Method for extracting neorevemedesmyl from leaves and stems of arisaema erubescens and application thereof

Abstract

The application belongs to the field of pharmaceutical chemistry, and relates to comprehensive utilization of Yunnan starwort. The application provides a method for extracting new sesquiterpenes from stems and leaves of Yunnan starwort and application. In the experiment, 95% ethanol is used for heating reflux extraction. Two new sesquiterpenes, Pedatisectaene C (1) and Pedatisectaene D (2), are separated and identified from ethyl acetate parts of stems and leaves of Yunnan starwort. β 25‑35 Induced PC-12 cell damage.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to the comprehensive utilization of *Gynostemma pentaphyllum*. Background Technology

[0002] Yu Nanxing is a plant of the genus Pinellia in the family Araceae, namely Pinellia palmata (Palm-leaved Pinellia). Pinellia pedatisecta The tuber of *Schott*, also known as tiger paw or palm-leaf pinellia, is warm in nature, bitter and pungent in taste, and slightly toxic. It has the effects of drying dampness and resolving phlegm, dispelling wind and relieving spasms, and dispersing nodules and reducing swelling. *Arisaema heterophyllum* is mainly produced in Yuzhou City, Henan Province, and is known as one of the "Four Great Yu Medicines" along with *Angelica dahurica*, *Aconitum carmichaelii*, and *Pinellia ternata*. Due to its large yield and high quality, it sells well overseas. However, in the process of large-scale use, the stems and leaves of *Arisaema heterophyllum* are discarded, resulting in serious resource waste. The state has clearly proposed to strengthen the comprehensive utilization research of the "non-medicinal parts" of Chinese medicinal materials, and to consider the resource utilization rate of stems and leaves. To clarify the pharmacodynamic material basis of the stems and leaves of *Arisaema heterophyllum* and improve its utilization rate...

[0003] Research on sesquiterpenes has largely focused on medicinal plants; application CN112920038A discloses sesquiterpenoid compounds with antibacterial activity found in *Chimonanthus praecox*. Studies have also discovered a series of anti-hepatocellular carcinoma active ingredients in *Artemisia annua*. The complex ring systems of sesquiterpenes and their derivatives, the low content of sesquiterpenes in traditional Chinese medicine, and the difficulty in isolation all pose significant challenges to the performance research of novel sesquiterpenes. Furthermore, existing data indicate that no novel sesquiterpenoid compounds have yet been isolated from *Arisaema heterophyllum*. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method and application for extracting novel sesquiterpenes from the stems and leaves of Arisaema heterophyllum.

[0005] The technical solution of this invention is implemented as follows:

[0006] A method for extracting novel sesquiterpenes from the stems and leaves of Arisaema heterophyllum, comprising the following steps:

[0007] (1) After crushing Yu Nanxing, the filtrate was refluxed with ethanol, filtered, and collected. The filtrate was concentrated under reduced pressure to obtain an extract. The extract was dissolved in water and extracted with petroleum ether, ethyl acetate and n-butanol in sequence to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction, respectively.

[0008] (2) After the ethyl acetate fraction is concentrated and dried, it is subjected to gradient elution I with a methanol aqueous solution with a volume ratio of 10%-100%. After elution, the fractions of the methanol aqueous solution with a volume ratio of 20% are combined as component E2. In this step, macroporous resin packing is used to initially separate the large weight of ethyl acetate fraction and select the 20% methanol aqueous solution fraction with suitable polarity.

[0009] (3) After component E2 is applied to a silica gel column, gradient elution II is performed using eluents composed of petroleum ether and ethyl acetate in a volume ratio of 0-10:1, successively, and the fractions in which the volume ratio of petroleum ether and ethyl acetate is 1:1 are combined to obtain component E2-10; in this step, silica gel is used as the filler, and the compounds are eluted in order of increasing polarity according to the polarity of the eluent, and the compounds with a polarity around petroleum ether: ethyl acetate = 1:1 are collected.

