CN112830947A

CN112830947A - Stilbene compounds isolated from Rheum lhasaense and their use in treating nervous system diseases

Info

Publication number: CN112830947A
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: 2021-05-25
Anticipated expiration: 2040-12-21
Also published as: CN112830947B; CN115650940B; CN115650940A

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 stilbene compounds separated from plants and 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 lasagnum, a national herb, is often used for the treatment of gastric diseases and is called as Quza by the local people in Tibet. Early literature analysis shows 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, the research reports on the rheum lasagnum are few, and only two documents report that the rheum lasagnum contains 12 phytochemical components and are all stilbene components, including 6 resveratrol monomer derivatives, 4 resveratrol dimers and 2 resveratrol trimers. Stilbene components exhibit remarkable effects in the prevention and treatment of nervous system diseases, and resveratrol is currently 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 states that a decrease in acetylcholine levels in brain tissue is one of the important factors in causing neurodegenerative diseases, in which the degree of acetylcholine hydrolysis is enhanced by the overexpression of acetylcholinesterase, resulting in a decrease in acetylcholine levels. 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 as the rasa, and is an important treasure house for searching 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 Rheum lhasaense;

the second purpose of the invention is to apply the provided new stilbene compound to prepare the medicine for treating neurodegenerative diseases.

The above object of the present invention is achieved by the following technical solutions:

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) the extract is divided into chloroform part, ethyl acetate part, n-butanol part and water part by extraction; (3) loading the ethyl acetate part sample into an MCI filler chromatographic column, and respectively leaching with 30%, 50%, 70% and 100% ethanol solvents; 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 of each solvent in the step (3).

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

new compounds

1, 2 and 3) shown in formula I, formula II and formula III on acetylcholinesterase shows that the inhibitory activity of formula I (new compound 1) and formula II (new compound 2) on acetylcholinesterase shows moderate inhibitory effect, and formula III (new compound 3) shows 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 results 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 the 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 antioxidant capacity and can reverse the oxidative damage caused by the scopolamine. According to the experimental result of the neuroinflammation caused by the 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, 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 isolating the compound 3 (4' -methoxy-scirpusin a) from the 70% elution fraction.

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 treatments. According to the detection, the in vitro determination of the inhibitory activity of three new compounds (

compounds

1, 2 and 3) on acetylcholinesterase shows that the inhibitory activity of the

compounds

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

compounds

1, 2 and 3 is obviously higher than that of resveratrol (compound 4) and quzastilbene glucoside (compound 5) (p is less than 0.0001).

In vitro activity experiments show that the new compound 1 in the rheum lasagnum has good cholinesterase inhibition activity and nervous system protection effect, and the separated new compound 1 is relatively more in amount and good in vitro activity, so the new compound 1 is selected for 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 gastric lavage 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 capacity 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 scopolamine, the new compound 1 can effectively reverse the acetylcholine level reduction and acetylcholinesterase injury caused by 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 for new chemicals 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 model groups.

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 (1.5mg 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 group)<0.05,**<0.01,***<0.001,****<0.0001 compared to model set.

FIG. 5 determination of learning and memory impairment by Compound 1 preventing SCOP treatment by the Water maze experiment; (a) an escape latency period, (b) the number of times the target platform is worn, (c) the swimming speed, (d) the number of times the target quadrant is crossed, (e) the swimming trajectory of the mouse in the water tank; the red small circle represents the hidden platform position and the green curve represents the motion trajectory^-1)；DNPZ:donepezil (3mg·kg^-1) (ii) a Compound 1_ L Compound 1(100mg kg)^-1)(ii) a Compound 1_ H Compound 1(400mg 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; (a) intracerebral acetylcholine level (b) intracerebral acetylcholinesterase activity (c) intracerebral ChAT activity T. model group, scopolamine (1.5mg kg^-1)；DNPZ:donepezil(3mg·kg^-1) (ii) a Compound 1_ L Compound 1(100mg kg)^-1)(ii) a Compound 1_ H Compound 1(400mg 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) SOD activity in brain (b) CAT activity in brain (c) GSH level in brain (d) MDA level in brain model group scopolamine (1.5mg kg)^-1)；DNPZ:donepezil(3mg·kg^-1) (ii) a Compound 1_ L Compound 1(100mg kg)^-1)(ii) a Compound 1_ H Compound 1(400mg 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 (1.5mg kg. kg)^-1)；DNPZ:donepezil(3mg·kg^-1) (ii) a Compound 1_ L Compound 1(100mg kg)^-1)(ii) a Compound 1_ H Compound 1(400mg kg)^-1) Experimental values are expressed as mean ± SEM (n ═ 15per group)<0.05,**<0.01,***<0.001,****<0.0001 compared to model group. .

