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

Abstract

The invention relates to a method for extracting sesquiterpenoids from stems and leaves of rhizoma arisaematis and application of the sesquiterpenoids, the sesquiterpenoids G and I can be effectively extracted from the stems and leaves of rhizoma arisaematis, and the problem of preparing medicines for treating Alzheimer's Disement.The preparation method comprises the steps that the dried stems and leaves of rhizoma arisaematis are taken to be smashed, heated and refluxed, filtered, concentrated under reduced pressure and dissolved with water, and a mixture is obtained; sequentially and respectively extracting with petroleum ether, ethyl acetate and n-butyl alcohol, concentrating and drying; the method is easy to operate, scientific and reasonable, the sesquiterpenoids G and I can be effectively extracted from the stems and leaves of the rhizoma arisaematis, the sesquiterpenoids G and I can remarkably improve PC-12 cell damage induced by Abeta25-35, new application of active ingredients in the stems and leaves of the rhizoma arisaematis is developed, and the sesquiterpenoids G and I are obtained. The commercial and use values of the rhizoma arisaematis are improved, meanwhile, a new way of the medicine for treating the Alzheimer's disease is developed, and the economic and social benefits are huge.

Description

Technical Field

[0001] The invention relates to the field of medicine, in particular to a method for extracting sesquiterpenoid compounds from stems and leaves of Arisaema confusa and application thereof. Background Art

[0002] Yunanxing is the tuber of Pinellia pedatisecta Schott, a plant of the genus Pinellia in the Araceae family. It is also known as tiger palm and palm leaf Pinellia. It was first recorded in Shennong's Herbal Classic and has the effects of drying dampness and resolving phlegm, dispelling wind and stopping spasm, dispersing nodules and reducing swelling. Yunanxing has been a traditional high-quality Chinese medicinal material since Shennong's Herbal Classic. Yunanxing produced in Yuzhou City, Henan Province, an authentic production area, is popular both at home and abroad and has good development prospects. The tuber of Yunanxing is widely used as a medicinal part, but its above-ground part is discarded, resulting in a waste of resources, which greatly limits the value development of the non-medicinal parts of Yunanxing. With the development of modernization of traditional Chinese medicine, it is a very important and urgent task to strengthen the comprehensive development and utilization of the non-medicinal parts of Yunanxing and improve the economic value of Yunanxing.

[0003] Alzheimer's disease (AD) is a degenerative disease of the central nervous system that mainly occurs in the elderly or pre-elderly. The main characteristics of the disease include progressive cognitive dysfunction, behavioral impairment, aphasia, apraxia, agnosia, and visual-spatial impairment, which will be accompanied by changes in personality and behavior and damage the patient's abstract thinking and calculation ability. It is the most common form of dementia, which may account for 60-70% of the number of cases. Alzheimer's disease is the most common type of dementia in the elderly, and the risk of the disease increases with age.

[0004] In order to further enrich the medicinal material basis of the stems and leaves of Arisaema confusa and expand the drug source, the present invention separated two sesquiterpenoid compounds Pedatisectaene G and PedatisectaeneI from the 95% ethanol heating reflux extract of the stems and leaves of Arisaema confusa. Activity experiments showed that the compounds had a significant improvement effect on Aβ25-35-induced PC-12 cell damage, providing a basis for further development of anti-Alzheimer's disease drugs. However, no public reports on related research have been seen so far. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting sesquiterpenoid compounds from the stems and leaves of Arisaema confusa and its application, which can effectively extract sesquiterpenoid compounds Pedatisectaene G and Pedatisectaene I from the stems and leaves of Arisaema confusa, and can effectively solve the problem of preparing medicines for treating Alzheimer's disease.

