Iridoid compound with anti-aging activity in rhododendron simsii as well as preparation method and application of iridoid compound

Abstract

The invention discloses an iridoid compound with anti-aging activity in rhododendron simsii as well as a preparation method and application of the iridoid compound, and belongs to the technical field of natural product chemistry. According to the present invention, the two iridoid compounds such as pacyodiridoid A and splendoside with anti-aging activity are separated from the rhododendron simsii for the first time, and the experiment results show that in the caenorhabditis elegans model, the two compounds can improve the physiological status of caenorhabditis elegans from multiple dimensions, can significantly improve the anti-oxidative stress ability of caenorhabditis elegans, and can provide the anti-aging activity for the caenorhabditis elegans, such that the anti-aging activity of the caenorhabditis elegans can be significantly improved, and the anti-aging activity of the caenorhabditis elegans can be significantly improved; the two compounds have obvious effects in the aspects of aging-related indexes (life, exercise ability and food intake) and growth and development indexes, fully prove that the two compounds have excellent anti-aging activity, lay a new material basis and theoretical basis for the research of an anti-aging mechanism, and open up a new direction for the development of novel anti-aging drugs at the same time. According to the present invention, the structure of the compound pachypodidoid A is novel, such that the new lead compound is provided for the research and the development of the anti-aging drug, and the important application value is provided;

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product chemistry, and particularly relates to a labdane-type iridoid compound with anti-aging activity in Rhododendron delavayi and a preparation method and application thereof. BACKGROUND

[0002] Aging is a natural process in which the functions of an organism gradually decline with age and eventually lead to death, accompanied by a significantly increased risk of various diseases such as cardiovascular disease, cancer, and neurodegenerative disease. China is rapidly moving towards an aging society, with 264 million people aged 60 and above, accounting for 18.70% of the total population. It is estimated that the number of elderly people will reach nearly 500 million by 2050. The deepening of the aging degree has brought a series of serious problems, and the incidence of aging-related diseases such as Alzheimer's disease, cardiovascular disease, and diabetes has increased significantly. For example, Alzheimer's disease not only seriously affects the quality of life of patients, but also causes a heavy burden on families and society. According to estimates, the social and economic cost of Alzheimer's disease in China will reach 320 billion yuan in 2030, and 1190 billion yuan in 2050. The above data show that finding effective anti-aging means, reducing the risk of aging-related diseases, and improving the quality of life of the elderly have become major social and medical problems that need to be solved urgently.

[0003] With the increasing problem of aging, there has been a surge in the research of anti-aging drugs worldwide. Currently, some anti-aging drugs have shown potential in research. In preclinical studies, SRN-901, jointly developed by Senolytic, Harvard University, Mayo Clinic, Scripps Research, and others, successfully extended the remaining lifespan of middle-aged animals by more than 1 / 3 in the latest preclinical trial, and significantly slowed down the decline in cognitive function of elderly animals. Its test data is better than the rapamycin control group under the same conditions. Among the drugs already on the market, the National Medical Products Administration has approved some traditional Chinese medicines with certain anti-aging effects, such as Bazi Bushen Capsules, which can delay the shortening of telomeres by regulating kidney deficiency, improving superoxide dismutase activity, and reducing malondialdehyde levels, and are suitable for early aging symptoms such as soreness of the waist and knees and early graying of hair. Compound Danshen Dropping Pills, composed of Danshen and Sanqi, can improve microcirculation disorders, reduce blood viscosity, and inhibit platelet aggregation, and are effective for related aging symptoms caused by atherosclerosis. In addition, there are new drugs for Alzheimer's disease, such as Aricept (donepezil injection), which is used to treat mild cognitive impairment and mild dementia caused by Alzheimer's disease in adults, and can significantly slow down the progression of the disease. However, overall, existing anti-aging drugs still have certain limitations in effectiveness, safety, and scope of application, and cannot fully meet social needs, so it is urgent to explore more candidate drugs with novel structures and clear mechanisms of action.

[0004] The present application aims to provide a kind of iridoid compounds with anti-aging activity in Rhododendron simsii and its preparation method, and further provide new material sources and technical approaches for the research and development of anti-aging drugs. SUMMARY

[0005] The first object of the present application is to provide a kind of iridoid compounds with anti-aging activity in Rhododendron simsii and its preparation method, and the second object of the present application is to provide the application of the iridoid compounds.

[0006] The first object of the present application is achieved by a kind of iridoid compounds with anti-aging activity in Rhododendron simsii, the structural formula is as shown in formula I or formula II: I II.

