CN114057822B - Extraction method and medical application of ketosteroid compound - Google Patents

Extraction method and medical application of ketosteroid compound Download PDF

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CN114057822B
CN114057822B CN202111386183.1A CN202111386183A CN114057822B CN 114057822 B CN114057822 B CN 114057822B CN 202111386183 A CN202111386183 A CN 202111386183A CN 114057822 B CN114057822 B CN 114057822B
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methanol
yeast
autophagy
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CN114057822A (en
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向兰
戚建华
刘亚楠
刘倩
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The invention provides an extraction method and medical application of a ketosteroid compound, which are obtained by extracting powder of dry roots of gentiana rigescens with methanol, distributing the powder with n-hexane, ethyl acetate, n-butanol and water solvent to obtain a crude extract of a water layer, and separating and purifying the crude extract by a positive-phase and negative-phase open column and HPLC. The sterone compound provided by the invention is a known compound, and the extraction method is simple and easy to implement. The sterone compound provided by the invention has significant autophagy induction activity in YOM38 yeast and PC12 cells, and can be applied as an autophagy inducer. Meanwhile, the method provides a basis for research and development and basic research of anti-aging drugs, foods and health-care foods for preventing neurodegenerative diseases, and has important practical significance.

Description

Extraction method and medical application of ketosteroid compound
Technical Field
The invention belongs to the technical field of medicines, relates to an extraction method and medical application of a ketosteroid compound, and is medical application serving as an autophagy inducer and having an anti-aging effect.
Background
Autophagy is a process whereby vesicles encapsulate self-cytoplasmic proteins or organelles and fuse with lysosomes, degrading the encapsulated contents, thereby fulfilling the metabolic needs of the cell itself and the renewal of certain organelles. Under the oxidative stress state, autophagy can relieve the oxidative damage state of cells and play a role in protecting the cells. Disruption of autophagy leads to accumulation of reactive oxygen species and inhibition of ubiquitination, leading to an imbalance in cell homeostasis, promoting the development of age-related diseases. Evidence from studies in model experiments with a variety of organisms (including yeast, worms, fruit flies, mice, etc.) suggests that inducing autophagy can slow the aging process and prolong life. In addition, autophagy can regulate the functions of pancreatic beta cells and insulin target tissues (skeletal muscle, liver and adipose tissue), enhance autophagy by fasting, can reverse pre-diabetes, and allow effective control of blood glucose. Autophagy can also help our brain cells to clear toxic proteins such as tau and amyloid beta, helping to reduce the risk of neurodegenerative diseases. Autophagy is a double-edged sword for cancer, on the one hand, autophagy eliminates damaged proteins that lead to tumor formation, and on the other hand, autophagy also increases the survival rate of cancer cells. In addition, increasing research has shown that autophagy can clear damaged proteins and organelles from heart cells, thereby reducing the risk of heart disease and other cardiovascular diseases.
Autophagy is classified into selective autophagy and nonselective autophagy. According to the target phagocytes, the selective autophagy is classified into mitochondrial autophagy, endoplasmic reticulum autophagy, nuclear autophagy, and the like. To date, researchers have identified about 40 autophagy-related (Atg) proteins in saccharomyces cerevisiae, where the core Atg protein, a subset of autophagy-related proteins, plays an important role in the formation and expansion of autophagosomes. The Atg8 binding system is a functional group of the core Atg protein. GFP-Atg8 fusion proteins are often used as biomarkers for detecting autophagosome formation. When the autophagy process is successful, GFP-Atg8 is cleaved by yeast vacuolar hydrolase to release the free GFP moiety. Free GFP is relatively stable and accumulates in vacuoles during autophagy. Studies have shown that ubiquitin is tagged as a mitochondrial autophagy substrate by binding to damaged mitochondria. At the same time, ubiquitin chains can facilitate the capture of autophagosomes. Selective autophagy requires the recruitment of other autophagy-related factors to specific organelles by specific receptor proteins. Atg32 is reported to anchor at the surface of mitochondria and is a specific receptor protein for mitochondrial autophagy. Atg32, acting as a mitochondrial autophagy receptor, interacts with Atg11 and Atg8, recruiting mitochondria to the site of phagocyte assembly. The Atg2-Atg18 complex also belongs to the core Atg protein, in yeast autophagy from phagocyte assembly site to recover Atg9. In addition, the Atg2-Atg18/Atg9 autophagy complex can maintain the integrity of Drosophila mitochondria.
