CN112830947B

CN112830947B - Stilbene compounds isolated from Rheum lhasaense and their use in treating nervous system diseases

Info

Publication number: CN112830947B
Application number: CN202011543207.5A
Authority: CN
Inventors: 何春年; 刘青; 申洁; 李珮; 肖培根
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2022-10-04
Anticipated expiration: 2040-12-21
Also published as: CN112830947A; CN115650940A; CN115650940B

Abstract

The invention discloses a stilbene compound separated from Lhasa rhubarb and an application thereof in treating nervous system diseases. The invention separates three new stilbenes compounds from the rhubarb of Lhasa, the structural formula of which is shown as formula I, formula II or formula III. The invention further discloses a method for separating and obtaining a stilbene compound from the Rheum lasagni. The result of in vitro determination of the inhibitory activity of the compound on acetylcholinesterase shows that the compound has stronger inhibitory effect on acetylcholinesterase. The invention further carries out animal experiments and behavioral experiments NOR, MWM, PAT, cholinergic detection, oxidative stress detection and the like on the compound shown in the formula I to evaluate the improvement effect of the compound shown in the formula I on the cognitive disorder induced by the scopolamine, and the experimental result shows that the compound shown in the formula I can obviously improve the cognitive disorder induced by the experimental animals on the scopolamine. The stilbene compound provided by the invention can be used for preparing a medicament for treating neurodegenerative diseases.

Description

Stilbene compounds isolated from Rheum lhasaense and their use in treating nervous system diseases

Technical Field

The invention relates to a stilbene compound, in particular to a stilbene compound separated from Lhasa rhubarb, and further relates to the stilbene compound and application thereof in preparing a medicament for treating nervous system diseases, belonging to the field of the stilbene compound separated from plants and the application thereof.

Background

Rheum lhasaense A.J.Li et P.K.Hsiao is a Bolete group plant of Rheum genus of Polygonaceae family, and is a unique species in China. Its wild species are mainly distributed in Tibet, and at present, artificial cultivation is started in some areas of Sichuan and Yunnan provinces. Rheum lapachense, a national medicine, is often used for treating stomach diseases and is called as quzao by local people in Tibet. Early literature analysis finds that the Lasa rhubarb does not contain anthraquinone and anthrone derivatives which are characteristic components of plants in the genus of Rheum, and shows great particularity on chemical components. At present, there are few research reports about rheum lasagnum, and only two documents report that rheum lasagnum contains 12 phytochemical components and is a stilbene component, including 6 resveratrol monomer derivatives, 4 resveratrol dimers and 2 resveratrol trimers. Stilbene components show remarkable effects in the prevention and treatment of nervous system diseases, and at present, resveratrol is the most deeply studied stilbene component. Some clinical data show that resveratrol is effective in improving the symptoms of patients with alzheimer's disease.

The choline hypothesis considers that a decrease in the level of acetylcholine in brain tissue is one of the important factors causing neurodegenerative diseases, in which the degree of hydrolysis of acetylcholine is enhanced by overexpression of acetylcholinesterase, resulting in a decrease in the level of acetylcholine. The acetylcholinesterase inhibitor is regarded as an important drug source for preventing and treating neurodegenerative diseases such as Alzheimer's disease, and two drugs such as donepezil and galantamine have good treatment effect on Alzheimer's disease by acting on cholinergic pathway. In the current report, the stilbene component is considered to be the main component of the rhubarb of the lassa, and is an important treasure house for preventing and treating neurodegenerative diseases such as Alzheimer disease and the like.

Disclosure of Invention

It is an object of the present invention to provide a novel stilbene compound isolated from Lhasa rhubarb;

the invention also aims to apply the provided new stilbene compound to the preparation of the medicament for treating the neurodegenerative diseases.

The above purpose of the invention is realized by the following technical scheme:

the invention firstly provides a novel toluylene compound separated from Lhasa rhubarb, and the structural formula of the toluylene compound is shown as formula I, formula II or formula III:

acid addition salts, hydrates or prodrugs of the compounds of formula I, formula II or formula III are also included in the invention; the acid addition salt of the compound is preferably a pharmaceutically acceptable suitable acid (e.g., hydrochloric acid, acetic acid, sulfuric acid) to form a non-toxic salt, and other salts besides pharmaceutically acceptable salts are also included in the present invention.

For reference, the present invention provides a process for isolating a compound of formula I, formula II or formula III from Rheum lhasaense, which comprises the steps of:

(1) Pulverizing Rheum palmatum L, extracting with 95% ethanol, and concentrating under reduced pressure to obtain extract; (2) Extracting the extract to obtain chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction; (3) Loading an ethyl acetate part sample into an MCI filler chromatographic column, and leaching with 30%, 50%, 70% and 100% ethanol solvents respectively; wherein, the compound of formula I and the compound of formula II are separated from 50% ethanol elution fraction, and the compound of formula III is separated from 70% ethanol elution fraction.

Wherein, the ethanol in the step (1) is preferably 40-98% ethanol, and most preferably 95% ethanol;

and (4) rinsing 4-5 column volumes by each solvent in the step (3).

