CN113336811A - Iridoid glycoside compounds, preparation and application thereof - Google Patents

Iridoid glycoside compounds, preparation and application thereof Download PDF

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CN113336811A
CN113336811A CN202110610263.4A CN202110610263A CN113336811A CN 113336811 A CN113336811 A CN 113336811A CN 202110610263 A CN202110610263 A CN 202110610263A CN 113336811 A CN113336811 A CN 113336811A
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戚建华
王颖
向兰
潘彦君
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Abstract

The invention discloses iridoid glycoside compounds and preparation and application thereof. The iridoid glycoside compound has a structure shown in the following formula (I):
Figure DDA0003095504410000011
in formula (I): r2Is H, R1is-CH2CH3、‑CH2CH2CH3、‑CH(CH3)2、‑CH2(CH2)2CH3、‑CH2(CH2)3CH3、‑CH2CH2CH(CH3)2or-CH2CH＝C(CH3)2(ii) a Or, R1is-CH2CH2CH(CH3)2，R2is-COOCH2CH3. The iridoid glycoside compound is prepared through geniposide esterification reaction or further glycosylation reaction. The iridoid glycoside compound can be used for preparing antioxidant stress and/or autophagy inducing preparations, and medicines, foods, health products and the like for resisting aging, resisting brain aging and preventing and treating neurodegenerative diseases including Alzheimer's disease.

Description

Iridoid glycoside compounds, preparation and application thereof
Technical Field
The invention relates to the technical field of medicines, and particularly relates to iridoid glycoside compounds and preparation and application thereof.
Background
The development of modern medical standards extends the average life of humans and also significantly increases the number of elderly people. With age, the risk of development of age-related diseases (e.g., alzheimer's disease, diabetes, and cardiovascular disease) continues to increase. Aging has become a risk factor for diseases associated with aging. Thus, the discovery of anti-aging molecules and drugs is an important strategy for the prevention and treatment of geriatric diseases.
In 1956, haman proposed a "free radical theory" with respect to aging, and it was thought that endogenous oxygen free radicals produced by cells in the human body would damage cellular components and cause aging and aging-related diseases. Reactive Oxygen Species (ROS) in cells are mainly derived from mitochondria. Excessive ROS production will lead to the production of harmful substances by the cells, such as Malondialdehyde (MDA), which has a detrimental effect on the physiological and pathological processes of the body. The complex antioxidant defense system (including antioxidant and non-antioxidant enzymes) eliminates ROS. Antioxidant enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPx) and Catalase (CAT). Wherein, the SOD catalyzes the conversion of superoxide anion to hydrogen peroxide, and the hydrogen peroxide is decomposed into water by GPx and CAT to achieve the effect of antioxidation. Non-antioxidant enzymes refer to some non-enzymatic antioxidants in the body, such as glutathione, ascorbic acid, pyruvic acid, flavonoids. The antioxidant system in the body provides an effective idea for anti-aging research. Under the condition of high-concentration oxygen, the over-expression of GPx can improve the oxidation resistance of drosophila and prolong the service life of drosophila. Increasing expression of SOD and CAT also increased stress resistance and prolonged life span of Drosophila.
Autophagy is an evolutionarily highly conserved process of cellular metabolism. It can phagocytose and digest senescent organelles, abnormally folded proteins or pathogens that invade the body, which are the defense mechanisms of the cell itself. At least 40 autophagy-related genes (ATGs) have been reported to encode proteins involved in autophagy. ATG2 is one of the important genes involved in autophagy. The protein ATG2 encoded by ATG2 mediates direct transfer of lipids from the endoplasmic reticulum to a separation membrane to effect expansion of the separation membrane to form autophagosomes. In mammals, the autophagy process involves two Atg2 orthologs, Atg2A and Atg 2B. ATG32 is a gene encoding the transmembrane protein ATG32 of the mitochondrial membrane essential for mitochondrial autophagy. In yeast, the presence of Atg32 protein on the outer mitochondrial membrane can recognize and target unwanted or damaged mitochondria for degradation. Autophagy is a cytoplasmic recovery process that counteracts the accumulation of age-related damaged organelles and proteins, thereby increasing the metabolic adaptation of the cell. An increase in autophagy can significantly extend the longevity of mammals.
Aging models are important tools for studying aging, and there are many types of aging models including yeast, nematodes, drosophila, zebrafish, mice, and the like. The selection of a suitable model according to the characteristics of the model is the key for research. Yeast is unicellular eukaryote, has short growth cycle, easy operation and low cost and can be used for large-scale sample screening. Therefore, the determination of the replicative life span of yeast is a more ideal biological activity evaluation system for screening anti-aging molecules from natural products. Resveratrol is a well-known anti-aging active molecule, can significantly prolong the replicative life of yeast, and is commonly used as a positive control in anti-aging activity evaluation and mechanism research using yeast systems. Therefore, the single cell yeast is used as a biological model for aging research, which lays a foundation for the anti-aging mechanism research of higher organisms.
Furthermore, the PC12 cell line, derived from rat adrenal pheochromocytoma cells, is one of the most prominent models for studying the nervous system at the cellular level. PC12 cells were also used to evaluate the anti-aging activity of molecules based on the association between aging and neurodegenerative diseases.
China is rich in natural resources, and researches on new components and new pharmacological activities in natural products are increasingly widespread. Researches report that Gardenia (Gardenia jasminoides Ellis.) has the effects of oxidation resistance, inflammation resistance, neuroprotection and angiogenesis resistance. In addition, gardenia has abundant chemical components including iridoid glycosides, triterpenoid saponins, flavonoids, organic acid esters, sterols, and pigment compounds. Iridoid glycoside is the main component of Gardenia jasminoides Ellis, and can exert various pharmacological effects, such as antidiabetic, antioxidant, neuroprotective, and hepatoprotective effects. However, studies on anti-aging active molecules derived from gardenia have been reported only in limited amounts.
Disclosure of Invention
Aiming at the technical problems and the defects existing in the field, the invention utilizes a three-level comprehensive biological evaluation system to discover and research anti-aging drugs, namely uses a yeast mutant strain biological activity identification system with short experimental period, low cost and easy operation for preliminary screening and preliminary action mechanism research of active compounds, utilizes a mammal cell PC12 cell model to verify and research action mechanism, further uses an animal model to verify the strategy of in vivo function and mechanism research again to research Chinese medicaments, discovers a lead compound with anti-aging function, and develops anti-brain aging drugs or foods, thereby achieving the purpose of preventing and treating neurodegenerative diseases, in particular Alzheimer's disease. A biological activity identification system is established by using the K6001 yeast mutant strain, two compounds (1 and 2) are obtained by separating and purifying the gardenia extract, the chemical structure is determined, the compound 2 can prolong the replicative life of yeast, and the activity of the compound 1 is not obvious. Eight iridoid glycoside compounds with new chemical structures are designed and synthesized on the basis of the structure-effect relationship research, and finally the compound 8 is determined to be a lead compound through the structure-effect relationship research to carry out the research on anti-aging activity, functions and action mechanisms.
A kind of iridoid glycoside compounds, have the following formula (I) structure:
Figure BDA0003095504390000031
in formula (I):
R2is H, R1is-CH2CH3、-CH2CH2CH3、-CH(CH3)2、-CH2(CH2)2CH3、-CH2(CH2)3CH3、 -CH2CH2CH(CH3)2or-CH2CH＝C(CH3)2Sequentially marked as compounds 3-9; alternatively, the first and second electrodes may be,
R1is-CH2CH2CH(CH3)2，R2is-COOCH2CH3And is denoted as compound 10.
The iridoid glycoside compounds provided by the invention have obvious anti-aging activity in a yeast model, wherein the compound 8 has the best activity, the anti-aging activity and the action mechanism of the iridoid glycoside compounds are researched in the yeast model and a mammalian cell model, and a foundation is laid for the research and development of subsequent related medicaments.