[0010] (4) Component E2-10 is continuously applied to a silica gel column, gradient elution III is performed using eluents composed of petroleum ether and ethyl acetate in a volume ratio of 0-5:1, successively, and the fractions in which the volume ratio of petroleum ether and ethyl acetate is 2:1 are combined to obtain component E2-10-4; in this step, silica gel is used as the filler, and the small amount of component E2-10 is subdivided, and the compounds with a polarity around petroleum ether: ethyl acetate = 2:1 are collected.

[0011] (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 sesquiterpene compound 1 and new sesquiterpene compound 2.

[0012] In the above step (1), the ethanol used for reflux is 95% ethanol; the extraction is performed 20-30 times.

[0013] In the above step (2), the flow rate of gradient elution I is 8-10 ml / min, and the time is 5-7 days.

[0014] In the above step (3), the flow rate of gradient elution II is 5-7 ml / min, and the time is 2-3 days.

[0015] In the above step (4), the flow rate of gradient elution III is 2-4 ml / min, and the time is 2-3 days.

[0016] In the above step (5), the mobile phase for semi-preparative HPLC separation is a methanol aqueous solution with a volume fraction of 25%, and the solvent water in the methanol aqueous solution contains 3 parts per million of trifluoroacetic acid; the flow rate for semi-preparative HPLC separation is 2 ml / min.

[0017] The structural formula of the above new sesquiterpene compound 1 is: ; and the structural formula of the new sesquiterpene compound 2 is: .

[0018] The second aspect also provides a pharmaceutical composition containing any one of the following compounds:

[0019] or .

[0020] The third aspect provides the above-mentioned pharmaceutical composition in the preparation of treatment of A β 25-35 The application of the medicine in the treatment of the disease caused by the induced PC-12 cell damage.

[0021] Further, the above-mentioned disease is Alzheimer's disease.

[0022] The present application has the following beneficial effects:

[0023] 1. In this study, 95% ethanol was heated and refluxed to extract it. Two new sesquiterpenes, Pedatisectaene C (1) and Pedatisectaene D (2) were isolated and identified from the ethyl acetate fraction of the stems and leaves of Yunnan star. The experimental results show that the two compounds can significantly improve A β 25-35 The application of the medicine in the treatment of the disease caused by the induced PC-12 cell damage.

[0024] 2. No one has ever systematically isolated the chemical components of Yunnan star stems and leaves. This study is the first to isolate and purify the sesquiterpenes in Yunnan star stems and leaves. It opens up the medicinal value and commercial value of Yunnan star stems and leaves, and has significant economic and social benefits.

[0025] 3. In this study, a variety of chromatographic fillers are used in cross-use mode, which is more rapid and efficient in obtaining new sesquiterpene compounds and meeting the industry implementation requirements. DETAILED DESCRIPTION

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0027] Figure 1 The structure of the compound.

[0028] Figure 2 The structure of the compound 1. 1 H NMR spectrum (500MHz, CD3OD).

[0029] Figure 3 The structure of the compound 1. 13 C NMR spectrum (500MHz, CD3OD).

[0030] Figure 4 The structure of the compound 2. 1H NMR spectrum (500MHz, CD3OD).

[0031] Figure 5 is compound 2 13 C NMR spectrum (500MHz, CD3OD). DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative effort belong to the scope of protection of the present application.

[0033] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0034] Instruments and reagents

[0035] Nuclear magnetic resonance: Bruker AVANCE III 500 nuclear magnetic resonance instrument (TMS internal standard) (Bruker);

[0036] Infrared spectrum: Nicolet is 10 Microscope Spectrometer (Thermo Scientific, USA);

[0037] High-resolution mass spectrometry: Bruker maxis HD mass spectrometer;

[0038] Ultraviolet spectrum: Shimadzu UV-2401PC apparatus;

[0039] LC50 type high-pressure preparation liquid chromatograph;

[0040] UV200 type ultraviolet detector [Spectrum Arcs (Beijing) Science and Technology Co., Ltd];

[0041] YMC-Pack ODS-A chromatographic column (250x10 mm. D. S-5 mm, 12 mm) (YMC Co., Ltd.);

