CN115650940B - Compounds isolated from Rheum emodi and their use in the treatment of neurological disorders - Google Patents

Compounds isolated from Rheum emodi and their use in the treatment of neurological disorders Download PDF

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CN115650940B
CN115650940B CN202211297942.1A CN202211297942A CN115650940B CN 115650940 B CN115650940 B CN 115650940B CN 202211297942 A CN202211297942 A CN 202211297942A CN 115650940 B CN115650940 B CN 115650940B
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compound
scopolamine
rheum
mice
treatment
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CN115650940A (en
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何春年
刘青
申洁
李珮
肖培根
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Institute of Medicinal Plant Development of CAMS and PUMC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/80—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products

Abstract

The invention discloses an isolated compound in Lhasa rhubarb and its use in treating neurological diseases. The invention discloses a new compound separated from Rheum lassa, which has a structural formula shown in a formula III. The invention further discloses a method for separating the compound from the rheum lassa. The in vitro test result of the inhibitory activity of the compound on acetylcholinesterase shows that the compound shown in the formula III has a stronger inhibitory effect. The compound provided by the invention can be used for preparing medicines for treating neurodegenerative diseases.

Description

Compounds isolated from Rheum emodi and their use in the treatment of neurological disorders
The present application is a divisional application of the invention patent application with the application number of 202011543207.5 and the application date of 2020, 12 months and 21 days, and the invention name of stilbene compounds separated from the Rheum lassa and the application of the stilbene compounds in treating nervous system diseases.
Technical Field
The invention relates to a stilbene compound, in particular to a stilbene compound separated from Rheum lassa, and further relates to a 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 Rheum emodi leaf group plant of Polygonaceae, and is a special species in China. The wild species are mainly distributed in Tibet, and at present, artificial cultivation is started in some areas of Sichuan and Yunnan. Rheum palmatum is commonly used as a ethnic medicine for treating stomach diseases, and is called as a campaigns by Tibetan local people. Early literature analysis found that rheum officinale does not contain anthraquinone and anthrone derivatives, which are characteristic components of rheum plants, and shows great specificity in chemical components. At present, few studies on rheum officinale have been reported, and only two documents report that the rheum officinale contains 12 phytochemicals and are stilbene components, including 6 resveratrol monomer derivatives, 4 resveratrol dimers and 2 resveratrol trimers. Stilbene components have remarkable effects in preventing and treating nervous system diseases, and resveratrol is currently the most deeply studied stilbene component. Some clinical data show that resveratrol can effectively improve symptoms of patients with alzheimer's disease.
The choline hypothesis suggests that a decrease in the level of acetylcholine in brain tissue is one of the important factors causing neurodegenerative diseases, wherein overexpression of acetylcholinesterase can enhance the degree of acetylcholine hydrolysis, resulting in a decrease in the level of acetylcholine. Acetylcholinesterase inhibitors are considered as important sources of drugs for preventing and treating neurodegenerative diseases such as Alzheimer's disease, and two drugs, namely donepezil and galantamine, exhibit good therapeutic effects on Alzheimer's disease by acting on the cholinergic pathway. In terms of the prior reports, the stilbene component is considered to be the main component of the rheum officinale, and is an important treasury for preventing and treating neurodegenerative diseases such as Alzheimer disease.
Disclosure of Invention
It is an object of the present invention to provide novel stilbenes isolated from Rheum lansium;
the second purpose of the invention is to apply the provided novel stilbene compounds to the preparation of medicaments for treating neurodegenerative diseases.
The above object of the present invention is achieved by the following technical solutions:
the invention firstly provides a novel stilbene compound separated from the Rheum lassa, and the structural formula of the stilbene 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 present invention; the acid-addition salts of the compounds are preferably pharmaceutically acceptable suitable acids (e.g., hydrochloric acid, acetic acid, sulfuric acid) to form non-toxic salts, and other salts besides pharmaceutically acceptable salts are included in the present invention.
For reference, the invention provides a method for separating a compound shown as a formula I, a formula II or a formula III from rheum officinale, which comprises the following steps:
(1) Pulverizing Rheum emodi, extracting with 95% ethanol, concentrating under reduced pressure to obtain extract; (2) The extract is divided into a chloroform part, an ethyl acetate part, an n-butanol part and a water part by extraction; (3) Loading the ethyl acetate part sample into an MCI packing chromatographic column, and eluting with 30%, 50%, 70% and 100% ethanol solvents respectively; wherein the compound of formula I and the compound of formula II are separated from a 50% ethanol elution fraction, and the compound of formula III is separated from a 70% elution fraction.
Wherein the ethanol in step (1) is preferably 40-98% ethanol, most preferably 95% ethanol;
4-5 column volumes per solvent rinse in step (3).