The invention also provides a preferable preparation method of the iridoid glycoside compound, and the preparation method of the compound 3-9 comprises the following steps: at room temperature, geniposidic acid is added into anhydrous alcohol solution containing N, N' -Dicyclohexylcarbodiimide (DCC) and DMAP (4-dimethylaminopyridine), the obtained mixture is stirred at room temperature to 55 ℃ to be fully reacted, then vacuum concentration is carried out, the concentrated product is extracted by EtOAc (ethyl acetate), and the organic phase is extracted by Na2SO4Drying and passing throughFiltering and concentrating, separating the crude product by silica gel column chromatography, and purifying by HPLC (high performance liquid chromatography) to obtain corresponding compound 3-9; the alcohol is HO-R1。
The preparation method of the compound 10 comprises the following steps: to a reaction mixture containing scandium trifluoromethanesulfonate (Sc (OTf))3) Adding the compound 8 into an anhydrous toluene/ethanol mixed solution of excessive diethyl pyrocarbonate (DEPC), heating and stirring the obtained mixture at 50-60 ℃ for full reaction, cooling to room temperature, and adding saturated NaHCO3The solution was quenched, filtered, concentrated in vacuo, the concentrate extracted with EtOAc and the organic phase over Na2SO4Drying, filtration and concentration, the crude material obtained was isolated by silica gel column chromatography and then purified by HPLC to give compound 10.
In the preparation of compounds 3-9: DCC and DMAP are used as catalysts, the use amount is adjustable, and in a preferred embodiment, the molar ratio of DCC to DMAP to geniposide is 4:1: 2-4; the alcohol serves as both a solvent and a reactant, generally in significant excess; stirring fully, and reacting for a long time, wherein the reaction time is generally one night or 12-24 hours; the EtOAc extraction can be performed several times.
For compounds 3-7, 9, silica gel column chromatography was performed using DCM (dichloromethane)/MeOH (methanol) in a volume ratio of 95: 5.
For compound 8, silica gel column chromatography was performed using EtOAc/MeOH in a volume ratio of 95: 5.
For the compound 3, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 30: 70-50: 502Performing gradient elution for 40min with O as mobile phase at flow rate of 3mL/min, detecting absorbance at wavelength of 210nm, and keeping for 16.3 min.
For the compounds 4 and 5, HPLC adopts C18 liquid chromatography column 5C18-AR-II phi 10 x 250mm, and adopts MeOH/H with volume ratio of 50: 50-100: 02Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 30min, detecting absorbance at wavelength of 210nm, and keeping for 10.7 min.
For Compound 6, HPLC was performed using a C18 liquid chromatography column 5C18-AR-II Φ 10X 250mm, using volumeMeOH/H in a ratio of 50:50 to 100:02Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 30min, detecting absorbance at wavelength of 210nm, and keeping for 12.9 min.
For the compound 7, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 50: 50-65: 352Performing gradient elution for 40min with O as mobile phase at flow rate of 3mL/min, detecting absorbance at wavelength of 210nm, and keeping for 23.3 min.
For the compound 8, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 30: 70-50: 502Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 45min, detecting absorbance at wavelength of 210nm, and keeping for 24.0 min.
For the compound 9, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 50: 50-65: 352Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 40min, detecting absorbance at wavelength of 210nm, and keeping for 14.1 min.
In the preparation of compound 10: diethyl pyrocarbonate is used as a reactant, and the amount of the diethyl pyrocarbonate is usually slightly excessive, and in a preferable embodiment, the molar ratio of the diethyl pyrocarbonate to the compound 8 is 1.01-1.3: 1; scandium trifluoromethanesulfonate is used as a catalyst, the dosage of the scandium trifluoromethanesulfonate is adjustable, and in a preferable embodiment, the molar ratio of the scandium trifluoromethanesulfonate to the compound 8 is 0.05: 1; the volume ratio of the toluene to the ethanol is adjustable, and in a preferred embodiment, the volume ratio of the toluene to the ethanol is 4: 1; in a preferred embodiment, the reaction temperature is 55 ℃; the time for stirring and fully reacting is generally not less than 5 hours; the EtOAc extraction can be carried out for many times; eluent adopted by silica gel column chromatography is DCM/MeOH with the volume ratio of 98: 2; HPLC adopts C18 liquid chromatography column 5C18-MS-II phi 10 x 250mm, and adopts MeOH/H with volume ratio of 50: 50-70: 302Performing gradient elution for 60min with O as mobile phase at flow rate of 3mL/min, detecting absorbance at 210nm wavelength, and keeping for 47 min.
The invention also provides application of the iridoid glycoside compound in preparation of a preparation for resisting oxidative stress and/or inducing autophagy.
The invention also provides application of the iridoid glycoside compound in preparation of drugs, foods and health products for resisting aging and brain aging and preventing and treating neurodegenerative diseases including Alzheimer's disease, wherein the iridoid glycoside compound has an action mechanism of resisting oxidative stress and/or inducing autophagy. The medicine, food and health care product can be a solid preparation or a liquid preparation, can be prepared according to the conventional production method in the fields of pharmacy, food and health care products, can be prepared into required dosage forms, can be added with pharmaceutically acceptable carriers and the like in the preparation process, and can be in various forms, such as tablets, capsules, oral liquid, small needles, infusion, ointment, freeze-dried powder injection, liniment or suppositories.
The food and the health-care product can be composed of the iridoid glycoside compound and a pharmaceutically acceptable carrier of the food or the health-care product. The pharmaceutically acceptable carrier as used herein refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as diluent, etc., filler such as sucrose, starch, etc.; adhesive such as hydroxypropyl cellulose, starch slurry, etc., humectant such as magnesium stearate, silica gel micropowder, etc., absorption enhancer such as polysorbate, lecithin, etc., and surfactant such as sorbitan fatty acid, poloxamer, etc. In addition, other adjuvants such as sweetener and flavoring agent can be added into the food and health product.
The drug is a drug in a gastrointestinal administration dosage form or a drug in a non-gastrointestinal administration dosage form, and can be administered in a unit dosage form. The mode of administration without gastrointestinal tract as a drug for external use includes injection, including intravenous injection, intraperitoneal injection, intramuscular injection, acupoint injection, subcutaneous injection, and the like.
The invention also provides a medicament, food and health care product, the iridoid glycoside compound is contained in the composition, has the effects of resisting oxidative stress and/or inducing autophagy, and can be used for resisting aging, resisting brain aging and preventing and treating neurodegenerative diseases including Alzheimer disease.
Compared with the prior art, the invention has the following remarkable technical effects: the invention synthesizes eight new iridoid glycoside compounds on the basis of two separated natural products, the anti-aging activity is shown to different degrees through the evaluation of a yeast model activity system, one compound (compound 8) with more obvious anti-aging effect is used as a lead compound for the action mechanism research, and the research finds that the compound improves the activity of SOD and CAT enzyme to play obvious anti-oxidative stress activity by inhibiting the content of ROS and MDA in a yeast model, thereby playing the anti-aging effect. The compound also has an anti-aging effect by inducing autophagy, and can be applied to the treatment and prevention of senile related diseases (such as Alzheimer disease and the like). In addition, the compound 8 has obvious antioxidant stress activity in PC12 cells derived from mammalian cells, can obviously prolong the time-sequence life of PC12 cells, and has an anti-aging effect in higher organisms.