Rapamycin is the most commonly used inducer of autophagy, with cell lines ranging from yeast to mammalian cells (including neuronal-like cells). Rapamycin induces autophagy characterized by accumulation of autophagic vesicles and stimulation of autophagic flux. Chloroquine inhibits lysosomal protease activity and, when used in combination with rapamycin, increases the rate of autophagosome formation.
The natural resources of China are rich, a ketosteroid compound is obtained by separating and purifying gentiana rigescens maxim which is a traditional Chinese medicine, and experimental results show that the compound induces mitophagy by regulating genes ATG32 and ATG2 of yeast and enhancing the expression of ubiquitin in mitochondria. Meanwhile, the compound can achieve the autophagy induction effect of the same level as rapamycin in PC12 cells at a lower concentration.
Disclosure of Invention
The invention aims to provide a method for extracting a ketosteroid compound, which is a method for extracting the ketosteroid compound from traditional Chinese medicine gentiana rigescens, and is realized by the following technical scheme: (the steps are combined into 2 pieces to correspond to the claims)
(1) Obtaining a crude extract: pulverizing dry root of Gentiana rigescens (500 g), extracting with 5L methanol at room temperature under shaking for 24 hr; filtering, and concentrating the supernatant to obtain crude extract. Dissolving the crude extract in water, and distributing with n-hexane, ethyl acetate and n-butanol to obtain n-hexane layer sample, ethyl acetate layer sample, n-butanol layer sample and water layer sample;
(2) Separation and purification: the aqueous layer samples were separated by a reversed-phase ODS open column (methanol: water =2, 8,3, 5,7, 1, 10); under the guidance of the K6001 yeast cell activity identification system, the active component was separated again through a silica gel normal phase open column, and the elution site where the methanol: water is 7 was collected and combined to obtain sample I-4, and subjected to a second separation through a silica gel open column (ethyl acetate: methanol =10, 1, 2,7, 5, 0; and (3) collecting and combining elution parts with ethyl acetate and methanol as 8 to obtain a sample II-3, carrying out HPLC purification (conditions are that a Cosmosil C18-AR-II packed column (phi 10/250 mm), the flow rate is 3mL/min, the detection wavelength is 210nm,30% -40% methanol aqueous solution is used, 70 min), collecting one component every five minutes, and if an obvious peak appears within five minutes, separately collecting the peak, and separating to obtain the target compound 1 (the retention time is 64 min).
The ketosteroid compound has the following structure:
Figure BDA0003367124350000021
the second purpose of the invention is to provide the application of the ketosteroid compound in the preparation of autophagy inducer.
The third purpose of the invention is to provide the application of the ketosteroid compound in preparing anti-aging and neurodegenerative disease prevention medicines.
The fourth purpose of the invention is to provide the application of the ketosteroid compound in preparing foods or health-care foods for assisting anti-aging and preventing neurodegenerative diseases.
In an anti-aging-related lifespan test experiment, compound 1 can significantly prolong the replicative and chronological lifespan of yeast cells, and can also prolong the yeast-like chronological lifespan of PC12 cells (see fig. 3,4, 5); in addition, compound 1 can induce neurogenesis and axonal elongation in primary neurons (see fig. 6); in the experiment of yeast and PC12 cells for inducing autophagy, the compound 1 can obviously improve the autophagy level of the yeast cells and achieve the optimal induction effect within 22 hours (see figures 7,8 and 9); the sterone compound induces mitophagy by regulating yeast ATG32 and ATG2 genes and enhancing the expression of ubiquitin in mitochondria (see figures 10, 11 and 12). In addition, the sterone compound was able to achieve the same level of autophagy-inducing effect as rapamycin in PC12 cells at lower doses (see fig. 13).
