CN115583981B - Eucalyptus alkane type sesquiterpene glycoside compound, erythrina ovalifolia extract containing compound and application of compound - Google Patents

Eucalyptus alkane type sesquiterpene glycoside compound, erythrina ovalifolia extract containing compound and application of compound Download PDF

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CN115583981B
CN115583981B CN202211234813.8A CN202211234813A CN115583981B CN 115583981 B CN115583981 B CN 115583981B CN 202211234813 A CN202211234813 A CN 202211234813A CN 115583981 B CN115583981 B CN 115583981B
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erythrina
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ethyl acetate
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CN115583981A (en
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张秀云
丁佳琪
高锦明
沙柏彤
张强国
关圣楠
黄蓉
张育凤
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Northwest A&F University
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Abstract

The invention belongs to the field of biological medicine, and relates to a eudesmane terpene glycoside compound, an erythrina extract containing the euonymus alatus, and application thereof. The eudesmane terpene glycoside compound has a structure shown in any one of formulas I-VIII. The eudesmane type sesquiterpene glycoside compound provided by the invention can obviously inhibit BV-2 microglial NO generation induced by LPS, has an IC50 value as low as 0.92 mu M, can inhibit NF- κB signal paths to inhibit downstream inflammatory proteins iNOS and COX-2, thereby exerting the anti-neuritis activity of the eudesmane type sesquiterpene glycoside compound and having development and application potential in the aspect of preventing and treating neurodegenerative diseases;

Description

Eucalyptus alkane type sesquiterpene glycoside compound, erythrina ovalifolia extract containing compound and application of compound
Technical Field
The invention belongs to the field of biological medicine, and relates to a series of eudesmane terpene glycoside compounds with unique structures, erythrina extract containing the euonymus alatus, and application thereof, in particular to application in preparing medicines for inhibiting nitric oxide synthase and cyclooxygenase-2 activity.
Background
With age, aging of the human brain may cause continued neurodegenerative disease and thus neurodegenerative disease, and human genetic and environmental factors together determine the progression of neurodegenerative disease. Neurodegenerative diseases are a series of diseases caused by progressive loss of a specific neuron population, mainly including Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), and the like. The clinical manifestations of AD are disorders of living memory and learning, mood swings, and behavioral disorders, which are complex in etiology and pathogenesis. Related studies in recent years have shown that neuroinflammation is a major pathological feature of neurodegenerative diseases, and that the correlation between neuroinflammation and neurodegenerative diseases has been confirmed many times. Excessive sustained activation of microglia (such as BV-2 microglial) and sustained elevation of pro-inflammatory factors and increased oxidative stress are evident in brain tissue of AD patients; inhibition of neuroinflammation has also been shown to prevent the progression of ataxia telangiectasia symptoms in ATM-deficient mice.
The pathogenesis of neurodegenerative diseases is complex, and neuroinflammation may also occur in the early stages of disease occurrence. The treatment and intervention of asymptomatic and prodromal phases are the first approaches for the development of the medicaments, and although basic research and clinical research have begun to explore the pathogenesis and development of neurodegenerative diseases, a plurality of problems remain unsolved at present, and the development of medicaments for treating neurodegenerative diseases is urgent. Natural products with anti-neuritis activity include apigenin, curcumin, cinnamaldehyde, etc. The related study on the biological activity of the Jatropha plant shows that the plant also contains natural active small molecules with stronger anti-inflammatory effect, such as guaiane type sesquiterpene from P.undulotum fruits, and has great effect on LPS-induced mouse megaphagyThe IC50 values of the cell (RAW 264.7) inflammation model are all about 10 mu M; the extract of viridifloum also shows a strong anti-inflammatory activity; prior Art A series of amygdalin sesquiterpene glycosides isolated from Mallotus japonicus (P.qinlingense) were screened for anti-neuritic activity by LPS-induced mouse microglial cell (BV-2) inflammation model, and the results indicate that some of the compounds have significant anti-neuritic activity (IC 50 ＝0.95～24.12μM)。
There are about 300 plants of the genus idesia (pittosporeae) of the family idecaceae (pittosporeae), the broad range Yu Dayang continents, the southeast Pacific islands, the southeast asia and the asia eastern subtropical regions. There are 44 varieties of 8 in China, including erythrina caerulea, erythrina brachycarpa, erythrina sylvestris, erythrina glauca, erythrina rupestris, erythrina angustifolia and erythrina, etc., which are mainly distributed in Sichuan, yunnan, gansu and Guangdong places. Researches show that the root, leaf and seed of the Chinese medicinal composition can be used as medicaments, and the root can dispel wind and activate collaterals, remove stasis and relieve pain, the leaf can detoxify and stop bleeding, and the seed can astringe intestines, arrest spontaneous emission and the like. The chemical components mainly comprise terpenes (monoterpenes, sesquiterpenes, triterpenes) and their glycosides, lignans (glycosides), carotenoid, flavone, phenylpropanoid, coumarin and other structural compounds, and the biological activities mainly comprise antibacterial, antitumor, antiinflammatory, antioxidant, neuroprotection, antiarrhythmic, anti-hemolysis, liver protecting and the like.