Drawings
FIG. 1 is a graph showing the structure (a) of compounds 1 to 10 and the effect on the replicative life of yeast at different concentrations;
FIG. 2 is a graph showing the effect of Compound 8(a) on the replicative longevity (b) and the chronological longevity (c) of yeast;
FIG. 3 is a graph showing the results of the effect of Compound 8 on the survival rate of yeast under oxidative stress conditions and on the antioxidant activity in yeast, wherein: (a) h at 9.5mM2O2Photographs of yeast growth after treatment with Compound 8 under induced oxidative stress conditions, (b) at 5mM H2O2(ii) a change in the viability of the yeast under oxidative conditions, (c-d) the effect of compound 8 on ROS and MDA levels, (e-h) a change in T-SOD, SOD1, CAT and GPx enzyme activity in the yeast after 48h treatment with compound 8;
FIG. 4 is a graph showing the effect of Compound 8 on autophagy in yeast, wherein: (a) fluorescence images (green) of YOM38 yeast containing free GFP after treatment with Resveratrol (RES) or compound 8 observed with two-photon confocal fluorescence microscopy, (b) percentage of YOM38 cells containing free GFP (green), seven pictures in each group containing over 60 cells for statistical analysis, (c) western blot analysis of GFP-Atg8 and free GFP in yeast after 22 hours of treatment of Resveratrol (RES) or compound 8 in SD medium, (d) numerical results of (c), (e) western blot analysis of GFP-Atg8 and free GFP in yeast after treatment with Resveratrol (RES) or 1 μ M compound 8, (f) numerical results of (e), (g-h) treatment of 10 μ M Resveratrol (RES) and 1 μ M compound 8, replicative life span of the Δ atg2 and Δ atg32 mutants of yeast;
FIG. 5 shows H in PC12 cells by Compound 82O2A graph of neuroprotective effects of induced oxidative damage; wherein: (a) with different concentrations of H2O2Relative viability of PC12 cells after 1H of treatment, following H2O2The survival rate is obviously reduced compared with the control group by increasing the concentration; (b) in the presence or absence of H2O2Compound 8 exerts neuroprotective effects at doses of 0.3, 1 and 3 μ M upon stimulation;
FIG. 6 shows the pair of compounds 8 and H2O2Results plot of effects of ROS, MDA, SOD enzyme activity in induced damaged PC12 cells; wherein: (a) compound 8 pairs with or without H as detected by a fluorescent microplate reader2O2Effect of induced ROS production in PC12 cells; (b) the MDA content level was measured by a corresponding assay kit, PC12 cells were pretreated with RES (10. mu.M) and different concentrations of 8(3 and 10. mu.M), respectively, for 48H and then placed in H2O2(0.8mM) for 1 h; (c-d) T-SOD and SOD1 levels were measured by corresponding assay kits, PC12 cells were pretreated with RES (10. mu.M) and different concentrations of 8(3 and 10. mu.M) for 48H, respectively, and then placed in H2O2(0.8mM) for 1 h;
FIG. 7 is a graph showing the effect of Compound 8 on the chronological lifespan of PC12 cells in pseudoyeast; wherein: (a) viable Colony Forming Units (CFU) of PC12 cells; (b) the corresponding number of cell colonies in (a).
Detailed Description
The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer. Unless otherwise specified, the ratios of reagents used in the separation and purification operations are volume ratios.
Compounds 1-10 are prepared as shown in the following structures:
Figure BDA0003095504390000071
1. extraction and structural identification of Compounds 1, 2
The method for obtaining the compounds 1 and 2 from the methanol extract of the gardenia comprises the following specific steps:
(1) gardenia jasminoides Ellis (dry weight 1.5 kg) was pulverized and extracted with methanol at room temperature for 24 hours with shaking. After filtration, the filtrate was concentrated in vacuo to give 203.2g of extract. The extract was dissolved in water, and the water-soluble substance was partitioned sequentially using n-hexane, dichloromethane, ethyl acetate and n-butanol.
(2) An active n-butanol layer (50.0g) was selected and separated by ODS column chromatography. The solvent system used was methanol/water (20/80, 40/60, 60/40, 80/20, 100/0). The fractions were then combined into 10 samples by TLC analysis.
(3) Active sample 3(24.1 g) from the methanol/water (40/60) elution system was again isolated using an ODS column and methanol/water solvent system (30/70, 35/65, 40/60, 45/55, 50/50, 70/30, 100/0), and the fractions were combined into 6 samples by TLC analysis.
(4) 2g of sample 2 (total 14.6g) obtained from the methanol/water (30/70) eluate was separated by silica gel column chromatography and methylene chloride/methanol elution system (100/0, 99/1, 95/5, 90/10, 85/15, 80/20, 70/30, 40/60, 0/100), and the fractions were combined into 13 samples by TLC analysis.
(5) Sample 6(260mg) from dichloromethane/methanol (80/20) was subjected to a first HPLC (5C18-AR-II Φ 10 × 250mm, mobile phase methanol: water ═ 10: 90-50: 50 gradient elution for 30min, mobile phase flow rate 3mL/min, absorbance detected at 210nm wavelength) to give sample a (120mg, retention time t:)R10.3 min). Sample a was purified by a second HPLC (5C18-AR-II Φ 10 × 250mm, mobile phase acetonitrile: water ═ 12:88, mobile phase flow rate 3mL/min, 210nm) three times to give Compound 1(60mg, t)R25.6 min). Identifying the structure by spectral analysis, and1h NMR and HR ESI-TOF-MS data were compared to literature.
(7) Sample 4(48.1mg) from dichloromethane/methanol (95/5) was taken 15mg and purified by HPLC (5C18-AR-II Φ 10 × 250mm, mobile phase methanol: water ═ 20: 80, mobile phase flow rate 3mL/min, 210nm) to give compound 2(11.2mg, t:, nR34.2 min). Identifying the structure by spectral analysis, and1h NMR and HR ESI-TOF-MS data were compared to literature.
The structures of the compounds 1 and 2 extracted by the method are identified:
the structure of the compound 1 is shown by HR ESI-MS,1H NMR and comparison with the literature. Compound 1, geniposide, white solid, [ alpha ]]16D+15.2(c 0.6,CH3OH),HR ESI-TOF-MS m/z 397.1095,calcd. for C16H22NaO10(M+Na)+397.1105.1H NMR(500MHz,methanol-d4):δ＝7.51(1H,s), 5.80(1H,brs),5.16(1H,d,J＝7.8Hz),4.72(1H,d,J＝7.9Hz),4.32(1H,d,J＝14.4Hz), 4.19(1H,d,J＝14.4Hz),3.84(1H,brd,J＝11.8Hz),3.64(1H,m),3.38(1H,m),3.17-3.29 (4H,m),2.84(1H,dd,J＝8.2,16.3Hz),2.72(1H,t,J＝7.8Hz),2.08(1H,dd,J＝8.2,16.3 Hz).
The structure of the compound 2 is shown by HR ESI-MS,1H NMR and the results of comparison with the literature. Compound 2 geniposide, white solid, [ alpha ]]16D+10.1(c 2.54,CH3OH),HR ESI-TOF-MS m/z 411.1269,calcd.for C17H24NaO10(M+Na)+411.1262.1H NMR(500MHz,methanol-d4):δ＝7.51(1H,s),5.80 (1H,brs),5.17(1H,d,J＝7.7Hz),4.71(1H,d,J＝7.9Hz),4.31(1H,d,J＝14.4Hz),4.18 (1H,d,J＝14.4Hz),3.86(1H,brd,J＝12.7Hz),3.71(3H,s),3.63(1H,dd,J＝5.0,12.7Hz), 3.22-3.40(4H,m),3.18(1H,m),2.82(1H,dd,J＝8.2,16.4Hz),2.72(1H,t,J＝7.7Hz), 2.10(1H,dd,J＝8.2,16.4Hz).
2. Synthesis and structural identification of compounds 3-10
At room temperature,to a solution of DCC (5.4mg, 0.026mmol) and DMAP (0.8mg, 0.0065mmol) in absolute ethanol (1mL) was added geniposidic acid (5mg, 0.013 mmol). The mixture was stirred at room temperature overnight and then the mixture was concentrated under high vacuum. The concentrate was extracted 3 times with EtOAc, 5mL each. The organic phase is passed through Na2SO4Drying, filtering and concentrating. The crude material was purified by column chromatography on silica gel (eluent DCM: MeOH ═ 95: 5) and then by HPLC (5C18-AR-II Φ 10X 250mm, mobile phase MeOH: H2O-30: 70-50: gradient elution with 50 deg.C for 40min, mobile phase flow rate of 3mL/min, absorbance at 210nm) to obtain compound 3(1.0mg, yield 19%, t)R＝16.3min)。
The synthesis, isolation and purification process of compound 4 is referred to compound 3, except that: geniposide (10mg), ethanol (1mL) is replaced by n-propanol (2mL), HPLC adopts C18 liquid chromatography column 5C18-AR-II phi 10 x 250mm, and adopts MeOH/H with volume ratio of 50: 50-100: 02Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 30min, detecting absorbance at wavelength of 210nm, and keeping for 10.7 min. Compound 43.6 mg is obtained in 32% yield.
The synthesis, isolation and purification process of compound 5 is referred to compound 4, except that: n-propanol was replaced by isopropanol. Compound 52.3 mg is obtained in 21% yield.