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only, and are not intended as limitations on 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 modifications may be within the scope of the invention.

Materials, medicaments and reagents

The roots and the roots of Rheum palmatum are collected in Linzhou county of autonomous region of Tibet, identified by professor Zhongguo (the center for researching natural resources of Chinese medicinal materials and national medicines in the university of medicine in Jiangxi), and the samples are stored in the kinship center of the institute of medicinal plant of Chinese medical science institute.

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

Scopolamine (aladdin, shanghai aladine biochemistry science and technology, 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 and separating stilbene compounds from herba Rheum Officinale

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 50% ethanol elution fraction, and compound 3 is separated from 70% elution fraction.

2. Structural analysis of novel Compound

Compound 1: brown powder, molecular ion peak M/z 595.1423 [ M + Na ] by (+) HRESIMS]⁺(calcd for 595.1428, see FIG. 1) determining its molecular formula as C₂₈H₂₈O₁₃The degree of unsaturation was 15. Process for preparation of Compound 1¹H NMR spectrum (see FIGS. 2-3): aromatic region hydrogen signal delta_H7.25(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, δ_HAX is 6.44(2H, d, J-2.2 Hz, H-2,6) and 6.17(1H, t, J-2.2 Hz, H-4)₂System, delta_H6.87(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 a piceatannol unit, δ_H7.15(2H, s, H-2',6') binding of delta_C166.18 (carbonyl signal) are identified as galloyl units. In addition, a methoxy (3.85, s,3H) and sugar signal was also observed. In that¹³28 carbon signals can be observed in the C NMR spectrum (see attached figures 4-6), and the basic structure can be determined to be piceatannol-3' -O-beta-_D-glucopyranoside. Methine proton of sugar at delta_H5.23(1H, J. 8.0Hz) is the triplet, binds the proton on C-2' of the saccharide identified in the HMBC spectrum, and this proton is associated with delta_C166.18 (carbonyl signal) and determining the carbon at the 2-position of the sugar at which the galloyl group is attached. The correlation () of methyl hydrogen with C-4' (Δ C139.90) can be observed in HMBC spectra. Finally, the compound 1 is determined to be piceatannol-3' -O-beta-_D- [2 "- (3, 5-dihydroxy-4-methoxybenzoyl)]-glucopyranoside.¹H NMR and¹³the C NMR data are shown in Table 1.

Compound 2: brown powder, molecular ion peak M/z 581.1265 [ M + Na ] by (+) HRESIMS]⁺(calcd for 581.1271)Determining the molecular formula as C₂₇H₂₆O₁₃The unsaturation degree was 15. Compound 2, other than lacking the methoxy signal¹H NMR and¹³the C NMR data were similar to those of Compound 1.¹H NMR spectra: contains the typical piceatannol signal an ABX system: delta_H7.23(1H, d, J ═ 2.0Hz),7.04(1H, dd, J ═ 8.3,2.0Hz) and 6.76(1H, d, J ═ 8.3Hz), and AX₂The system comprises the following steps: delta_H6.44(2H, d, J ═ 2.2Hz) and 6.16(1H, t, J ═ 2.2Hz), and the trans-olefin proton δ_H6.87(d, J ═ 16.2Hz) and 6.72(d, J ═ 16.3 Hz). Furthermore, via the methine hydrogen (delta) at sugar position 2 in the HMBC spectrum_H5.19) and carbonyl (. delta.) groups_C166.68) can determine that the galloyl is connected at the 2-position of glycosyl, which indicates that the compound 2 is piceatannol-3' -O-beta-_D- (2 "-galloyl) -glucopyranoside,¹h NMR and¹³the C NMR data are shown in Table 1.

