CN116589516A - Dendrobium candidum sesquiterpene glycoside A with Alzheimer disease treatment effect, and preparation method and application thereof - Google Patents
Dendrobium candidum sesquiterpene glycoside A with Alzheimer disease treatment effect, and preparation method and application thereof Download PDFInfo
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- CN116589516A CN116589516A CN202310221195.1A CN202310221195A CN116589516A CN 116589516 A CN116589516 A CN 116589516A CN 202310221195 A CN202310221195 A CN 202310221195A CN 116589516 A CN116589516 A CN 116589516A
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- Prior art keywords
- dendrobium candidum
- sesquiterpene glycoside
- glycoside
- sesquiterpene
- disease
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Abstract
The invention discloses a novel dendrobium candidum sesquiterpene glycoside A with an Alzheimer disease treatment effect, and a preparation method and application thereof. According to the invention, through carrying out systematic deep research on chemical components of dendrobium candidum, the analysis of data such as spectrum, mass spectrum and the like shows that a novel sesquiterpene glycoside compound (dendrobium candidum sesquiterpene glycoside A) is separated from dendrobium candidum. According to the invention, the dendrobium candidum sesquiterpene glycoside A is acted on the pathology model of the Alzheimer's disease caenorhabditis elegans, so that the dendrobium candidum sesquiterpene glycoside A has a remarkable treatment effect on the caenorhabditis elegans of the pathology model of the Alzheimer's disease, is shown to remarkably delay the muscle paralysis phenotype of the caenorhabditis elegans of the Alzheimer's disease, and has the potential of treating neurodegenerative diseases.
Description
Technical Field
The invention relates to the field of medicines, in particular to a novel natural extracted sesquiterpene glycoside compound separated from traditional Chinese medicine dendrobium clavatum (Dendrobium findlayanum Par et Rchb.f.), and application thereof in preparation of medicines for preventing or treating Alzheimer's disease.
Background
Alzheimer's Disease (AD) is a central nervous system degenerative disease which is mainly progressive cognitive dysfunction and memory impairment, and is the most common one of Alzheimer's disease. Clinically, the dementia is characterized by memory loss, cognitive dysfunction, language disorder, abnormal emotion control and the like.
Senile plaques formed by extracellular amyloid (aβ protein) aggregation and intracellular neurofibrillary tangles are two major pathological phenomena of AD. Modern medicine has been studying the pathogenesis of AD for many years, but due to its complex etiology, the pathogenesis of this disease has not yet been defined, and the "aβ cascade hypothesis" is one of the mechanisms currently widely accepted by the scientific community. The theory considers that Abeta deposited abnormally in the brain of a patient directly or indirectly acts on neurons and glial cells through a series of cascade reactions such as free radical reaction, mitochondrial oxidative damage, inflammatory reaction and the like, finally leads to abnormal neuron functions or death, causes cognitive injury and memory decline, and finally leads to dementia. Currently, aβ amyloid is one of the most accepted targets for screening anti-AD drugs.
The pathogenesis of AD is complex, the course of disease is long, the onset links are more, long-term administration is needed, and currently marketed anti-Alzheimer disease drugs comprise acetylcholinesterase inhibitors (such as rivastigmine) and N-methyl-D-aspartate receptor (NMDA receptor) antagonists (such as memantine), but the drugs are expensive, and the side effects of hallucinations, consciousness chaos, dizziness, headache, tiredness and the like are obvious after taking the drugs, and only the symptoms, but not the etiology, are aimed at. Thus, currently clinically applied anti-AD drugs only control but do not reverse the patient's condition. In this case, it is urgent to find more effective anti-AD drugs.