The synthesis, isolation and purification process of compound 6 is referred to compound 3, except that: geniposide (10mg), ethanol (1mL) replaced by n-butanol (2mL), HPLC using C18 liquid chromatography column 5C18-AR-II Φ 10 × 250mm, MeOH/H at volume ratio of 50: 50-100: 02Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 30min, detecting absorbance at wavelength of 210nm, and keeping for 12.9 min. Compound 65.0 mg was obtained in 44% yield.
The synthesis, isolation and purification process of compound 7 is referred to compound 3, except that: geniposide (10mg), ethanol (1mL) replaced by n-pentanol (3mL), HPLC using C18 liquid chromatography column 5C18-AR-II phi 10X 250mm, MeOH/H with volume ratio of 50: 50-65: 352Performing gradient elution with O as mobile phase for 40minThe flow rate of the mobile phase is 3mL/min, the absorbance is detected at the wavelength of 210nm, and the retention time is 23.3 min. Compound 73.2 mg was obtained in 27% yield.
The synthesis, isolation and purification process of compound 8 is referred to compound 3, except that: geniposide (50mg), replacing ethanol (1mL) with isoamylol (4mL), reacting at 55 deg.C, eluting with 95:5 volume ratio EtOAc/MeOH by silica gel column chromatography, separating with C18 liquid chromatography column 5C18-AR-II phi 10 × 250mm by HPLC, and eluting with 30: 70-50: 50 volume ratio MeOH/H2Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 45min, detecting absorbance at wavelength of 210nm, and keeping for 24.0 min. Compound 838.6 mg was obtained in 65% yield.
The synthesis, isolation and purification process of compound 9 is referred to compound 3, except that: geniposide (10mg), ethanol (1mL) is replaced by isopentenol (3mL), HPLC adopts C18 liquid chromatography column 5C18-AR-II phi 10 x 250mm, and adopts MeOH/H with volume ratio of 50: 50-65: 352Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 40min, detecting absorbance at wavelength of 210nm, and keeping for 14.1 min. Compound 94.2 mg is obtained in 36% yield.
To DEPC (1.2eq.15.5mg) and Sc (OTf) at room temperature3(0.05eq.1.97mg) of anhydrous toluene: compound 8(1eq.30mg) was added to the ethanol 4:1 mixed solution. After the mixture was heated at 55 ℃ and stirred for 5h, the reaction was stopped, cooled to room temperature and quenched with saturated NaHCO3The solution was quenched, filtered, and the mixture was concentrated under vacuum. The concentrate was extracted with EtOAc. The organic phase is passed through Na2SO4Drying, filtering and concentrating. The crude material was chromatographed on silica gel (eluent DCM: MeOH 98: 2) and then by HPLC (5C18-MS-II Φ 10X 250mm, mobile phase MeOH: H)2O50: 50-70: gradient elution is carried out for 60min at 30, the flow rate of a mobile phase is 3mL/min, and the absorbance is detected under the wavelength of 210nm) to purify the obtained product, thus obtaining a compound 10(5.2mg, the yield is 15 percent, t)R＝47.0min)。
Identifying the structure of the compound 3-10:
by analysis of HR ESI-MS and1h NMR data confirmationStructure of compound 3, compound 3: a white solid, a solid,
Figure BDA0003095504390000101
9.9(c 0.5,CH3OH),HR ESI-TOF-MS m/z 425.1415,calcd.for C18H26NaO10 (M+Na)+425.1418,1H NMR(500MHz,methanol-d4):δ＝7.51(1H,s),5.80(1H,brs),5.17 (1H,d,J＝7.6Hz),4.71(1H,d,J＝7.9Hz),4.31(1H,d,J＝14.4Hz),4.20(1H,d,J＝14.4 Hz),4.17(2H,m),3.86(1H,dd,J＝1.1,11.6Hz),3.64(1H,m),3.38(1H,m),3.27(1H,m), 3.28(1H,m),3.23(1H,m),3.18(1H,m),2.83(1H,dd,J＝8.2,16.2Hz),2.72(1H,t,J＝7.6 Hz),2.10(1H,dd,J＝8.2,16.2Hz),1.28(3H,t,J＝7.4Hz).
by analysis of HR ESI-MS and1h NMR data determined the structure of compound 4, compound 4: a white solid, a solid,
Figure BDA0003095504390000102
16.7(c 0.5,CH3OH),HR ESI-TOF-MS m/z 439.1572,calcd.for C19H28NaO10 (M+Na)+439.1574,1H NMR(500MHz,methanol-d4):δ＝7.52(1H,s),5.80(1H,brs),5.17 (1H,d,J＝7.7Hz),4.71(1H,d,J＝7.9Hz),4.32(1H,d,J＝14.5Hz),4.19(1H,d,J＝14.5 Hz),4.09(2H,m),3.86(1H,dd,J＝1.2,11.9Hz),3.64(1H,m),3.37(1H,m),3.28(1H,m), 3.27(1H,m),3.23(1H,m),3.19(1H,m),2.83(1H,dd,J＝8.2,16.4Hz),2.72(1H,t,J＝7.7 Hz),2.11(1H,dd,J＝8.2,16.4Hz),1.69(2H,m),0.98(3H,t,J＝7.6Hz).
by analysis of HR ESI-MS and1h NMR data determined the structure of compound 5, compound 5: a white solid, a solid,
Figure BDA0003095504390000103
13.1(c 0.5,CH3OH),HR ESI-TOF-MS m/z 439.1553,calcd.for C19H28NaO10 (M+Na)+439.1575,1H NMR(500MHz,methanol-d4):δ＝7.49(1H,s),5.80(1H,brs),5.15 (1H,d,J＝7.8Hz),5.03(1H,m),4.71(1H,d,J＝7.9Hz),4.32(1H,d,J＝14.8Hz),4.19 (1H,d,J＝14.8Hz),3.86(1H,dd,J＝1.2,11.9Hz),3.64(1H,m),3.37(1H,m),3.28(1H, m),3.27(1H,m),3.17(1H,m),2.83(1H,dd,J＝8.2,16.3Hz),2.71(1H,t,J＝7.8Hz),2.10 (1H,dd,J＝8.2,16.3Hz),1.26(6H,d,J＝6.2Hz).
by analysis of HR ESI-MS and1h NMR data determined the structure of compound 6, compound 6: a white solid, a solid,
Figure BDA0003095504390000104
10.4(c 0.5,CH3OH),HR ESI-TOF-MS m/z 453.1733,calcd.for C29H30NaO10 (M+Na)+453.1731,1H NMR(500MHz,methanol-d4):δ＝7.51(1H,s),5.80(1H,brs),5.17 (1H,d,J＝7.7Hz),4.71(1H,d,J＝7.9Hz),4.32(1H,d,J＝14.4Hz),4.19(1H,d,J＝14.4 Hz),4.12(2H,m),3.86(1H,d,J＝11.8Hz),3.64(1H,m),3.38(1H,m),3.28(1H,m),3.27 (1H,m),3.23(1H,m),3.18(1H,m),2.83(1H,dd,J＝8.2,16.4Hz),2.72(1H,t,J＝7.7Hz), 2.10(1H,dd,J＝8.2,16.4Hz),1.65(2H,m),1.42(2H,m),0.96(3H,t,J＝7.6Hz).
by analysis of HR ESI-MS and1h NMR data determined the structure of compound 7, compound 7: a white solid, a solid,
Figure BDA0003095504390000105
14.6(c 0.5,CH3OH),HR ESI-TOF-MS m/z 467.1877,calcd.for C21H32NaO10 (M+Na)+467.1888,1H NMR(500MHz,methanol-d4):δ＝7.51(1H,s),5.80(1H,brs),5.17 (1H,d,J＝7.6Hz),4.71(1H,d,J＝7.9Hz),4.32(1H,d,J＝14.4Hz),4.19(1H,d,J＝14.4 Hz),4.12(2H,m),3.86(1H,dd,J＝1.2,12.0Hz),3.64(1H,m),3.38(1H,m),3.28(1H,m), 3.27(1H,m),3.23(1H,m),3.19(1H,m),2.83(1H,dd,J＝8.2,16.4Hz),2.72(1H,t,J＝7.6 Hz),2.11(1H,dd,J＝8.2,16.4Hz),1.67(2H,m),1.38(2H,m),1.38(2H,m),0.93(3H,t,J ＝7.4Hz).