[0042] The rest has N-1100 type rotary evaporator (Shanghai Ailang Instrument Co., Ltd.);

[0043] A-1000S type water flow air extractor (Shanghai Ailang Instrument Co., Ltd.);

[0044] N-1111 type refrigerated water circulating device (Shanghai Ailing Instrument Co., Ltd.);

[0045] FDU-2110 type freeze dryer (Shanghai Ailing Instrument Co., Ltd.);

[0046] DFZ-60508 type vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.);

[0047] AB204-N one-thousandth precision analytical balance (METTLER TOLEDO);

[0048] Carbon dioxide 3111 type incubator (Thermo);

[0049] ECLPSE TS100 inverted microscope (Nikon);

[0050] Centrifuge-5804R high-speed centrifuge (Eppendorf);

[0051] Multiskan MK3 microplate reader (Thermo Fisher);

[0052] Advantage A10 ultrapure water instrument (Sartorius);

[0053] BCD-206TAS ultra-low temperature refrigerator (Haier);

[0054] DZF-6050B vacuum drying oven (Beijing Hengtai Fengke Test Equipment Co., Ltd.);

[0055] HVA-85 high-pressure sterilization pot (Hirayama).

[0056] Highly differentiated rat adrenal medulla pheochromocytoma cell line (PC-12);

[0057] A β 25-35 Freeze-dried powder (Shanghai Sunway Biotech Co., Ltd.);

[0058] Fetal bovine serum (Hangzhou Sijiqing Biological Engineering Co., Ltd.);

[0059] DMEM medium (Gibco Invitrogen Co., Ltd.);

[0060] MTT (Beijing Solabio Technology Co., Ltd.);

[0061] DMSO (Shanghai Maikelin Biological Technology Co., Ltd.).

[0062] Yunaxing stems and leaves were collected from Yuzhou, Henan in November 2023 and identified as Yunaxing by Professor Chen Suiqing and Professor Dong Chengming of Henan University of Chinese MedicinePinellia pedatisecta Dried stems and leaves of Schott.

[0063] Example 1

[0064] A method for extracting neorevemedes from stems and leaves of Yunnansheng, the steps are as follows:

[0065] (1) Take 20 kg of dried stems and leaves of Yunnansheng, crush them, and heat them to reflux twice with 95% ethanol, each time for 2 hours. Filter and combine the filtrates, then concentrate them under reduced pressure to obtain 1.8 kg of extract. Dissolve the extract in 3 L of water, and then extract it with 4 L of petroleum ether, ethyl acetate, and n-butanol, respectively, for 20 times to obtain the petroleum ether fraction, the ethyl acetate fraction, the n-butanol fraction, and the water fraction.

[0066] (2) Concentrate and dry each fraction, and then take the ethyl acetate fraction E (60 g) and load it on an ODS pressure column. Elute it with 10%-100% methanol containing water, respectively, at a flow rate of 8 ml / min, and detect it every 250 ml. Determine the amount of each gradient mobile phase by anisaldehyde-concentrated sulfuric acid thin layer detection. Elute for 5 days, and then combine the 20% methanol fractions and mark them as component E2.

[0067] (3) Dissolve component E2 in methanol, load it on a silica gel column, and mix the sample with silica gel at a ratio of 1:1. Load the column with 200-300 mesh silica gel, and elute it with petroleum ether:ethyl acetate as the mobile phase in a gradient manner at a flow rate of 6 ml / min. The proportions used are petroleum ether:ethyl acetate=10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. Determine the amount of each gradient mobile phase by anisaldehyde-concentrated sulfuric acid thin layer detection. Elute for 2 days, and then combine the petroleum ether:ethyl acetate=1:1 fractions and mark them as component E2-10.

[0068] (4) Continue to load component E2-10 on a silica gel column, mix the sample with silica gel at a ratio of 1:1, load the column with 200-300 mesh silica gel, and elute it with petroleum ether:ethyl acetate as the mobile phase in a gradient manner at a flow rate of 3 ml / min. The proportions used are petroleum ether:ethyl acetate=5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. Perform anisaldehyde-concentrated sulfuric acid thin layer detection. Elute for 2 days, and then combine the petroleum ether:ethyl acetate=2:1 fractions and mark them as component E2-10-4.