The erythrina variegata has the academic name Pittosporum ovoideum Gowda, evergreen shrubs, she Husheng, all edges or wavy teeth, and is produced on the branches in a usual way, mainly grows in the mountain evergreen forest of limestone, mainly distributes in the North Guangxi and southeast Guizhou, and has obvious regional distribution characteristics. The Chinese medicinal composition has a long medicinal history, and can be used as a medicament for branches, leaves, roots, seeds and fruits, and the branches, leaves can stop bleeding, detoxify and resist bacteria; the root has the effects of dispersing blood stasis, relieving pain, dispelling wind, activating collaterals, protecting tumors and the like. However, the prior art for the component with the anti-neuritis activity in the erythrina oomycete has not been reported yet.
Disclosure of Invention
In view of the above technical problems, the invention obtains a series of eudesmane type sesquiterpene glycoside structural analogues from the ethanol extract of erythrina lunata leaves by directional separation, and identifies the structures of all compounds by a spectrum method. The activity test results show that the compounds have certain anti-neuritis activity.
The invention provides a eudesmane terpene glycoside compound, which is extracted from erythrina ovata, and has a structure shown in any one of formulas I-VIII:
in a second aspect of the present invention, there is provided an extract of erythrina superba containing a compound of any one of formulae I to VIII.
In a third aspect of the present invention, there is provided a method for preparing the erythrina procumbens extract, comprising the steps of:
extracting erythrina variegata with ethanol, and concentrating to obtain crude extract;
sequentially extracting the crude extract with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract phase, ethyl acetate extract phase and n-butanol extract phase respectively;
and drying the ethyl acetate extract phase to obtain the erythrina ovata extract.
Preferably, the specific operation process of ethanol extraction of the erythrina ovata comprises the following steps: extracting erythrina ovata by using 95% ethanol solution with a mass fraction as a solvent for 4-6 times at 75 ℃ by adopting a heating reflux method, 4-5 hours each time, combining the extracting solutions, and concentrating to obtain the extract;
wherein the dosage ratio of the dried product of the erythrina ovata to the 95% ethanol solution by mass fraction is 1 g:4-6 mL.
Preferably, the specific operation process of extracting the crude extract by petroleum ether, ethyl acetate and n-butanol sequentially comprises the following steps: after the crude extract is uniformly dispersed by water, petroleum ether, ethyl acetate and n-butanol are sequentially used for extraction for 3 to 6 times, and the volume ratio of the aqueous dispersion of the crude extract to the petroleum ether, the ethyl acetate or the n-butanol is 1:1 to 1.5.
In a fourth aspect, the invention provides a pharmaceutical composition comprising at least one compound of formulae I to VIII, and a pharmaceutically acceptable adjuvant or carrier.
In a fifth aspect, the invention provides an application of a eudesmane-type terpene glycoside compound, an erythrina extract or a pharmaceutical composition in preparing medicines for preventing and treating diseases related to neuroinflammation.
Preferably, the eudesmane-type terpene glycoside compound, the erythrina extract or the pharmaceutical composition is used for preparing medicines for inhibiting the activity of nitric oxide synthase and/or cyclooxygenase-2.