Caenorhabditis elegans (Caenorhabditis elegans, c.elegans) is a very useful platform for the screening of multifunctional drugs and the study of the mechanism of action of the drugs. It is cheap and easy to culture; the breeding speed is high, the life cycle is short, the number of offspring is large, a large number of individuals with consistent background can be obtained, the experimental repeatability is ensured, and the experiment is performed by adopting a large sample size, so that the influence of individual difference is eliminated; neurotransmitters that are highly conserved in gene and molecular pathways with higher organisms and that are similar to humans. As a bridge from cell in-vitro level drug primary screening to mouse in-vivo level drug secondary screening, the novel compound can be widely applied to the research of neurodegenerative diseases such as aging resistance, AD, parkinsonism and the like. The humanized caenorhabditis elegans AD pathological model is prepared from human A beta 1-42 The gene is transferred into downstream of myoglobin promoter and expressed under the control of temperature, and said nematode strain can be normally grown at 15 deg.C, when transferred into 25 deg.C condition, Aβ 1-42 Expressed and accumulated in its muscle, causing the nematodes to lose their ability to exercise and paralyze. The addition of the test drug delays the muscle paralysis phenotype, the result being expressed as a percentage of the number of muscle paralysis nematodes in the population of test nematodes. The higher the ratio of unparalysis nematodes, the more pronounced the anti-AD effect of the drug. This model has long been used for the study of the mechanism of occurrence of anti-AD disease and the mechanism of action of anti-AD drugs and candidates.
Dendrobium (Dendrobium) is the second largest genus of Orchidaceae (Orchidaceae), about 1500 species worldwide, mainly distributed in Asian tropical and subtropical, australian and Pacific islands, and China is mainly distributed in southwest, south China, etc. The dendrobium nobile is a fresh or dried stem of dendrobium nobile Dendrobium nobile, dendrobium huoshanense D.huoshanense, dendrobium chrysotoxum D.fimbriatum or dendrobium fimbriatum cultivated by Orchidaceae (Orchidaceae) in 2020 edition of Chinese pharmacopoeia, and similar plant species, and comprises the dendrobium nobile D.findlayanum. In the traditional Chinese medicine, the dendrobium is a common precious medicinal material, has long medicinal history, and has the effects of benefiting stomach, promoting fluid production, nourishing yin, clearing heat and the like. Researches show that the dendrobium plant chemical components have various structural types and mainly contain sesquiterpene glycoside, alkaloid, stilbene compounds and the like. Modern pharmacological researches have proved that Dendrobium has the effects of resisting tumor, relieving inflammation, enhancing immunity, resisting AD and treating cataract. Currently there are few reports of monomeric compounds of dendrobe plants against AD.
Disclosure of Invention
The invention aims to: the invention aims to provide a novel medicine or health care product for preventing or treating AD.
The technical scheme is as follows: in order to achieve the aim, the invention provides a dendrobium candidum sesquiterpene glycoside A (11, 15-dihydroxypichroxane 15-di-O-beta-D-glucopyranoside), which is found for the first time and the application of the dendrobium candidum sesquiterpene glycoside A in pharmacy is also found for the first time.
The pharmaceutical application is specifically embodied in the preparation of medicines or health care products for preventing or treating AD.
Compared with the prior art, the invention has the following advantages:
the invention confirms that the invention has the following pharmacological researches on the monomer compound dendrobium candidum sesquiterpene glycoside A
The paralytic phenotype of the caenorhabditis elegans with the Alzheimer's disease is obviously delayed, the caenorhabditis elegans with the Alzheimer's disease has the effect of resisting the Alzheimer's disease, lays a foundation for subsequent drug effect research, further clinical research and guiding drug administration, and expands the variety of Alzheimer's disease treatment drugs.