The in vitro determination of the inhibitory activity of three new compounds (a

new compound

1,2,3) shown in the formula I, the formula II and the formula III on acetylcholinesterase shows that the inhibitory activity of the compound shown in the formula I (a new compound 1) and the compound shown in the formula II (a new compound 2) on acetylcholinesterase shows a moderate inhibitory effect, and the compound shown in the formula III (a new compound 3) shows a strong inhibitory effect.

The invention further carries out animal experiments on the novel compound 1, and researches and evaluates the improvement effect of the novel compound 1 on cognitive disorder induced by scopolamine through behavioral experiments NOR, MWM, PAT and cholinergic detection and oxidative stress detection. According to the experimental result of the mouse new object recognition capability, the new compound 1 can enhance the memory of the mouse to a familiar object and can remarkably improve the scopolamine-induced short-term and non-spatial learning memory impairment. According to the experimental result of the passive avoidance ability of the mice, the novel compound 1 can improve the injury caused by scopolamine, so that the dark avoidance latency of the C57 mice is prolonged, and the error frequency is reduced. According to the experimental result of learning and memory ability in the mouse water maze experiment, the new compound 1 can obviously improve the damage caused by scopolamine. According to the experimental result of improving the cholinergic injury caused by scopolamine, the new compound 1 can effectively reverse the acetylcholine level reduction and acetylcholinesterase injury caused by scopolamine. According to the experimental result of the oxidative damage caused by the scopolamine, the new compound 1 has good oxidation resistance and can reverse the oxidative damage caused by the scopolamine. According to the experimental result of the neuroinflammation caused by scopolamine, the compound 1 has obvious inflammation inhibition effect and can weaken inflammatory injury.

The invention also provides a pharmaceutical composition for treating neurodegenerative diseases, which is prepared by matching a compound shown in formula I, formula II or formula III or pharmaceutically acceptable salts thereof with a pharmaceutically acceptable carrier, wherein the compound is a compound shown in formula I, formula II or formula III; after the compound shown in the formula I, the formula II or the formula III with the pharmaceutically acceptable dosage is matched with a pharmaceutically acceptable carrier or auxiliary material, the compound is prepared into any suitable pharmaceutical composition according to the conventional preparation method in the field. The compositions are generally suitable for oral administration and for administration by injection, as are other methods of administration. The composition can be in the form of tablet, capsule, powder, granule, lozenge, suppository or oral liquid. Depending on the method of administration, the pharmaceutical compositions according to the invention may contain from 0.1% to 99% by weight, preferably from 10% to 60% by weight, of a compound of the formula I, II or III

Wherein, the auxiliary materials can be antioxidant complexing agent, filling agent, framework material and the like; the pharmaceutically acceptable carrier is one or more of xylitol, mannitol, lactose, fructose, dextran, glucose, polyvinylpyrrolidone, low molecular dextran, sodium chloride, calcium gluconate or calcium phosphate, and preferably mannitol or lactose.

Wherein the neurodegenerative disease comprises Alzheimer Disease (AD), parkinson Disease (PD) or Huntington Disease (HD).

Detailed description of the invention

Pulverizing Rheum palmatum L, extracting with 95% ethanol, and concentrating under reduced pressure to obtain extract. The extract is divided into chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction. The ethyl acetate fraction samples were loaded onto MCI packed chromatography columns and eluted with 30%, 50%, 70% and 100% ethanol solvents, respectively, each eluting 4-5 column volumes. Wherein the compound 1 (piceatannol-3' -O-beta- _D - [2 "- (3,5-dihydroxy-4-methoxybenzoyl)]Glucopyranoside) and the new compound 2 (piceatannol-3' -O-beta- _D - (2 "-galloyl) -glucopyranoside), and separating from 70% of the eluate fraction to obtain the compound 3 (4' -methoxy-scirpusin A).

Since a decrease in the level of acetylcholine in brain tissue is one of the important factors for causing neurodegenerative diseases such as alzheimer's disease, acetylcholinesterase inhibitors are an important candidate for the search of preventive and therapeutic remedies. The results of the in vitro determination of the inhibitory activity of three new compounds (

compound

1,2,3) on acetylcholinesterase show that

compound

1,2 shows a moderate inhibitory effect on the inhibitory activity of acetylcholinesterase, and compound 3 shows a strong inhibitory effect. The activity of the

compounds

1,2 and 3 is significantly higher than that of resveratrol (compound 4) and quzastilbene glycoside (compound 5) (p < 0.0001).

The in vitro activity experiment shows that the new compound 1 in the rheum lasagnum has good cholinesterase inhibition activity and nervous system protection effect, and the amount of the new compound 1 obtained by separation is relatively large, and the in vitro activity is good, so the new compound 1 is selected to carry out animal experiments, and the improvement effect of the new compound 1 on scopolamine-induced cognitive disorder is researched and evaluated through behavior experiments NOR, MWM, PAT and cholinergic detection and oxidative stress detection.