Compound 3: brown powder with molecular formula C determined by UPLC-Q-TOF-MS₂₉H₂₄O₇ (m/z 485.1601[M+H]⁺Calculation formula 485.1556), in combination¹³C and¹analysis of the H NMR data showed 18 degrees of unsaturation.¹Aromatic region of H NMR at delta_HA group of AA 'BB' coupled protons at 6.88(2H, d, J ═ 8.4Hz, H-3, 5) and 7.30(2H, d, J ═ 8.3Hz, H-2,6) indicate that the structure contains a para-substituted phenyl group. At delta_H6.67(1H, d, J ═ 2.1Hz, H-14) and 6.31(1H, d, J ═ 2.1Hz, H-12) are a group of aromatic protons of the AX type; there are other aromatic signals: delta_H 6.10(3H，s，2′，4′，6′)，δ_H6.74(2H, H-10 ', 13 ') and 6.62(1H, dd, J ═ 8.1, 2.1Hz, H-14 '). Delta_HAliphatic protons and δ at 5.31(1H, d, J ═ 4.9Hz, H-8 ') and 4.44(1H, d, J ═ 4.9Hz, H-7')_HThe protons of the trans double bonds at 6.94(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_H3.74(3H, s). Process for preparation of Compound 3¹³The C NMR spectrum showed a 29 carbon signal,which contains one methoxy group, 16 methyl groups and 12 quaternary carbons. Of compounds 1, except for one more methoxy group¹H and¹³the C NMR data agreed with those of spirpusin A. H-7' (delta)_H4.4) with C-2 ',6' (delta)_C105.9) presence of HMBC correlation, H-8' (delta)_H5.31) was associated with 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 protons (. delta.) are observed in HMBC spectra_H3.74) and C-4 (. delta.))_C159.5) there is a correlation, leading to the conclusion that the methoxy group is located 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 oligomers¹As 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) has a positive Cotton effect at 237nm and (-) -epsilon viniferin (7 'R, 8' R) has a negative Cotton effect. The CD spectrum of compound 3 showed 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 2¹H NMR and¹³c NMR data

^bData are shown measured in deuterated methanol.

^*Indicating multiple overlapsPeak(s).

TABLE 2 preparation of Compound 3¹H NMR and¹³c NMR data

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

1. Test method

Acetylcholinesterase inhibitory activity compounds 1 and 2 were tested for AChE inhibition 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 in 1% SDS buffer (1g SDS in PBS (ph 8.0)). The determination steps are as follows: first, 60. mu.L of PBS (pH8.0), 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 stop 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 acetylcholine esterase. Wherein each sample was replicated three times. Cell viability (%)

Inhibition ratio (%) ═ 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 two comparisons 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 treatments. The in vitro determination of the inhibitory activity of three new compounds (

compounds

1, 2 and 3) on acetylcholinesterase shows that the inhibitory activity of the

compounds

1 and 2 on acetylcholinesterase shows a moderate degree of inhibition, while the compound 3 shows a strong inhibition. The activity of the

compounds

1, 2 and 3 is significantly higher than that of resveratrol (compound 4) and quzastilbene glucoside (compound 5) (p is less than 0.0001), and the results are shown in table 3 and figure 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 35 inhibition results of the stilbene Components on the 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 Schbefu, Beijing Biotechnology, Inc.) aged 8 weeks were fed in plastic cages with free access to food and water at room temperature of 25 + -2 deg.C and humidity of 55 + -10% maintaining a circadian rhythm of 12h light and 12h dark.Male mice were randomized 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) And (4) Low dose group (Compound 1, Scopolamine + comp.1,70 mg. kg^-1) And (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 post-administration, with the exception of the blank group, were injected intraperitoneally with scopolamine, and the behavioural experiment was started 0.5h post-modelling.

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: 40 cm; width: 40 cm; height: 40cm) 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 5 min. After an interval of 30min, a test period was started, one of the familiar objects was changed to a new one, the other one was kept unchanged, and the animals were again placed in the laboratory for 5 min. The time of the mice's exploration for new and old familiar objects during the test period was recorded and the learning and memory ability of the animals was evaluated by the relative Discrimination Index (DI). The relative discrimination index is calculated by the formula DI ═ N-F)/(N + F) × 100%, where N (new) is the time for the animal to explore the new object and F (family) is the time for the animal to explore the 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: 100 cm; height: 50cm), a black platform (diameter: 9 cm; height: 15cm) 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 fixed sailing period from 1 st to 5 th, each mouse is trained 1 time in the morning and afternoon, and the mice are respectively placed from two fixed position surfaces. 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 to 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 aveoid 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 to the arched door for freely exploring for 3min, then the study is started, the mouse is placed in the bright room with the back facing to the arched door, and when the mouse enters a dark room, an electric shock of 0.36mA is given for 5 min. 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 the first time entering the dark room 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. The total protein concentration was determined by rapid homogenization with ice-cold physiological saline 9 times the weight of the tissue, centrifugation at 2500 rpm-1 for 10min at 4 ℃, collection of the supernatant, and use of a BCA protein quantification kit (beijing kang, century biotechnology ltd, 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^-1The GSH level is expressed as umol mgprot^-1As shown, MDA level is represented by nmol mgprot-1, and ACh level is represented by ug mgprot^-1And (4) showing. The detection of the levels of inflammatory factors IL-1 beta, IL-4, IL-6 and TNF-a in the serum samples is carried out according to the corresponding ELISA detection kit (Beijing Huaying Biotech institute, Beijing, China) and in ng.mL^-1And (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 the cognitive disorder induced by the scopolamine is researched and evaluated through behavioral experiments NOR, MWM, PAT and cholinergic detection and oxidative stress detection.