The in vitro test of the invention shows that three novel compounds (novel compounds 1,2 and 3) shown in the formulas I, II and III have inhibitory activity on acetylcholinesterase, and the result shows that the inhibitory activity of the novel compound 1 shown in the formula I and the novel compound 2 shows moderate inhibitory effect on acetylcholinesterase, and the novel compound 3 shown in the formula III shows stronger 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 scopolamine-induced cognitive impairment through the behavioural experiments NOR, MWM, PAT and cholinergic detection and oxidative stress detection. According to the experimental result of the recognition capability of the new object of the mouse, the novel compound 1 can enhance the memory of the mouse on the familiar object, and can remarkably improve the short-time and non-space learning memory injury induced by scopolamine. According to the experimental result of the passive escape ability of the mice, the novel compound 1 can improve the damage caused by scopolamine, so that the dark-avoiding latency period of the C57 mice is prolonged, and the error frequency is reduced. According to the experimental results of learning and memory ability in the mouse water maze experiment, the novel compound 1 can obviously improve the damage caused by scopolamine. According to the experimental results of improving cholinergic injury caused by scopolamine, it is seen that the novel compound 1 can effectively reverse the reduction of the level of acetylcholine and the injury of acetylcholinesterase caused by scopolamine. According to the experimental result of the oxidative damage caused by scopolamine, the novel compound 1 has good antioxidation capability and can reverse the oxidative damage caused by scopolamine. According to the experimental result of the neuroinflammation caused by scopolamine, the compound 1 has obvious inflammation inhibition effect and reduces inflammatory injury.
Another object of the present invention is to provide a pharmaceutical composition for treating neurodegenerative diseases, which is prepared by compounding a prophylactically or therapeutically effective amount of a compound of formula i, formula ii or formula iii, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier; the compound shown in the formula I, the formula II or the formula III with pharmaceutically acceptable dosage is matched with pharmaceutically acceptable carriers or auxiliary materials, and then is prepared into any one of proper pharmaceutical compositions according to a preparation method conventional in the art. Generally, the compositions are suitable for oral administration and injection administration, as well as other methods of administration. The composition can be in the form of liquid preparation such as tablet, capsule, powder, granule, lozenge, suppository or oral liquid. The pharmaceutical compositions according to the invention may contain, according to the different methods of administration, from 0.1% to 99% by weight, preferably from 10% to 60% by weight, of a compound of formula I, formula II or formula III
Wherein the auxiliary materials can be antioxidant complexing agent, filler, 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's Disease (AD), parkinson's Disease (PD) or Huntington's Disease (HD).
Detailed description of the technical scheme
Pulverizing radix et rhizoma Rhei, extracting with 95% ethanol, and concentrating under reduced pressure to obtain extract. The extraction is divided into chloroform part, ethyl acetate part, n-butanol part and water part. Ethyl acetate fraction samples were loaded onto MCI packed chromatography columns, rinsed with 30%, 50%, 70% and 100% ethanol solvents, respectively, 4-5 column volumes per solvent rinse. Wherein the compound 1 (piceatannol-3' -O-beta-) -is separated from 50% ethanol elution fraction D - [2 "- (3, 5-dihydroxy-4-methoxybenzoyl)]Glucopyranoside) and novel compounds 2 (piceatannol-3' -O-beta- D - (2 "-galloyl) -glucopyranoside), and the compound 3 (4' -methoxy-scirpusin a) is isolated from the 70% eluted fraction.
The reduction of the level of acetylcholine in brain tissue is one of the important factors causing neurodegenerative diseases such as Alzheimer's disease, and thus acetylcholinesterase inhibitors are finding an important treasury for preventive treatment. The in vitro test of the invention shows that three novel compounds (compounds 1,2 and 3) have inhibitory activity on acetylcholinesterase, and the results show that the inhibitory activity of the compounds 1 and 2 on acetylcholinesterase shows moderate inhibitory effect, and the compound 3 shows stronger inhibitory effect. Compounds 1,2 and 3 were significantly more active than resveratrol (compound 4) and Qu Za glycoside (compound 5) (p < 0.0001).
According to the in vitro activity experiment, the novel compound 1 in the rheum officinale has good cholinesterase inhibition activity and nervous system protection effect, and the amount of the novel compound 1 obtained by separation is relatively large, and the in vitro activity is good, so that animal experiments are carried out by selecting the novel compound 1, and the improvement effect of the novel compound 1 on scopolamine-induced cognitive dysfunction is researched and evaluated through the behavioural experiments NOR, MWM, PAT and cholinergic detection and oxidative stress detection.