by analyzing the HR ESI-MS,1h NMR and13c NMR data determined the structure of compound 8, compound 8: a white solid, a solid which is,
Figure BDA0003095504390000111
11.1(c 0.5,CH3OH),HR ESI-TOF-MS m/z 467.1875,calculated for C21H32NaO10(M+Na)+467.1887.1H NMR(500MHz,methanol-d4):δ＝7.50(1H,s),5.80 (1H,brs),5.17(1H,d,J＝7.8Hz),4.71(1H,d,J＝7.9Hz),4.32(1H,m),4.19(1H,m),4.16 (2H,m),3.86(1H,m),3.64(1H,m),3.38(1H,m),3.28(1H,m),3.27(1H,m),3.23(1H, m),3.18(1H,m),2.82(1H,dd,J＝8.3,16.4Hz),2.72(1H,t,J＝7.8Hz),2.10(1H,dd,J ＝8.3,16.4Hz),1.72(1H,m),1.57(2H,m),0.95(6H,t,J＝6.6Hz).13C NMR(125MHz, methanol-d4):δ＝22.8,26.4,36.7,38.6,39.8,47.0,61.4,62.7,63.7,71.5,74.9,77.9,78.4, 98.29,100.3,112.8,128.3,144.9,153.2and 169.2.
by analysis of HR ESI-MS and1h NMR data determined the structure of compound 9, compound 9: a white solid, a solid,
Figure BDA0003095504390000112
10.9(c 0.5，CH3OH),HR ESI-TOF-MS m/z 465.1729,calcd.for C21H30NaO10 (M+Na)+465.1731,1H NMR(500MHz,methanol-d4):δ＝7.50(1H,s),5.80(1H,brs),5.37 (1H,t,J＝7.2Hz),5.17(1H,d,J＝7.8Hz),4.71(1H,d,J＝7.9Hz),4.63(2H,m),4.31(1H, d,J＝14.4Hz),4.18(1H,d,J＝14.4Hz),3.86(1H,dd,J＝1.2,12.0Hz),3.64(1H,m),3.38 (1H,m),3.28(1H,m),3.27(1H,m),3.23(1H,m),3.18(1H,m),2.82(1H,dd,J＝8.2,16.2 Hz),2.72(1H,t,J＝7.8Hz),2.10(1H,dd,J＝8.2,16.2Hz),1.75(6H,d,J＝14.9Hz).
by analysis of HR ESI-MS and1h NMR data determined the structure of compound 10, compound 10: white solid, HR ESI-TOF-MS m/z 539.2063, calculated for C24H36NaO12(M+Na)+539.2099.1H NMR(500MHz,methanol-d4):δ＝7.51(1H,s),5.81(1H,brs),5.02(1H,d,J＝7.8Hz), 4.72(1H,d,J＝7.9Hz),4.62(1H,m),4.40(1H,m),4.27(1H,m),4.25(1H,m),4.20(1H, m),4.17(1H,m),4.15(2H,m),3.47(1H,m),3.40(1H,m),3.37(1H,m),3.24(1H,m), 3.18(1H,m),2.83(1H,dd,J＝8.3,16.4Hz),2.73(1H,t,J＝7.8Hz),2.09(1H,dd,J＝8.3, 16.4Hz),1.72(1H,m),1.57(2H,m),1.27(3H,m),0.95(6H,d,J＝6.6Hz).
3. Biological activity of compounds 1-10 in yeast model
Method for determining yeast replicative life: the K6001 yeast strain was taken out from the freezer at-30 ℃ and washed with PBS, inoculated into a galactose liquid medium (YPG, 1% yeast extract, 2% hippopeptone, 3% galactose) and cultured at 28 ℃ for 24 hours with shaking at 180 rpm. After the yeast cells reached the logarithmic growth phase, 4,000 yeast cells were inoculated into glucose solid medium (YPD, 2% glucose, 2% hippopeptone, 1% yeast extract and 2% agar) to which a sample was previously added. Incubated at 28 ℃ for 48 hours. The 40 mother cells were randomly selected under the microscope, the surrounding daughter cells were counted and finally analyzed using statistical software.
The effect of compounds 1-10 on yeast replicative life at a concentration of 1 μ M was determined using the K6001 bioactive system, and the results are shown in FIG. 1. Compound 2, a natural source, showed significant anti-aging activity (p <0.01) compared to compound 1. The replicative life of yeast is obviously prolonged to different degrees for the new compounds 3-10 obtained by chemical synthesis. Wherein isoamyl (compound 8) is introduced into position 11 of geniposidic acid, and the anti-aging activity is optimal (p is less than 0.001) at 1 mu M, which shows that the anti-aging effect of yeast can be influenced by the esterification of position 11 and the length and spatial configuration of the introduced carbon chain. The compound 8 is selected as a lead compound for the next research on anti-aging action mechanisms, including antioxidant stress and autophagy induction.
The method for measuring the time-sequence life of the yeast comprises the following steps: the YOM36 yeast strain was removed from the-30 ℃ freezer, washed with PBS, and then inoculated with about 200 yeast cells in glucose solid medium, cultured at 28 ℃ for 48h, and then a single colony was picked and inoculated into SD medium (0.17% yeast nitrogen base without amino acid and ammonium sulfate, 0.5% ammonium sulfate, and 2% glucose) and cultured with shaking at 28 ℃ and 180rpm for 24 hours. On day 0, when the OD value of the culture solution was 0.01, yeast cells were seeded in new 100mL of SD medium, treated with 0 and 1. mu.M of Compound 8, respectively, and cultured with shaking at 160rpm at 28 ℃. On the third day, about 200 yeast cells were plated on glucose solid medium, cultured at 28 ℃ for 48 hours, and then Colony Forming Units (CFU) of each plate were counted, and the CFU on the third day was expressed as 100% survival rate. These steps were repeated every two days and the survival rate of each group (CFU per plate/CFU of the same plate on the third day x 100%) was calculated until the survival rate dropped below 5%.
As previously described, compound 8 had a significant effect on the replicative life span of the K6001 yeast at 1 μ M. The replication lifetimes of the negative and positive control (10. mu.M RES) were 7.8. + -. 0.54 and 11.05. + -. 0.73, respectively (p < 0.001). The replicative lifetime of compound 8 at a concentration of 1 μ M was 11.08 ± 0.63(p < 0.001). In addition, a chronological lifespan analysis of YOM36 yeast was also performed to evaluate the anti-aging activity of compound 8, which significantly improved the survival rate of yeast at a concentration of 1 μ M (p < 0.001). These results are shown in figure 2, indicating that compound 8 shows anti-aging effects on yeast.
For the qualitative test of Compound 8 against oxidative stress, a BY4741 yeast strain was inoculated into glucose liquid medium, 0, 1 and 3. mu.M of Compound 8 or 10. mu.M of positive control Resveratrol (RES) were added to the medium when the initial value of OD600 was 0.1, and incubated at 28 ℃ and 160rpm with shaking for 24 hours. H at 9.0mM2O2The solutions were inoculated with 5. mu.L of yeast medium, respectively, and incubated at 28 ℃ for 48 or 72 hours. Yeast growth was observed and recorded.
For quantitative testing of Compound 8 against oxidative stress, the BY4741 yeast strain in sample-treated glucose liquid medium was shake-cultured at 28 ℃ and 160rpm for 12 h. Preparation of blank control Medium (0 mM H in glucose solid Medium)2O2) And hydrogen peroxide group medium (5.5 mM H in glucose solid Medium)2O2) 200 yeast cells of different sample groups were plated and cultured at 28 ℃ for 48 hours. The number of colonies per group was then calculated, and the survival rate (number of colonies of hydrogen peroxide group/number of colonies of blank control group x 100%) of each group was calculated, plotted and analyzed.