Preferably, the neuroinflammation-related disorder includes a neurodegenerative disorder.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention extracts and separates 8 eudesmane type sesquiterpene glycoside compounds from the first erythrina. The experimental result proves that 8 eudesmane type sesquiterpene glycoside compounds separated from the erythrina extract have good anti-neuritis activity and show obvious correlation of structure and activity, so that the eudesmane type sesquiterpene glycoside compounds provided by the invention can be used as anti-neuritis micromolecule medicaments and have development and application potential in the aspect of preventing and treating neurodegenerative diseases.
2. The eudesmane type sesquiterpene glycoside compound provided by the invention can obviously inhibit BV-2 microglial NO generation induced by LPS, has an IC50 value as low as 0.92 mu M, and can inhibit NF- κB signal channels to inhibit downstream inflammatory proteins iNOS and COX-2, thereby exerting anti-neuritis activity.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 is a UV spectrum of Pitovocoside A;
FIG. 2 is an IR spectrum of Pitovocoside A;
FIG. 3 is a high resolution mass spectrum of Pitovocoside A;
FIG. 4 is Pitovocoside A 1 H spectrogram;
FIG. 5 is Pitovocoside A 13 C NMR spectrum;
FIG. 6 is Pitovocoside B 1 H spectrogram;
FIG. 7 is Pitovocoside B 13 C NMR spectrum;
FIG. 8 is Pitovocoside C 1 H spectrogram;
FIG. 9 is Pitovocoside C 13 C NMR spectrum;
FIG. 10 is Pitovocoside D 1 H spectrogram;
FIG. 11 is Pitovocoside D 13 C NMR spectrum;
FIG. 12 is Pitovocoside E 1 H spectrogram;
FIG. 13 is Pitovocoside E 13 C NMR spectrum;
FIG. 14 is Pitovocoside F 1 H spectrogram;
FIG. 15 is Pitovocoside F 13 C NMR spectrum;
FIG. 16 is Pitovocoside G 1 H spectrogram;
FIG. 17 is Pitovocoside G 13 C NMR spectrum;
FIG. 18 is Pitovocoside H 1 H spectrogram;
FIG. 19 is a diagram of Pitovocoside H 13 C NMR spectrum;
FIG. 20 shows cell viability of Pitovocoside A-H at 6.25, 12.5, 25 and 50. Mu.M concentrations, respectively;
FIG. 21 is a Western blot analysis of the effect of Compound 1 on the expression levels of iNOS, COX-2; A. iNOS, B, COX-2;
FIG. 22 is a Western blot of the effect of Compound 1 on expression of iNOS, COX-2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It should be apparent that the embodiments described below are only some, but not all embodiments of the present invention, and the present invention is not limited in any way, and all embodiments using the technical solutions of the present embodiment, including simple changes, fall within the scope of the present invention.
The target compound of the invention, 8 eudesmane type sesquiterpene glycoside compounds with anti-neuritis activity, is directionally separated from ethanol extracts of erythrina ovata leaves. The plant sample erythrina leaves were collected from Kunming plant classification biotechnology limited company, hechi, guangxi Zhuang nationality, hechi city, fengshan county, 5 months 2021, identified by the company Wu Jun, with the specimen number PO-004, and stored in the national product chemical and biological important laboratory specimen laboratory of the northwest agriculture and forestry university. The batch was used after drying in the shade at room temperature.
The reagents and instruments used in the invention are as follows:
common organic solvents: chloroform, methanol, ethyl acetate, petroleum ether, acetone and the like are industrial reagents and are used after being steamed again. Organic solvent: the chromatographic methanol, chromatographic acetonitrile and the like are used as the analytical or chromatographic pure reagents depending on the actual use. The following is a volume ratio of the reagents unless otherwise specified.