[0007] The preparation method of the compound I is achieved by the following steps: 1) After removing the stamens of dry Rhododendron simsii, it is crushed, and refluxed with methanol at 70℃ for 3 times, each for 2 hours; 2) The extract is concentrated under reduced pressure, and the extract is sequentially extracted with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract (1.1 kg) and n-butanol extract (2.5 kg), respectively; 3) The ethyl acetate extract is concentrated under reduced pressure to obtain the extract, and then subjected to silica gel column chromatography: the extract is mixed with silica gel powder, and eluted with chloroform-methanol solution with a gradient of 100:1-90:1-80:1-70:1-60:1-50:1-40:1-30:1-20:1-10:1-1:1 to obtain 8 components Fr. A-H; 4) Component Fr. D is subjected to RP-C18 reverse phase column chromatography and Sephadex LH-20 gel column chromatography to obtain component Fr. D2; 5) Component Fr. D2 is subjected to silica gel column chromatography, and eluted with petroleum ether: ethyl acetate with a volume ratio of 5:1 to obtain compound I.

[0008] After filtering the n-butanol extract in step 2), gradient elution is carried out by medium-high pressure column chromatography with ethanol-water solution with a gradient of 10:90-20:80-30:70-40:60-50:50, and the target segment components are collected, and then subjected to RP-C18 reverse phase column chromatography, Sephadex LH-20 gel column chromatography and silica gel column chromatography to obtain compound II.

[0009] The second object of the present application is achieved by the application of the iridoid compounds in the preparation of anti-aging drugs.

[0010] The present application firstly separates two iridoid compounds pachypodiridoid A and splendoside with anti-aging activity from Rhododendron anthopogonoides. Experiments prove that the two compounds can improve the physiological state of Caenorhabditis elegans from multiple dimensions, can significantly improve the antioxidant stress capacity of Caenorhabditis elegans, and also show significant effects on aging-related indicators (life span, movement ability, food intake) and growth and development (body length, body width) indicators, fully proving that the two compounds have excellent anti-aging activity, laying a new material foundation and theoretical basis for anti-aging mechanism research, and opening up a new direction for the development of new anti-aging drugs. Among them, the splendoside prepared by the method of the present application has high yield and good anti-aging activity. The compound pachypodiridoid A has a complex and unique polycyclic peroxo-bridged heterocyclic skeleton, belongs to an iridoid compound with a novel structure, and can be used as a lead compound for anti-aging drugs, and has important application value in the field of innovative drug development. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a chemical structural formula of the compound I and II in the present application; Figure 2 is a chemical structural formula of the compound I in the present application; 1 is an H NMR spectrum of the compound I in the present application; Figure 3 is a C NMR spectrum of the compound I in the present application; 13 is an H NMR spectrum of the compound I in the present application; Figure 4 is an H NMR spectrum of the compound I in the present application; 1 is an H NMR spectrum of the compound I in the present application; 1 is an H COSY spectrum of the compound I in the present application; Figure 5 is a nuclear magnetic resonance HSQC spectrum of the compound I in the present application; Figure 6 is a nuclear magnetic resonance HMBC spectrum of the compound I in the present application; Figure 7 is a nuclear magnetic resonance ROESY spectrum of the compound I in the present application; Figure 8 is a nuclear magnetic resonance mass spectrum of the compound I in the present application; Figure 9 is an electronic circular dichroism spectrum of the compound I in the present application; Figure 10 is a chemical structural formula of the compound II in the present application; 1 is an H NMR spectrum of the compound II in the present application; Figure 11 is a C NMR spectrum of the compound II in the present application; 13 is a C NMR spectrum of the compound II in the present application; Figure 12Figure of the effect of the compound I of the present invention on the survival curve of the life span of C. elegans; Figure 13 Figure of the effect of the compound II of the present invention on the survival curve of the life span of C. elegans; Figure 14 Figure of the effect of the compounds I and II of the present invention on the survival curve of the normal life span of C. elegans at a concentration of 0.1 mg / mL; Figure 15 Figure of the effect of the compound I of the present invention on the survival curve of the normal life span of C. elegans under oxidative stress conditions; Figure 16 Figure of the effect of the compound II of the present invention on the survival curve of the normal life span of C. elegans under oxidative stress conditions; Figure 17 Figure of the effect of the compounds I and II of the present invention on the survival curve of the normal life span of C. elegans under oxidative stress conditions at a concentration of 0.1 mg / mL; Figure 18 Figure of the feeding and phagocytosis rate of C. elegans after 7 days and 14 days of action of the compound I of the present invention; Figure 19 Figure of the feeding and phagocytosis rate of C. elegans after 7 days and 14 days of action of the compound II of the present invention; Figure 20 Figure of the feeding and phagocytosis rate of C. elegans after 7 days and 14 days of action of the compounds I and II of the present invention at a concentration of 0.1 mg / mL; Figure 21 Figure of the motility of C. elegans after 7 days and 14 days of action of the compound I of the present invention; Figure 22 Figure of the motility of C. elegans after 7 days and 14 days of action of the compound II of the present invention; Figure 23 Figure of the motility of C. elegans after 7 days and 14 days of action of the compounds I and II of the present invention at a concentration of 0.1 mg / mL; Figure 24 Figure of the body length of C. elegans after 10 days of action of the compounds I and II of the present invention; Figure 25 Figure of the body length of C. elegans after 10 days of action of the compounds I and II of the present invention at a concentration of 0.1 mg / mL; Figure 26 Figure of the body width of C. elegans after 10 days of action of the compounds I and II of the present invention; Figure 27 Figure of the body width of C. elegans after 10 days of action of the compounds I and II of the present invention at a concentration of 0.1 mg / mL. DETAILED DESCRIPTION