According to the experimental result that the new compound 1 improves the recognition capability of a new object of a mouse, the administration of the new compound 1 through intragastric administration can enhance the memory of the mouse to a familiar object and can obviously improve the short-term and non-spatial learning memory impairment induced by scopolamine. According to the experimental result that the novel compound 1 improves the passive avoidance ability of mice, the compound 1 can improve the injury caused by scopolamine, so that the dark avoidance latency of C57 mice is prolonged, and the error frequency is reduced. According to the experimental result that the new compound 1 improves the learning and memory ability in the mouse water maze experiment, the new compound 1 can obviously improve the damage caused by the scopolamine. According to the experimental result that the new compound 1 improves the cholinergic injury caused by the scopolamine, the new compound 1 can effectively reverse the acetylcholine level reduction and acetylcholinesterase injury caused by the scopolamine under the administration of low dose and high dose. According to the experimental result that the novel compound 1 improves the oxidative damage caused by the scopolamine, the novel compound 1 has good antioxidant capacity and can reverse the oxidative damage caused by the scopolamine. According to the experimental result that the novel compound 1 improves neuroinflammation caused by scopolamine, the compound 1 has obvious inflammation inhibition effect and reduces inflammatory injury.

Drawings

FIG. 1 shows the main HMBC related schematic of the

compounds

1,2, 3.

Figure 2 IC50 values of new chemistry isolated from rasa rhubarb (×, p < 0.0001).

FIG. 3 detection of learning and memory disorders that novel Compound 1 prevents SCOP treatment by mouse neoformant recognition ability assay; model group: scopolamine (1.5 mg. Kg-1); DNPZ donepezil (3 mg. Kg-1); compound 1_L (100 mg. Kg-1); compound 1_H (400 mg. Kg-1). Experimental values are expressed as mean ± SEM (n =15per group). X <0.05,. X <0.01,. X <0.001,. X <0.0001 compared to the model group.

FIG. 4 measures learning and memory impairment by inhibition of SCOP treatment by Compound 1 via a passive avoidance ability assay; (a) Latency into the dark Chamber (b) number of charged errors into the dark Chamber model group scopolamine (1.5 mg kg. Kg) ^-1 )；DNPZ:donepezil(3mg·kg ^-1 ) (ii) a Compound 1_L (100 mg kg) ^-1 ) (ii) a Compound 1_H (400 mg kg) ^-1 ) Experimental values are expressed as mean ± SEM (n =15per gro)up).*<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.

FIG. 5 determination of learning and memory impairment by Compound 1 preventing SCOP treatment by the Water maze experiment; the swimming speed, (d) the number of times of crossing target quadrants, (e) the swimming trajectory of a mouse in a water tank; red circles indicate hidden platform positions, green curves indicate motion trajectories model set scopolamine (1.5 mg. Kg) ^-1 )；DNPZ:donepezil(3mg·kg ^-1 ) (ii) a 1_L Compound 1 (100 mg kg) ^-1) (ii) a Compound 1_H Compound 1 (400 mg kg) ^-1 ) Experimental values are expressed as mean ± SEM (n =15per group)<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.

FIG. 6 is an experiment on the effect of the novel compound 1 on improving the cholinergic nervous system of SCOP mice; intracerebral acetylcholine level, (b) intracerebral acetylcholinesterase activity, (c) intracerebral ChAT activity T. Model group, scopolamine (1.5 mg kg ^-1 )；DNPZ:donepezil(3mg·kg ^-1 ) (ii) a Compound 1_L Compound 1 (100 mg kg) ^-1) (ii) a Compound 1_H Compound 1 (400 mg kg) ^-1 ) Experimental values are expressed as mean ± SEM (n =15per group)<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.

FIG. 7 Experimental results of novel Compound 1 for improving oxidative stress status in SCOP treated mice; (a) brain SOD activity, (b) brain CAT activity, (c) brain GSH level, (d) brain MDA level model group, scopolamine (1.5 mg kg ^-1 )；DNPZ:donepezil(3mg·kg ^-1 ) (ii) a Compound 1_L Compound 1 (100 mg kg) ^-1) (ii) a Compound 1_H Compound 1 (400 mg kg) ^-1 ) Experimental values are expressed as mean ± SEM (n =15per group)<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.

FIG. 8 shows that compound 1 improves neuroinflammation caused by scopolamine; (a) IL-1 β level in serum, (b) IL-6 level in serum, (c) TNF-a level in serum M model group, scopolamine (1.5 mg kg) ^-1 )；DNPZ:donepezil(3mg·kg ^-1 ) (ii) a Compound 1_L Compound 1 (100 mg kg) ^-1) (ii) a Compound 1_H Compound 1 (400 mg kg) ^-1 ) Experiment ofValues are expressed as mean ± SEM (n =15per group)<0.05,**<0.01,***<0.001,****<0.0001 compared to model group. .

Detailed Description

The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

Materials, medicaments and reagents

The roots and the roots of Rheum palmatum are collected in forest prefecture of Tibetan autonomous region, identified by professor Zhong Guoyue (the research center for natural resources of Chinese medicinal materials and national medicines of Jiangxi Chinese medicinal university), and the samples are stored in the kinship center of the institute of medicinal plants of Chinese academy of medicine.

Acetylcholinesterase (AChE, 1002652214), butyrylcholinesterase (BuChE, 1002399384), 5,5-dithiobis [ 2-nitrobenzoic acid ] (DNTB, 1002473536), thioacetylcholine iodide (ATCI, 101792824), thiobutyrylcholine iodide (BTCI, 101758768) and tacrine (1002020029) were purchased from SigmaAldrich, respectively. The enzyme solution was prepared with ph8.0 phosphate buffer (50 mM) (Hyclone Laboratories, USA). Sodium Dodecyl Sulfate (SDS) was obtained from the national pharmaceutical group chemical agents Co.