Common instruments: polarimeter Rudolph Autopol type iii; high performance liquid chromatograph: waters 1525; ultraviolet spectrometer: thermo Evolution-300; nuclear magnetic resonance: bruker Avance III 500 (TMS internal standard); low resolution mass spectrometer: thermo Fisher LTQ Fleet type. Rotary evaporator: B-chiRotavapor R-101, R-3HB type; cryogenic cooling liquid circulation pump: DLSB-10/20 type (Zhengzhou great wall Co., ltd.); circulating water type multipurpose vacuum pump: SHB-iii (zheng great wall family industry and trade limited); ultra-clean bench: SW-OJ-2F type (Sujing group, soy air technologies Co., ltd.); vertical steam sterilizer (Shanghai Boqing Uygur autonomous medical equipment factory); field emission scanning electron microscope: nova Nano SEM-450 type (FEI company, usa); full-automatic critical point desiccator: EM CPD300 (Leica company, germany); ion sputtering instrument: EM ACE600 (Leica company, germany); temperature control rotary table oscillator: ZWY-240 (Shanghai Zhi City analytical instruments Co., ltd.).
Column chromatography silica gel (100-200 mesh, 200-300 mesh and 300-400 mesh) and thin layer chromatography silica gel (silica gel H) are all produced by Qingdao ocean chemical plants; liquid chromatographic column: hypersil BDS 5 μm C18 (250×4.6and 250×10; thermo); the hydroxypropyl dextran gel Sephadex LH-20 and RP-C18 reverse silica gel are both manufactured by Merck company.
Example 1
Extraction and separation of eudesmane type sesquiterpene glycoside compounds
1. Experimental materials
Blade of erythrina ovata: fresh erythrina indica leaves were dried in the shade at room temperature and weighed to 10kg.
2. Method of
Extracting extract: pulverizing dried fresh erythrina ovata leaves to about 40 meshes, adding 95% ethanol solution with a mass fraction of 5 times of that of the dried erythrina ovata leaves, extracting for 5 times at 75 ℃ by adopting a heating reflux method, filtering the extracting solution for 4 hours each time to separate solid from liquid, combining the 5 extracting solutions, and concentrating under reduced pressure by a rotary evaporator to obtain ethanol total extract (with the number of PO);
dispersing and kneading the extract in water, and extracting with petroleum ether, ethyl acetate and n-butanol respectively, wherein the specific method comprises the following steps: placing the extract water dispersion in a separating funnel, adding petroleum ether with equal volume, shaking, standing, transferring the petroleum ether extract at the upper layer after layering, adding petroleum ether with equal volume again, repeating for 5 times, mixing the extracts, and concentrating under reduced pressure on a rotary evaporator to obtain petroleum ether extract phase with number of POA; then sequentially carrying out the same treatment on the aqueous dispersion liquid by using ethyl acetate and n-butanol to obtain corresponding extract phases with the numbers of POB and POC; after the development of thin-layer chromatography, the target component is judged to be mainly positioned in an acetic acid extraction part (named POB) by using a natural product general color developing agent and combining a sesquiterpene special color developing agent for color development. The specific judgment basis is as follows: the universal color developing agent adopts 5% sulfuric acid-ethanol solution, a thin layer chromatography plate after a sample is unfolded by using a solvent is sprayed with a proper amount of color developing agent, a hot air gun is used for heating to more than 105 ℃, the change condition of spot color is observed, and target components show orange red, orange yellow, light brown and other colors in the sulfuric acid-ethanol color developing agent; the special terpene color developing agent adopts 1% vanillin-sulfuric acid-glacial acetic acid solution, the color developing agent is sprayed by the same method, the change of spot color is observed after heating, and the target component shows bright red or pink.