According to the experimental result of improving the recognition capability of the novel compound 1 on the novel object of the mice, the novel compound 1 can enhance the memory of the mice on the familiar object by the gastric lavage administration, and can remarkably improve the short-time and non-space learning memory injury induced by scopolamine. According to the experimental result of the novel compound 1 for improving the passive escape ability of mice, the compound 1 can improve the damage caused by scopolamine, so that the dark-avoiding latency period of the C57 mice is prolonged, and the error frequency is reduced. According to the experimental result of the novel compound 1 for improving the learning and memory capacity in the mouse water maze experiment, the novel compound 1 can obviously improve the injury caused by scopolamine. According to the experimental results of the novel compound 1 for improving cholinergic injury caused by scopolamine, the novel compound 1 can effectively reverse the reduction of the level of acetylcholinesterase and the injury of acetylcholinesterase caused by scopolamine under the condition of low-dose and high-dose administration. According to the experimental result of improving the oxidative damage caused by scopolamine by the novel compound 1, the novel compound 1 has good antioxidation capability and can reverse the oxidative damage caused by scopolamine. According to the experimental result of improving the nerve inflammation caused by scopolamine by the novel compound 1, the compound 1 has obvious inflammation inhibition effect and reduces inflammatory injury.
Drawings
The main HMBC-related schematic of compounds 1,2,3 of figure 1.
Fig. 2 shows IC50 values (××p < 0.0001) of new chemistries isolated from pizza rhubarb.
FIG. 3 detection of learning and memory disorders of novel compounds 1 preventing SCOP treatment by a mouse new object recognition capability experiment; model group: scopolamine (1.5 mg.kg) -1 )；DNPZ:donepezil(3mg·kg -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_L (100 mg kg- 1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_H (400 mg.kg) -1 ). Experimental values are expressed as mean ± SEM (n=15 per group)<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.
FIG. 4 determination of learning and memory disorders of compound 1 that prevent SCOP treatment by a passive evasion ability experiment; (a) Latent period of entering darkroom, (b) number of errors of being electrified into darkroom, model group: scopolamine (1.5 mg. Kg) -1 )；DNPZ:donepezil(3mg·kg -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_L (100 mg kg) -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_H (400 mg.kg) -1 ) Experimental values are expressed as mean ± SEM (n=15 per group) & x<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.
FIG. 5 determination of learning and memory disorders of compound 1 that prevented SCOP treatment by water maze test; (a) escape latency, (b) number of platform penetration of the target platform, (c) swimming speed, (d) number of target quadrant crossing, and (e) swimming track of mice in the water tank; the red small circles indicate the hidden platform positions, and the green curves indicate the motion trajectories, model set: scopolamine (1.5 mg. Kg) -1 )；DNPZ:donepezil(3mg·kg -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_L Compound 1 (100 mg kg) -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_H Compound 1 (400 mg kg) -1 ) Experimental values are expressed as mean ± SEM (n=15 per group) & x<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.
FIG. 6 experiment of the effect of novel compound 1 on improving the SCOP mouse biliary energy nervous system; (a) levels of acetylcholine in the brain, (b) acetylcholinesterase activity in the brain, (c) ChAT activity in the brain, T, model set: sconolamine (1.5 mg. Kg) -1 )；DNPZ:donepezil(3mg·kg -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_L Compound1(100mg·kg -1) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_H Compound 1 (400 mg kg) -1 ) Experimental values are expressed as mean ± SEM (n=15 per group) & x<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.
FIG. 7 results of an oxidative stress test for improving SCOP-treated mice with novel compound 1; (a) SOD activity in brain, (b) CAT activity in brain, (c) GSH level in brain, (d) MDA level in brain, model group: scopolamine (1.5 mg. Kg) -1 )；DNPZ:donepezil(3mg·kg -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_L Compound 1 (100 mg kg) -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_H Compound 1 (400 mg kg) -1 ) Experimental values are expressed as mean ± SEM (n=15 per group) & x<0.05,**<0.01,***<0.001,****<0.0001 compared to model group.
FIG. 8 experiment of novel compound 1 to improve neuroinflammation caused by scopolamine; (a) IL-1. Beta. Levels in serum, (b) IL-6 levels in serum, (c) TNF-a levels in serum, M model group: scopolamine (1.5 mg. Kg) -1 )；DNPZ:donepezil(3mg·kg -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_L Compound 1 (100 mg kg) -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Compound 1_H Compound 1 (400 mg kg) -1 ) Experimental values are expressed as mean ± SEM (n=15 per group) & x<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 advantages and features of the invention will become apparent from the description. It should be understood that the embodiments described are exemplary only and should not be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.
Materials, medicaments and reagents
The root and rhizome of Lasa rhubarb are collected in the county of the Tibetan autonomous region, and are identified by Zhong Guoyue professor (Chinese medicinal materials of the university of Jiangxi and national medical natural resource research center), and the sample is stored in the related center of the medical plant research center of the national academy of medical science.
Acetylcholinesterase (AChE, 1002652214), butyrylcholinesterase (BuChE, 1002399384), 5-dithiobis2-nitrobenzoic acid (DNTB, 1002473536), thiocholine iodide (ATCI, 101792824), thiobutyrylcholine iodide (BTCI, 101758768) and tacrine (1002020029) were purchased from sigma aldrich, respectively. An enzyme solution was prepared with phosphate buffer (50 mM) ph8.0 (Hyclone Laboratories, USA). Sodium Dodecyl Sulfate (SDS) was from national pharmaceutical group chemical company, inc.