[0012] The application will be further described in connection with the accompanying drawings and examples, but not in any way limited to the application, any transformation or improvement based on the teaching of the application falls within the protection scope of the application.

[0013] The instruments and materials used in the application are shown below, the reagents not specifically mentioned are the conventional reagents in the field, and the methods not mentioned are the conventional methods in the field.

[0014] Jasco-P-1020 polarimeter; Horiba SEPA-300 polarimeter; Bruker Tensor 27 FT-IR spectrometer; Bruker AV-600MHz nuclear magnetic resonance spectrometer; waters Autospec Primier P776 spectrometer; API QSTAR pulsar I spectrometer; silica gel (200-300 mesh, Qingdao Haigong Co., Ltd.); Sephadex LH-20 gel (Amersham Biosciences, Sweden), RP-C18 reversed-phase silica gel (40-75 μm, Fujixiluichemical Co., Ltd.). The fractions were monitored by TLC, and the spots were visualized by silica gel plate with sulfuric acid color reagent (5% sulfuric acid).

[0015] The iridoid compounds with anti-aging activity in the application are pachypodiridoid A and splendoside respectively, and the structural formulae are shown as formula I or formula II: I II.

[0016] Pachypodiridoid A and splendoside have the activities of prolonging the average life span of Caenorhabditis elegans, improving the antioxidant stress resistance, feeding and swallowing rate, movement ability, body length and body width of Caenorhabditis elegans at a concentration of 0.1 mg / mL.

[0017] The application further provides the use of the iridoid compounds in the preparation of anti-aging drugs for Caenorhabditis elegans.

[0018] The application further provides the use of the iridoid compounds in the preparation of anti-aging drugs.

[0019] The application further provides a preparation method of the iridoid compounds, and the specific method is realized according to the following steps: 1) After removing the stamens of dried Rhododendron anthopogon flowers, the flowers are crushed, and methanol is used for reflux extraction at 70℃ for 3 times, each time for 2 hours; 2) The extract was concentrated under reduced pressure, and the extract was extracted with petroleum ether, ethyl acetate and n-butanol successively to obtain petroleum ether extract, ethyl acetate extract (1.1 kg) and n-butanol extract (2.5 kg), respectively; 3) The ethyl acetate extract was concentrated under reduced pressure to obtain an extract, and the extract was subjected to silica gel column chromatography. The extract was mixed with silica gel powder, and eluted with chloroform-methanol solution with a gradient of 100:1-90:1-80:1-70:1-60:1-50:1-40:1-30:1-20:1-10:1-1:1 to obtain eight fractions Fr. A-H. 4) Fraction Fr. D was subjected to RP-C18 reverse phase column chromatography and Sephadex LH-20 gel column chromatography to obtain fraction Fr. D2. 5) Fraction Fr. D2 was subjected to silica gel column chromatography, and eluted with petroleum ether: ethyl acetate (5:1, by volume) to obtain compound I.

[0020] In step 4), the RP-C18 reverse phase column chromatography was gradient eluted with methanol-water solution with a gradient of 10:90-20:80-30:70-40:60-50:50-60:40-70:30-80:20-90:10-100:0, and the Sephadex LH-20 gel column chromatography was eluted with methanol.

[0021] The n-butanol extract in step 2) was filtered, and gradient eluted with ethanol-water solution with a gradient of 10:90-20:80-30:70-40:60-50:50 through a medium-high pressure column chromatography tower. The target fraction was collected, and subjected to RP-C18 reverse phase column chromatography, Sephadex LH-20 gel column chromatography and silica gel column chromatography to obtain splendoside. The RP-C18 reverse phase column chromatography was gradient eluted with methanol-water solution with a gradient of 10:90-20:80-30:70, the Sephadex LH-20 gel column chromatography was eluted with methanol, and the silica gel column chromatography was eluted with dichloromethane-methanol solution with a gradient of 10:1.