Scopolamine (aladdin, shanghai aladine biochem ltd); donepezil hydrochloride (aladdin, shanghai alading biochemistry science and technology, ltd); sodium carboxymethylcellulose (solarbio, beijing solibao science and technology ltd); physiological saline (Shijiazhuang four drugs Co., ltd.).

Example 1 isolation of stilbene Compounds from Rheum Lhasa and characterization thereof

1. Extracting stilbene compounds from Rheum lhasaense

Pulverizing Rheum palmatum L, extracting with 95% ethanol, and concentrating under reduced pressure to obtain extract. The extract is divided into chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction. The ethyl acetate fraction samples were loaded onto MCI packed chromatography columns and eluted with 30%, 50%, 70% and 100% ethanol solvents, respectively, each eluting 4-5 column volumes. Wherein compounds 1 and 2 are separated from the 50% ethanol elution fraction and compound 3 is separated from the 70% elution fraction.

2. Structural analysis of novel Compound

Compound 1: brown powder, molecular ion peak given by (+) HRESIMS m/z 595.1423[ M ] +Na] ⁺ (calcd for 595.1428) determining its molecular formula as C ₂₈ H ₂₈ O ₁₃ The unsaturation degree was 15. Process for preparation of Compound 1 ¹ H NMR spectra: aromatic region hydrogen signal delta _H 7.25 (1H, d, J =2.0Hz, H-10), 7.05 (1H, dd, J =8.4,2.0Hz, H-14) and 6.77 (1H, d, J =8.3Hz, H-13) are ABX systems, delta _H 6.44 (2H, d, J =2.2Hz, H-2,6) and 6.17 (1H, t, J =2.2Hz, H-4) are AX ₂ System, δ _H 6.87 (1H, d, J =16.2Hz, H-8) and 6.73 (1H, d, J =16.3Hz, H-7) are trans-olefin protons, these proton signals indicating that Compound 1 contains piceatannol units, delta _H 7.15 (2H, s, H-2', 6') binding of delta _C 166.18 (carbonyl signal) is identified as a galloyl unit. In addition, a methoxy (3.85,s, 3H) and sugar signal was also observed. In that ¹³ 28 carbon signals can be observed in a C NMR spectrum, and the basic structure of piceatannol-3' -O-beta- _D -glucopyranoside. Methine proton of sugar at delta _H 5.23 (1H, J =8.0 Hz) is the triplet, combines with the proton on C-2' of the saccharide determined in the HMBC spectrum, and this proton is associated with delta _C 166.18 (carbonyl signal) correlation, determining the carbon at the 2-position of the galloyl linkage to the sugar. Methyl hydrogen was observed to correlate with C-4' (δ C139.90) in the HMBC spectra. Finally, the compound 1 is determined to be piceatannol-3' -O-beta- _D - [2 "- (3,5-dihydroxy-4-methoxybenzoyl)]-glucopyranoside. ¹ H NMR and ¹³ the C NMR data are shown in Table 1.

Compound 2: brown powder, molecular ion peak given by (+) HRESIMS m/z 581.1265[ m ] +Na +] ⁺ (calcd for 581.1271) determination of its molecular formulaIs C ₂₇ H ₂₆ O ₁₃ The unsaturation degree was 15. Compound 2, other than lacking the methoxy signal ¹ H NMR and ¹³ the C NMR data were similar to those of Compound 1. ¹ H NMR spectra: contains the typical piceatannol signal an ABX system: delta _H 7.23 (1H, d, J = 2.0Hz), 7.04 (1H, dd, J =8.3, 2.0Hz) and 6.76 (1H, d, J = 8.3Hz), and AX ₂ The system comprises: delta _H 6.44 (2h, d, j = 2.2hz) and 6.16 (1h, t, j = 2.2hz), and trans-olefin proton δ _H 6.87 (d, J =16.2 Hz) and 6.72 (d, J =16.3 Hz). Furthermore, via the methine hydrogen (delta) at sugar position 2 in the HMBC spectrum _H 5.19 With a carbonyl group (. Delta.) _C 166.68 Correlation can confirm that the galloyl group is linked at the 2-position of the glycosyl group, indicating that the compound 2 is piceatannol-3 '-O-beta-D- (2' -galloyl) -glucopyranoside, ¹ h NMR and ¹³ the C NMR data are shown in Table 1.