Scopolamine (aladin, shanghai Ala Biochemical technologies Co., ltd.); donepezil hydrochloride (aladin, shanghai Ala Biochemical technologies Co., ltd.); sodium carboxymethylcellulose (solarbio, beijing solebao technologies); normal saline (Shijizhuang four-medicine limited).
Example 1 isolation of stilbenes from Rheum lassa and characterization thereof
1. Stilbene compounds extracted and separated from Rheum lassa
Pulverizing radix et rhizoma Rhei, extracting with 95% ethanol, and concentrating under reduced pressure to obtain extract. The extraction is divided into chloroform part, ethyl acetate part, n-butanol part and water part. Ethyl acetate fraction samples were loaded onto MCI packed chromatography columns, rinsed with 30%, 50%, 70% and 100% ethanol solvents, respectively, 4-5 column volumes per solvent rinse. Wherein, compounds 1 and 2 are separated from 50% ethanol elution fraction, and compound 3 is separated from 70% elution fraction.
2. Structural resolution of novel compounds
Compound 1: brown powder, molecular ion peak m/z 595.1423[ M+Na ] given by (+) HRESIMS] + (calcd for 595.1428) determining the molecular formula as C 28 H 28 O 13 The unsaturation was 15. Compound 1 1 H NMR spectrum: aromatic zone hydrogen signal delta H 7.25 (1 h, d, j=2.0 hz, h-10), 7.05 (1 h, dd, j=8.4, 2.0hz, h-14) and 6.77 (1 h, d, j=8.3 hz, h-13) are ABX systems, δ 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, delta H 6.87 (1H, d, J=16.2 Hz, H-8) and 6.73 (1H, d, J=16.3 Hz, H-7) are trans-olefinsHydrocarbon protons, whose signals indicate that compound 1 contains piceatannol units, delta H 7.15 (2H, s, H-2', 6') binding delta C 166.18 (carbonyl signal) is determined as galloyl units. In addition, a methoxy group (3.85, s, 3H) and a sugar signal were also observed. At the position of 13 28 carbon signals can be observed in the C NMR spectrum, and the basic structure of the white piceatannol-3' -O-beta-room can be determined by the carbon and hydrogen signals and literature values D -glucopyranoside. The methine proton of the sugar is at delta H 5.23 (1 h, j=8.0 Hz) is the triplet, binding to a proton on C-2 "which is determined to be a sugar in HMBC spectra, and this proton is associated with δ C 166.18 (carbonyl signal) and the galloyl group was linked to the carbon at the 2-position of the sugar. The correlation of methyl hydrogen with C-4' (δC 139.90) was observed in the HMBC spectra. Finally, compound 1 was identified as piceatannol-3' -O-beta- D - [2 "- (3, 5-dihydroxy-4-methoxybenzoyl)]-glucopyranoside. 1 H NMR 13 The C NMR data are shown in Table 1.
Compound 2: brown powder, molecular ion peak m/z 581.1265[ M+Na ] given by (+) HRESIMS] + (calcd for 581.1271) determining the molecular formula as C 27 H 26 O 13 The unsaturation was 15. Compound 2 lacks methoxy signal, other than 1 H NMR 13 C NMR data were similar to compound 1. 1 H NMR spectrum: contains a typical piceatannol signal-ABX system: delta H 7.23 (1 h, d, j=2.0 Hz), 7.04 (1 h, dd, j=8.3, 2.0 Hz) and 6.76 (1 h, d, j=8.3 Hz), and AX 2 The system comprises: delta H 6.44 (2h, d, j=2.2 Hz) and 6.16 (1h, t, j=2.2 Hz), and trans-olefin protons δ H 6.87 (d, j=16.2 Hz) and 6.72 (d, j=16.3 Hz). Furthermore, the sugar 2-position methine hydrogen (. Delta.) was found by HMBC spectrometry H 5.19 Carbonyl group (delta) C 166.68 Related to the determination that galloyl is attached at the 2-position of the glycosyl group, the compound 2 is piceatannol-3' -O-beta- D - (2' -galloyl) -glucopyranoside, 1 h NMR 13 The C NMR data are shown in Table 1.