[0006] The technical solution provided by the present invention is to extract a sesquiterpenoid compound G (Pedatisectaene G) (1) and a sesquiterpenoid compound I (Pedatisectaene I) (2) from the stems and leaves of Arisaema yunnanensis, and the chemical molecular structure formula is as follows:

[0007]

[0008] The preparation method is:

[0009] The dried stems and leaves of Yunanxing are crushed, heated and refluxed with 95% ethanol by volume, filtered, and concentrated under reduced pressure to obtain an extract, which 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, which are concentrated and dried;

[0010] The concentrated and dried ethyl acetate fraction was loaded onto an ODS medium pressure column and eluted with a gradient of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol in sequence, and the fractions with 30% methanol were combined to obtain component E;

[0011] Dissolve component E with methanol, mix with silica gel, and perform gradient elution with a volume ratio of petroleum ether:ethyl acetate = 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. Combine the fractions with petroleum ether:ethyl acetate = 3:1 to obtain component E1;

[0012] Dissolve component E1 in methanol, further separate, elute with 70% methanol by volume, flow rate 1 ml / min, dosage 500 ml, combine 100-200 ml and 200-300 ml fractions, and 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 sample with retention time t R = The fraction from 20.1 to 22.0 min was concentrated and dried to obtain sesquiterpenoid compound G (Pedatisectaene G);

[0014] Component E1-2 was separated by semi-preparative HPLC, loaded onto a chromatographic column, and the retention time t R = The fraction from 15.6 to 18.0 min was concentrated and dried to obtain sesquiterpenoid compound I (Pedatisectaene I).

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

[0016] The preparation method of the invention is easy to operate, scientific and reasonable, and can effectively extract sesquiterpenoid compound G and sesquiterpenoid compound I from the stems and leaves of Arisaema confusa. The compounds can significantly improve Aβ25-35-induced PC-12 cell damage, open up new uses of active ingredients in the stems and leaves of Arisaema confusa, save resources, improve the commercial value and use value of Arisaema confusa, and at the same time open up a new way of treating Alzheimer's disease drugs, with huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The chemical molecular structure diagrams of the sesquiterpenoid compound G (Pedatisectaene G) (1) and the sesquiterpenoid compound I (Pedatisectaene I) (2) of the present invention;

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

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

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

[0021] Figure 5 is the compound Pedatisectaene I of the present invention 13 C NMR spectrum (125 MHz, Acetone-d6). DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described in detail below in combination with specific situations.

[0023] The present invention can be specifically implemented by the following examples.

[0024] A method for extracting sesquiterpenoid compound G (Pedatisectaene G) and sesquiterpenoid compound I (Pedatisectaene I) from the stems and leaves of Arisaema confusa, comprising the following steps:

[0025] Take 20kg of dried stems and leaves of Yunanxing, crush them, heat and reflux them with 95% ethanol by volume for 2 times, add 200L of 95% ethanol by volume each time and reflux for 2h, filter, combine the two filtrates, and concentrate under reduced pressure to obtain 1.8kg of extract, dissolve it with 3L of water, and extract it with petroleum ether, ethyl acetate, and n-butanol 20 times respectively, each time using 4L, to obtain petroleum ether part, ethyl acetate part, n-butanol part and water part, and concentrate and dry each part;

[0026] The concentrated and dried ethyl acetate fraction was loaded onto an ODS medium-pressure column and eluted with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol gradient elution in sequence. Each gradient elution was 1 L, the flow rate was 8 ml / min, and the elution was detected once every 250 ml. The elution was completed in 5 days, and the fractions with 30% methanol were combined to obtain component E.

[0027] Dissolve component E in methanol, mix with 100-200 mesh silica gel, the amount of sample to silica gel is 1:1, apply to 200-300 mesh silica gel column, and gradient elution is performed with volume ratio of petroleum ether: ethyl acetate = 20:1, 15:1, 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 is 3 times the column volume, the flow rate is 6ml / min, and the detection is performed once every 200ml. The detection is performed with anisaldehyde-concentrated sulfuric acid thin layer. After elution is completed in 3d, the fractions with petroleum ether: ethyl acetate = 3:1 are combined to obtain component E1;

[0028] Dissolve component E1 in methanol, further separate it through Sephadex LH-20, elute with 70% methanol by volume, flow rate 1 ml / min, dosage 500 ml, detect with anisaldehyde-concentrated sulfuric acid thin layer chromatography, combine 100-200 ml and 200-300 ml fractions, and 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 with a size of 250×10 mm, a particle size of 5 μm, and a pore size of 12 nm. The mobile phase was a volume ratio of methanol to trifluoroacetic acid aqueous solution of 35:65, with a mass concentration of trifluoroacetic acid aqueous solution of 0.03 ppm, a flow rate of 2 ml / min, and a collection retention time of t R = The fraction from 20.1 to 22.0 min was concentrated and dried to obtain sesquiterpenoid compound G (Pedatisectaene G);