Compound 3: brown powder with molecular formula C determined by UPLC-Q-TOF-MS ₂₉ H ₂₄ O ₇ (m/z 485.1601[M+H] ⁺ The calculation formula is 485.1556), in combination ¹³ C and ¹ analysis of the H NMR data showed 18 degrees of unsaturation. ¹ Aromatic region of H NMR, at delta _H 6.88 A group of AA 'BB' coupled protons was present at (2H, d, J =8.4Hz, H-3,5) and at 7.30 (2H, d, J =8.3Hz, H-2,6), indicating that the structure contains one para-substituted phenyl group. At delta _H 6.67 (1H, d, J =2.1Hz, H-14) and 6.31 (1H, d, J =2.1Hz, H-12) are a group of aromatic protons of AX type; there are other aroma signals: delta _H 6.10(3H，s，2′，4′，6′)，δ _H 6.74 (2H, H-10',13 ') and 6.62 (1H, dd, J =8.1,2.1Hz, H-14 '). Delta _H 5.31 Aliphatic protons and δ at (1H, d, J=4.9Hz, H-8 ') and 4.44 (1H, d, J=4.9Hz, H-7') _H 6.94 The trans double bond protons at (1H, d, J =16.4Hz, H-7) and 6.71 (1H, d, J =16.4Hz, H-8) were considered as typical protons of the stilbene dimer component. Compound 3 has, in addition to the above proton, a methoxy group delta _H 3.74 (3H, s). Process for preparation of Compound 3 ¹³ C NMR spectrum showed 29 carbon signals containing one methoxy group, 16 methyl groups and12 quaternary carbons. Of compounds 1, other than having one more methoxy group ¹ H and ¹³ the C NMR data agreed with those of spirpusin A. H-7' (delta) _H 4.4 To C-2', 6' (delta) _C 105.9 Existence of HMBC correlation, H-8' (delta) _H 5.31 Related to the presence of HNBC at C-10 '(113.2), C-14' (117.3), indicating that the structure of Compound 3 is consistent with scirpusin A. In addition, methyl proton (. Delta.) was observed in HMBC spectrum _H 3.74 Is with C-4 (. Delta.) _C 159.5 There is a correlation to conclude that the methoxy group is at C-4. The dihydrobenzofuran skeleton of compound 3 is two chiral centers at C-7 'and 8', and the coupling constant (J) of the two protons at C-7 'and 8' is 4.9Hz, indicating trans. According to some resveratrol oligomers ¹ As can be seen from HNMR data, the coupling constant should be around 8.0Hz when oriented in cis. And there is no NOESY correlation between H-7 'and H-8', further indicating the opposite directionality of H-7 'and H-8'. The configuration of the dihydrobenzofuran skeleton at C-7 'and 8' can be distinguished by the Cotton effect in the region of 220-240nm, for example, (+) -epsilon viniferin (7 'S,8' S) is positive Cotton effect at 237nm, and (-) -epsilon viniferin (7 'R,8' R) is negative Cotton effect. The CD spectrum of Compound 3 exhibited a negative Cotton effect (Δ ε, -13.17) at 231nm, indicating that the absolute configurations at C-7 'and C-8' are 7'R,8' R. Therefore, the structure of compound 3 is elucidated as 4'-methoxy-scirpusin a (4' -methoxy-scirpusin a).

TABLE 1 preparation of Compound 1 and Compound 2 ¹ H NMR and ¹³ c NMR data

^b Data are shown measured in deuterated methanol.

^* Multiple overlapping peaks are indicated.

TABLE 2 preparation of Compound 3 ¹ H NMR and ¹³ c NMR data

Experimental example 1 screening experiment for inhibitory Activity of acetylcholinesterase (AChE)

1. Test method

Acetylcholinesterase inhibitory activity was determined on AChE inhibition by

compounds

1 and 2 in 96-well plates after modification based on the Ellman method. The reagents used in the experiment included the reaction substrate ATCI, the developer DTNB and the reaction terminator 1% by weight of the SDS buffer content (1 g SDS in PBS (ph 8.0)). The determination steps are as follows: first, 60. Mu.L of PBS (pH 8.0), 10. Mu.L of 3mM DTNB, 20. Mu.L of the test compound and 20. Mu.L of 0.50U/mL AChE solution were mixed in a 96-well plate and incubated at 37 ℃ for 10 minutes to activate the enzyme; next, 10. Mu.L of substrate (ATCI) was added to the microplate and incubated at 37 ℃ for 15 minutes. Third, 80. Mu.L of 1% SDS solution was added to terminate the reaction. Finally, the absorbance was measured at 405nm using a multifunctional microplate reader (Tecan Infinite M200, switzerland Tecan tracing co., ltd). Blank samples were used as a blank sample with 20 μ l PBS instead of sample solution to record blank absorption, and tacrine was used as a positive control for inhibition of acetylcholinesterase. Wherein each sample was replicated three times. Cell viability (%)

Inhibition (%) =1- (sample a-background)/(blank a-background) × 100 (1)

Data processing

Statistical analysis of the data was performed using IBM SPSS Statistics V23.0 software and the results are expressed as mean + -Standard Error Mean (SEM). Performing One-way ANOVA (One-way ANOVA) on the data conforming to the normal distribution, and performing pairwise comparison on the difference between groups through LSD analysis; data that do not satisfy normal distributions are tested non-parametrically. The results of the analysis are graphed using GraphPad Prism software 8.0.

2. Test results

Since a decrease in the level of acetylcholine in brain tissue is one of the important factors for neurodegenerative diseases such as alzheimer's disease, acetylcholinesterase inhibitors are an important candidate for the search of preventive and therapeutic remedies. The in vitro determination of the inhibitory activity of three new compounds (

compound

1,2,3) on acetylcholinesterase shows that

compound

1,2 shows moderate inhibitory effect on the inhibitory activity of acetylcholinesterase, and compound 3 shows strong inhibitory effect. The activity of the

compounds

1,2 and 3 is significantly higher than that of resveratrol (compound 4) and quzastilbene glycoside (compound 5) (p < 0.0001), and the results are shown in table 3 and fig. 2. From the chemical structure, the

compounds

1 and 2 are generated after acylation reaction on glycosyl on the basis of the compound 5, and the structural change obviously improves the inhibition effect on acetylcholinesterase and provides reference for structural modification. In a word, two new compounds separated from the root part of Rheum laneum lhasaense show obvious inhibition effect on acetylcholinesterase, and are potential drugs expected to be used for preventing diseases such as Alzheimer disease and the like.