The ketosteroid compound may be prepared into pharmaceutically acceptable carriers or diluents according to any conventional procedure. The pharmaceutically acceptable carrier as used herein refers to a pharmaceutical carrier which is conventional in the pharmaceutical field, such as diluents, excipients and the like; fillers such as sucrose, starch, and the like; binders such as hydroxypropylcellulose, starch slurry, etc.; wetting agents such as magnesium stearate, aerosil and the like; absorption enhancers polysorbates, lecithins, etc.; the surfactant is sorbitan fatty acid, poloxamer, etc., and other adjuvants such as sweetener, flavoring agent, etc. can be added into the composition.
The sterone compounds of the present invention may be administered in unit dosage form, both enterally and parenterally.
The ketosteroid compound can be mixed with one or more carriers to form a pharmaceutical composition, and the pharmaceutical composition is prepared into required dosage forms according to a conventional production method in the pharmaceutical field.
The parenteral route of administration described herein may be by intravenous injection. The injection includes intravenous injection, intraperitoneal injection, intramuscular injection, acupoint injection and subcutaneous injection.
The pharmaceutical dosage form related to the intestinal administration route can be solid preparation, capsule or liquid preparation, including tablet, capsule, oral liquid, small needle, transfusion, ointment, freeze-dried powder injection, liniment or suppository.
The invention has the advantages that: (1) A ketosteroid compound is obtained through a series of processes such as solvent distribution, chromatographic column separation, high Performance Liquid Chromatography (HPLC) purification and the like, and the preparation method has the advantages of simplicity, rapidness, high purity of the obtained compound and the like. (2) The steroid ketone compound has remarkable autophagy induction activity and can be used as an autophagy inducer. (3) As a small molecular compound, the naturally separated active compound shows obvious anti-aging and nerve growth factor activity simulation at the PC12 cell level in yeast and PC12 cells. The compound is used as a guide to optimize the structure, and has important practical significance for basic research on developing drugs, foods or health-care foods for resisting aging and preventing neurodegenerative diseases.
Drawings
Compound 1 hydrogen spectrum of figure 1 (deuterated methanol as solvent).
Compound 1 carbon spectrum of figure 2 (deuterated methanol as solvent).
Figure 3 effect of compound 1 on replicative life span of K6001 yeast cells. In the figure: the average life span of the negative control group K6001 was 6.4 + -0.43, the average life span of the 10. Mu.M resveratrol-treated K6001 was 8.6 + -0.48 (P < 0.001), the average life span of the compound 1-treated K6001 was 7.7 + -0.67 at 0.1. Mu.M, 8.0 + -0.61 at 0.3. Mu.M (P < 0.05), 10.1 + -0.67 at 1. Mu.M (P < 0.001), 8.5 + -0.65 at 3. Mu.M (P < 0.01), and 7.7 + -0.63 at 10. Mu.M.
Figure 4 effect of compound 1 on time-ordered lifespan of YOM36 yeast cells. In the figure: the maximum survival time for the negative control group YOM36 was 13 days, and the maximum survival time for the YOM36 after compound 1 treatment was as follows: 1 μ M for 15 days (P < 0.001) and 3 μ M for 17 days (P < 0.001).
FIG. 5 Effect of Compound 1 on the yeast-like time-series longevity of PC12 cells. In the figure: (A) Colony forming units of PC12 cells after compound 1 treatment. (B) Image J was a digitized result of the quantification of the colony-forming units of PC12 cells. P <0.05, P <0.01.
Figure 6 effect of compound 1 on neurogenesis of primary neuronal cells. In the figure: fluorescence pictures of PC12 cells taken with a fluorescence microscope after treatment with nerve growth factor (10 ng/mL) or 0.1,0.3. Mu.M Compound 1 for 72 hours and staining with Neuro stain. Arrows indicate neuronal cell bodies.