[0069] (5) Separate component E2-10-4 by semi-preparative HPLC, load it on a YMC-Pack ODS-AA chromatographic column with a specification of 250×10 mm, a particle size of 5 μ m, and a pore size of 12 nm. The mobile phase is methanol:water (containing 0.03% trifluoroacetic acid) 25:75, and the flow rate is 2 ml / min. Collect the fractions with retention times tR = 16.9~18.0 min and t R = 20.0~22.5 min, concentrated and dried to give compound 1 (Pedatisectaene C (1)) and compound 2 (Pedatisectaene D (2)).

[0070] Structure identification

[0071] Pedatisectaene C (1): Yellow oil. HR-ESI-MS gave the quasi-molecular ion peak at m / z 229.1065 [M+H] m / z 229.1065 [M+H] + , (calcd for C 11 H 17 O5, 229.1070), determined its molecular formula as C 11 H 16 O5; UV (MeOH) λ max : 212 nm; IR (iTR) ν max : 3377, 1744, 1680, 1209, 1143 cm -1 ; its 1H NMR, 13C NMR spectra were shown in Figure 2 and Figure 3 .

[0072] Pedatisectaene D (2): Yellow oil. HR-ESI-MS gave the quasi-molecular ion peak at m / z 213.1116 [M+H] m / z 213.1116 [M+H] + , (calcd for C 11 H 17 O4, 213.1121), determined its molecular formula as C 11 H 16 O4; UV (MeOH) λ max : 212 nm; IR (iTR) ν max : 3426, 2923, 1731, 1678, 1202, 1141 cm -1 ; its 1H NMR, 13C NMR spectra were shown in Figure 4 and Figure 5 .

[0073] NMR data were shown in Table 1:

[0074] Table 1. NMR data of compounds in CD3OD

[0075] .

[0076] Example 2

[0077] A method for extracting new nor-seesquiterpenes from the stems and leaves of Podophyllum emodi Wall, comprising the following steps:

[0078] (1) Take 20 kg of dried stems and leaves of Podophyllum emodi Wall, crush them, and reflux them twice with 95% ethanol for 2 hours each time. Filter the mixture, combine the filtrates, and concentrate them under reduced pressure to obtain 1.8 kg of extract. Dissolve the extract in 3 L of water, and sequentially extract it with 4 L of petroleum ether, ethyl acetate, and n-butanol for 20 times to obtain the petroleum ether fraction, the ethyl acetate fraction, the n-butanol fraction, and the water fraction.

[0079] (2) Concentrate and dry each fraction, take the ethyl acetate fraction E (60 g), and load it on an ODS pressure column. Sequentially elute it with 10%-100% methanol containing water at a flow rate of 9 ml / min, and detect it every 250 ml. Determine the amount of each gradient mobile phase by anisaldehyde-concentrated sulfuric acid thin layer detection. Elute for 6 days, combine the 20% methanol fractions, and mark them as component E2.

[0080] (3) Dissolve component E2 in methanol, load it on a silica gel column, mix the sample with silica gel at a ratio of 1:1, and load the mixture on a 200-300 mesh silica gel column. Elute it with petroleum ether: ethyl acetate as the mobile phase at a flow rate of 5 ml / min, and sequentially use petroleum ether: ethyl acetate = 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 as the gradient elution. Determine the amount of each gradient mobile phase by anisaldehyde-concentrated sulfuric acid thin layer detection. Elute for 3 days, combine the fractions of petroleum ether: ethyl acetate = 1:1, and mark them as component E2-10.

[0081] (4) Continue to load component E2-10 on a silica gel column, mix the sample with silica gel at a ratio of 1:1, and load the mixture on a 200-300 mesh silica gel column. Elute it with petroleum ether: ethyl acetate as the mobile phase at a flow rate of 4 ml / min, and sequentially use petroleum ether: ethyl acetate = 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 as the gradient elution. Determine the amount of each gradient mobile phase by anisaldehyde-concentrated sulfuric acid thin layer detection. Elute for 3 days, combine the fractions of petroleum ether: ethyl acetate = 2:1, and mark them as component E2-10-4.