Compound 3: brown powder with molecular formula C as determined by UPLC-Q-TOF-MS 29 H 24 O 7 (m/z 485.1601[M+H] + 485.1556) and is combined with 13 C and C 1 H NMR data analysis, unsaturation 18. 1 The aromatic region of H NMR, at delta H 6.88 A set of AA 'BB' coupled protons at (2H, d, J=8.4 Hz, H-3, 5) and 7.30 (2H, d, J=8.3 Hz, H-2, 6) indicate that the structure contains a para-substituted phenyl group. At delta H 6.67 A group of aromatic protons of the AX type (1 h, d, j=2.1 hz, h-14) and 6.31 (1 h, d, j=2.1 hz, h-12); there are other fragrance signals: delta H 6.10(3H，s，2′，4′，6′)，δ H 6.74 (2 h, h-10',13 ') and 6.62 (1 h, dd, j=8.1, 2.1hz, h-14 '). Delta H 5.31 Aliphatic protons and δ at (1 h, d, j=4.9 hz, h-8 ') and 4.44 (1 h, d, j=4.9 hz, h-7') H 6.94 The trans double bond protons at (1 h, d, j=16.4 hz, h-7) and 6.71 (1 h, d, j=16.4 hz, h-8) are considered typical protons of the stilbene dimer component. In addition to the protons mentioned above, compound 3 has a methoxy group delta H 3.74 (3H, s). Compound 3 13 The C NMR spectrum showed 29 carbon signals containing one methoxy group, 16 methyl groups and 12 quaternary carbons. In addition to one more methoxy group, compound 1 1 H and 13 the C NMR data were consistent with that of scirpusin A. H-7' (delta) H 4.4 And C-2', 6' (delta) C 105.9 HMBC related, H-8' (delta) H 5.31 Regarding the presence of HNBC in C-10 '(113.2) and C-14' (117.3), the structure of Compound 3 was consistent with scirpusin A. In addition, methyl protons (δ) are observed in HMBC spectra H 3.74 And C-4 (delta) C 159.5 A correlation exists and a conclusion is made 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 oligomers 1 HNMR data shows that the coupling constant value in the cis-orientation should be around 8.0 Hz. And there is no NOESY correlation between H-7 'and H-8', further indicating the opposite direction of H-7 'and H-8'. The configuration of the dihydrobenzofuran skeleton at C-7 'and 8' can be determined by the Cotton effect in the 220-240nm regionDiscrimination, such as (+) -epsilon viniferin (7 'S,8' S) is positive at 237nm and (-) -epsilon viniferin (7 'R,8' R) is negative. The CD spectrum of Compound 3 showed a negative Cotton effect (Δε, -13.17) at 231nm, indicating absolute configurations at C-7 'and C-8' of 7'R,8' R. Thus, the structure of compound 3 was elucidated as 4'-methoxy-scirpusin a (4' -methoxy-scirpusin a).
TABLE 1 Compounds 1 and 2 1 H NMR 13 C NMR data
b Data are shown as measured in deuterated methanol.
* Representing multiple overlapping peaks.
TABLE 2 Compound 3 1 H NMR 13 C NMR data
Experimental example 1 AChE (AChE) inhibitory Activity screening experiment
1. Test method
Acetylcholinesterase inhibition activity was modified based on the Ellman method and the AChE inhibition by compounds 1 and 2 was measured in 96-well plates. Reagents used in the experiments included reaction substrate ATCI, chromogenic agent DTNB and 1% SDS buffer containing (1 g PBS of SDS (ph 8.0)). The measurement steps are as follows: first, 60. Mu.L of PBS (pH 8.0), 10. Mu.L of 3mM DTNB, 20. Mu.L of 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, the reaction was stopped by adding 80. Mu.L of 1% SDS solution. Finally, absorbance was measured at 405nm using a multifunctional microplate reader (Tecan Infinite M200, switzerland Tecan Trading co., ltd). A20. Mu.l sample solution was used as a blank sample instead of PBS to record blank absorption, with tacrine as a positive control for acetylcholinesterase inhibition. Wherein each sample was repeated 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 V23.0.0 software and the results were expressed in terms of mean± Standard Error Mean (SEM). Data conforming to normal distribution are subjected to One-way ANOVA (One-way ANOVA), and differences among groups are compared pairwise through LSD analysis; data that does not satisfy normal distribution employs non-parametric testing. Analysis results are plotted with GraphPad Prism software 8.0.8.0.
2. Test results
The reduction of the level of acetylcholine in brain tissue is one of the important factors causing neurodegenerative diseases such as Alzheimer's disease, and thus acetylcholinesterase inhibitors are finding an important treasury for preventive treatment. The in vitro test shows that three new compounds (compounds 1,2 and 3) have inhibitory activity on acetylcholinesterase, and the results show that the inhibitory activity of the compounds 1 and 2 on acetylcholinesterase shows moderate inhibitory effect, and the compound 3 shows stronger inhibitory effect. Compounds 1,2 and 3 were significantly more active than resveratrol (compound 4) and Qu Za glycoside (compound 5) (p < 0.0001), the results are shown in table 3 and figure 2. From the chemical structure point of view, the compounds 1 and 2 are generated after the acylation reaction on glycosyl groups on the basis of the compound 5, and the structural change obviously improves the inhibition effect on acetylcholinesterase and provides a reference for structural modification. In a word, three novel compounds separated from the root of the rheum officinale of the Lhasa have obvious inhibition effect on acetylcholinesterase, and are potential medicaments for preventing diseases such as Alzheimer's disease.