Pretreating ethyl acetate extract (POB, 440 g) by column chromatography, mixing with equal amount of silica gel, drying in the shade or oven drying at a temperature, and grinding into fine powder;
the ethyl acetate extract was subjected to silica gel column chromatography using successive elutions of petroleum ether-ethyl acetate (10:1, 8:1, 5:1, 3:1, 2:1, 1:1, v/v, 60L total eluent per gradient 10L), methylene chloride-ethyl acetate (1:1, v/v,1000 mL) and methylene chloride-methanol (10:1, 8:1, 5:1, 1:1, v/v, 10L total 40L total eluent) with the aid of thin-layer chromatography f Value) is the segment basis, R is f The components with the same or similar values are combined to obtain 13 fractions (the numbers are POB-1 to POB-13);
the second fraction, POB-2 (9.79 g), was eluted with normal phase silica gel column chromatography using methylene chloride-ethyl acetate (20:1, 10:1,5:1, 1:1, v/v, 4L each gradient, 16L total eluent) using the same thin layer chromatography guidance as above to give 7 secondary fractions in sequence. The 2 nd fraction was purified by gel chromatography (chloroform-methanol 1:1, total 2L) using petroleum ether-ethyl acetate (10:1, isocratic elution, total 5L) to give compounds 5 (i.e., compound of formula V) and 6 (i.e., compound of formula VI). The 4 th fraction was eluted with methylene chloride-methanol (15:1, isocratic elution, 3L total) to give compounds 1 (i.e., compounds of formula I) and 2 (i.e., compounds of formula II).
The third fraction, POB-3 (16.48 g), was first subjected to normal phase silica gel column chromatography, eluting with n-hexane-ethyl acetate (15:1 isocratic elution, total 8L), using the same thin layer chromatography guidance method described above, to give 7 secondary fractions in sequence. The 4 th fraction (6.5 g) was purified by reverse phase silica gel column chromatography (methanol/water volume ratio 4:1, total 3L), gel column chromatography (chloroform, 1.5L), normal phase silica gel column chromatography (dichloromethane-ethyl acetate volume ratio 10:1,1.5L) and preparative HPLC technique (methanol/water volume ratio 2:1, total 1.5L) to give compound 4 (i.e., compound of formula IV) and 8 (i.e., compound of formula VIII). The 6 th fraction (522 mg) was subjected to gel column chromatography (chloroform, 1.0L) and HPLC (methanol/water volume ratio: 2:1, total 750 mL) was prepared to give compound 3 (i.e., the compound of formula III). Subjecting the 7 th component to gel column chromatography (dichloromethane-methanol volume ratio of 1: 1,2L), normal phase silica gel column chromatography (dichloromethane-methanol volume ratio of 10:1,1.5L), and preparing HPLC (methanol/water volume ratio of 3:1, 750 mL) to obtain compound 7 (i.e. compound of formula VII).
The structures of the compounds 1 to 8 are shown in the formulas I to VIII in sequence:
3. physicochemical Properties of Compounds 1 to 8
The physicochemical properties of the eudesmane type sesquiterpene glycosides 1-8 of the invention are respectively as follows:
(1) Compound 1, named as erythrinoside A (Pitovocoside A), has chemical formula C 28 H 44 O 7 Colorless crystals, HR-ESIMS (positive) m/z 515.2968[ M+Na ]] + (calculated value C) 28 H 44 O 7 Na + ，515.2985)。Ultraviolet spectroscopic data (MeOH); lamax (log ε) 291 (1.65) nm; infrared spectroscopic data (KBr) v max 3425,1710,1721,1651cm -1 。
(2) Compound 2, named as erythrinoside B (Pitovocoside B), has chemical formula C 28 H 44 O 8 Colorless crystals, HR-ESIMS (positive) m/z 531.2966[ M+Na ]] + (calculated value C) 28 H 44 O 8 Na + ，531.2934)。Ultraviolet spectroscopic data (MeOH);lamax (log ε) 277 (2.12) nm; infrared spectroscopic data (KBr) v max 3429,1721,1710,1652cm -1 。
