Method for extracting yunnanshentere E and F from yunnanshentere stem and leaf and application thereof

Abstract

A method for extracting yunnanternes E and F from yunnanternes stem and leaf and application, can effectively separate the epimer E and F from yunnanternes stem and leaf, solve the problem of treating alzheimer's disease, the steps are: take the dry stem and leaf of yunnanternes, crush, heat reflux, filter, reduce pressure concentration, dissolve in water, and extract with petroleum ether, ethyl acetate and n-butanol in turn, then concentrate and dry, take the concentrated and dried ethyl acetate part, column, elute, purify, elute, collect the flow part in the retention time, concentrate and dry, then get, the preparation method is easy to operate, scientific and reasonable, can effectively extract the epimer compound E and F from yunnanternes stem and leaf, the compound can significantly improve the A beta 25-35 induced PC-12 cell damage, be effectively used for preparing the medicine for treating alzheimer's disease, develop the medicinal and commercial value of yunnanternes stem and leaf, improve the utilization rate, increase the drug source, the economic and social benefits are remarkable.

Description

TECHNICAL FIELD

[0001] The present application relates to medicine, in particular to a method and application for extracting pedatisectaene E and F from Pinellia pedatisecta Schott. BACKGROUND

[0002] Pinellia pedatisecta Schott is the tuber of Pinellia pedatisecta Schott of Araceae Pinellia, also known as palm leaf Pinellia, which is warm in nature, bitter and pungent in taste, and slightly toxic. Pinellia pedatisecta Schott is similar to tiger's palm and the tuber is accompanied by a large number of tubers. Tiger's palm was first recorded in Shennong's Herbal Classic: "Tiger's palm: bitter, warm. Main heart pain, cold and heat, accumulation, accumulation, injury, flaccidity, stiffness, waterway, mountain valley." Pinellia pedatisecta Schott is a geographical indication product, and the quality and genuineness of medicinal materials have excellent medicinal value and economic value, which should be further developed. It is found by checking the literature that the current research on Pinellia pedatisecta Schott mainly focuses on tubers and anti-tumor directions, and there is little research on Pinellia pedatisecta Schott leaves.

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

[0004] With the development of science and technology, in order to further enrich the medicinal material basis of Pinellia pedatisecta Schott leaves, the present application separates the epimer Pedatisectaene E (1) and Pedatisectaene F (2) from the ethyl acetate part of Pinellia pedatisecta Schott leaves by heating reflux with 95% ethanol. Activity experiments show that the compound can significantly improve the cell viability of PC-12 injury induced by Aβ 25-35 , and has activity in treating Alzheimer's disease, but there is no public report so far. SUMMARY

[0005] In view of the above situation, in order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a method and application for extracting pedatisectaene E and F from Pinellia pedatisecta Schott, which can effectively separate epimer E (1) and Pedatisectaene F (2) from Pinellia pedatisecta Schott, and solve the problem of using medicine to treat Alzheimer's disease.

[0006] The technical scheme solved by the present application is a method for extracting Pedatisectaene E and Pedatisectaene F from stems and leaves of Pedatisecta.

[0007]

[0008] The preparation method is as follows:

[0009] 20 kg of dried stems and leaves of Pedatisecta are pulverized, and 95% ethanol by volume is used for heating reflux twice, each time for 2 hours, and then filtered; the filtrates are combined and concentrated under reduced pressure to obtain an extract, which is dissolved in water and sequentially extracted with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether part, ethyl acetate part, n-butanol part and water part, which are respectively concentrated and dried;

[0010] The concentrated and dried ethyl acetate part is subjected to gradient elution on an ODS medium pressure column with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90% and 100% methanol by volume, and the 20% methanol elution fraction is combined to obtain component E;

[0011] The component E is dissolved in methanol and subjected to gradient elution on a silica gel column with 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 by volume, and the petroleum ether: ethyl acetate = 3:1 elution fraction is combined to obtain component E1;

[0012] The component E1 is dissolved in methanol and subjected to elution on a Sephadex LH-20 column, and the 100-180 ml elution fraction is combined to obtain component E2;

[0013] The component E2 is separated by semi-preparative HPLC, and the elution fractions with retention time t R = 20.1-22.0 min and t R = 25.6-27.2 min are collected, concentrated and dried to obtain Pedatisectaene E and Pedatisectaene F, respectively.