[0022] Preparation of compound pachypodiridoid A in Example 1 1. 38.0 kg of dried flower parts of P. sinensis (with corolla removed) were pulverized, and then extracted with industrial methanol at 70°C for 3 times, each for 2 hours. The solvent was recovered under reduced pressure to obtain 10.0 kg of extract. The extract was extracted with petroleum ether, ethyl acetate and n-butanol successively to obtain petroleum ether extract, ethyl acetate extract (1.1 kg) and n-butanol extract (2.5 kg), respectively.

[0023] 2. The ethyl acetate extract fraction was concentrated under reduced pressure to give 1.1 kg of extract, which was separated by silica gel column chromatography (10 cm x 200 cm). The sample was mixed with silica gel powder (200-300 mesh, 800 g), and the column was filled with 4.0 kg of silica gel. The column was eluted with chloroform-methanol solutions (100:1-90:1-80:1-70:1-60:1-50:1-40:1-30:1-20:1-10:1-1:1) in gradient elution, and 1000 mL of eluate was collected as one fraction. According to TLC detection, a total of 8 fractions (Fr. A-H) were obtained.

[0024] 3. The Fr. D fraction was concentrated under reduced pressure, and then eluted by RP-C18 reverse phase column chromatography with chloroform-methanol solutions (100:1-90:1-80:1-70:1-60:1-50:1-40:1-30:1-20:1-10:1-1:1) in gradient elution and Sephadex LH-20 gel column chromatography (eluted with methanol). The eluate was concentrated under reduced pressure to give fraction D2, which was purified by silica gel column chromatography (eluted with 5:1 petroleum ether-ethyl acetate solution) to obtain compound pachypodiridoid A (34 mg).

[0025] Compound structure identification and analysis: colorless oil, showing [M+Na]⁺ ion peak in high-resolution electrospray ionization mass spectrometry (HRESIMS) with mass-to-charge ratio (m / z) of 281.0998, corresponding to the molecular formula of C 12 H 18 O6, indicating that the unsaturation degree is 4. The absorption peaks in infrared spectroscopy show the presence of hydroxyl group (3476 cm - ¹) and carbonyl group (1739 cm - ¹). In hydrogen nuclear magnetic resonance spectroscopy (¹HNMR) ( Figure 2 ), combined with heteronuclear multiple quantum coherence spectroscopy (HMQC), it was found that there are two methoxy groups [δH 3.30 (3H, singlet, 1-OMe), 3.74 (3H, singlet, 11-OMe)] and two hemiacetal methine groups [δH 5.15 (1H, doublet, coupling constant J = 3.0 Hz, δC 96.6), 5.40 (1H, doublet, coupling constant J = 3.0 Hz, δC 89.6)]. Carbon nuclear magnetic resonance spectroscopy (¹³CNMR) ( Figure 3 ) and distortionless enhancement by polarization transfer spectroscopy (DEPT) data show that there are 12 carbons, including two methoxy carbons, three methylene carbons, five methine carbons, and two quaternary carbons (including one carbonyl carbon). Based on the unsaturation degree and one-dimensional nuclear magnetic resonance data (Table 1), the compound is a cyclic iridoid.

[0026] Table Compound pachypodiridoid A 1 H NMR (600 MHz) and 13 C NMR (600 MHz) data (CD3OD)

[0027] The skeleton of the iridoid was further confirmed by two-dimensional nuclear magnetic resonance (2D NMR) data analysis. In the hydrogen-hydrogen correlation spectrum (1H-1H COSY), the correlation signals of H-3 with H-4, H-4 with H-5, H-5 with H-6, H-5 with H-9, H-6 with H-7, and H-9 with H-1 showed the key spin system as shown in FIG. 2. In the heteronuclear multiple bond correlation spectrum (HMBC), the correlation signal of δH 3.30 with C-1 (δC 96.6) indicated that the 1-methoxy group was connected to C-1; the correlation signal of δH 3.74 with C-11 (δC 171.0) indicated the position of the methoxy group (δC 52.1). The carbon signal of C-10 (δC 66.3) appeared a low-field shift, indicating that it was connected with a hydroxyl group. The HMBC cross peaks of H-10 with C-7 (δC 34.5), C-8 (δC 83.4), and C-9 (δC 43.6) indicated that C-10 was connected to C-8. The HMBC correlation signals of H-3 (δH 5.40), H-4 (δH 2.55), and H-5 (δH 3.20) with C-11 (δC 171.0) confirmed the position of C-11.

[0028] The remaining one unsaturation in pachypodiridoid A was speculated to be an oxygen bridge bond. Combined with the HMBC correlation signal of H-3 with C-8, and the low-field chemical shifts of C-3 and C-8, it was inferred that the oxygen bridge bond was located between C-3 and C-8. The relative configuration of compound 1 was determined by rotating frame Figure 9 ) analysis, and the absolute configuration was determined by comparison of experimental electronic circular dichroism (ECD) ( Figure 1 ) with density functional theory (DFT) calculated spectrum.