FIG. 7 Effect of Compound 1 on free GFP formation in YOM38 yeast cells. In the figure: (A) YOM38 yeast (containing plasmid pRS316-GFP-ATG 8) fluorescence images taken with a two-photon confocal fluorescence microscope after treatment with 300. Mu.M resveratrol or 0,0.1, 0.3, 1. Mu.M Compound 1 for 22 hours. (B) Percentage of YOM38 cells containing free GFP (green). Number of pictures per group: n =10. Each picture contained 60 more cells for statistical analysis. * P <0.01, P <0.001.
Figure 8 expression levels of free GFP in compound 1 dose experiments. In the figure: (A) Western blot analysis of GFP-Atg8 and free GFP in YOM38 yeast after 22 hours of treatment with 300. Mu.M resveratrol or 0,0.1, 0.3, 1. Mu.M Compound 1. And (B) the result of the digitization of the diagram (A). * P <0.05, P <0.01.
Figure 9 expression levels of free GFP in compound 1 time course experiments. In the figure: (A) Western blot analysis of GFP-Atg8 and free GFP in YOM38 yeast after various times of treatment with 0.3. Mu.M Compound 1. And (B) the digitized results of the graph (A). * P <0.05, P <0.001.
FIG. 10 Effect of Compound 1 on co-localization of free GFP and mitochondria in YOM38 yeast cells. In the figure: (A) A300. Mu.M resveratrol or 0,0.1, 0.3, 1. Mu.M Compound 1 fluorescence image of YOM38 yeast (containing plasmid pRS316-GFP-ATG 8) treated for 22 hours and stained with Mito-Tracker Red CMXRos taken with a two-photon confocal fluorescence microscope. (B) Percentage of YOM38 cells with free GFP (green) co-localized with MitoTracker Red CMXRos (Red). Number of pictures per group: n =10. Each picture contained 60 more cells for statistical analysis. * P <0.05, P <0.001.
FIG. 11 Effect of Compound 1 on ubiquitin expression in mitochondria of YOM38 yeast cells. In the figure: western blot analysis of YOM38 yeast cell mitochondria in vivo ubiquitin after 22 hours of treatment with 300. Mu.M resveratrol or 0,0.1, 0.3, 1. Mu.M Compound 1.
Figure 12 effect of compound 1 on Δ atg32, Δ atg2 replicative life. In the figure: the mean life of the control group K6001 was 7.95 ± 0.66, whereas the mean life of K6001 after treatment with 10 μ M resveratrol and 0.3 μ M compound 1 was 10.75 ± 0.79 (P < 0.01), 11.58 ± 0.70 (P < 0.001), respectively. (A) The average lifetime of control Δ atg32 was 8.63 ± 0.55, while the average lifetimes of Δ atg32 after 10 μ M resveratrol and 0.3 μ M compound 1 treatment were 8.85 ± 0.52 and 7.10 ± 0.52, respectively. (B) The average lifetime of control group Δ atg2 was 8.38 ± 0.59, while the average lifetimes of Δ atg2 after 10 μ M resveratrol and 0.3 μ M compound 1 treatment were 8.38 ± 0.59 and 8.55 ± 0.54, respectively.
Figure 13 effect of compound 1 on autophagosome formation in PC12 cells. In the figure: fluorescence pictures of PC12 cells taken with a two-photon confocal fluorescence microscope after 18 hours of treatment with rapamycin (500 nM) + chloroquine (10. Mu.M) or compound 1 at 0,0.01, 0.03. Mu.M. Green highlights indicate autophagosomes.
Detailed Description
The present invention is described in further detail with reference to the drawings and examples, but it should not be understood that the scope of the subject matter described above is limited to the following examples, and any technique that can be implemented based on the above description of the present invention is within the scope of the present invention.