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

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

[0032] The preparation method of the invention is easy to operate, scientific and reasonable, and can effectively extract sesquiterpenoid compound G (Pedatisectaene G) and sesquiterpenoid compound I (Pedatisectaene I) from the stems and leaves of Arisaema yunnanensis. The compound can significantly improve Aβ 25-35 Induced PC-12 cell damage, and carried out characteristic structure determination and activity test of active ingredients on the extract, all of which achieved very good beneficial technical effects. The relevant experimental data are as follows:

[0033] 1. Instruments and reagents

[0034] Nuclear magnetic resonance was performed using a Bruker AVANCEⅢ500 nuclear magnetic resonance instrument (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 pressure preparative liquid chromatograph, UV200 ultraviolet detector [Saipu Ruisi (Beijing) Technology Co., Ltd.], YMC-PackODS-A chromatographic column (250×10mm.DS-5mm,12mm) (YMC Co., Ltd.), the rest are N-1100 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), A-1000S water flow vacuum pump (Shanghai Ailang Instrument Co., Ltd.), N-1111 chilled water circulation device (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 1 / 10,000 precision analytical balance (METTLER TOLEDO), carbon dioxide 3111 incubator (Thermo), ECLPSE TS100 inverted microscope (Nikon), Centrifuge-5804R high-speed centrifuge (Eppendorf), Multiskan MK3 microplate reader (Thermo Fisher), Advantage A10 ultrapure water meter (Sartorius), BCD-206TAS ultra-low temperature refrigerator (Haier Company), DZF-6050B vacuum drying oven (Beijing Hengtai Fengke Testing Equipment Co., Ltd.), HVA-85 high pressure sterilizer (Hirayama).

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

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

[0037] 2 Structural characterization

[0038] Pedatisectaene G(1): Yellow oil. HR-ESI-MS gave the quasi-molecular ion peak m / z 263.1257[M+H] + ,(calcd for C 13 H 21 O4,263.1254), and its molecular formula is 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 , and its NMR data are shown in Table 1;

[0039] Pedatisectaene I (2): yellow oil. HR-ESI-MS gave a quasi-molecular ion peak of m / z 247.0943 [M+Na] + ,(calcd for C 12 H 15 O4Na, 247.0941), and its molecular formula is 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 , and its 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 compounds in Acetone-d6

[0043]

[0044]

[0045] 3 Activity experiments

[0046] 3.1Aβ 25-35 Lyophilized powder preparation

[0047] Aβ 25-35 The freeze-dried powder was taken out of the -20℃ refrigerator and placed at room temperature for half an hour. Aβ 25-35The powder was prepared into a mother solution with a concentration of 1 M, placed in a 37°C, 5% CO2 incubator for aging for 7 days, sealed and labeled, and stored in a -20°C refrigerator for later use.

[0048] 3.2 Cell culture

[0049] PC-12 cells were cultured in a culture dish containing DMEM medium containing 10% FBS in a 37°C constant temperature incubator with 5% CO2. The cells were passaged when 80% of the cells were confluent in the dish, and were passaged every 2 days.

[0050] 3.3 MTT assay to detect the effects of compounds in Yunanxing on Aβ 25-35 Experiment on inducing cell viability of PC-12 cells

[0051] PC-12 cells were cultured in a 37°C, 5% CO2 incubator until the logarithmic growth phase. 4 / mL were inoculated in a 96-well plate and divided into a normal group (CON), a model group (M, Aβ 25-35 , 0.1μM) and each drug group (10μM+Aβ25-35, 0.1μM). After 24h of continuous culture, 20μL MTT solution (5mg / mL) was added to each well and incubated for 4h. The liquid in the well was discarded, 150μL DMSO was added to each well, and oscillated for 10min to completely dissolve it. The OD value of each well was measured at a wavelength of 490nm using an ELISA reader, and repeated 3 times to calculate the cell viability. Cell viability = OD value of each group / OD value of the normal group.