[0029] Example 2 Preparation of compound splendosid 1. Take 50.57 g of the n-butanol extract fraction (2.5 kg total) from Example 1, filter through a Buchner funnel, and concentrate the filtrate to dryness. Elute the residue with 600 mL of 10%, 20%, 30%, 40%, and 50% ethanol in water through a medium pressure chromatography column. Combine the eluate from fractions 5-17, and concentrate the combined eluate to dryness under reduced pressure to obtain 11.9604 g of a crude extract.

[0030] 2. Elute the crude extract with 600 mL of 10%, 20%, and 30% methanol in water through an RP-C18 reverse phase chromatography column. Combine the eluate from fractions 9-56, and concentrate the combined eluate to dryness under reduced pressure to obtain 3.9064 g of Component 1.

[0031] 3. Elute Component 1 with 600 mL of methanol through a Sephadex LH-20 gel chromatography column. Combine the eluate from fractions 40-67, and concentrate the combined eluate to dryness under reduced pressure to obtain 3.3081 g of Component 2.

[0032] 4. Elute Component 2 with 600 mL of 10%, 20%, and 30% methanol in water through an RP-C18 reverse phase chromatography column. Combine the eluate from fractions 43-86, and concentrate the combined eluate to dryness under reduced pressure to obtain 2.2324 g of Component 3.

[0033] 5. Elute Component 3 with 1200 mL of 10% methanol in water through an RP-C18 reverse phase chromatography column. Combine the eluate from fractions 14-33, and concentrate the combined eluate to dryness under reduced pressure to obtain 1.7213 g of Component 4.

[0034] 6. Elute Component 4 with 600 mL of methanol through a Sephadex LH-20 gel chromatography column. Combine the eluate from fractions 54-63, and concentrate the combined eluate to dryness under reduced pressure to obtain 1.3066 g of Component 5. Elute Component 5 with 600 mL of dichloromethane:methanol (10:1) through a silica gel chromatography column. Combine the eluate from fractions 30-61, and concentrate the combined eluate to dryness under reduced pressure to obtain 1.0436 g of Compound splendosid. By calculation, 2.5 kg of the n-butanol extract fraction yields 51.59 g of Compound splendosid.

[0035] Compound splendosid: white powder, soluble in methanol. The molecular formula is C 17 H 16 O 11 , with 10 degrees of unsaturation. By 1 H NMR, it is speculated that the compound contains a methoxy group, with the methoxy group at δ H 3.71. In addition, 13 H proton signals were observed. By 13The CNMR and DEPT spectra suggested that the compound contained 17 carbon atoms, of which 1 methoxyl, 4 methylene (2 oxygen-containing substituted -CH2-, 2 non-oxygen-containing substituted -CH2-), 10 methine (3 oxygen-containing substituted -CH-, 7 non-oxygen-containing substituted -CH-), 1 carbonyl, suggesting that the compound was a monoterpenoid compound. Again, in combination with the 1H NMR and 13C NMR data, it was consistent with the reference data, and it was determined that the compound was splendosid.

[0036] 1H NMR (600 MHz, CD3OD) δ H : 7.48 (1H, d, J = 1.2 Hz, H-3), 5.44 (1H, d, J = 5.6 Hz, H-1), 4.73 (1H, d, J = 8.1 Hz, H-1'), 3.71 (3H, s, -COOMe), 3.51 (2H, s, H-10), 2.28 (1H, dd, J = 9.0, 5.5 Hz, H-9); 13C NMR (150 MHz, CD3OD) δC: 96.6 (C-1), 153.5 (C-3), 112.5 (C-4), 35.7 (C-5), 31.9 (C-6), 36.9 (C-7), 83.3 (C-8), 46.3 (C-9), 69.6 (C-10), 169.6 (C-11), 51.9 (-OMe), 100.9 (C-1'), 74.8 (C-2'), 78.0 (C-3'), 71.4 (C-4'), 78.5 (C-5'), 62.7 (C-6'). 1 H and 13 C NMR data (δ in ppm, J in Hz).

[0037] Table 2. 1H NMR (600 MHz) and 1 H NMR (600 MHz) and 13 C NMR (600 MHz) data (CD3OD)

[0038] Example 3 Preparation of compound splendosid 1. Take 10.08 g of the n-butanol extract fraction from Example 1, filter through a regular funnel, and concentrate the filtrate to dryness. Then elute the dry residue with 10%, 20%, 30%, 40%, and 50% ethanol-water solutions, each at a flow rate of 600 mL, through a medium-high pressure chromatography column. Combine the eluate from the 11th to 23rd fractions, and concentrate the combined eluate to dryness under reduced pressure to obtain 2.3213 g of a crude extract.