(3) Compound 3, named as erythrinoside C (Pitovocoside C), has chemical formula C 28 H 44 O 8 Pale yellow oil, HR-ESIMS (positive) m/z 531.2920[ M+Na ]] + (calculated value C) 28 H 44 O 8 Na + ,531.2934)。Ultraviolet spectroscopic data (MeOH); lamax (log ε) 291 (2.41) nm; infrared spectroscopic data (KBr) v max 3512,1722,1705,1644cm -1 。
(4) Compound 4, named as erythrinoside D (Pitovocoside D), has chemical formula C 28 H 44 O 8 Pale yellow oil, HR-ESIMS (positive) m/z 531.2938[ M+Na ]] + (calculated value C) 28 H 44 O 8 Na + ,531.2934)。Ultraviolet spectroscopic data (MeOH); lamax (log ε) 291 (2.45) nm; infrared spectroscopic data (KBr) v max 3512,1718,1709,1623cm -1 。
(5) Compound 5, named as erythrinoside E (Pitovocoside E), has chemical formula C 28 H 44 O 8 Pale yellow oil, HR-ESIMS (positive) m/z 531.2921[ M+Na ]] + (calculated value C) 28 H 44 O 8 Na + ,531.2934)。Ultraviolet spectroscopic data (MeOH); λmax (log ε) 290 (3.30) nm; infrared spectroscopic data (KBr) v max 3511,1721,1704,1645cm -1 。
(6) Compound 6, named as erythrinoside F (Pitovocoside F), has chemical formula C 28 H 44 O 9 Pale yellow oil, HR-ESIMS (positive) m/z 547.2868[ M+Na ]] + (calculated value C) 28 H 44 O 8 Na + ,547.2878)。Ultraviolet spectroscopic data (MeOH); lamda max (log epsilon) 285 (1.56) nm; infrared spectroscopic data (KBr) v max 3516,1728,1721,1561cm -1 。
(7) Compound 7, named as erythrinoside G (Pitovocoside G), has chemical formula C 28 H 44 O 9 Pale yellow oil, HR-ESIMS (positive) m/z 547.2864[ M+Na ]] + (calculated value C) 28 H 44 O 9 Na + ,547.2878)。Ultraviolet spectroscopic data (MeOH); lamda max (log epsilon) 287 (1.30) nm; infrared spectroscopic data (KBr) v max 3516,1728,1721,1561cm -1 。
(8) Compound 8, named as erythrinoside H (Pitovocoside H), has chemical formula C 28 H 44 O 9 Pale yellow oil, HR-ESIMS (positive) M/z547.2872[ M+Na ]] + (calculated value C) 28 H 44 O 9 Na + ,547.2878)。Ultraviolet spectroscopic data (MeOH); lamda max (log epsilon) 285 (1.14) nm; infrared spectroscopic data (KBr) v max 3516,1728,1721,1561cm -1 。
13 C-NMR spectra 1 The H-NMR spectrum data are shown in tables 1-2, and the nuclear magnetism, mass spectrum and other spectrum spectra are shown in figures 1-19.
TABLE 1 Compounds 1 to 8 13 C NMR spectroscopic data
a: at 100MHz frequency in CDCl 3 -d; b: at a frequency of 100MHz in MeOH-d 4 Is recorded.
TABLE 2 Compounds 1 to 8 1 H NMR spectroscopic data [ delta ] H in ppm,multi(Jin Hz)]
a: at 400MHz frequency in CDCl 3 -d; b: at 400MHz frequency in MeOH-d 4 Recording; c: the signals overlap.
Example 2
Anti-neuritic activity of eudesmane-type sesquiterpene glycosides
By using microglial cells (BV-2) as a nerve cell model and the action of BV-2 microglial cell inflammatory response induced by Lipopolysaccharide (LPS), the MTT method is used for measuring the cell survival rate in experiments, and detecting whether the eudesmane type sesquiterpene glycoside compound has anti-nerve inflammatory activity under the induction of LPS or not, wherein the positive medicine is Quercetin (Quercetin).
1. Culture of BV-2 microglial cells
BV-2 microglia cells were resuscitated prior to culture. The specific operation process is as follows:
the temperature of the water bath kettle is set to be 37 ℃, then the freezing tube is taken out of the liquid nitrogen tank, rapidly shaken in the water bath at 37 ℃ for thawing, and then centrifuged by a centrifuge. After centrifugation, the supernatant containing the cryopreservation agent was aspirated with a pipette, and the bottom cells were then blown down with freshly prepared DMEM complete medium (containing 10% fbs and 1% diab) and transferred to petri dishes at 5% co 2 Culturing and passaging in a cell culture box at 37 ℃.