EXAMPLE 1 preparation of ketosteroid Compounds
The specific separation and purification steps for obtaining the compound 1 from the gentiana rigescens comprise:
(1) Obtaining a crude extract: pulverizing dried root of Gentiana rigescens (500 g), extracting with 5L methanol under shaking (180 rpm) at room temperature for 24 hr, spin drying the crude extract, dissolving in water again, and partitioning with n-hexane, ethyl acetate and n-butanol to obtain n-hexane layer sample (1.3 g), ethyl acetate layer sample (1.9 g), n-butanol layer sample (8.5 g) and water layer sample (17.8 g);
(2) Separation and purification: the aqueous layer crude (17.8 g) was separated by a reversed-phase ODS open column (methanol: water =2, 8,3, 7,5,7; collecting and combining methanol, wherein the elution part (300 mg) of water is 7; this sample was separated again a second time through a silica gel open column (ethyl acetate: methanol =10: 1,8; and collecting elution parts (38.8 mg) of a combined ethyl acetate and methanol-8 (8) sample II-3, carrying out HPLC purification (conditions are that a Cosmosil C18-AR-II packed column (phi 10/250 mm), the flow rate is 3mL/min, the detection wavelength is 210nm, 30-40% methanol water solution is 70 min), collecting one component every five minutes, if an obvious peak appears within five minutes, independently collecting the peak, and keeping the retention time for 64min to obtain the target compound 1 (2.5 mg).
Example 2 structural identification of compound 1 obtained in example 1:
the structure of the compound 1 is shown by LC-MS, 1 H NMR and 13 c NMR and literature comparison (FIG. 1-2). Compound 1, HRESI-TOF-MS m/z 480.3131, calculated for C 27 H 45 O 7 (M+H) + 481.3226. 1 H NMR(500MHz, CD 3 OD):δ＝1.78(1H,dd,J＝13.26,4.56Hz,H-1α),1.42(1H,dd,J＝8.31,17.53Hz,H-1β), 3.83(1H,m,H-2),3.94(1H,m,H-3),1.71～1.77(2H,m,H-4),2.37(1H,dd,J＝12.80,4.39Hz,H-5), 5.79(1H,d,J＝2.38Hz,H-7),3.15(1H,m,H-9),1.71～1.81(2H,m,H-11),2.12(1H,td,J＝13.02, 4.84Hz,H-12α),1.88(1H,ddd,J＝12.11,3.42Hz,H-12β),1.97(1H,m,H-15α),1.59(1H,m,H-15β), 1.97(1H,m,H-16α),1.74(1H,m,H-16β),2.37(1H,dd,J＝9.5,8.0Hz,H-17),3.33(1H,m,H-22), 1.19(1H,m,H-23b),1.66(1H,m,J＝13,12,4.2,1.8Hz,H-23a),1.1～1.71(2H,m,H-24),1.6(1H,m, H-25),0.88(3H,s,H-18),0.95(3H,s,H-19),1.16(3H,s,H-21),3.46(1H,dd,J＝10.8,5.6,H-26), 3.34(1H,dd,J＝10.8,6.8,H-26),0.93(3H,d,J＝6.7,H-27)。 13 C NMR(125MHz,CD 3 OD): δ＝37.35(C-1),68.70(C-2),68.51(C-3),32.89(C-4),51.79(C-5),206.44(C-6),122.15(C-7), 167.93(C-8),35.09(C-9),39.27(C-10),21.50(C-11),32.52(C-12),49～(C-13),85.25(C-14), 31.77(C-15),21.5(C-16),50.48(C-17),18.04(C-18),24.41(C-19),77.82(C-20),21.01(C-21), 78.22(C-22),30.17(C-23),32.10(C-24),37.06(C-25),68.14(C-26),17.51(C-27)。
EXAMPLE 3 Effect of Compound 1 on the replicative Life of K6001 Yeast cells
The experimental method comprises the following steps:
after the K6001 yeast cells stored at-30 ℃ were thawed at room temperature, an appropriate amount of PBS solution was added, centrifuged at 1500rpm, for 3min, and washing was repeated three times. Removing supernatant, adding 1mL PBS, mixing, adding 300 μ L galactose liquid culture medium (w/v, 3% galactose, 2% polypeptone, 1% yeast extract) to 5mL, and shake culturing for 24h (28 deg.C, 180 rpm); after the yeast cells reached the logarithmic growth phase, 1mL of galactose yeast culture was washed three times by centrifugation using PBS (1500rpm, 3min). Removing supernatant, adding 10mL PBS, mixing by vortex, counting by a cell counting plate, and diluting yeast cells to 100/mu L by using PBS; the sample was dissolved with absolute ethanol to make the desired concentration, compound 1 final concentrations were 0.1,0.3, 1,3, 10 μ M, resveratrol was used as a positive control, 10 μ M was used, absolute ethanol was used as a negative control. Adding 150 μ L of the sample into a glucose solid medium (w/v, 2% glucose, 2% peptone, 1% yeast extract, 2% agar), adding 40 μ L of diluted yeast cells after the solvent on the medium is evaporated, uniformly coating the diluted yeast cells on the medium without liquid by using a coater, and then placing the medium in a constant temperature incubator for culturing for 48h (28 ℃); the proliferation and division of yeast cells were observed using an upright microscope, 40 mother cells were randomly selected per glucose solid medium, and daughter cells around each mother cell were counted. Finally, analysis and mapping were performed using statistical software.