TABLE 3 inhibition results of stilbene Components on acetylcholinesterase Activity

Experimental example 2 animal experiments and behavioral experiments of Compound 1

1 method of experiment

1.1 animal experiments

Animal experiments mainly research the new compound 1 (comp.1, piceatannol-3' -O-beta- _D - [2 "- (3,5-dihydroxy-4-methoxybenzoyl)]Glucopyranoside) for the prevention of cognitive disorders. 60 SPF grade C57 male mice (purchased from Strobilants Bei Fu, beijing Biotechnology, inc.) at 8 weeks of age were fed in plastic mouse cages, with free access to food and water, at room temperature 25 + -2 deg.C and humidity 55 + -10%, maintaining a circadian rhythm of 12h light and 12h dark. The male mice were randomly divided into 5 groups (12 mice per group): (1) Blank Control group (Control, vehicle), and Model group (Model, scopolamine,1.5 mg. Kg) ^-1 ) And (3) a group of positive drugs (DNP, scopolamine + Donepezil,3.0 mg/kg ^-1 ) (4) Low dose group (Compound 1, scopolamine +comp.1,70mg·kg ^-1 ) (5) high dose group (Compound 1, scopolamine + comp.1, 350mg. Kg) ^-1 ). Mice were gavaged with compound 1 for 4 weeks, on the day of the behavioural experiment, at 0.5h after dosing, with the exception of the blank group, were injected intraperitoneally with scopolamine, and the behavioural experiment was started 0.5h after molding.

1.2 behavioural experiments

1.2.1 New object recognition test (NOR)

The new object recognition experiment is an important method for evaluating the learning and memory ability of mice and plays an important role in the research of neurodegenerative diseases. The experimental apparatus consisted of a white laboratory box (length: 40cm; width: 40cm; height: 40 cm) and an animal behavioural video analysis system (SuperMaze, shanghai Xin Soft information technology Co., ltd.). The experimental process is divided into 3 stages: acclimation period, familiarity period, and testing period. The adaptation period is 1 day, the animals are put into the experimental box with their back facing the box body in sequence and then adapted to the experimental box environment for 5min, and the instrument is cleaned with 70% ethanol to eliminate residual odor. The next day the familiarity and testing periods were performed. In the familiarity period, two identical objects are placed in the experimental box, the distance between the two objects and the wall is kept consistent, and the animals are placed in the experimental box to freely explore for 5min. After an interval of 30min, a test period was started, one of the familiar objects was changed to a new one, the other one remained unchanged, and the animals were again placed in the laboratory for 5min. The time of the mice's exploration for new and old familiar objects during the test period was recorded and the relative Discrimination Index (DI) was used to evaluate the learning and memory abilities of the animals. The relative discrimination index is calculated by the formula DI = (N-F)/(N + F). Times.100%, wherein N (new) is the search time of the animal for a new object, and F (family) is the search time of the animal for an original familiar object.

1.2.2 Moris Water Maze (MWM)

Spatial learning memory was assessed by the Morris water maze experiment, which was modified with reference to the Morris water maze classical protocol. The Morris water maze animal behavior analysis system comprises a black water pool (diameter: 100cm; height: 50 cm), a black platform (diameter: 9cm; height: 15 cm) and an animal behavior video analysis system. The water pool is divided into four quadrants (southeast quadrant, northeast quadrant, southwest quadrant and northwest quadrant), and colored paper with different shapes is stuck to the middle point of the wall of the water pool of the quadrants and is used as a mark for a mouse to find a platform. The platform was located 1.5cm below the center of the target quadrant, and the position was held constant throughout the training period. To keep the mice from seeing the platform below the water surface, milk was added to the pool and homogenized.

The MWM experiment is performed in a positioning navigation stage from 1 st to 5 th days, each mouse is trained 1 time in the morning and afternoon, and the mice are respectively placed in two fixed position face walls. Allowing 60s of time for each mouse to search for the platform, if the platform is found successfully, allowing the mouse to stay on the platform for 15s and then cage, and automatically recording the latency of reaching the platform by the system; if no platform is found, the animal is guided to the platform and stays for 15s (the latency period is recorded as 60 s).

And 6, taking a space exploration stage, removing the platform, and detecting the space memory capacity of the animal on the position of the platform. The animal face wall is put into water from the middle point of the opposite angle quadrant of the platform, and the system automatically records the times of crossing the target quadrant and the platform of the animal within 60s, and the indexes of swimming time ratio, running ratio and the like of the platform quadrant.

1.2.3 dark avoidance experiment (Pat, passive avidity test)

The darkness-avoiding experimental facility consists of a bright room and a dark room which are connected through an arch. The experiment is divided into two stages of darkness-avoiding learning and darkness-avoiding consolidation. Before the experiment, each mouse is placed in a bright room with the back facing the arch for freely exploring for 3min, then the study is started, the mouse is placed in the bright room with the back facing the arch, and when the mouse enters a dark room, an electric shock of 0.36mA is given for 5min. After learning for 24h, a consolidation experiment is carried out, the mice are still put into a bright room according to the original mode, and the number of errors entering a dark room and the latency of entering the dark room for the first time within 5min are recorded.