Table 35 results of inhibition of acetylcholinesterase Activity by stilbene components
Experimental example 2 animal experiments and behavioural experiments for Compound 1
1 Experimental method
1.1 animal experiments
Animal experiments mainly studied novel compound 1 (comp.1, piceatannol-3' -O-beta- D - [2 "- (3, 5-dihydroxy-4-methoxybenzoyl)]-glucopyranoside) on cognitive disorders. SPF class C57 male mice at 8 weeks of age, 60 (purchased from St Bei Fu (Beijing) Biotechnology Co., ltd.), were fed in plastic cages, fed ad libitum, fed with water, at room temperature 25.+ -. 2 ℃ and humidity 55.+ -. 10%, maintaining a circadian rhythm of 12h light and 12h darkness. Male mice were randomly divided into 5 groups (12 mice per group): (1) Control (Vehicle), (2) Model group (Model, scopolamine,1.5 mg. Kg) -1 ) (3) Positive drug group (DNP, scopolamine+Donepenzil, 3.0mg 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 ). The mice were given compound 1 by intragastric administration for 4 weeks, and on the day of behavioural experiments, scopolamine was injected intraperitoneally except for the blank group after 0.5h of administration, and the behavioural experiments were started after modeling for 0.5 h.
1.2 behavioural experiments
1.2.1 New object identification (Novel object recognition test, NOR)
New object recognition experiments are important methods for evaluating learning and memory abilities of mice, and play an important role in research of neurodegenerative diseases. The experimental device consists of a white experimental 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 procedure was divided into 3 stages: adaptation period, familiarity period, testing period. The adaptation period is 1 day, after the animals are placed back to the middle of the experiment box in sequence, the animals are adapted to the environment of the experiment box for 5min, and the instrument is cleaned by 70% ethanol to eliminate residual odor. The next day a familiarity period and a testing period are performed. In the familiarity period, two identical objects are placed in an experiment box, the distance between the two objects and a wall is kept consistent, and animals are placed in the experiment box to be freely explored for 5min. After 30min interval, the test period is started, one of the familiar objects is changed into a new object, the other familiar object is kept unchanged, and the animal is put into the experiment box again for exploration for 5min. The time of exploration of the mice for new and old familiar objects during the test period was recorded and the relative discrimination index (discrimination index, DI) was used to evaluate the learning and memory ability of the animals. The relative discrimination index calculation formula is di= (N-F)/(n+f) ×100%, where N (new) is the exploration time of the animal for the new object and F (family) is the exploration time of the animal for the original familiar object.
1.2.2 Morris water maze (Morris water maze test, MWM)
Spatial learning memory was assessed by the Morris water maze test, which referenced the Morris water maze classical protocol and improved. The Morris water maze animal behavior analysis system comprises a black water tank (diameter: 100cm; height: 50 cm), a black platform (diameter: 9cm; height: 15 cm) and an animal behavior video analysis system. The pool is divided into four quadrants (southeast quadrant, northeast quadrant, southwest quadrant and northwest quadrant), and colored paper with different shapes is stuck at the middle point of the pool wall of the quadrants to be used as a mark for a mouse to find a platform. The platform was positioned 1.5cm under the center of the target quadrant, keeping the position unchanged throughout the training period. Milk was added to the pool and homogenized in order to prevent the mice from seeing the platform below the water surface.
MWM experiments were performed at the positioning voyage stage from day 1 to day 5, each mouse trained 1 time each day in the morning and afternoon, and mice were placed from two fixed-position walls, respectively. Each mouse is allowed to search for a platform in 60s, if the platform is successfully found, the mouse stays on the platform for 15s and returns to the cage, and the system automatically records the latency period reaching the platform; if no platform was found, the animals were guided to the platform and left for 15s (latency noted 60 s).
Day 6 is the space exploration phase, the platform is removed, and the space memory capacity of animals on the platform position is detected. The animal face wall is put into water from the midpoint of the diagonal quadrant of the platform, and the system automatically records the indexes such as the number of times that the animal crosses the target quadrant, the number of times that the animal crosses the platform within 60s, the swimming time ratio of the platform quadrant, the swimming Cheng Bi and the like.
1.2.3 dark avoidance experiments (Passive avoidance test, PAT)
The darkness-avoiding experimental device consists of a bright room and a dark room, and the two rooms are connected through an arch. The experiment is divided into two stages of dark avoidance study and dark avoidance consolidation. Before the experiment, each mouse was placed back to the arch in the open room for free exploration for 3min, then learning was started, and when it entered the darkroom, 0.36mA electric shock was given for 5min. After 24h of study, a consolidation experiment is carried out, the mice are still placed in a darkroom in an original mode, and the number of errors entering the darkroom within 5min and the incubation period of entering the darkroom for the first time are recorded.