The technical scheme adopted by the invention is as follows:
a novel sesquiterpene glycoside compound with AD resisting effect, namely a dendrobium candidum sesquiterpene glycoside A, has the following structural formula:
an extraction and separation method of dendrobium candidum sesquiterpene glycoside A with the effect of treating Alzheimer's disease comprises the following steps:
(1) Pulverizing dried stem of Dendrobium Officinale, extracting with ethanol, filtering, mixing extractive solutions, and recovering ethanol under reduced pressure until no ethanol smell; adding proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, concentrating the extracts under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Taking the n-butanol extract in the step (1), performing MCI gel CHP 20P column chromatography, performing ethanol-water gradient elution, and detecting and combining the same fractions by TLC to obtain dendrobium candidum total sesquiterpene glycoside;
(3) Dividing the dendrobium candidum total sesquiterpene glycoside obtained in the step (2) by RP-18 column chromatography, eluting with methanol-water gradient, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; subjecting Fraction A to silica gel column chromatography, eluting with ethyl acetate-methanol, and mixing to obtain 4 fractions A1-A4; separating Fractiona1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions, wherein Fractiona 1.1.1-Fractiona 1.1.3; the Fractiona1.1.1 is purified by semi-preparative HPLC to obtain the compound of the invention, dendrobium sesquiterpene glycoside A.
As a preferred scheme, the extraction and separation method of the dendrobium candidum sesquiterpene glycoside A with the effect of treating Alzheimer's disease comprises the following steps of:
(1) Taking dry dendrobium candidum stems, crushing, extracting for 2-4 times by using 95% ethanol in a cold leaching way, stirring every time for 1-5 days, filtering, and recovering ethanol under reduced pressure until no alcohol smell exists; adding a proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, extracting each time with 8L of organic solvent for 3 times, concentrating under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Subjecting the n-butanol extract obtained in the step (1) to MCI gel CHP 20P column chromatography, sequentially eluting with ethanol-water gradient of volume ratio of 10:90,30:70,50:50 and 70:30, and detecting and combining the same fractions by TLC to obtain herba Dendrobii total sesquiterpene glycoside;
(3) Taking the dendrobium candidum total sesquiterpene glycoside obtained in the step (2), separating by RP-18 column chromatography, sequentially eluting with methanol-water gradient with the volume ratio of 20:80,40:60,60:40 and 80:20, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; separating the Fraction A by silica gel column chromatography, eluting with ethyl acetate-methanol with volume ratio of 11:1, and mixing to obtain 4 Fraction A1-A4; separating the Fraction A1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions A1.1.1-A1.1.3; taking part Fr.A1.1 of the compound and purifying the part Fr.A1.1 by semi-preparative HPLC to obtain the compound; the semi-preparative HPLC purification conditions were: the mobile phase is methanol-water with the volume ratio of 42:58, the flow rate is 3mL/min, and the detection wavelength is 210nm. The structure of the structure is shown in figure 1,
preferably, the extraction and separation method of the dendrobium candidum sesquiterpene glycoside A with the effect of treating the Alzheimer's disease in the step (1) comprises a cold leaching method, a percolation method, a microwave extraction method, an ultrasonic extraction method, a reflux extraction method or a continuous reflux extraction method.
The dendrobium candidum sesquiterpene glycoside A with the AD resisting function is prepared into tablets, capsules, injection, powder injection, granules, fat emulsion, microcapsules, dripping pills, ointment or transdermal controlled release patch and other dosage forms by using the dendrobium candidum sesquiterpene glycoside A with the AD resisting function, wherein the 8 beta-hydroxy-cyclopacampran-5-one 8-O-beta-D-glucopyranoside and a pharmaceutically acceptable carrier.
When the dendrobium candidum sesquiterpene glycoside A provided by the invention is prepared into tablets, the dendrobium candidum sesquiterpene glycoside A and lactose or corn starch are added with a lubricant magnesium stearate when needed, uniformly mixed, granulated and tabletted to prepare the tablets.
When the dendrobium candidum sesquiterpene glycoside A provided by the invention is prepared into capsules, the dendrobium candidum sesquiterpene glycoside A and a carrier lactose or corn starch are uniformly mixed, granulated and then encapsulated into capsules.
When the dendrobium candidum sesquiterpene glycoside A provided by the invention is prepared into granules, the dendrobium candidum sesquiterpene glycoside A and a diluent lactose or corn starch are uniformly mixed, granulated, dried and prepared into granules.
The dendrobium candidum sesquiterpene glycoside A provided by the invention is prepared by adding a carrier when preparing a powder injection and an injection according to a conventional pharmaceutical method.