1.3 Biochemical index detection

After the behavioral experiments were completed, all mice were anesthetized by ether inhalation. Blood was collected from the angular veins in the eye and serum samples were isolated by centrifugation at 3500rpm for 10 minutes at 4 ℃. The mice were then decapitated and brain tissue was removed on ice and weighed. Using 9 times of the weight of the tissueAn amount of ice-cold physiological saline was quickly homogenized and centrifuged at 2500 r.min-1 for 10min at 4 ℃, the supernatant was collected, and the total protein concentration was measured using BCA protein quantification kit (beijing kang, century biotechnology limited, beijing, china). The detection of AChE, chAT, SOD and CAT activities and the levels of ACh, GSH and MDA are all operated according to the corresponding detection kit (Nanjing institute of bioengineering, nanjing, china). Wherein the AChE, chAT, SOD and CAT activities are represented by U.mgprot ^-1 The GSH level is expressed as umol mgprot ^-1 As shown, MDA level is represented by nmol mgprot-1, and ACh level is represented by ug mgprot ^-1 And (4) showing. The detection of the levels of inflammatory factors IL-1 beta, IL-4, IL-6 and TNF-a in the serum sample is carried out according to the corresponding ELISA detection kit (Beijing Hua Ying Biotechnology institute, beijing, china) and in ng.mL ^-1 And (4) showing.

1.4 data processing

2 results of the experiment

2.1 novel Compound 1 ameliorates cognitive impairment caused by scopolamine

The in vitro activity experiment of the experimental example 1 shows that the new compound 1 in the rheum lasagnum has good cholinesterase inhibition activity and nervous system protection effect. Because the amount of the new compound 1 obtained by separation is relatively large and the in vitro activity is good, the new compound 1 is selected to carry out animal experiments, and the improvement effect of the new compound 1 on cognitive disorder induced by scopolamine is researched and evaluated through behavioral experiments NOR, MWM, PAT and cholinergic detection and oxidative stress detection.

2.2 novel Compound 1 improves recognition of mouse Neon

In the experiment, the short-term and non-spatial learning and memory abilities of the C57 mouse are evaluated through a new object recognition experiment. As can be seen from the results, the cognitive impairment induced by scopolamine mice had diminished ability to remember and discriminate between familiar and novel objects, and the relative discrimination index of the mice in the model group was relatively low. The relative discrimination index of the blank group mice was significantly greater than that of the model group (P < 0.05), indicating that normal C57 mice had memory of the familiar object and were able to discriminate between the familiar object and the new object; the relative discrimination index of the DNPZ group was significantly increased compared to the model group (P < 0.0001); as shown in fig. 3, the DI values of the low dose group (comp.1 _ L) and the high dose group (comp.1 _ L) were significantly greater than 0 after administration of the novel compound 1, compared to the model group, indicating that the administration of the novel compound 1 through gavage can enhance the memory of mice to familiar subjects and significantly improve scopolamine-induced short-term, non-spatial learning memory impairment.

2.3 New Compound 1 improves Passive escape Capacity in mice

The darkness-avoiding experiment utilizes the habit design of darkness-avoiding of rodent, when the animal enters the darkroom, it is shocked by foot bottom, after several times, it forms conditioned reflex. The experiment is used for detecting the passive escape capability of the rodent, and takes the latency of entering a darkroom and the number of electrified errors of entering the darkroom as main evaluation indexes. In the dark avoidance experiment, the dark avoidance latency of the model group mice was significantly reduced and the number of errors was significantly increased (P) compared to the blank group<0.0001 ); the dark avoidance latencies were significantly increased for both the DNPZ and comp.1_ L and comp.1_ H groups compared to the model group (P)<0.0001 The number of errors for comp.1_ L group is significantly reduced (p)<0.05 Comp.1_ H group error times are reduced, but no significant difference occurs, as shown in fig. 4 (a), (b). The results showed 1.5 mg.kg ^-1 Scopolamine significantly impaired the passive avoidance ability of mice in the avoidance experiment. The compound 1 can improve the injury caused by scopolamine, so that the dark-avoiding latency of C57 mice is prolonged, and the error frequency is reduced.

2.4 New Compound 1 improves learning and memory Capacity in mouse Water maze experiment

The Morris water maze experiment is a classic detection method for researching space learning and memory abilities of rodents, and is commonly used for investigating long-term and space reference memory abilities of the rodents, including positioning flight ranksSegment and space exploration two phases. In the positioning navigation stage, the platform searching latency is the most classical evaluation index; in the space exploration stage, the number of times of platform crossing and the number of times of target quadrant crossing are main indexes for evaluating the space exploration capacity. As shown in FIG. 5 (e), in the 5-day positioning navigation training of the C57 mice, the time required for the model group mice to search for the platform is obviously longer than that of the blank group mice, and in the space exploration phase, the latency of the model group mice for reaching the platform area for the first time is obviously increased, and the times of crossing the target quadrant and the platform area are obviously reduced (p)<0.01 As shown in fig. 5 (a), (b), (c), and (d). New compound 1 at 100 and 400mg kg ^-1 The latency period (p) of the mice reaching the plateau area for the first time can be obviously reduced when the drug is administrated<0.0001 400 mg/kg) of the composition ^-1 Significantly increased target quadrant crossing times (p) upon administration<0.05 And number of plateaus (p)<0.05). There was no significant difference in swimming speed between the groups of mice, as shown in FIG. 5 (c). The results of this study showed that 1.5 mg.kg ^-1 The scopolamine obviously damages the spatial learning and memory ability of mice in a water maze experiment, the new compound 1 can obviously improve the damage caused by the scopolamine, 100 mg/kg and 400mg/kg ^-1 The effect is equivalent when the medicine is administrated.