1.3 Biochemical index detection
At the end of the behavioural experiment, all mice were anesthetized with ether inhalation. Blood was collected from the canthus vein and serum samples were isolated by centrifugation at 3500rpm for 10 minutes at 4 ℃. The mice were then cervical and cephalized, and brain tissue was removed on ice and weighed. The total protein concentration was measured using BCA protein quantification kit (beijing, century biotechnology, beijing, china) by rapid homogenization with ice-cold physiological saline at 9 times the tissue weight and centrifugation at 2500r min-1 for 10min at 4 ℃. AChE, chAT, SOD, CAT activity and ACh, GSH, MDA level are all carried out by referring to the instruction book of the corresponding detection kit (Nanjing, china). Wherein AChE, chAT, SOD, CAT is active as U.mgprot -1 Expressed as umol.mgprot for GSH level -1 The MDA level is expressed as nmol mgprot-1, and the ACh level is expressed as ug mgprot -1 And (3) representing. The detection of the levels of inflammatory factors IL-1 beta, IL-4, IL-6 and TNF-a in serum samples was performed with reference to the corresponding ELISA detection kit instructions (Beijing Huaying Biotechnology institute, beijing, china) and was performed in ng.mL -1 And (3) representing.
1.4 data processing
Statistical analysis of the data was performed using IBM SPSS Statistics V23.0.0 software and the results were expressed in terms of mean± Standard Error Mean (SEM). Data conforming to normal distribution are subjected to One-way ANOVA (One-way ANOVA), and differences among groups are compared pairwise through LSD analysis; data that does not satisfy normal distribution employs non-parametric testing. Analysis results are plotted with GraphPad Prism software 8.0.8.0.
2 experimental results
2.1 novel Compounds 1 improve scopolamine-induced cognitive dysfunction
The in vitro activity experiment of experiment example 1 shows that the novel compound 1 in the rheum officinale has good cholinesterase inhibition activity and nervous system protection effect. Because the amount of the novel compound 1 obtained by separation is relatively large and the activity in vitro is good, animal experiments are carried out by selecting the novel compound 1, and the improvement effect of the novel compound 1 on scopolamine-induced cognitive impairment is researched and evaluated through the behavioural experiments NOR, MWM, PAT and cholinergic detection and oxidative stress detection.
2.2 New Compounds 1 improving New object recognition ability in mice
The experiment evaluates the short-time and non-space learning and memory capacity of the C57 mouse through a new object recognition experiment. As a result, it was found that scopolamine-induced cognitive impairment mice had reduced memory and discrimination ability for familiar and new subjects, and that the relative discrimination index of mice in the model group was relatively low. The relative discrimination index of the mice in the blank group was significantly greater than that of the model group (P < 0.05), indicating that normal C57 mice had memory for the familiar objects and were able to discriminate between the familiar objects and new objects; the relative discrimination index of the DNPZ group was significantly elevated (P < 0.0001) compared to the model group; experimental results 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 administration of the novel compound 1 by gavage can enhance the memory of the mice to the familiar object and can significantly improve scopolamine-induced short-term, non-spatial learning memory impairment.
2.3 novel Compounds 1 improve the Passive escape ability of mice
The darkness test is to utilize the habit design of rodent to avoid darkness, and form conditions after the animal enters darkroom, i.e. is shocked by sole and repeatedly used for several timesAnd (5) reflection. The experiment is used for detecting the passive evasion ability of rodents, and takes the latency period of entering a darkroom and the number of errors of being electrified entering the darkroom as main evaluation indexes. In the dark avoidance experiments, the dark avoidance latency of mice in the model group was significantly reduced and the number of errors was significantly increased compared to the blank group (P<0.0001 A) is provided; the dark-avoidance latency was significantly increased in both the DNPZ group and comp.1_l and comp.1_h compared to the model group (P<0.0001 The number of errors of comp.1_l group is significantly reduced (p<0.05 The number of comp.1_h group errors was reduced, but no significant difference occurred, as shown in fig. 4 (a), (b). The results showed 1.5 mg.kg -1 Scopolamine significantly impairs the passive evasion ability of mice in dark-avoidance experiments. Compound 1 was able to ameliorate scopolamine-induced injury, allowing C57 mice to have prolonged dark-avoidance latency and reduced error times.