2. Cell viability assay
This experiment utilizes tetrazolium salt (MTT) colorimetry to determine toxicity of compounds to BV-2 microglia. The basic principle is that succinic dehydrogenase in living cell mitochondria can reduce exogenous MTT into insoluble blue-violet crystal, and after the exogenous MTT is dissolved by dimethyl sulfoxide (DMSO), the absorbance of the crystal is measured at 490nm by an enzyme-labeled instrument, so that the number of living cells can be indirectly reflected, and the evaluation of cytotoxicity can be realized. The specific operation process is as follows:
BV-2 microglia were first grown at 2X 10 5 The culture medium is sucked up after the culture medium is placed in a culture box for static culture for 24 hours, 100 mu L of DMEM culture medium containing a certain concentration of drugs (generally, the primary screening concentration is set to be 20 mu M, five gradients of 20 mu M, 10 mu M, 5 mu M, 2.5 mu M and 1.25 mu M are screened again for continuous culture, after the drugs act for 24 hours, 10 mu LMTT solution (5 mg/mL) is added to each well for continuous culture for 4 hours, then 100 mu L of DMSO is carefully sucked up and added, and after the purple crystals are completely dissolved, the light absorption value at the wavelength of 490nm is measured by an enzyme-labeling instrument, so that the cell survival rate is calculated.
Cell viability/% = absorbance of test group/absorbance of control group x 100.
FIG. 20 shows cell viability of compounds 1-8 at 6.25, 12.5, 25 and 50. Mu.M concentrations, respectively. The results show that the series of compounds have no toxicity to BV-2 microglia cells and meet the requirements of anti-neuritis activity test.
3. Evaluation of anti-neuritis Activity at cellular level
mu.L of the cell suspension (density 2X 10) 5 Inoculating to 96-well plate, standing in incubator for 24 hr, carefully removing supernatant, adding DMEM medium containing preset concentration (20, 10, 5, 2.5 and 1.25 μm) of compound and LPS, culturing for 24 hr, transferring 50 μl supernatant from each well to new 96-well plate, sequentially adding 50 μl Griess reagent I and II, shaking, and measuring absorbance at 540nm wavelength with enzyme marker instrument (A 540 )。
The compound was initially screened at 20. Mu.M and the regreen was set at five gradients of 20, 10, 5, 2.5 and 1.25. Mu.M. Three replicates were set for each treatment with quercetin (20. Mu.M) as positive control, LPS (1. Mu.g/mL) as negative control, and DMSO as blank.
The experiment utilizes a nitric oxide detection kit (Biyun) to detect the concentration of Nitric Oxide (NO) generated by a BV-2 inflammation model. Due to NO conversion in vivo or in aqueous solution to NO 2 - Reacts with a color-developing agent to generate the light red azo compound with NO 2 - The content of the fluorescent dye has good linear relation within a certain range (1-100 mu M), and the absorbance (A) at 540nm is measured by a colorimetric method 540 ) The NO content can be reflected indirectly. So the NaNO in the kit is utilized 2 Standard (1M) A was measured after preparation of a series of 40, 20, 10, 5 and 2.5. Mu.M gradients 540 A standard curve for NO concentration calculation can be obtained and used to calculate the NO concentration in the test sample.
The results of the study show that the series of eudesmane type sesquiterpene glycosides have anti-neuritis activity and show structural differences, and part of compounds have activity with the same order of magnitude as that of the medicament contrast, and the results are shown in Table 3.
TABLE 3 inhibition of LPS-induced BV-2 microglial NO production by Compounds 1 to 8
Example 3
Effect of Compound 1 (Pitovocoside A) on COX-2 and iNOS expression levels
The protein expression is detected by adopting an immunoblotting method, and the specific steps are as follows:
(1) Protein sample preparation: the treated cell samples were placed on ice, 200. Mu.L of RIPA solution (containing 2. Mu.L of PMSF) was added, and the cells were allowed to come into full contact with the solution once every ten minutes with shaking. Standing on ice for 30min, centrifuging at 11000rpm and 4deg.C for 5min, collecting supernatant, and diluting protein sample 5 times with PBS buffer.