The experimental results are as follows:
the replicative life of yeast cells of K6001 after treatment with 0.3, 1 and 3 μ M of compound 1 was significantly prolonged compared to the control, where compound 1 had a comparable effect to 10 μ M resveratrol at a concentration of 1 μ M (fig. 3).
The research result shows that the compound 1 can remarkably prolong the replicative life span of the K6001 yeast cell at the action concentration of 1,3 mu M.
EXAMPLE 4 Effect of Compound 1 on the chronological Life-span of YOM36 Yeast cells
The experimental method comprises the following steps:
for convenience, the time for starting the culture of the sample with the SD culture solution was defined as day 0.
After the YOM36 strain stored in a refrigerator at-30 ℃ was taken out on day-3, and washed by repeated centrifugation with PBS three times (1500 rpm,5 min), the supernatant was removed, and after adding PBS and mixing, counting by a cell counting plate was performed, and yeast cells were diluted to 5 cells/. Mu.l with PBS. Uniformly coating the diluted cells on a glucose solid culture medium until the surface has no liquid, and then placing the cells in a constant temperature incubator for culturing for 48h (28 ℃); on day-1, single colonies growing on glucose solid medium were picked up in synthetic medium (w/v, 2% glucose, 0.5% ammonium sulphate and 0.17% yeast nitrogen base) and placed in shaker for 24h (28 ℃,180 rpm); on day 0, after taking appropriate amount of yeast culture solution and measuring absorbance, adding a certain amount of yeast into 100mL of synthetic medium to make initial OD of synthetic medium 600 Is 0.01. Then, absolute ethyl alcohol as a negative control and compound 1 with a final concentration of 1,3 μ M were added, mixed well and incubated in a shaker (28 ℃,180 rpm); on day 3, 50. Mu.L of yeast in the synthetic medium was removed, counted on a cell counting plate, and then the cells were diluted to 5 cells/. Mu.L with PBS, and then 40. Mu.L of the diluted yeast was dropped onto a glucose solid plate, and after being uniformly spread on the surface without liquid using a spreader, the plate was placed in a constant temperature incubator and cultured for 48 hours (28 ℃); on day 5, macroscopic yeast colonies on the glucose solid medium were counted and recorded; from day 3, repeat the sampling count every two days andcoating until the survival rate of yeast is lower than 5%; the survival rate of the number of colonies on day 3 was calculated as 100%, and the remaining survival rates (survival rate = the number of colonies on day n/the number of colonies on day 3 of the corresponding group × 100%) were calculated and plotted.
The experimental results are as follows:
the time-ordered lifespan of the YOM36 yeast cells after treatment with 1 and 3 μ M compound 1 was significantly extended compared to the control group (fig. 4).
Research data show that the compound 1 can remarkably prolong the time-sequence life of the YOM36 yeast cells when the acting concentration is 1,3 mu M.