FIG. 3(a) shows Compound 8 vs 9.0mM H2O2Influence of yeast growth under induced oxidative stress. H2O2Yeast growth was inhibited in the negative control group, resulting in a significant reduction in viable cells, whereas yeast in the 10 μ M RES and compound 8 treated groups grew relatively normally with distinct and dense colonies, indicating that 1 μ M and 3 μ M compound 8 can significantly improve the survival of yeast in oxidative stress environments. To quantify the viability of the yeast, 200 yeast cells were plated containing 0 or 5.5mM H2O2And the survival rate of the yeast of each group was calculated by quantifying the number of yeast colonies, as shown in FIG. 3 (b). The survival rate of each group of yeast is respectively as follows: negative control: 52.70 ± 1.23%, positive control (10 μ M RES): 64.28 + -2.09% (p)<0.001)，1μM 8：66.00±1.09％ (p<0.001))，3μM 8：64.18±2.78％(p<0.01). Thus, compound 8 exhibits anti-aging activity by inhibiting oxidative stress.
ROS are the major source of free radicals in the body. Effectively eliminating ROS is beneficial to protecting organelles and molecules from damage, maintaining cell homeostasis and delaying senescence. ROS, through peroxidation with lipids on biological membranes, can produce toxic end products Malondialdehyde (MDA), destroying the integrity and normal physiological functions of biological membranes, and further cause protein misfolding, DNA modification and accelerated aging. Therefore, our study evaluated the antioxidant effect of compound 8 by measuring its effect on ROS and MDA content.
And (3) ROS determination method: briefly, the BY4741 yeast strain was inoculated into glucose liquid medium, treated with 0, 1, 3. mu.M Compound 8 or 10. mu.M RES, and cultured at 28 ℃ and 160rpm with shaking for 12 h. Each group was washed with PBS and 2', 7' -dichloro-dihydrofluorescein diacetate (DCFH-DA) fluorescent probe was added to a final concentration of 10 μ M, incubated for 1 hour with shaking in the dark, then washed with PBS, and DCF fluorescence was measured with a microplate reader (BioTek, floret, usa) at 488nm excitation and 525nm emission wavelength.
And (3) an MDA determination method: briefly, the BY4741 yeast strain was inoculated into glucose liquid medium, treated with 0, 1, 3. mu.M Compound 8 or 10. mu.M RES, and cultured at 28 ℃ and 160rpm with shaking for 12 h. Then, the supernatant was washed with PBS, sonicated on ice for 5min, and then centrifuged at 4 ℃ and 12,000rpm for 10min to obtain a protein sample. Protein concentration was determined with BCA kit (cown Biotech, beijing, china). The MDA content of each group was determined according to the MDA kit (bio engineering company, tokyo, south beige, china).
ROS levels are shown in fig. 3 (c). After 24 hours of co-incubation with the samples, the mean fluorescence of each group was: negative control: 1.10 ± 0.05, positive control (10 μ M RES): 0.44 ± 0.08(p <0.001), 1 μ M8: 0.28 ± 0.06(p <0.001), 3 μ M8: 0.23 ± 0.06(p < 0.001). MDA levels are shown in FIG. 3 (d). The average MDA for each group was: negative control: 0.56 ± 0.04, positive control (10 μ M RES): 0.37 ± 0.03(p <0.01), 1 μ M8: 0.39 ± 0.03(p <0.01), 3 μ M8: 0.39 ± 0.04(p < 0.01). Compound 8 can reduce MDA levels in cells at concentrations of 1 and 3. mu.M. Based on the above results, compound 8 exerts antioxidant effects by reducing the levels of ROS and MDA in yeast cells.
Methods for determining the activity of SOD, GPx and CAT enzymes: sample handling and protein extraction methods for BY4741 Yeast were the same as described for MDA measurements. After determining the concentration of the protein, SOD, CAT and GPx activities were determined using SOD assay kit (Tokyo institute of bioengineering, Nanjing, China), CAT and GPx assay kit (Beyotime Biotechnology Limited, Shanghai, China). The detailed procedures are described with reference to the kit.
The antioxidant enzyme system can effectively eliminate active oxygen generated by organism metabolism and reduce the oxidative damage of organisms. Thus, we evaluated the enzymatic activities of T-SOD, SOD1, CAT and GPx in yeast after 24 hours of treatment with different concentrations of Compound 8. As shown in FIGS. 3(e) to 3(h), the activity of T-SOD, SOD1 and CAT enzymes was significantly increased after treatment with 1 and 3. mu.M of Compound 8. GPx also had some increase in enzymatic activity, but no significant difference. Thus, compound 8 exhibited anti-aging effects by modulating the enzymatic activities of T-SOD, SOD1 and CAT.
Autophagy of compound 8: yeast strain YOM38, containing the pR316-GFP-Atg8 plasmid, was inoculated into glucose liquid medium and treated with doses of 0, 0.3, 1 and 3. mu.M Compound 8 or 300. mu.M RES for 22h when the initial value of OD600 was 0.1. The cells were then washed three times with PBS and stained with DAPI staining solution (1 μ g/μ L) in the dark, after 10 minutes, the yeast was washed three times with PBS and observed using a vertical two-photon confocal fluorescence microscope (olympus FV1000BX-51, tokyo, japan). Yeast strain YOM38, containing the pR316-GFP-Atg8 plasmid, was inoculated into glucose liquid medium and treated with doses of 0, 0.3, 1 and 3. mu.M Compound 8 or 300. mu.M RES for 22h when the initial value of OD600 was 0.1. Different groups of yeast cells were collected and sonicated for 5 minutes. The cell lysate was centrifuged at 12,000g for 15 minutes to obtain the supernatant for western blot analysis. Protein concentration was measured with BCA protein assay kit (cown Biotech, beijing, china). Approximately 20. mu.g of protein was separated by SDS-PAGE and transferred to PVDF membrane. The membrane is incubated with a primary antibody and then with a secondary antibody. The primary antibody used was as follows: anti-GFP (Medical & Biological Laboratories, Minggu, Japan), anti- β -actin (CoWin Biotech, Beijing, China), using secondary antibodies as follows: horseradish peroxidase-linked anti-rabbitit IgGs (CoWin Biotech, Beijing, China). The antigen was visualized using the eECL Western Blot kit (cown Biotech, beijing, china) and digitized using ImageJ software (rockvier, national institute of health, maryland, usa).
Autophagy is a degradation process that degrades cellular components to cycle into amino acids and other metabolites. Autophagy is closely related to aging. Thus, the effect of compound 8 on autophagy was investigated. ATG8 is a LC3 and gamma-aminobutyric acid receptor related protein in mammals, and is one of important ubiquitin-like systems for autophagosome formation and maturation during autophagy. I used YOM38-GFP-Atg8 yeast strains that expressed GFP-Atg8 at physiological levels and detected free GFP levels after treatment with Compound 8 by fluorescence microscopy. The fluorescence image is shown in fig. 4(a), and the numerical result is shown in fig. 4 (b). Compound 8 can significantly increase the percentage of cells with free GFP from 13.00 ± 1.29% (p <0.01), 24.50 ± 0.99% (p <0.001), 34.00 ± 2.03% (p <0.001), 22.40 ± 0.93% (p <0.01) at concentrations of 0, 0.3, 1, 3 and 10 μ M. The percentage of positive control RES below 300. mu.M increased from 13.00. + -. 1.29% to 40.00. + -. 1.07% (p < 0.001). In addition, western blot analysis was performed to test the production of free GFP released from autophagy access into vacuoles. The results of the Western blot analysis are shown in FIG. 4 (c-d). The expression levels of free GFP protein were significantly increased in compound 8 at concentrations of 0, 0.3, 1 and 3 μ M (p <0.05, p <0.001, p < 0.01). Among them, compound 8 showed the best effect at 1 μ M and was used to examine the time course of GFP after treatment. The released GFP was time-dependent, as shown in FIG. 4 (e-f). Since ATG2 and ATG32 are two important genes involved in autophagy, we used yeast mutant strains Δ ATG2 and Δ ATG32 to measure the replicative life of compound 8. The result of K6001 replication lifetime is: negative control: 7.80 ± 0.54, positive control (10 μ M RES): 11.05 ± 0.73(p <0.001), 1 μ M8: 11.08 ± 0.64(p < 0.001). The average lifetime of Δ atg2 in the control group was 6.78. + -. 0.47, whereas the average lifetimes at 10 μ M for RES and Compound 8 were 6.78. + -. 0.46 and 6.65. + -. 0.53, respectively. The mean lifetime of Δ atg32 in the control group was 6.30. + -. 0.46, whereas the mean lifetimes at 10 μ M for RES and Compound 8 were 6.58. + -. 0.51 and 6.72. + -. 0.50, respectively (FIG. 4 (g-h)). These results demonstrate that autophagy of compound 8 is associated with both genes. Compound 8 showed anti-aging effects by increasing autophagy in yeast.