[0039] 2. Elute the crude extract obtained in the preceding step with 10%, 20%, and 30% methanol-water solutions through an RP-C18 reverse-phase chromatography column, each at a flow rate of 600 mL. Combine the eluate from the 46th to 55th fractions, and concentrate the combined eluate to dryness under reduced pressure to obtain 463.3 mg of Fraction 1.

[0040] 3. Elute Fraction 1 with methanol through a Sephadex LH-20 gel chromatography column. Combine the eluate from the 40th to 56th fractions, and concentrate the combined eluate to dryness under reduced pressure to obtain 354.9 mg of Fraction 2.

[0041] 4. Elute Fraction 5 with dichloromethane:methanol (10:1) through a silica gel chromatography column. Combine the eluate from the 30th to 61st fractions, and concentrate the combined eluate to dryness under reduced pressure to obtain 252.60 mg of compound splendosid. Based on the calculation, 2.5 kg of the n-butanol extract fraction can yield 62.6 g of compound splendosid.

[0042] Example 4. Preparation of compound splendosid 1. Take 5.36 g of the n-butanol extract fraction from Example 1, filter through a regular funnel, and concentrate the filtrate to dryness. Then elute the dry residue with methanol through a Sephadex LH-20 gel chromatography column. Combine the eluate from the 60th to 73rd fractions, and concentrate the combined eluate to dryness under reduced pressure to obtain 1.8446 mg of Fraction 1. 2. Elute Fraction 1 obtained in the preceding step with 10% and 20% methanol-water solutions through an RP-C18 reverse-phase chromatography column, each at a flow rate of 600 mL. Combine the eluate from the 19th to 31st fractions, and concentrate the combined eluate to dryness under reduced pressure to obtain 177.7 mg of Fraction 2.

[0043] 3. Elute Fraction 2 with dichloromethane:methanol (10:1) through a silica gel chromatography column. Combine the eluate from the 38th to 58th fractions, and concentrate the combined eluate to dryness under reduced pressure to obtain 148 mg of compound splendosid. Based on the calculation, 2.5 kg of the n-butanol extract fraction can yield 69 g of compound splendosid.

[0044] Example 5 Either of the compounds pachypodiridoid A and splendosid prepared in Examples 1-4 can be formulated into an injection solution by adding an injection solvent, filtering, and then aseptically filling and sterilizing the filtered solution.

[0045] Example 6 Each of the compounds pachypodiridoid A and splendoside prepared in Examples 1-4, together with various pharmaceutical excipients, can be formulated into tablets by conventional methods.

[0046] Using each of the compounds pachypodiridoid A and splendoside as a pharmaceutically active ingredient, using several excipients as auxiliary ingredients for preparing a combined pharmaceutical tablet, and formulating them in a certain ratio to prepare tablet samples containing 1-100 mg of the pharmaceutical ingredient per tablet.

[0047] Example 7 Each of the compounds pachypodiridoid A and splendoside prepared in Examples 1-4, together with various pharmaceutical excipients, can be formulated into capsules by conventional methods: Preparation of a pharmaceutical combined capsule formulation containing each of the compounds pachypodiridoid A and splendoside as an effective ingredient, using each of the compounds pachypodiridoid A and splendoside prepared in Example 1 as a pharmaceutically active ingredient, using several excipients as auxiliary ingredients for preparing a combined pharmaceutical capsule, and formulating them in a certain ratio to prepare capsule formulations containing 1-100 mg of the compound ingredient per capsule.

[0048] Example 8 Take 1 part of each of the compounds pachypodiridoid A and splendoside prepared by the method of Examples 1-4, 10 parts of vegetable butter powder, mix well, and prepare a solid beverage by conventional methods.

[0049] Experimental Example 1: Anti-aging activity detection of two compounds on Caenorhabditis elegans I. Experimental method 1. OP50 strain culture: After streak culture of the OP50 strain, a single colony was picked and cultured in LB medium at 37°C, 180 r / min to the logarithmic growth phase and stored at 4°C for use as nematode food.

[0050] 2. Synchronization of nematode culture: Prepare a 2 mol / L NaOH solution, and mix with a 10% NaClO solution by volume ratio 1:2 to prepare a nematode lysis solution (freshly prepared). The specific operation is as follows: use M9 buffer to wash the nematodes from the plate after activation culture, collect in a centrifuge tube and mix with the lysis solution 1:1, lysis for 5-8 min, wash with M9 buffer for 3 times, and incubate the eggs at 20°C for 72 h to grow to L4 stage, and synchronization is completed.

[0051] 3. Nematode experiment grouping: the control group was coated with 50 μL OP50 of the food culture plate. The treatment group was coated with 100 μL of compound pachypodiridoid A and splendoside with a final mass concentration of 1.00 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.1 mg / mL and 50 μL OP50 mixture of the food culture plate. OP50 was used as a blank control, and 400 μmol / L β-nicotinamide mononucleotide (NMN) was used as a positive control.