The dendrobium candidum sesquiterpene glycoside A provided by the invention is prepared by adding a carrier into the preparation formulations such as fat emulsion, ointment or transdermal controlled release patch according to a conventional pharmaceutical method.
Of course, the dendrobium candidum sesquiterpene glycoside A is extracted from dendrobium candidum or can be artificially synthesized by adopting a drug synthesis method.
The beneficial effects are that:
1. the invention discloses a preparation method for extracting and separating dendrobium candidum sesquiterpene glycoside A from dendrobium candidum for the first time. Cold soaking at room temperature, separating by chromatography and semi-preparative HPLC.
2. According to the invention, the dendrobium candidum sesquiterpene glycoside A is a novel compound which is obtained by first separating and identifying dendrobium candidum through data analysis and identification such as spectrum and mass spectrum.
3. Cell experiments prove that the dendrobium nobile sesquiterpene glycoside A obtained by separation can obviously delay the paralytic phenotype of the Alzheimer's disease caenorhabditis elegans, and has obvious inhibition effect on the paralytic phenotype caused by the intramuscular A beta over-expression of the AD caenorhabditis elegans. Therefore, the dendrobium candidum sesquiterpene glycoside A provided by the invention has potential of resisting AD, and can be applied to preparing medicines for preventing or treating AD.
Drawings
FIG. 1 is a structural diagram of dendrobe sesquiterpene glycoside A provided by the invention;
FIG. 2 shows the effect of different concentrations of dendrobe sesquiterpene glycoside A on delaying paralysis of the AD caenorhabditis elegans according to the invention;
FIG. 3 shows the herba Dendrobii sesquiterpene glycoside A of the present invention 1 H NMR spectroscopy;
FIG. 4 shows a herba Dendrobii sesquiterpene glycoside A of the present invention 13 C NMR spectrum;
FIG. 5 is a HSQC spectrum of Dendrobium candidum sesquiterpene glycoside A of the present invention;
FIG. 6 is an HMBC spectrum of dendrobe sesquiterpene glycoside A of the present invention;
FIG. 7 is a COSY spectrum of Dendrobium candidum sesquiterpene glycoside A of the present invention;
FIG. 8 is a ROESY spectrum of Dendrobium candidum sesquiterpene glycoside A of the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1 selection of pathological models
The invention uses the transgenic caenorhabditis elegans strain CL4176 as a pathological model for screening and preventing or treating Alzheimer's disease to evaluate the dendrobium candidum sesquiterpene glycoside A for preventing or treating Alzheimer's disease. The nematode strain can grow normally at 15 ℃, and Aβ when transferred to 25 DEG C 1-42 Expressed and accumulated in its muscle, resulting in nematodesLoss of exercise capacity and paralysis. The addition of the test drug delays the muscle paralysis phenotype, the result being expressed as a percentage of the number of muscle paralysis nematodes in the total number of the test nematode population. The higher the ratio of unparalysis nematodes, the more pronounced the anti-AD effect of the drug.
Example 2 extraction separation of preparation of Dendrobium candidum sesquiterpene glycoside A
(1) Taking 5kg of dried dendrobium candidum stems, crushing, carrying out cold leaching extraction for 4 times by using 95% ethanol for 5 days each time, stirring at intervals, filtering, and recovering ethanol under reduced pressure until no ethanol smell exists; adding proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, extracting with 8L of organic solvent each time for 3 times, concentrating under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract for later use.
(2) Subjecting the n-butanol extract obtained in the step (1) to MCI gel CHP 20P column chromatography, and subjecting to ethanol/water (volume ratio 10:90,30:70,50:50, 70:30) gradient elution, and combining the same fractions by TLC detection to obtain herba Dendrobii total sesquiterpene glycoside (310 g).