4. Research on anti-aging action mechanism of compound 8 at PC12 cell level
Compound 8 to H2O2The neuroprotective effect of the injured induced PC12 cells is shown in fig. 5: firstly, recovering PC12 cells, quickly taking out the cells frozen in liquid nitrogen, and thawing in a water bath at 37 ℃ until the cells appear ice crystal shape for about 1-2 min. Then, the cell fluid in the frozen tube was quickly transferred completely to a 15mL centrifuge tube to which 4mL of CM medium had been added, blown up and mixed well, centrifuged (800rpm,3min), and the supernatant was decanted off. 5mL of CM medium was added, and after repeating the procedure, the pellet was left, 1mL of CM medium was added to the centrifuge tube, and after carefully pipetting the mixture, the hemacytometer plate was counted. Finally, 200,000 PC12 cells were inoculatedIn a 100mm petri dish containing 10mL of CM medium, the medium was changed after 24 hours, the medium was changed again after 72 hours, and the cells were subcultured after 72 hours. CM medium and PBS solution were pre-warmed in a 37 ℃ water bath for half an hour. Thereafter, the CM medium was removed from the dish, the dish was washed twice with 10mL PBS solution, and 10mL PBS was added to the dish in 5% CO2After culturing in a cell culture box at 37 ℃ for 10min, taking out, purging, transferring to a 15mL disposable centrifuge tube, centrifuging (800rpm,3min), adding 1mL CM medium, carefully blowing and mixing uniformly, and counting by using a blood ball counting plate. Add 10mL of CM Medium and 200,000 cells to the original petri dish, put in CO2Culturing in an incubator.
Antioxidant stress is one of the important ways to prevent degeneration of nerve cells. Based on the remarkable antioxidant activity of compound 8 in yeast, a PC12 cell line derived from mammalian cells was used as a bioassay system to confirm the antioxidant stress effect of compound 8 in higher organisms. Using H2O2PC12 cells were induced to produce oxidative stress and the effect of compound 8 on cell viability was assessed by the MTT method. After cell subculture approximately 50,000 PC12 cells were seeded in each well of a 24-well plate and 5% CO at 37 deg.C2And culturing for 24 hours. The medium was then replaced with 1mL serum free DMEM (Thermo Scientific, shanghai, china) containing the different test samples. At H2O2In a dose-dependent experiment, cells were treated with 0.5% Dimethylsulfoxide (DMSO) for 48 hours and with different concentrations of H2O2The culture was carried out for 1 hour. To investigate the neuroprotective effect of the compounds, PC12 cells were treated with resveratrol (RES, 10. mu.M) or Compound 8 for 48H and 0.8mM H2O2And treating for 1 h. The medium was replaced with 500. mu.L of serum-free DMEM containing 200. mu.g/mL 3- (4, 5-dimethylterazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) and incubated for an additional 2 hours. The medium was completely removed and replaced with 200 μ L DMSO into each well to dissolve formazan crystals formed. The absorbance was detected at 570nm by using a microplate reader (Bio-Tec Instruments Inc., Winooski, VT, USA). The results are shownShows that with H2O2The cell viability decreased dose-dependently with increasing concentrations from 0.2mM to 1 mM. The results show that 0.8mM H is used2O2After 1 hour of treatment, about 50% of the cells died or were in an undesirable state. According to H2O2Induced cell viability, 0.8mM H2O2As the optimal concentration for inducing oxidative stress of PC12 cells, compound 8 at 3 μ M and Resveratrol (RES) at 10 μ M significantly increased the survival rate of PC12 cells. These results demonstrate that compound 8 exhibits neuroprotective effects on PC12 cells.
Compound 8 to H2O2The effect of ROS, MDA, SOD enzyme activity in induced damaged PC12 cells is shown in figure 6: ROS are one of the leading causes of many age-related diseases (e.g., AD). In addition, ROS are effectors of oxidative stress in cells. Thus, the present subject investigated Compound 8 vs H2O2Induces protection from ROS produced by PC12 cells. Intracellular formation of ROS in PC12 cells was monitored by using the DCFH-DA assay. When non-fluorescent DCFH-DA reacts with ROS, it can be oxidized to green fluorescent species (DCF). To determine ROS levels in PC12 cells, approximately 50,000 PC12 cells were seeded into each well of a 24-well plate. Cells were treated with RES (10. mu.M) or compound, respectively, for 24 hours, followed by 0.8mM H2O2And treating for 1 h. DCFH-DA (2', 7' -dichlorodihydrofluorescein diacetate, final concentration 10. mu.M) was then added to each well and incubated for 30 minutes. Cells were washed with PBS to remove extracellular DCFHDA, and intercellular ROS were detected by using a SpectraMax M3 multimodal microplate reader (Molecular Devices Corporation, California, USA) at an excitation wavelength of 488nm and an emission wavelength of 525 nm. Meanwhile, DCF was observed in PC12 cells using a fluorescence microscope (HCS, Thermo Fisher, Mass., USA). The results show that 0.8mM H is used2O2After 1h of treatment, the fluorescence intensity increased. However, after addition of 1, 3 and 10. mu.M of Compound 8, H2O2The increased fluorescence intensity decreased. These results indicate that compound 8 is counteracting H2O2Induced oxidative damage of cells by highly reactive ROS productionInjury is of great importance.
Lipids belong to the biomacromolecules targeted by oxidative substances. Lipid oxidation results in the formation of a number of metabolites, mainly aldehydes. Metabolites produced by lipid peroxidation can interact with other macromolecules, such as nucleic acids and proteins, and this interaction often results in irreversible impairment of cell function. MDA is the most studied product of polyunsaturated fatty acid peroxidation and is generally considered as a biomarker of oxidative stress. Thus, compound 8 was evaluated for H2O2Protection of MDA levels in oxidative damage induced in PC12 cells. Approximately 2,000,000 PC12 cells were seeded into 60mm dishes containing 5mL of DMEM medium and incubated for 24 hours. PC12 cells were then treated with RES (10. mu.M) or compound, respectively, for 24H, followed by 0.8mM H2O2After 1h of treatment, cells were collected by cell lysis, and the supernatant was used as a protein sample to evaluate the MDA content. MDA content was determined using an MDA assay kit (tokyo institute of bioengineering, tokyo, china) according to the manufacturer's instructions. Three sets of blank, standard and measurement tubes were set. Prepare reagent I (20 mL. times.1 bottles of liquid, stored at room temperature), reagent II (12 mL. times.1 bottles of liquid, add 340mL of MiliQ water to each bottle and mix well, refrigerated at 4 ℃ C.), reagent III (powdered form dissolve 60mL of hot MiliQ water at 90-100 ℃ C. in advance, add 100. mu.L of 10 nmol/mL of standard solution to standard tube, add 100. mu.L of absolute ethanol to blank tube, add 100. mu.L of sample to measurement tube, then add 100. mu.L of reagent I to each set and mix well, then add 375. mu.L of reagent II and 125. mu.L of reagent III (protected from light) and vortex well, seal with sealing film, heat in 95 ℃ water bath for 40 minutes, then cool with tap water, then centrifuge at 12000r/min for 10 minutes, then put 200. mu.L of supernatant into 96 well plate, repeat 2 wells one tube, OD was measured at 532nm with a microplate reader, and PBS was used as a blank control. MDA content is (measurement tube OD value-blank tube OD value)/(standard tube OD value-blank tube OD value) × standard concentration/protein concentration of the sample. The results show that H is compared to the untreated control2O2Treated ofMDA levels in cells are significantly increased. But with H2O2The MDA level was significantly reduced in compound 8 compared to the treatment group. This result demonstrates that pretreatment with compound 8 can inhibit lipid peroxidation, reduce the level of MDA formation, and protect cells from damage.