2.4 novel Compounds 1 improving learning and memory Capacity in mouse Water maze experiments
Morris water maze test is a classical test method for studying the spatial learning and memory ability of rodents, and is commonly used for examining the long-term, spatial reference memory ability of animals, including two stages of positioning navigation and spatial exploration. In the positioning navigation stage, the platform searching latency is the most classical evaluation index; in the space exploration stage, the number of channel crossing times and the number of target quadrant crossing times are main indexes for evaluating the space exploration capability. As shown in FIG. 5 (e), in the 5-day positioning voyage training, the model mice took significantly more time to find the platform than the blank mice, and in the space exploration phase, the latency of the model mice reaching the platform region for the first time was significantly increased, and the number of crossing the target quadrant and the platform region was significantly reduced (p<0.01 As shown in fig. 5 (a), (b), (c), and (d). New compound 1 at 100 and 400 mg.kg -1 Can significantly reduce the latency period (p<0.0001 400 mg/kg) -1 The number of times of crossing the target quadrant (p<0.05 Number of plateau times (p)<0.05). There was no significant difference in the swimming speed of each group of mice, as shown in fig. 5 (c). The result of the study shows that 1.5 mg.kg -1 Scopolamine significantly impairs spatial learning and memory ability of mice in water maze experiments, novel compoundsThe compound 1 can significantly improve injury caused by scopolamine, 100 mg.kg and 400 mg.kg -1 The effect of the drug administration is equivalent.
2.5 New Compounds 1 improve cholinergic injury caused by scopolamine
The effect of novel compound 1 on cholinergic injury by scopolamine was evaluated by measuring acetylcholine transferase (AChE), acetylcholine transferase (CHAT) activity 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 the brain tissue of C57 mice (p<0.05 A) is provided; oral compound 1 can significantly increase ACh level and AChE activity (p<0.05). The change in AChT activity in mouse brain tissue was not particularly pronounced, but compound 1 also significantly increased AChT activity at high doses compared to the model group (p<0.05 As shown in fig. 6 (c). The study result shows that the oral administration of the C57 mouse is 1.5 mg.kg -1 Scopolamine causes significant decrease in acetylcholine levels in brain tissue and abnormalities in acetylcholinesterase and acetylcholinesterase activities. Compound 1 was effective in reversing the decline in acetylcholine levels and the impairment of acetylcholinesterase caused by scopolamine at both low and high doses.
2.6 New Compounds 1 improve oxidative damage caused by scopolamine
The brain is a tissue that is extremely vulnerable to oxygen radicals, and oxidative stress injury is therefore also a major factor in the development of AD. In the study, scopolamine in the model group reduces the antioxidant activity of the brain tissue of the mice, reduces CAT and SOD activities and GSH levels, increases MDA levels, and increases the degree of free radical injury of the brain tissue. Compound 1 administration can increase the antioxidant activity of the brain tissue of mice to various degrees and reduce the MDA level. Wherein CAT activity was significantly increased in the low dose group and the high dose group relative to the model group (p<0.05 The result is shown in FIG. 7 (a). DNP and low dose groups had significantly elevated GSH levels (p<0.01 The GSH level was significantly elevated in the high dose group, but no significant difference occurred, and the results are shown in fig. 7 (b). SOD activity was significantly increased in DNP and high dose groups (p<0.05 While each of the administration groups had a tendency to lower MDA levels in brain tissue, no significant differences occurred, as shown in FIG. 7 (c),(d) As shown. The above results indicate 1.5 mg.kg -1 The scopolamine causes oxidative stress injury, and meanwhile, the compound 1 has good antioxidant capacity, and can effectively reverse the oxidative injury caused by the scopolamine.
2.7 novel Compounds 1 improve the neuroinflammation caused by scopolamine
In this study, the intraperitoneal injection of scopolamine significantly increased IL-1 beta, IL-6, TNF-a levels in the serum of model group C57 mice relative to the blank group (p < 0.0001), and the intraperitoneal injection of scopolamine also caused different degrees of inflammatory injury in the C57 mice of each of the dosing groups. Compared with the model group, the administration of the compound 1 at 400mg/kg can significantly reduce the IL-1 beta, IL-6 and TNF-a in the serum of the C57 mice (p < 0.05). IL-1. Beta., IL-6, TNF-a levels were reduced but not significantly reduced when administered at low doses, as shown in FIGS. 8 (a), (b), and (c). Experimental results show that the compound 1 has remarkable inflammation inhibition effect at high dosage, and reduces inflammatory injury.

Claims (6)

1. A compound isolated from rheum lassa, characterized by the structural formula shown in formula iii:
2. a process for the isolation of a compound according to claim 1 from pizza radix et rhizoma Rhei, comprising:
(1) Pulverizing Rheum emodi, extracting with 95% ethanol, concentrating under reduced pressure to obtain extract; (2) The extract is divided into a chloroform part, an ethyl acetate part, an n-butanol part and a water part by extraction; (3) Loading the ethyl acetate part sample into an MCI packing chromatographic column, and eluting with 30%, 50%, 70% and 100% ethanol solvents respectively; wherein the compound of formula III is isolated from a 70% elution fraction.
3. The process according to claim 2, wherein 4 to 5 column volumes are rinsed per solvent in step (3).
4. Use of a compound according to claim 1 for the preparation of an acetylcholinesterase inhibitor.
5. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of neurodegenerative diseases.
6. A pharmaceutical composition for the treatment of neurodegenerative diseases, which is prepared by combining a prophylactically or therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier.
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