(3) Dividing the dendrobium candidum total sesquiterpene glycoside obtained in the step (2) by RP-18 column chromatography, and performing methanol/water gradient elution (volume ratio is 20:80,40:60,60:40, 80:20), wherein TLC detection is performed to combine identical Fractions to obtain 4 fraction sections of Fractions A-D. And performing silica gel column chromatography on the Fraction A, eluting with ethyl acetate/methanol in a volume ratio of 11:1, and combining to obtain 4 Fraction sections, wherein the Fractions A1-A4 are obtained. The Fractions A1 is taken and separated by Sephadex LH-20 column chromatography, eluted by methanol, and 3 Fractions are obtained by combining A1.1.1-A1.1.3. Fr.A1.1.1 (145 mg)
The preparation is partially carried out by semi-preparative liquid chromatography, and the chromatographic conditions are as follows: chromatographic column: YMC-Pack ODS-A (250 mm. Times.10 mm,5 μm, japan YMC Co.); mobile phase: methanol/water, 42:58 (V/V); detection wavelength: 210nm; flow rate: 3ml/min; column temperature: purifying at 30deg.C to obtain herba Dendrobii sesquiterpene glycoside A (8 mg, t) R ＝53min)。
The structural identification of the dendrobium candidum sesquiterpene glycoside A is as follows:
dendrobium candidum sesquiterpene glycoside A, white amorphous powder, is easily dissolved in methanol. By high resolution mass spectrometry (+) The excimer ion peak given by HR-ESI-MS is at m/z 419.2101 (C 21 H 32 O 7 Na, calculated: 419.2103 Determining the molecular formula as C 21 H 32 O 7 The unsaturation was 6. Hydroxyl groups (3426 cm) were observed from IR spectrum –1 ) And carbonyl (1690 cm) –1 ) Is not shown in the figure). Hydrolysis of the compound with 10% HCl in methanol, detection of glucose from thin layer chromatography in the hydrolysate, and further passage 1 The coupling constant of the terminal protons in the H NMR spectrum (j=7.8 Hz) determines that there is a β -D-glucosyl group in the compound. 13 C NMR, DEPT and HSQC spectra showed 21 carbon signals: 4 methyl groups, 2 methylene groups (1 of which is linked to oxygen), 12 methine groups (6 of which is linked to oxygen), and 3 quaternary carbons, which contain a set of carbon atom signals [ delta ] for glucosyl groups C 106.3(C-1′),75.7(C-2′),77.7(C-3′),71.7(C-4′),78.4(C-5′),62.8(C-6′)]The compound may be a sesquiterpene glycoside. And from 1 Two sets of unimodal methyl signals delta were observed in the H NMR spectrum (Table 1) H 1.16(3H,s,H 3 -14) and 1.34 (3H, s, H 3 -15), two sets of bimodal methyl signals delta H 0.95(3H,d,J＝7.2Hz,H 3 -12) and 0.97 (3 h, d, j=7.2 hz, h 3 -13), and a sugar end group proton signal delta H 4.28(1H,d,J＝7.8Hz,H-1′)。
The C-12 and C-15 positions of the compound of the invention have no hydroxyl substitution; the carbonyl carbon at position C-5 and the linking of the glycosyl group to C-8 is demonstrated by the HMBC related signals of the glycosyl end group protons H '-1 and C-8, as demonstrated by the related signals of H-12 and C-10, C-11, C-13, H-15 and C-2, C-4, C-7, and H-9, H-14, H' -1 and C-8 in the HMBC spectra of the compounds of the invention.
The relevant signals for H-8/H-9α, H-8/H-10, H-9β/H-2, H-2/H-11, H-8/H-14, H-8/H-6, H-14/H-6, H-2/H-15, H-15/H-4 in the ROESY spectrum indicate that H-1, H-6, H-8, H-10, C-14 are in the α -configuration and H-2, H-4, C-15 are in the β -configuration in the compounds of the invention. Therefore, the structure of the compound is determined to be 8 beta-hydroxy-cycloparaffin-5-one 8-O-beta-D-glucopyranoside, and the compound is not reported and is a novel compound, namely dendrobium candidum sesquiterpene glycoside A.
A: 2.0mg (purity 99%) of dendrobium candidum sesquiterpene glycoside A is weighed, 50.0 mu L of dimethyl sulfoxide (DMSO) is added, and 100mM (40 mg/ml) of liquid medicine is prepared for standby.