[0052] 4. Anti-aging activity detection 4.1 Determination of nematode lifespan activity: after synchronization, L4 stage C. elegans were inoculated into NGM plates containing 1 mg / mL, 0.5 mg / mL, 0.25 g / mL, and 0.1 mg / mL of the drug, with OP50 as a blank control and 400 μmol / L β-nicotinamide mononucleotide (NMN) as a positive control. Each treatment had 60 nematodes. Each treatment was repeated 3 times. The survival time of nematodes was counted from the day of transfer. The growth of nematodes was observed every day, the number of dead nematodes was recorded, and the dead nematodes were picked out. Until all nematodes died, the average lifespan of nematodes was calculated, and the lifespan curve was drawn.

[0053] 4.2 Determination of nematode antioxidant stress capacity: nematodes synchronized to L4 stage were inoculated into NGM medium containing 1 mg / mL, 0.5 mg / mL, 0.25 g / mL, and 0.1 mg / mL of the drug, with OP50 as a blank control and 60 per plate, and each treatment was repeated 3 times. After 3 days of constant temperature culture at 20 °C, transfer to NGM medium containing 0.25% H2O2, count the number of dead nematodes every 1 hour, until all nematodes died, draw the survival curve of lifespan.

[0054] 4.3 Determination of nematode phagocytosis frequency: after synchronization, L4 stage C. elegans were inoculated into 1 mg / mL, 0.5 mg / mL, 0.25 g / mL, and 0.1 mg / mL of the drug, and incubated at 20 °C, with OP50 as a blank control. Randomly select 30 C. elegans at 7 days and 14 days to determine the pharyngeal pump contraction frequency of C. elegans for 30 s, and draw a bar chart.

[0055] 4.4 Evaluation of the movement ability of nematodes: After synchronization, L4 stage C. elegans were incubated in drugs at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 g / mL, and 0.1 mg / mL, with OP50 as a control. At 7 days and 14 days, 30 C. elegans were randomly selected to observe the movement ability of the nematodes. The movement of nematodes in each group was recorded according to the following standards to draw a stacked graph: movement ability A (nematodes moved spontaneously), movement ability B (nematodes moved only in the environment around the stimulus), and movement ability C (nematodes only moved the head and tail when stimulated).

[0056] 4.5 Measurement of the length and width of nematodes: After synchronization, L4 stage C. elegans were incubated in drugs at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 g / mL, and 0.1 mg / mL, with OP50 as a control. At 10 days, 30 C. elegans were randomly selected to measure the length and width of the nematodes.

[0057] II. Experimental results 1. The effects of different concentrations of pachypodiridoid A and splendoside on the normal average lifespan of C. elegans are as follows: Figures 12-14 ): pachypodiridoid A: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the normal average lifespan of nematodes was increased by 30.4%, 23.4%, 21.7%, and 20.8%, respectively.

[0058] splendoside: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the normal average lifespan of nematodes was increased by 29.8%, 30.8%, 22.7%, and 21.3%, respectively.

[0059] 2. The effects of different concentrations of pachypodiridoid A and splendoside on the average lifespan of C. elegans under oxidative stress induced by 0.25% H2O2 are as follows: Figures 15-17 ): pachypodiridoid A: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the average lifespan of nematodes was increased by 13.9%, 15.2%, 30.1%, and 33.8%, respectively.

[0060] splendoside: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the average lifespan of nematodes was increased by 34.1%, 30.9%, 41.1%, and 30.0%, respectively.

[0061] 3. The effects of different concentrations of pachypodiridoid A and splendoside on the feeding pharyngeal pumping rate of C. elegans are as follows: Figures 18-20 pachypodiridoid A: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the feeding pharyngeal pumping rate of C. elegans was increased by 4.2%, 10.9%, 2.9%, and 16.1% on day 7, and by 22.3%, 20.4%, 6.4%, and 27.2% on day 14, respectively. splendoside: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the feeding pharyngeal pumping rate of C. elegans was increased by 13.0%, 9.2%, 22.0%, and 17.9% on day 7, and by 23.4%, 4.9%, 14.7%, and 25.2% on day 14, respectively.

[0062] 4. The effects of different concentrations of pachypodiridoid A and splendoside on the movement ability of C. elegans are as follows: Figures 21-23 pachypodiridoid A: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the proportion of C. elegans with A-level movement ability was 36.8%, 31.7%, and 27.2% on day 14, which was significantly better than the 15.5% of the blank control group. At a concentration of 0.1 mg / mL, the proportion of C. elegans with A-level movement ability was 40.6%, which was significantly better than the 21.7% of the blank control group.

[0063] splendoside: at concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL, the proportion of C. elegans with A-level movement ability was 33.6%, 34.5%, and 31.4% on day 14, and at a concentration of 0.1 mg / mL, the proportion of C. elegans with A-level movement ability was 40.9%, which was significantly better than the blank control group.