[0052] The results showed that the PC-12 cell viability was significantly improved compared with the model group (P<0.01), as shown in Table 2, indicating that both compounds can improve Aβ 25-35 Induce PC-12 cell damage.

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

[0054]

[0055]

[0056] In summary, the present invention adopts the method of heating and refluxing 95% ethanol to extract the stems and leaves of Arisaema confusa, separates and identifies the sesquiterpenoid compounds Pedatisectaene G and Pedatisectaene I from the ethyl acetate part of the extract, and through Aβ 25-35 PC-12 cells were induced to establish an in vitro model, and the activity of the 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 material basis of the efficacy of Yunanxing, which can be further used to prepare drugs for the treatment of Alzheimer's disease, opening up the medicinal value of Yunanxing and new ways to treat Alzheimer's disease, with significant social and economic benefits.

Claims

1. A method for extracting sesquiterpenoid compound G (Pedatisectaene G) (1) and sesquiterpenoid compound I (Pedatisectaene I) (2) from the stems and leaves of Arisaema confusa, wherein the chemical molecular structure is as follows:

2. The method for extracting Pedatisectaene G and Pedatisectaene I from the stems and leaves of Arisaema confusa according to claim 1, characterized in that: Take 20 kg of dried stems and leaves of Arisaema confusa, crush them, heat and reflux them with 95% ethanol by volume for 2 times, add 200 L of 95% ethanol by volume each time and reflux for 2 h, filter, combine the two filtrates, and concentrate under reduced pressure to obtain 1.8 kg of extract, dissolve it with 3 L of water, and extract it with petroleum ether, ethyl acetate, and n-butanol 20 times respectively, each time using 4 L, to obtain petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction, and concentrate and dry each fraction; The ethyl acetate fraction was loaded onto an ODS medium-pressure column and eluted with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol gradient elution in sequence. Each gradient elution was 1 L, and the flow rate was 8 ml / min. The fraction was detected every 250 ml and detected with anisaldehyde-concentrated sulfuric acid thin layer chromatography. The elution was completed in 5 days, and the fractions with 30% methanol were combined to obtain component E. Dissolve component E in methanol, mix with 100-200 mesh silica gel, the amount of sample to silica gel is 1:1, apply to 200-300 mesh silica gel column, and perform gradient elution with volume ratio of petroleum ether: ethyl acetate = 20:1, 15:1, 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 is 3 times the column volume, the flow rate is 6 ml / min, detect once every 200 ml, detect with anisaldehyde-concentrated sulfuric acid thin layer, elution is completed in 3 days, combine the fractions with petroleum ether: ethyl acetate = 3:1, and obtain component E1; Dissolve component E1 in methanol, further separate it on Sephadex LH-20, elute with 70% methanol by volume, flow rate 1 ml / min, use 500 ml, detect with anisaldehyde-concentrated sulfuric acid thin layer chromatography, combine 100-200 ml and 200-300 ml fractions, and obtain components E1-1 and E1-2 respectively; Component E1-1 was separated by semi-preparative HPLC. The upper specification model was: 250×10mm, particle size 5 μ m, YMC-Pack ODS-AA column with a pore size of 12 nm, the mobile phase was methanol: trifluoroacetic acid aqueous solution with a volume ratio of 35:65, the mass concentration of trifluoroacetic acid aqueous solution was 0.03%, the flow rate was 2 ml / min, and the collection retention time was t R = The fraction from 20.1 to 22.0 min was concentrated and dried to obtain compound Pedatisectaene G; Component E1-2 was separated by semi-preparative HPLC. The upper specification model was: 250×10mm, particle size 5 μ m, YMC-Pack ODS-AA column with a pore size of 12 nm, the mobile phase was methanol: trifluoroacetic acid aqueous solution with a volume ratio of 35:65, the mass concentration of trifluoroacetic acid aqueous solution was 0.03%, the flow rate was 2 ml / min, and the collection retention time was t R = 15.6 ~ 18.0min fractions were concentrated to dryness to obtain compound Pedatisectaene I.

3. Use of the sesquiterpenoid compounds Pedatisectaene G and Pedatisectaene I according to claim 1 in the preparation of drugs for treating Alzheimer's disease.

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