B: 10. Mu.L of solution A was taken and diluted in a gradient to prepare a 10mM (4.0 mg/ml) 4000. Mu.g/ml solution.
C: 10. Mu.L of the solution B was diluted in a gradient to prepare a 1mM (0.40 mg/ml) 40. Mu.g/ml solution.
Table 1: the dendrobium candidum sesquiterpene glycoside A of the invention 1 H NMR 13 Data assignment by C NMR (methanol-d) 4 ,600MHz；δin ppm；J in Hz)
Example 3 Dendrobium candidum sesquiterpene glycoside A dose-dependently delays muscle paralysis of AD caenorhabditis elegans
1. Biological material
(1) AD nematode strain CL4176 was purchased from Caenorhabditis Genetics Center (CGC); for transgenic lines, human aβ is specifically expressed by muscle at 25 ℃ under induction, and the aβ is gathered in muscle tissues to finally lead to nematode paralysis, and the caenorhabditis elegans CL4176 strain is used as a pathological model for screening anti-AD drugs in the embodiment.
(2) Coli OP50 (uracil leaky mutant), purchased from Caenorhabditis Genetics Center
(CGC) as food for caenorhabditis elegans.
2. Reagent(s)
(1) Memantine hydrochloride, chemical name: 1-amino-3, 5-dimethyladamantanamide hydrochloride, of formula: c (C) 12 H 21 N.hcl, purchased from belvedere technology, CAS:41100-52-1; memantine hydrochloride is a non-competitive NMDA receptor antagonist for the treatment of moderately severe alzheimer's disease patients, which the literature reports significantly inhibiting the paralytic phenotype of transgenic nematode strain CL4176, which was used as a comparative positive drug in this example.
(2) Solid NGM (Nematode Growth Medium) medium composition and preparation (1 liter for example):
| composition of the components | Content of |
| NaCl | 3.01g |
| K 2 HPO 4 | 2.32g |
| KH 2 PO 4 | 17.05g |
| Peptone | 2.65g |
| Agar-agar | 17.08g |
| Make up H 2 O to | 1000mL |
After the solid NGM medium was prepared, the mixture was autoclaved at 121℃for 20min, and 5mg/mL cholesterol 1mL,1M MgSO were added to the sterile console 4 1mL，1M CaCl 2 1mL, shake well, pour hot into sterilized 9cm plates, about 20 mL/plate, and leave stand waiting for the medium to cool and solidify.
(3) M9 buffer formulation
| Composition of the components | Content of |
| NaCl | 4.99g |
| Na 2 HPO 4 | 5.98g |
| KH 2 PO 4 | 3.00g |
| 1M MgSO 4 | 1.00mL |
| Make up H 2 O to | 1000mL |
(4) Preparing a lysate: 6.4% NaClO solution and 1M NaOH solution were mixed in a volume ratio of 1:1.
(5) Preparing memantine hydrochloride solution with optimal concentration of drug effect: 2.0mg of memantine hydrochloride is weighed, 186.04 mu L of dimethyl sulfoxide (DMSO) is added, and 50Mm (10.75 mg/ml) of liquid medicine is prepared for standby.
3. Preparing NGM flat plate
(1) Preparing NGM culture medium (without agar, magnesium sulfate, calcium chloride and cholesterol) according to NGM formula, packaging the culture medium into 6 conical flasks, recording into 6 groups, adding 20ml NGM culture medium liquid into each conical flask, adding 0.343g agar into each conical flask, sealing with sealing film, and sterilizing at 121deg.C for 20 min.