2.5 New Compound 1 ameliorates cholinergic injury caused by scopolamine

The effect of the novel compound 1 on cholinergic injury caused by scopolamine is evaluated by detecting the activity of acetylcholine transferase (AChE), acetylcholine transferase (CHAT) and acetylcholine (Ach) levels in mouse brain tissue. As shown in FIGS. 6 (a), (b), scopolamine caused a significant decrease in ACh levels and AChE activity in brain tissues of C57 mice (p)<0.05 ); compound 1 can significantly increase ACh levels and AChE activity (p) upon oral administration<0.05). The change in the activity of AChT in mouse brain tissue was not particularly significant, but compound 1 showed a significant increase in AChT activity at higher doses than the model group (p)<0.05 As shown in fig. 6 (c). The research result shows that C57 mice take 1.5 mg/kg orally ^-1 Scopolamine causes a significant decrease in acetylcholine levels in brain tissues and abnormalities in acetylcholinesterase and acetylcholinesterase activities. Compound 1 can effectively reverse acetylcholine level caused by scopolamine under low-dose and high-dose administrationReducing acetylcholinesterase damage.

2.6 novel Compound 1 ameliorates oxidative damage caused by scopolamine

The brain is a tissue that is highly vulnerable to oxygen radicals, and thus oxidative stress injury is also a major factor in AD. In the research, the scopolamine model group reduces the antioxidant activity of the brain tissue of a mouse, reduces the CAT and SOD activities and the GSH level, and simultaneously increases the MDA level and increases the free radical damage degree of the brain tissue. The compound 1 can improve the antioxidant activity of the brain tissue of mice to different degrees and reduce the MDA level. In which CAT activity was significantly increased in the low dose group and the high dose group as compared with the model group (p)<0.05 The results are shown in FIG. 7 (a). Significant increase in GSH levels (p) in DNP and low dose groups<0.01 There was a significant increase in GSH levels in the high dose group, but no significant difference was seen, with the results shown in fig. 7 (b). SOD activity was significantly increased in DNP group and high dose group (p)<0.05 Meanwhile, the MDA level of the brain tissue tends to be decreased in each administration group, but no significant difference was observed, and the results are shown in fig. 7 (c) and (d). The above results show that 1.5 mg/kg ^-1 The scopolamine causes oxidative stress injury, and the compound 1 has good oxidation resistance and can effectively reverse the oxidative injury caused by the scopolamine.

2.7 novel Compound 1 ameliorates neuroinflammation caused by scopolamine

In the study, the levels of IL-1 beta, IL-6 and TNF-a in the serum of the model group C57 mice are obviously increased relative to the blank group (p is less than 0.0001) by injecting scopolamine intraperitoneally, and the C57 mice of each administration group are also subjected to inflammatory injury with different degrees by injecting scopolamine intraperitoneally. Compared with the model group, the compound 1 can obviously reduce the level of IL-1 beta, IL-6 and TNF-a in the serum of C57 mice when being administrated at 400mg/kg (p is less than 0.05). When administered at low doses, the levels of IL-1 β, IL-6, TNF-a were reduced, but not significantly, as shown in FIGS. 8 (a), (b), (c). The experimental result shows that the compound 1 has obvious inflammation inhibition effect under high dosage and reduces inflammatory injury.

Claims

1. Stilbene compounds separated from Lhasa rhubarb are characterized in that the structural formula of the stilbene compounds is shown as a formula I or a formula II:

2. a method for separating the stilbene compound of claim 1 from Rheum lhasaense, which comprises:

(1) Pulverizing Rheum palmatum L, extracting with 40-98% ethanol, and concentrating under reduced pressure to obtain extract; (2) Extracting the extract to obtain chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction; (3) Loading an ethyl acetate part sample into an MCI filler chromatographic column, and leaching with 30%, 50%, 70% and 100% ethanol solvents respectively; wherein the compound of formula I and the compound of formula II are separated from the fraction eluted by 50% ethanol.

3. The method of claim 2, wherein the ethanol in step (1) is 95% ethanol.

4. The method of claim 2, wherein in step (3) 4-5 column volumes are eluted per solvent.

5. Use of a stilbene compound as defined in claim 1 in the preparation of acetylcholinesterase inhibitors.

6. Use of the stilbene compound of claim 1 in the preparation of a medicament for the treatment of neurodegenerative diseases.

7. A pharmaceutical composition for treating neurodegenerative diseases, which comprises a prophylactically or therapeutically effective amount of the stilbene compound or pharmaceutically acceptable salt thereof according to claim 1 in combination with a pharmaceutically acceptable carrier; the neurodegenerative disease comprises Alzheimer disease, parkinson disease or Huntington disease.