SOD is an important endogenous free radical scavenger in mammalian cells. Thus, compound 8 was evaluated for H2O2Protection of SOD levels in oxidative damage induced to PC12 cells. For SOD enzyme activity assays, protein samples were obtained in the same manner as the protein extraction method mentioned above for the MDA assay. The extracted protein samples were sampled, 2. mu.g of protein was taken for each sample, and the activity of superoxide enzyme was determined using SOD enzyme assay kit. The process is as follows: first, each sample was mixed with reagent 7, reacted for 1 minute to lose the enzymatic activity of SOD2 in the sample, and centrifuged to obtain a supernatant as a sample. Reagent I, blank, sample and reagent 7 treated sample were added to 96-well plates, respectively. Then, reagent II, reagent III and reagent IV were added, mixed well and incubated at 37 ℃ for 40 minutes. Finally, the a550 absorbance value of the sample was measured after adding the developer and standing at room temperature for 10 minutes. The activity of the SOD enzyme was calculated according to the following formula: [ OD value of control group-OD value of measurement group]OD value of control group/50X total volume of reaction solution/sample volume/protein concentration of sample to be tested. Results show H2O2The total SOD activity of the treated group decreased. In cells treated with compound 8, the total SOD activity was significantly increased. However, SOD1 activity in PC12 cells was not affected by compound 8. These results indicate that compound 8 can significantly reduce oxidative damage of PC12 cells.
The effect of compound 8 on the chronological lifespan of PC12 cells in pseudoyeast is shown in fig. 7: the anti-aging activity of compound 8 has been previously validated on a yeast model, and although yeast has many important features that are evolutionarily conserved in mammalian cells, the anti-aging effect of compound 8 in mammalian cells must be confirmed to assess the anti-aging activity of the molecule. Therefore, the effect of compound 8 on the chronological lifespan of PC12 cells in pseudoyeast was experimentally determined. First, 80,000 PC12 cells were seeded in each well of a 96-well plate and cultured for 24 hours. The medium was then changed to 1mL serum-free DMEM containing different concentrations of test sample or 0.5% DMSO. Cells were incubated for six consecutive days and every two days the medium was replaced with new serum-free DMEM containing a dose of sample or DMSO (0.5%). Rapamycin inhibits the mTOR signaling pathway and has beneficial effects on the health and longevity of all cellular and biological systems. Thus, rapamycin was used as a positive control in the experiment. Cells treated in 96-well plates were subsequently trypsinized and 5% aliquots were plated on six-well plates filled with fresh medium. Finally, colony formation on the plate after 15 days was stained with crystal violet and photographed. The experimental results show that compound 8 can significantly increase the viability of PC12 cells at concentrations of 1 and 3 μ M compared to the control group. The compound 8 is proved to have an anti-aging effect on mammalian cells.
The invention discovers a molecule with better anti-aging activity through a yeast model and a mammalian cell model. The iridoid glycoside compound provided by the invention can obviously improve the replicative life and the time sequence life of yeast in an anti-aging in-vitro screening model, and can be applied to the preparation of anti-aging and anti-Alzheimer's disease drugs, foods and health products. The invention provides basis for research and development of new anti-aging and anti-Alzheimer's disease drugs and basic research, and has important practical significance.
Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above description of the present invention, and such equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. An iridoid glycoside compound is characterized by having a structure shown in the following formula (I):
Figure FDA0003095504380000011
in formula (I):
R2is H, R1is-CH2CH3、-CH2CH2CH3、-CH(CH3)2、-CH2(CH2)2CH3、-CH2(CH2)3CH3、-CH2CH2CH(CH3)2or-CH2CH＝C(CH3)2Sequentially marked as compounds 3-9; alternatively, the first and second electrodes may be,
R1is-CH2CH2CH(CH3)2，R2is-COOCH2CH3And is denoted as compound 10.
2. A method for preparing iridoid glycoside compounds according to claim 1, wherein the method for preparing compounds 3 to 9 comprises: at room temperature, adding geniposide into anhydrous alcohol solution containing N, N' -dicyclohexylcarbodiimide and DMAP, stirring the obtained mixture at room temperature to 55 ℃ for full reaction, then concentrating in vacuum, extracting the concentrated product with EtOAc, and passing the organic phase through Na2SO4Drying, filtering and concentrating, separating the crude sample by silica gel column chromatography, and purifying by HPLC to obtain corresponding compound 3-9; the alcohol is HO-R1；
The preparation method of the compound 10 comprises the following steps: adding a compound 8 into an anhydrous toluene/ethanol mixed solution containing scandium trifluoromethanesulfonate and excessive diethyl pyrocarbonate at room temperature, heating and stirring the obtained mixture at 50-60 ℃ for full reaction, cooling to room temperature, and adding saturated NaHCO3The solution was quenched, filtered, concentrated in vacuo, the concentrate extracted with EtOAc and the organic phase over Na2SO4Drying, filtration and concentration, the crude material obtained was isolated by silica gel column chromatography and then purified by HPLC to give compound 10.
3. The process according to claim 2, wherein the process for the preparation of compounds 3 to 9 comprises:
for compounds 3-7, 9, the eluent used for silica gel column chromatography was DCM/MeOH at a volume ratio of 95: 5;
for compound 8, silica gel column chromatography was performed using EtOAc/MeOH in a volume ratio of 95: 5.
4. The process according to claim 2 or 3, wherein the process for the preparation of compounds 3 to 9 comprises:
for the compound 3, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 30: 70-50: 502Performing gradient elution for 40min by taking O as a mobile phase, wherein the flow rate of the mobile phase is 3mL/min, the absorbance is detected under the wavelength of 210nm, and the retention time is 16.3 min;
for the compounds 4 and 5, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and adopts MeOH/H with the volume ratio of 50: 50-100: 02Performing gradient elution for 30min by taking O as a mobile phase, wherein the flow rate of the mobile phase is 3mL/min, the absorbance is detected under the wavelength of 210nm, and the retention time is 10.7 min;
for the compound 6, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and adopts MeOH/H with the volume ratio of 50: 50-100: 02Performing gradient elution for 30min by taking O as a mobile phase, wherein the flow rate of the mobile phase is 3mL/min, the absorbance is detected under the wavelength of 210nm, and the retention time is 12.9 min;
for the compound 7, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 50: 50-65: 352Performing gradient elution for 40min by taking O as a mobile phase, wherein the flow rate of the mobile phase is 3mL/min, the absorbance is detected under the wavelength of 210nm, and the retention time is 23.3 min;
for the compound 8, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 30: 70-50: 502Performing gradient elution for 45min by taking O as a mobile phase, wherein the flow rate of the mobile phase is 3mL/min, the absorbance is detected under the wavelength of 210nm, and the retention time is 24.0 min;
for the compound 9, HPLC adopts a C18 liquid chromatographic column 5C18-AR-II phi 10 multiplied by 250mm and MeOH/H with the volume ratio of 50: 50-65: 352Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 40minThe absorbance was measured at a wavelength of 210nm and the retention time was 14.1 min.
5. The process of claim 2, wherein the compound 10 is prepared by silica gel column chromatography using DCM/MeOH in a volume ratio of 98: 2.
6. The method of claim 2 or 5, wherein the compound 10 is prepared by HPLC using C18 liquid chromatography column 5C18-MS-II Φ 10X 250mm with MeOH/H at a volume ratio of 50:50 to 70:302Performing gradient elution with O as mobile phase at flow rate of 3mL/min for 60min, detecting absorbance at 210nm wavelength, and keeping for 47 min.
7. Use of iridoid glycosides according to claim 1 for the preparation of a preparation against oxidative stress and/or inducing autophagy.
8. The use of iridoid glycosides compounds of claim 1 in the preparation of drugs, foods and health products for anti-aging, anti-aging of the brain, and prevention and treatment of neurodegenerative diseases including alzheimer's disease, wherein the iridoid glycosides compounds have antioxidant stress and/or autophagy-inducing effects.
9. The use according to claim 8, wherein the medicament, food, health product is a solid formulation or a liquid formulation;
the medicine is a medicine with a gastrointestinal administration dosage form or a medicine with a non-gastrointestinal administration dosage form.
10. A pharmaceutical, food or health product comprising the iridoid glycoside compound according to claim 1, which has antioxidant stress and/or autophagy-inducing effects, and is useful for anti-aging, and preventing and treating neurodegenerative diseases including Alzheimer's disease.
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