(2) When the temperature of the medium was reduced to 80 ℃, at each ofInto the Erlenmeyer flasks were each charged 20. Mu.L of 1M magnesium sulfate, 20. Mu.L of 1M calcium chloride, 20. Mu.L of 5mg/mL cholesterol. Then, 2mL of deionized water sterilized and cooled to room temperature, 20. Mu.L of dimethyl sulfoxide DMSO, 20. Mu.L of the 100mM dendrobe sesquiterpene glycoside A liquid medicine, 20. Mu.L of the 10mM dendrobe sesquiterpene glycoside A liquid medicine, 20. Mu.L of the 1mM dendrobe sesquiterpene glycoside A liquid medicine, and 20. Mu.L of the 50mM memantine hydrochloride solution were added to each of the conical flasks, respectively. 1.98mL of sterilized and cooled deionized water at room temperature was thoroughly mixed and then equally divided into 3cm diameter dishes, each of which had about 4mL of medium, each of which had 4 dishes, and each of which was designated as a control group (H) 2 O), a control group (DMSO), a dendrobium candidum sesquiterpene glycoside A high concentration group (DF-H), a dendrobium candidum sesquiterpene glycoside A medium concentration group (DF-M), a dendrobium candidum sesquiterpene glycoside A low concentration group (DF-L) and a positive control group (MJG).
Standing and waiting for the culture medium to solidify. Liquid E.coli OP50 was uniformly spread on the medium as a food for nematodes.
4. Implementation steps
(1) Culturing nematodes:
the caenorhabditis elegans are grafted on a solid NGM plate coated with an E.coli OP50, and then placed in an incubator at 20℃for cultivation, and the synchronization treatment is carried out when the nematodes grow into adults.
(2) Nematode synchronization:
an NGM medium containing a large number of adults and having a portion of the nematode eggs hatched is selected, the nematodes are washed from the medium with M9 buffer, transferred to a 15mL centrifuge tube, allowed to stand to allow the nematodes to freely settle to the bottom of the tube, and the supernatant discarded. Adding the lysate into the centrifuge tube according to the amount of nematodes, oscillating for 5-7 minutes on a vortex stirrer, stopping vortex after the nematodes are completely lysed, subpackaging the nematode in a 1.5mL centrifuge tube, and washing eggs with M9 solution three times.
(3) Nematode paralysis count:
nematode eggs split into centrifuge tubes after synchronization are transferred to prepared culture dishes of different groups, each culture dish is used for culturing 60 nematodes, 4 culture dishes are arranged in each group as a parallel, and the culture is carried out for 3 days at 15 ℃ until the nematode L3 phase. To allow the nematodes to express aβ protein, L3-phase nematodes were transferred to 25 ℃ for induction and 34 hours later the number of nematode paralysis was counted. Once every two hours until the nematode paralyzes to about 80% (paralysis means that the nematode is unable to move or only moves its head when it is mechanically stimulated to the body). The results are shown in FIG. 2 as the unparalysis percentage of C.elegans CL4176 by different concentrations of dendrobe sesquiterpene glycoside A. The ordinate of figure 2 shows the percentage of caenorhabditis elegans without paralysis, and at the same time point, a greater value indicates a greater proportion of caenorhabditis elegans without paralysis in the treatment group, i.e. a greater effect of the drug on delaying paralysis.
Wherein, the final concentration of each group of drug-containing culture medium is as follows: the high concentration group (DF-H) of the dendrobium candidum sesquiterpene glycoside A contains the dendrobium candidum sesquiterpene glycoside A of 100 mu M; the concentration group (DF-M) of the dendrobium candidum sesquiterpene glycoside A contains the dendrobium candidum sesquiterpene glycoside A of 10 mu M; the low concentration group (DF-L) of the dendrobium candidum sesquiterpene glycoside A contains the dendrobium candidum sesquiterpene glycoside A of 1 mu M; memantine control (MJG) contained 50 μm memantine hydrochloride.
Experimental results show that the DMSO solvent does not have extra influence on the experiment, the dendrobium candidum sesquiterpene glycoside A-containing drug group with the dendrobium candidum sesquiterpene glycoside A concentration of 100 mu M, 10 mu M and 1 mu M has obvious inhibition effect on the paralysis phenotype caused by the intramuscular Abeta over-expression of the AD caenorhabditis elegans, and compared with the group with the highest drug effect, the dendrobium candidum sesquiterpene glycoside A high concentration and the medium concentration group have obvious effect on delaying the paralysis phenotype of the Alzheimer's disease caenorhabditis elegans, have obvious capability of treating the Alzheimer's disease and have higher medicinal value.