[0082] (5) Separate component E2-10-4 by semi-preparative HPLC, load it on a YMC-Pack ODS-AA chromatographic column with a specification of 250 x 10 mm, a particle size of 5 μ m, and a pore size of 12 nm. Use methanol: water (containing 0.03% trifluoroacetic acid) 25:75 as the mobile phase at a flow rate of 2 ml / min, and collect the retention time t R= 16.9 ~ 18.0 min and t R = 20.0 ~ 22.5 min, concentrated and dried to obtain compound 1 (Pedatisectaene C (1)) and compound 2 (Pedatisectaene D (2)).

[0083] Pedatisectaene C (1): yellow oil. Its characterization data are the same as in Example 1.

[0084] Pedatisectaene D (2): yellow oil. Its characterization data are the same as in Example 1.

[0085] Example 3

[0086] A method for extracting new nor-sesquiterpenes from the stems and leaves of Podophyllum hexandrum Royle, comprising the following steps:

[0087] (1) 20 kg of dried stems and leaves of Podophyllum hexandrum Royle were pulverized, heated and refluxed with 95% ethanol twice, each for 2 h, filtered, and the filtrates were combined and concentrated under reduced pressure to obtain 1.8 kg of extract, which was dissolved in 3 L of water and sequentially extracted with 4 L of petroleum ether, ethyl acetate and n-butanol, each for 20 times, to obtain the petroleum ether fraction, the ethyl acetate fraction, the n-butanol fraction and the water fraction.

[0088] (2) After the fractions were concentrated and dried, the ethyl acetate fraction E (60 g) was subjected to ODS pressure column elution with 10%-100% methanol gradient, at a flow rate of 8.5 ml / min, and each 250 ml was detected once, and the amount of each gradient mobile phase was determined by anisaldehyde-concentrated sulfuric acid thin layer detection. After 7 d of elution, the 20% methanol fractions were combined and labeled as component E2.

[0089] (3) After the component E2 was dissolved in methanol, it was subjected to silica gel column chromatography, with 100-200 mesh silica gel as the sample, the sample and silica gel in a ratio of 1:1, and 200-300 mesh silica gel as the column, and eluted with petroleum ether: ethyl acetate gradient, at a flow rate of 7 ml / min, and the proportions used were 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, respectively, and the amount of each gradient mobile phase was determined by anisaldehyde-concentrated sulfuric acid thin layer detection. After 2 d of elution, the petroleum ether: ethyl acetate = 1:1 fractions were combined and labeled as component E2-10.

[0090] (4) Component E2-10 continues to the silica gel column, 100-200 mesh silica gel is mixed with the sample, the sample and silica gel dosage is 1:1, 200-300 mesh silica gel is packed, petroleum ether: ethyl acetate is used as the mobile phase for gradient elution, the flow rate is 4 ml / min, the proportions used are petroleum ether: ethyl acetate = 5:1, 4:1, 3:1, 2:1, 1:1, 0:1 in turn, anisaldehyde-concentrated sulfuric acid thin layer detection, 2.5 d elution is completed, the flow fractions of petroleum ether: ethyl acetate = 2:1 are combined, and are marked as component E2-10-4.

[0091] (5) Component E2-10-4 is separated by semi-preparative HPLC, and a YMC-Pack ODS-AA chromatographic column with a specification of 250x10mm, a particle size of 5 μ m, and a pore size of 12 nm is used, the mobile phase is methanol: water (the content of trifluoroacetic acid is 0.003%), 25:75, the flow rate is 2 ml / min, the flow fractions with retention times t R =16.9~18.0min and t R =20.0~22.5min are collected, and are concentrated and dried to obtain compound 1 (Pedatisectaene C (1)) and compound 2 (Pedatisectaene D (2)).

[0092] Pedatisectaene C (1): yellow oil. Its characterization data are the same as those of Example 1.