[0014] The epimers Pedatisectaene E and Pedatisectaene F prepared by the method of the present application are used for preparing drugs for treating Alzheimer's disease.

[0015] The preparation method is easy to operate, scientific and reasonable, can effectively extract epimeric compounds Pedatisectaene E and Pedatisectaene F from the stems and leaves of Yunnan star, the compounds can significantly improve the A beta 25-35 induced PC-12 cell damage, are effectively used for preparing Alzheimer's disease treating drugs, develop the medicinal value and commercial value of the stems and leaves of Yunnan star, improve the utilization rate, increase the drug source, and have remarkable economic and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a chemical molecular structural formula drawing of Pedatisectaene E (1) and Pedatisectaene F (2) of the application;

[0017] Figure 2 It is the H NMR spectrum (500MHz, CD3OD) of Pedatisectaene E of the application; 1

[0018] Figure 3 It is the C NMR spectrum (125MHz, CD3OD) of Pedatisectaene E of the application; 13

[0019] Figure 4 It is the H NMR spectrum (500MHz, CD3OD) of Pedatisectaene F of the application; 1

[0020] Figure 5 It is the C NMR spectrum (125MHz, CD3OD) of Pedatisectaene F of the application; 13 DETAILED DESCRIPTION

[0021] The specific embodiments of the application are described in detail in combination with specific cases.

[0022] In the specific implementation of the application, the following examples can be given.

[0023] The method for extracting epimeric compounds Pedatisectaene E and Pedatisectaene F from the stems and leaves of Yunnan star provided by the application has the following steps:

[0024] ​​​​Take 20 kg of dry stems and leaves of Pedatiseta grandiflora B1. pulverize, heat reflux with 95% ethanol twice, each time add 200 L of 95% ethanol, reflux for 2 h, filter, combine the filtrates, and concentrate under reduced pressure to obtain 1.8 kg of extract, dissolve with 3 L of water, and sequentially extract with petroleum ether, ethyl acetate, and n-butanol, each for 20 times, each time using 4 L, to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, and respectively concentrate and dry;

[0025] Take the ethyl acetate fraction (60 g) and load it on an ODS pressure column, and sequentially elute with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, and 100% methanol aqueous solution, each gradient elution being 1 L, the flow rate being 8 ml / min, and detecting every 250 ml, and using anisaldehyde-concentrated sulfuric acid thin layer detection, 5 d elution is completed, and the 20% methanol fractions are combined to obtain component E;

[0026] Dissolve component E with methanol, load it on a silica gel column, and use 100-200 mesh silica gel to mix the sample, the sample and silica gel being used in a ratio of 1:1, load it on a 200-300 mesh silica gel column, and elute with petroleum ether:ethyl acetate in a ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, each gradient mobile phase being 3 times the volume of the column, the flow rate being 6 ml / min, and using anisaldehyde-concentrated sulfuric acid thin layer detection, 2 d elution is completed, and the petroleum ether:ethyl acetate = 3:1 fractions are combined to obtain component E1;

[0027] Dissolve component E1 with methanol, pass it through a Sephadex LH-20 column, elute with 70% methanol as the mobile phase, the flow rate being 1 ml / min, and the mobile phase being 400 ml, and using anisaldehyde-concentrated sulfuric acid thin layer detection, and the 100-180 ml fractions are combined to obtain component E2;

[0028] Separate component E2 by semi-preparative HPLC, load it on a YMC-Pack ODS-AA column with a specification of 250*10 mm, a particle size of 5 μm, and a pore size of 12 nm, the mobile phase being methanol:trifluoroacetic acid aqueous solution in a ratio of 35:65, the mass concentration of the trifluoroacetic acid aqueous solution being 0.003, the flow rate being 2 ml / min, and collecting the fractions with retention times of t R = 20.1-22.0 min and t R = 25.6-27.2 min, and respectively concentrating and drying to obtain the epimeric Pedatisectaene E and Pedatisectaene F.