Claims (6)
1. A dendrobium candidum sesquiterpene glycoside A with the effect of treating Alzheimer's disease is characterized by having the following structural formula:
2. the dendrobium nobile sesquiterpene glycoside A with the effect of treating the Alzheimer's disease according to claim 1, wherein the dendrobium nobile sesquiterpene glycoside A and a pharmaceutically acceptable carrier are prepared into a medicine in the form of tablets, capsules, injections, powder injections, granules, fat emulsions, microcapsules, dripping pills, ointments or transdermal controlled release patches.
3. The extraction and separation method of dendrobium candidum sesquiterpene glycoside A with the effect of treating Alzheimer's disease as claimed in claim 1, which is characterized by comprising the following steps:
(1) Pulverizing dried stem of Dendrobium Officinale, extracting with ethanol, filtering, mixing extractive solutions, and recovering ethanol under reduced pressure until no ethanol smell; adding proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, concentrating the extracts under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Taking the n-butanol extract in the step (1), performing MCI gel CHP 20P column chromatography, performing ethanol-water gradient elution, and detecting and combining the same fractions by TLC to obtain dendrobium candidum total sesquiterpene glycoside;
(3) Dividing the dendrobium candidum total sesquiterpene glycoside obtained in the step (2) by RP-18 column chromatography, eluting with methanol-water gradient, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; subjecting Fraction A to silica gel column chromatography, eluting with ethyl acetate-methanol, and mixing to obtain 4 fractions A1-A4; separating Fractiona1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions, wherein Fractiona 1.1.1-Fractiona 1.1.3; taking the Fractiona1.1.1, and purifying by semi-preparative HPLC to obtain the dendrobium candidum sesquiterpene glycoside A.
4. The method for extracting and separating dendrobium candidum sesquiterpene glycoside A with the effect of treating Alzheimer's disease according to claim 3, which is characterized by comprising the following steps:
(1) Taking dry dendrobium candidum stems, crushing, extracting for 2-4 times by using 95% ethanol in a cold leaching way, stirring every time for 1-5 days, filtering, and recovering ethanol under reduced pressure until no alcohol smell exists; adding a proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, extracting each time with 8L of organic solvent for 3 times, concentrating under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Subjecting the n-butanol extract obtained in the step (1) to MCI gel CHP 20P column chromatography, sequentially eluting with ethanol-water gradient of volume ratio of 10:90,30:70,50:50 and 70:30, and detecting and combining the same fractions by TLC to obtain herba Dendrobii total sesquiterpene glycoside;
(3) Taking the dendrobium candidum total sesquiterpene glycoside obtained in the step (2), separating by RP-18 column chromatography, sequentially eluting with methanol-water gradient with the volume ratio of 20:80,40:60,60:40 and 80:20, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; separating the Fraction A by silica gel column chromatography, eluting with ethyl acetate-methanol with volume ratio of 11:1, and mixing to obtain 4 Fraction A1-A4; separating the Fraction A1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions A1.1.1-A1.1.3; taking the Fr.A1.1.1 part of the extract, and purifying by semi-preparative HPLC to obtain Dendrobium candidum sesquiterpene glycoside A; the semi-preparative HPLC purification conditions were: the mobile phase is methanol-water with the volume ratio of 42:58, the flow rate is 3mL/min, and the detection wavelength is 210nm.
5. The method for extracting and separating the dendrobium candidum sesquiterpene glycoside A with the effect of treating the Alzheimer's disease according to claim 3 or 4, wherein the extraction method in the step (1) comprises a cold leaching method, a percolation method, a microwave extraction method, an ultrasonic extraction method, a reflux extraction method or a continuous reflux extraction method.
6. Use of dendrobium candidum sesquiterpene glycoside a of claim 1 in the preparation of a medicament for treating alzheimer's disease.
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