2.2 novel Compound 1 improves mouse neologism recognition ability

The short-term and non-spatial learning and memory abilities of the C57 mouse were evaluated by 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 novel Compound 1 enhances Passive escape Capacity of 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 period 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 sets is significantly reduced (p)<0.05), the number of comp.1_ H group errors is reduced, but no significant difference occurs, as shown in fig. 4(a), (b). The results showed 1.5 mg.kg^-1Scopolamine 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 darkness-avoiding latency of C57 mice is prolonged, and the damage is reducedThe number of errors.

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

The Morris water maze experiment is a classical detection method for researching the space learning and memory abilities of rodents, and is commonly used for inspecting the long-term and space reference memory abilities of the rodents, and comprises a positioning navigation stage and a space exploration stage. 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 significantly 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 significantly increased, and the times of crossing the target quadrant and the platform area are significantly reduced (p)<0.01) as shown in FIGS. 5(a), (b), (c), and (d). New compound 1 at 100 and 400mg kg^-1The 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), at 400mg/kg^-1Significantly 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^-1Scopolamine obviously damages the spatial learning and memory ability of mice in the water maze experiment, and the new compound 1 can obviously improve the damage caused by the scopolamine, 100 and 400 mg.kg^-1The 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 resulted in a significant decrease in ACh levels and AChE activity in brain tissue 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 activity of AChT in mouse brain tissue was not particularly significant, but compound 1 was at a higher dose than the model groupCan obviously increase the activity of AChT (p)<0.05), as shown in fig. 6 (c). The research result shows that C57 mice take 1.5mg kg orally^-1Scopolamine causes a significant decrease in the level of acetylcholine in brain tissue and abnormalities in acetylcholinesterase and acetylcholinesterase activities. The compound 1 can effectively reverse the decrease of acetylcholine level and acetylcholinesterase damage caused by scopolamine under the administration of low dose and high dose.

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 the mouse and reduce the MDA level in different degrees after being administrated. In which CAT activity was significantly increased in the low dose group and the high dose group as compared with that in 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 observed, and the results are shown in fig. 7 (b). SOD activity was significantly increased in DNP group and high dose group (p)<0.05), while each administration group had a tendency to lower the brain tissue MDA level, 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^-1The 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 research, the intraperitoneal injection of scopolamine can obviously increase the levels of IL-1 beta, IL-6 and TNF-a in the serum of the mice of the model group C57 (p is less than 0.0001) relative to the blank group, and the intraperitoneal injection of scopolamine can also cause inflammatory injuries of different degrees to the mice of C57 of each given group. Compared with the model group, the compound 1 can significantly 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, a formula II or a formula III:

2. a stilbene compound according to claim 1 wherein the stilbene compound comprises an acid addition salt, a hydrate or a prodrug thereof.

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

(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 the ethyl acetate part sample into an MCI filler chromatographic column, and respectively leaching with 30%, 50%, 70% and 100% ethanol solvents; 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.

4. A process according to claim 3, wherein the ethanol in step (1) is 40-98% ethanol, preferably 95% ethanol.

5. A method according to claim 3, wherein in step (3) 4-5 column volumes are eluted per solvent.

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

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

8. A pharmaceutical composition for treating neurodegenerative diseases, which comprises a prophylactically or therapeutically effective amount of the stilbene compound or stilbene compound of claim 1 or stilbene compound of claim 2, or stilbene compound or stilbene.

9. The pharmaceutical composition according to claim 8, which is prepared into any suitable pharmaceutical preparation according to a preparation method conventional in the art.

10. The pharmaceutical composition of claim 8, wherein the neurodegenerative disease comprises alzheimer's disease, parkinson's disease, or huntington's disease.