(2) BCA assay for protein concentration: the BSA standard with the concentration of 2mg/mL is subjected to gradient dilution to prepare a standard curve (0, 0.00625, 0.0125, 0.25, 0.5, 1 and 2 mg/mL), BCA working solution is prepared in advance according to the quantity of the BSA standard and the sample to be tested, namely reagent A and reagent B are uniformly mixed according to the volume ratio of 50:1, 100 mu L of BCA working solution is added into each hole, the mixture is incubated for about 5 minutes at 37 ℃ in a dark place, the optical density of each sample at the wavelength of 562nm is measured by using an enzyme-labeled instrument, and the protein concentration of each sample is measured according to the standard curve.
(3) Protein sample treatment: the protein sample was diluted to the same concentration and then added to the loading buffer and heated at 97℃for 7min.
(4) Preparation of SDS-PAGE gel plates: preparing 10% separating gel and 5% concentrating gel, and standing for more than 30 min.
(5) Loading and electrophoresis: assembling an electrophoresis tank, adding an electrophoresis buffer solution, adding each experimental sample and protein Maker into a sample loading hole, and carrying out electrophoresis for 90min under the condition of constant pressure of 90V.
(6) Transferring: after electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane (PVDF membrane) and transferred to the membrane for 120min under a constant current of 300 mA.
(7) Closing: the PVDF film was removed, and 5% skim milk powder was added in about 5mL and the mixture was sealed at room temperature for 2 hours.
(8) Incubating primary antibodies: the skim milk powder was discarded, and the primary antibody was diluted in the corresponding proportion with 5% skim milk powder in advance at 4℃overnight.
(9) Washing the film: the primary antibody was recovered and the membrane was washed three times with TBST for ten minutes each.
(10) Incubating a secondary antibody: incubation for 1h at room temperature or 2-3h at 4℃using pre-diluted secondary antibody.
(11) Washing and developing: washed 3 times with TBST for 5 min/time, developed by ECL chemiluminescence and visualized using a gel imager.
The effect of compound 1 on the expression levels of two inflammation-associated proteins, namely cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), in LPS-induced BV-2 cells at a depth of 6.25-25. Mu.M was tested by Western blot, and quercetin was used as a positive control. The results show that the pyrrole alkaloid shows concentration dependence on the expression of two proteins in BV-2 cells induced by LPS, and the results are shown in figures 21-22.
In conclusion, the series of eudesmane type sesquiterpene glycoside compounds show good anti-neuritis activity in the aspect of preventing and treating neurodegenerative diseases, in particular to the compound 1 (Pitovocoside A), and can be developed as a potential lead compound.
The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Claims (7)

1. The eudesmane terpene glycoside compound is characterized by being extracted from erythrina ovata, and has a structure shown in any one of formulas I-VIII:
。
2. an extract of erythrina ovalifolia containing a compound of any one of formulae i to viii in claim 1.
3. A method for preparing the erythrina procumbens extract according to claim 2, comprising the steps of:
extracting erythrina variegata with ethanol, and concentrating to obtain crude extract;
sequentially extracting the crude extract with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract phase, ethyl acetate extract phase and n-butanol extract phase respectively;
and drying the ethyl acetate extract phase to obtain the erythrina ovata extract.
4. The preparation method according to claim 3, wherein the specific operation process of extracting the erythrina ovata with ethanol is as follows: extracting erythrina ovata by using 95% ethanol solution with a mass fraction as a solvent for 4-6 times at 75 ℃ by adopting a heating reflux method, 4-5 hours each time, and mixing and concentrating the extracting solutions to obtain the extract;
wherein the dosage ratio of the dried erythrina procumbens to the 95% ethanol solution is 1g to 4-6 mL.
5. The preparation method according to claim 4, wherein the specific operation process of extracting the crude extract sequentially by petroleum ether, ethyl acetate and n-butanol is as follows: and after the crude extract is uniformly dispersed by water, extracting for 3-6 times by petroleum ether, ethyl acetate and n-butanol in sequence, wherein the volume ratio of the aqueous dispersion of the crude extract to the petroleum ether, ethyl acetate or n-butanol is 1:1-1.5.
6. A pharmaceutical composition comprising at least one compound of formulae i to viii in claim 1, and pharmaceutically acceptable excipients.
7. Use of the eudesmane-type terpene glycoside compound according to claim 1, the erythrina procumbens extract according to claim 2 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for preventing and treating neurodegenerative diseases.
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