pedatisectaene D (2): Yellow oil. Its characterization data is the same as that in Example 1.

[0061] Example of implementation effect: Activity screening experiment 1. A β 25-35 Preparation of freeze-dried powder Take the A β 25-35 freeze-dried powder out of the -20 °C refrigerator and let it stand at room temperature for half an hour. In the laminar flow hood, dissolve the A β 25-35 powder with sterile double-distilled water to prepare a stock solution with a concentration of 1 M, and place it in a 37 °C, 5% CO 2 incubator for 7 days. Seal it with a sticker, label it and store it in the -20 °C refrigerator for later use.

[0062] 2. Cell culture PC-12 cells were cultured in a culture dish containing DMEM medium with 10% FBS in a 37 °C constant temperature incubator with 5% CO 2 . When the cells covered 80% of the dish, subculture was carried out, and subculture was performed every 2 days.

[0063] 3. Detection of the effect of compounds in Arisaema heterophyllum Blume on the cell viability of A β 25-35 induced PC-12 cells by MTT method Place the PC-12 cells in a 37 °C, 5% CO 2 incubator and culture them until the logarithmic growth phase. Inoculate them into a 96-well plate at a cell density of 5×10 4 cells / mL. After 24 h, divide them into a normal group (CON), a model group (M, Aβ 25-35 0.1 μ M) and each administration group (10 μ M + A β 25 - 35, 0.1 μ M). After continuous culture for 24 h, 20 μ μL MTT solution (5 mg / mL) was added to each well and incubated for 4 h. The liquid in the wells was discarded, and 150 μ μL DMSO was added to each well, and it was shaken for 10 min to completely dissolve it. The OD value of each well was measured using an enzyme - linked immunosorbent assay (ELISA) reader at a wavelength of 490 nm, repeated 3 times, and the cell viability was calculated.

[0064] Cell viability = OD value of each group / OD value of the normal group. Table 2 shows that compared with the model group, the cell viability of PC - 12 cells can be significantly increased ( P <0.01), indicating that both compounds can improve Aβ 25-35 -induced PC - 12 cell damage.

[0065] Table 2 Effects of compounds on A β 25-35 -induced PC - 12 cell damage ( ± sd , n = 4) In summary, in this study, the aerial parts of Arisaema heterophyllum Blume were extracted by heating under reflux with 95% ethanol, and Pedatisectaene C (1) and Pedatisectaene D (2) were isolated and identified from the ethyl acetate fraction of its extract. Through the A β 25-35 -induced PC - 12 cell damage experiment to evaluate whether the compounds have potential anti - Alzheimer's disease effects. The results show that the compounds can significantly improve A β 25-35 -induced PC - 12 cell damage, further enriching the pharmacodynamic material basis of the aerial parts of Arisaema heterophyllum Blume, improving its resource utilization rate, and also providing more possibilities for the development of anti - Alzheimer's disease drugs.

[0066] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa, characterized in that: Here are the steps: (1) After crushing the yunnanxing, the ethanol is refluxed, filtered, and the filtrate is collected. The obtained filtrate is concentrated under reduced pressure to obtain an extract. The obtained extract is dissolved in water and extracted with petroleum ether, ethyl acetate and n-butanol in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, an n-butanol fraction and a water fraction, respectively; (2) After the ethyl acetate fraction was concentrated and dried, it was gradient eluted with a 10%-100% methanol-water solution by volume. After elution, the fractions with a 20% methanol-water solution by volume were combined as component E2. (3) After component E2 was applied to a silica gel column, gradient elution II was performed using an eluent consisting of petroleum ether and ethyl acetate in a volume ratio of 0-10:1, and the fractions with a volume ratio of petroleum ether and ethyl acetate of 1:1 were combined as component E2-10; (4) Component E2-10 was continuously loaded onto a silica gel column and gradient eluted with an eluent consisting of petroleum ether and ethyl acetate in a volume ratio of 0-5:

1. The fractions with a volume ratio of petroleum ether and ethyl acetate of 2:1 were combined to obtain component E2-10-4. (5) Component E2-10-4 was separated by semi-preparative HPLC and the samples with retention time t R =16.9-18.0min and t R = 20.0-22.5min fraction was concentrated and dried to obtain new norsesquiterpene compound 1 and new norsesquiterpene compound 2.

2. The method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa according to claim 1, characterized in that: In the step (1), the ethanol used for ethanol reflux is 95% ethanol; and the number of extractions is 20 to 30 times.

3. The method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa according to claim 2, characterized in that: In the step (2), the flow rate of gradient elution I is 8-10 ml / min and the time is 5-7 days.

4. The method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa according to claim 3, characterized in that: In step (3), the flow rate of gradient elution II is 5-7 ml / min and the time is 2-3 days.

5. The method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa according to claim 4, characterized in that: In the step (4), the flow rate of gradient elution III is 2-4 ml / min and the time is 2-3 days.

6. The method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa according to claim 5, characterized in that: The mobile phase for semi-preparative HPLC separation in step (5) is a 25% by volume methanol-water solution, wherein the solvent water of the methanol-water solution contains 0.03 parts by volume of trifluoroacetic acid; the flow rate for semi-preparative HPLC separation is 2 ml / min.

7. The method for extracting new norsesquiterpenes from the stems and leaves of Arisaema confusa according to any one of claims 1 to 6, characterized in that: The structural formula of the new norsesquiterpene compound 1 is: ; The structural formula of the new norsesquiterpene compound 2 is: .

8. A pharmaceutical composition, characterized in that Contains any of the following compounds: or .

9. The pharmaceutical composition according to claim 8 is used in the preparation of a β 25-35 Application of drugs that induce PC-12 cell damage-induced diseases.

10. The use according to claim 9, characterized in that: The disease is Alzheimer's disease.

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