[0093] Pedatisectaene D (2): yellow oil. Its characterization data are the same as those of Example 1.

[0094] Example 4

[0095] A method for extracting new nor-seesquiterpenes from the stems and leaves of Podophyllum hexandrum Royle, the steps are as follows:

[0096] (1) 20 kg of dried stems and leaves of Podophyllum hexandrum Royle are crushed, heated and refluxed with 95% ethanol for 2 hours each time, filtered, and the filtrates are combined and concentrated under reduced pressure to obtain 1.8 kg of extract, which is dissolved in 3L of water and sequentially extracted with 4L of petroleum ether, ethyl acetate, and n-butanol for 20 times to obtain the petroleum ether fraction, the ethyl acetate fraction, the n-butanol fraction, and the water fraction.

[0097] (2) After the fractions are concentrated and dried, the ethyl acetate fraction E (60 g) is loaded onto an ODS medium pressure column, and gradient elution is performed with 10%-100% methanol containing water, the flow rate is 10 ml / min, the sample is detected every 250 ml, the amount of each gradient mobile phase is determined by anisaldehyde-concentrated sulfuric acid thin layer detection, 5d elution is completed, the flow fractions of 20% methanol are combined, and are marked as component E2.

[0098] (3) After dissolving component E2 in methanol, it was loaded onto a silica gel column and mixed with 100-200 mesh silica gel. The ratio of sample to silica gel was 1:1. The column was packed with 200-300 mesh silica gel and 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 petroleum ether:ethyl acetate = 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. The amount of each gradient mobile phase was determined by thin-layer chromatography with anisaldehyde-concentrated sulfuric acid. Elution was completed in 3 days. The fractions of petroleum ether:ethyl acetate = 1:1 were combined and recorded as component E2-10.

[0099] (4) Component E2-10 was loaded onto a silica gel column. The sample was mixed with 100-200 mesh silica gel, and the ratio of sample to silica gel was 1:1. The column was packed with 200-300 mesh silica gel. The mobile phase was petroleum ether:ethyl acetate as the gradient elution, with a flow rate of 2 ml / min. The ratios used were petroleum ether:ethyl acetate = 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. The anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. The elution was completed in 3 days. The fractions with petroleum ether:ethyl acetate = 2:1 were combined and recorded as component E2-10-4.

[0100] (5) Component E2-10-4 was separated by semi-preparative HPLC. The specifications were: 250×10mm, particle size 5. μ A YMC-Pack ODS-AA column with a pore size of 12 nm was used. The mobile phase was methanol:water (trifluoroacetic acid content 0.03%) 25:75, the flow rate was 2 ml / min, and the collection retention time was t. R =16.9~18.0 min and t R =20.0~22.5 min fraction, concentrated and dried to obtain compound 1 (Pedatisectaene C (1)) and compound 2 (Pedatisectaene D (2)).

[0101] Pedatisectaene C (1): Yellow oil. Its characterization data are the same as those in Example 1.

[0102] Pedatisectaene D (2): Yellow oil. Its characterization data are the same as those in Example 1.

[0103] Example of implementation results: Activity screening experiment

[0104] 1. A β 25-35 Freeze-dried powder preparation

[0105] A β 25-35The freeze-dried powder was taken out of the -20 °C refrigerator and placed at room temperature for half an hour. In the clean bench, A β 25-35 The powder was prepared into a mother liquor with a concentration of 1 M, placed in a 37 °C, 5% CO2 incubator for aging for 7 days, sealed, labeled and stored in a -20 °C refrigerator for standby use.

[0106] 2. Cell culture

[0107] PC-12 cells were cultured in a culture dish containing 10% FBS in DMEM medium in a 5% CO2 37 °C constant temperature incubator. When the cell density reached 80%, the cells were subcultured every 2 days.