[0029] The epimeric Pedatisectaene E and F prepared by the method of the application are used for preparing drugs for treating Alzheimer's disease.

[0030] The above method extracts epimeric compounds Pedatisectaene E and Pedatisectaene F from the stems and leaves of Yunnan starwort, which can significantly improve the Aβ25-35-induced PC-12 cell damage and is effective for preparing a drug for treating Alzheimer's disease. The relevant experimental data are as follows:

[0031] 1. Instruments and reagents

[0032] Nuclear magnetic resonance was performed using a Bruker AVANCE III 500 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, ultraviolet spectroscopy was performed using a Shimadzu UV-2401PC apparatus, LC50 type high-pressure preparative liquid chromatograph, UV200 type ultraviolet detector [Spectrum Arxgen (Beijing) Science and Technology Co., Ltd.], YMC-Pack ODS-A chromatographic column (250×10 mm.D.S-5mm, 12mm) (YMC Co., Ltd.), and the remaining instruments were N-1100 type rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), A-1000S type water flow air extractor (Shanghai Ailang Instrument Co., Ltd.), N-1111 type refrigerated water circulating 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 one-hundredth precision analytical balance (METTLER TOLEDO), carbon dioxide 3111 type incubator (Thermo), ECLPSE TS100 inverted microscope (Nikon), Centrifuge-5804R high-speed centrifuge (Eppendorf), Multiskan MK3 enzyme marker (Thermo Fisher), Advantage A10 ultrapure water instrument (Sartorius), BCD-206TAS ultra-low temperature refrigerator (Haier), DZF-6050B vacuum drying oven (Beijing Hengtai Fengke Test Equipment Co., Ltd.), HVA-85 high-pressure sterilization pot (Hirayama).

[0033] Highly differentiated rat adrenal medulla pheochromocytoma cell line (PC-12), Aβ 25-35Freeze-dried powder (Shenguo Bioengineering Co., Ltd.), Fetal bovine serum (Hangzhou Four Seasons Green Co., Ltd.), DMEM medium (Gibco Invitrogen Co., Ltd.), MTT (Beijing Solabio Science and Technology Co., Ltd.), DMSO (Shanghai Maikelin Biotechnology Co., Ltd.).

[0034] The stems and leaves of Pinellia pedatisecta Schott were collected from Yuzhou, Henan in November 2023 and identified as the dried stems and leaves of P. pedatisecta by Professor Chen Suiqing and Professor Dong Chengming of Henan University of Chinese Medicine.

[0035] 2. Structural characteristics identification

[0036] Pedatisectaene E (1): yellow oil. HR-ESI-MS gave the quasi-molecular ion peak m / z 211.0956 [M+H] + , (calcd for C 11 H 15 O4, 211.0965), confirming its molecular formula as C 11 H 14 O4; UV (MeOH) λ max : 213 nm; IR (iTR) ν max : 3418, 2930, 1749 cm -1 , see Figures 2-3 , and its NMR data are shown in Table 1.

[0037] Pedatisectaene F (2): yellow oil. HR-ESI-MS gave the quasi-molecular ion peak m / z 211.0956 [M+H] + , (calcd for C 11 H 15 O4, 211.0965), confirming its molecular formula as C 11 H 14 O4; UV (MeOH) λ max : 213 nm; IR (iTR) ν max : 3418, 2930, 1749 cm -1 , see Figures 4-5 , and its NMR data are shown in Table 1.

[0038] The chemical molecular structural formulas of Pedatisectaene E (1) and Pedatisectaene F (2) are as follows:

[0039]

[0040] Table 1 NMR data of compounds in CD3OD

[0041]

[0042]

[0043] 3. Activity experiment

[0044] 3.1 Aβ 25-35 Freeze-dried powder preparation

[0045] Aβ 25-35 Freeze-dried powder was taken out of the -20°C refrigerator and placed at room temperature for half an hour. In the clean bench, Aβ 25-35 powder was prepared into a mother liquor with a concentration of 1M, placed in a 37°C, 5% CO2 incubator for aging for 7 days, sealed, labeled and stored in a -20°C refrigerator for standby.