[0064] 5. The effects of different concentrations of pachypodiridoid A and splendoside on the body width of C. elegans are as follows:​​ From Figures 24-25 It can be known that the two compounds can significantly improve the body width of C. elegans.

[0065] Pachypodiridoid A: at the concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.1 mg / mL, the body width of C. elegans at the 10th day is increased by 20.0%, 12.0%, 8.4% and 8.4% respectively.

[0066] Splendoside: at the concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.1 mg / mL, the body width of C. elegans at the 10th day is increased by 16.1%, 3.1%, 16.1% and 12.0% respectively.

[0067] 6, the effects of different concentrations of pachypodiridoid A and splendoside on the body length of C. elegans are as follows: From Figures 26-27 It can be known that the two compounds can significantly improve the body length of C. elegans.

[0068] Pachypodiridoid A: at the concentrations of 1 mg / mL, 0.5 mg / mL and 0.1 mg / mL, the body length of C. elegans at the 10th day is increased by 5.1%, 7.8% and 5.0% respectively.

[0069] Splendoside: at the concentrations of 1 mg / mL, 0.5 mg / mL and 0.25 mg / mL, the body length of C. elegans at the 10th day is increased by 4.4%, 5.0% and 10.4% respectively.

[0070] From the above, it can be known that the two compounds of pachypodiridoid A and splendoside can improve the physiological state of C. elegans from multiple dimensions, can significantly improve the antioxidant stress ability of C. elegans, and also show significant effects on aging-related indicators (life span, movement ability, food intake) and growth and development indicators (body length and body width), which indicates that the two compounds have excellent anti-aging activity. The present application not only provides a new lead compound for anti-aging drug research and development, but also lays a new material foundation and theoretical basis for anti-aging mechanism research, and at the same time opens up a new direction for the development of new anti-aging drugs.

Claims

1. A cyclopentadiene compound with anti-aging activity, the structural formula of which is shown in Formula I or Formula II: I II.

2. The iridoid compound with anti-aging activity according to claim 1, characterized in that, At a concentration of 0.1 mg / mL, compounds I and II significantly prolonged the average lifespan of *C. elegans* compared to the positive control at 0.134 mg / mL, and enhanced the oxidative stress resistance, feeding and swallowing rate, motility, and body length and width of *C. elegans*.

3. The use of the iridoid compound of claim 1 in the preparation of an anti-aging drug for Caenorhabditis elegans.

4. The use of the iridoid compound of claim 1 in the preparation of anti-aging drugs.

5. The method for preparing the cycloalkenyl ether compound according to claim 1, characterized in that, Follow these steps to achieve the following: 1) After removing the stamens from the dried Rhododendron yunnanense flowers, crush them and extract them by reflux with methanol at 70℃ for 2 hours each time, 3 times; 2) The extract was concentrated under reduced pressure, and then extracted with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether extract fraction, ethyl acetate extract fraction and n-butanol extract fraction respectively. 3) The ethyl acetate extract was concentrated under reduced pressure to obtain an extract, which was then subjected to silica gel column chromatography: the extract was mixed with silica gel powder and eluted with a chloroform-methanol solution with a gradient of 100:1-90:1-80:1-70:1-60:1-50:1-40:1-30:1-20:1-10:1-1:1 to obtain 8 fractions Fr. AH; 4) Component Fr. D was subjected to RP-C18 reversed-phase chromatography and Sephadex LH-20 gel chromatography to obtain component Fr. D2; 5) The component Fr. D2 was subjected to silica gel column chromatography and eluted with petroleum ether:ethyl acetate at a volume ratio of 5:1 to obtain compound I.

6. The preparation method according to claim 5, characterized in that, In step 4), the RP-C18 reversed-phase column is eluted with a gradient of methanol-water solution at a volume ratio of 10:90-20:80-30:70-40:60-50:50-60:40-70:30-80:20-90:10-100:0, and the Sephadex LH-20 gel chromatography column is eluted with methanol.

7. The preparation method according to claim 5, characterized in that, After filtering the n-butanol extract in step 2), the target fraction was eluted by gradient elution with an ethanol-water solution of 10:90-20:80-30:70-40:60-50:50 through a medium- and high-pressure chromatography column. The target fraction was collected and then subjected to chromatography on an RP-C18 reversed-phase column, a Sephadex LH-20 gel column, and a silica gel column to obtain compound II.

8. The preparation method according to claim 7, characterized in that, The RP-C18 reversed-phase column was eluted with a gradient of methanol-water solution at a volume ratio of 10:90-20:80-30:70, the Sephadex LH-20 gel chromatography column was eluted with methanol, and the silica gel column was eluted with a dichloromethane-methanol solution at a volume ratio of 10:1.

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