[0108] 3. MTT method for detecting the effect of compounds in Yunnan starwort on A β 25-35 Effect of inducing PC-12 cell viability

[0109] PC-12 cells were cultured in a 37 °C, 5% CO2 incubator to the logarithmic growth phase, and inoculated in a 96-well plate at a cell density of 5×10 4 6cells / mL. After 24 h, they were divided into a normal group (CON), a model group (M, A β 25-35 , 0.1 μ M) and each drug group (10 μ M + A β 25-35, 0.1 μ M). After 24 h of continuous culture, 20 μ L of MTT solution (5 mg / mL) was added to each well for 4 h of incubation. The liquid in the wells was discarded, 150 μ L of DMSO was added to each well, shaken for 10 min to completely dissolve it, and an enzyme marker was used to measure the OD value of each well at a wavelength of 490 nm, repeated 3 times, and the cell viability was calculated.

[0110] Cell viability = OD value of each group / OD value of normal group. Table 2 shows that compared with the model group, the PC-12 cell viability can be significantly improved P <0.01), indicating that both compounds can improve Aβ 25-35 induced PC-12 cell damage.

[0111] Table 2 Effect of compounds on A β 25-35 induced PC-12 cell damage ± sd , n =4)

[0112]

[0113] In conclusion, Pedatisectaene C (1) and Pedatisectaene D (2) were isolated from the ethyl acetate fraction of the extract of the stems and leaves of Pedatiisetum japonicum by heating reflux with 95% ethanol. β 25-35 The results showed that the compounds could significantly improve the A β 25-35 The induction of PC-12 cell damage further enriches the pharmacodynamic material basis of the stems and leaves of Pedatiisetum japonicum, improves the resource utilization rate, and also provides more possibilities for the development of anti-Alzheimer's drugs.

[0114] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for extracting a sesquiterpene from the leaves of Podophyllum hexandrum Royle, characterized by, The steps are as follows: (1) after the pulverization of the stems and leaves of Yunnan starwort, the stems and leaves are subjected to ethanol reflux, filtration, collection of the filtrate, concentration of the obtained filtrate under reduced pressure to obtain an extract, dissolution of the obtained extract in water, and extraction 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; (2) after the concentrated drying of the ethyl acetate fraction, gradient elution I is performed with 10%-100% methanol aqueous solution, and the eluate of 20% methanol aqueous solution is combined to obtain component E2; (3) after the silica gel column chromatography of component E2, gradient elution II is performed with eluent composed of petroleum ether and ethyl acetate in a volume ratio of 0-10:1 in sequence, and the eluate of petroleum ether and ethyl acetate in a volume ratio of 1:1 is combined to obtain component E2-10; (4) component E2-10 is subjected to silica gel column chromatography again, and gradient elution III is performed with eluent composed of petroleum ether and ethyl acetate in a volume ratio of 0-5:1 in sequence, and the eluate of petroleum ether and ethyl acetate in a volume ratio of 2:1 is combined to obtain component E2-10-4. (5) Component E2-10-4 is separated by semi-preparative HPLC, the fractions with retention time t R = 16.9-18.0 min and t R = 20.0-22.5 min are collected, concentrated and dried to obtain sesquiterpene compound 1 and sesquiterpene compound 2; the mobile phase for semi-preparative HPLC separation in the step (5) is 25% methanol aqueous solution, the solvent water of the methanol aqueous solution contains 3 parts per million trifluoroacetic acid; the flow rate for semi-preparative HPLC separation is 2 ml / min; The structural formula of the sesquiterpene compound 1 is: ; The structural formula of the sesquiterpene compound 2 is: .

2. The method of claim 1, wherein the extraction of the sesquiterpenes from the leaves of Aralia decaisneana is characterized by: In step (1), the ethanol used for ethanol reflux is 95% ethanol, and the extraction is performed for 20-30 times.

3. The method of claim 2, wherein the extraction of the sesquiterpenes from the leaves of A. arisanensis is characterized by: In step (2), the flow rate of gradient elution I is 8-10 ml / min, and the time is 5-7 days.

4. The method of claim 3, wherein the extraction of the sesquiterpenes from the leaves of A. arisanensis is characterized by: 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 of claim 4, wherein the extraction of the sesquiterpenes from the leaves of A. arisanensis is characterized by: In step (4), the flow rate of gradient elution III is 2-4 ml / min, and the time is 2-3 days.

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