[0046] 3.2 Cell culture

[0047] 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 culture dish was 80%, it was subcultured every 2 days.

[0048] 3.3 MTT method for detecting the effect of compounds in Yunnan star on Aβ 25-35 induced PC-12 cell viability

[0049] PC-12 cells were cultured in a 37°C, 5% CO2 incubator to the logarithmic growth phase, inoculated in a 96-well plate at a cell density of 5×10 4 After 24h, they were divided into a normal group (CON), a model group (M, Aβ 25-35 , 0.1 μM) and each drug group (10 μM + Aβ25-35, 0.1 μM). After 24h of continuous culture, 20 μL of MTT solution (5 mg / mL) was added to each well for 4h of incubation. The liquid in the well was discarded, 150 μL 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. Cell viability = OD value of each group / OD value of normal group.

[0050] The results showed that compared with the model group, it could significantly improve the PC-12 cell viability (P<0.01), as shown in Table 2, suggesting that both compounds could improve Aβ 25-35 induced PC-12 cell damage.

[0051] Table 2 Effect of compounds on Aβ 25-35 induced PC-12 cell damage n=4

[0052]

[0053] The experiment proves that the invention adopts the method of heating and refluxing 95% ethanol to extract the stems and leaves of Pedatisetae chinensis, separates and identifies a pair of sesquiterpene epimers from the ethyl acetate part of the stems and leaves of Pedatisetae chinensis, which are Pedatisectaene E and Pedatisectaene F, and the activity experiment shows that the compounds can significantly improve the Aβ 25-35 The induced PC-12 cell damage is expected to become a new drug for resisting Alzheimer's disease, and develops the medicinal value of Pedatisetae chinensis and a new way for treating Alzheimer's disease drugs, and has significant social and economic benefits.

Claims

1. A method for extracting Pedatisectaene E and Pedatisectaene F from Pedatiisecta leaves, wherein Pedatisectaene E (1) and Pedatisectaene F (2) have the following chemical molecular structures: The steps are as follows: 20 kg of dried Pedatiisecta leaves are pulverized, and then extracted twice by heating reflux with 95% ethanol, each time adding 200 L of 95% ethanol for refluxing 2 h, filtering, combining the filtrates, and concentrating under reduced pressure to obtain an extract, which is dissolved in 3 L of water and then extracted with petroleum ether, ethyl acetate, and n-butanol, each for 20 times, each time using 4 L, to obtain petroleum ether, ethyl acetate, n-butanol, and water fractions, which are concentrated and dried respectively; the ethyl acetate fraction is subjected to gradient elution on an ODS medium pressure column with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, and 100% methanol aqueous solutions, each time using 1 L, at a flow rate of 8 ml / min, and detecting every 250 ml, and then eluted for 5 d, and the 20% methanol eluate is combined to obtain component E; component E is dissolved in methanol and subjected to silica gel column chromatography, wherein the sample and silica gel are mixed at a ratio of 1:1, and then packed with 200-300 mesh silica gel, and eluted with 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, each time using 3 times the column volume of the mobile phase, at a flow rate of 6 ml / min, and detecting every 2 d, and then the petroleum ether: ethyl acetate = 3:1 eluate is combined to obtain component E1; component E1 is dissolved in methanol and subjected to Sephadex LH-20 column chromatography, eluted with 70% methanol as the mobile phase, at a flow rate of 1 ml / min, and the mobile phase is 400 ml, and detecting every 100-180 ml of the eluate, to obtain component E2. ； 2. The use of Pedatisectaene E and Pedatisectaene F prepared by the method of claim 1 in the preparation of a medicament for treating Alzheimer's disease. ​ ​ ​ ​ The component E2 was separated by semi-preparative HPLC with YMC-Pack ODS-AA column of 250x10mm, particle size 5 ​ m, pore size 12 nm, mobile phase of methanol: trifluoroacetic acid aqueous solution = 35:65 by volume, mass concentration of trifluoroacetic acid aqueous solution 3 / 10,000, flow rate 2 ml / min, and the fractions with retention time t R =20.1~22.0min and t R =25.6~27.2min were collected and concentrated to dryness, respectively, to obtain the epimeric yunansiterpenes E and F. ​

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