EXAMPLE 5 Effect of Compound 1 on Yeast-like time-series Life of PC12 cells
The experimental method comprises the following steps:
40X 10 cells were inoculated into 100mm dishes containing 10mL of DMEM medium (10% horse serum, 7.5% fetal bovine serum, 1% double antibody) 4 Two days later, the culture medium was replaced for each PC12 cell, and three days later, subculture was performed. Washing the cells twice with 5mL PBS, adding 10mL PBS to the petri dish, at 37 deg.C, 5% CO 2 The carbon dioxide incubator of (1) was incubated for 10 minutes, lightly purged, and transferred to a 15mL disposable centrifuge tube, and after centrifugation at 800rpm for 3 minutes, counted on a hemocytometer. The 96-well cell culture plate is added with 200 mu L of DMEM medium containing serum and double antibodies in advance into each well, and each well is connected with 8 multiplied by 10 4 Individual cell, CO 2 After the incubator is cultured for 24 hours, preparing to carry out activity test; DMSO at 0.5% was used as a negative control, rapamycin (1 μ M) was used as a positive control, and compound 1 was formulated with DMSO at the desired concentration. After replacing each well of the 96-well plate with 200. Mu.L of a DMEM solution (serum-free) containing 0.5% DMSO, rapamycin (1. Mu.M) and compound 1 at final concentrations of 0.003,0.01 and 0.03. Mu.M, the medium was incubated at 37 ℃ and 5% CO 2 The incubator of (2). Every two days, the original medium was replaced with a DMEM solution (without serum) containing the corresponding sample. On the fifth day, 5% of the cell amount was transferred to six-well plates, each of which was previously charged with 2mL of a DMEM solution (containing serum and double antibody). Put at 37 ℃ and 5% CO 2 The cells were cultured in an incubator for 15 days, and the colony formation was observed by a crystal violet staining method.
The experimental results are as follows:
the yeast-like time-series lifespan of PC12 cells after treatment with 0.003 and 0.01 μ M compound 1 was significantly prolonged compared to the control group and was comparable to the effect of 1 μ M rapamycin (fig. 5).
The data show that compound 1 acts at concentrations of 0.003 and 0.01. Mu.M to significantly extend the yeast-like time-series life of PC12 cells.
EXAMPLE 6 Effect of Compound 1 on neurogenesis in Primary neuronal cells
The experimental method comprises the following steps:
taking ICR mice pregnant for 15-17 days, taking out embryos after neck amputation and sacrifice. The embryos removed were transferred to a dish containing precooled HBSS dissecting fluid and placed on ice. In the superclean bench, brain tissue was removed from the embryo, and the cerebral cortex was isolated by removing vascular membranes. The removed cortex was cut into small pieces, transferred to 0.25% trypsin pre-warmed at 37 ℃ for digestion for 20min, and digestion was stopped with DMEM containing 10% FBS. Transferring the tissue into DMEM containing 0.2mg/mL DNase, lightly blowing with a gun, standing for 2min, sucking the supernatant, repeating the above steps for several times to obtain single cell suspension, centrifuging at 1000r/min for 4min, and collecting the precipitate. The cell pellet is resuspended in neuronal cell culture solution, after counting, the cells are inoculated into a 24-well cell plate coated with 0.1mg/mL polylysine and cultured, 10-12 ten thousand cells per well, containing 0.5mL neuronal cell culture solution. Cell input at 37 ℃ and 5% CO 2 The culture was carried out in the incubator of (1), and the plates were changed in volume after 2 hours. The next day after plating of cells, half the amount was loaded (0.25 mL of neuronal cell culture aspirated, 0.25mL of culture containing sample added), and DMSO (0.5%) negative control, or 10ng/mL nerve growth factor positive control, or 0.1,0.3. Mu.M Compound 1 was added to each 24-well plate. Observing cell morphology 24h and 48h after sample addition, staining neurons 72h after sample addition, directly adding Neuro staining agent (in dark place during storage) into the wells to make the final concentration 0.5 μ M, acting for 1h, then sucking out the culture medium, washing twice with PBS, finally adding 150 μ L PBS into each well, observing cells under a fluorescence microscope, and taking pictures.
The experimental results are as follows:
fluorescence picture results show that the number and length of primary neuron cell axons treated by 0.1 mu M and 0.3 mu M of compound 1 are remarkably improved compared with those of a control group, and the effect is equivalent to that of a nerve growth factor of 10ng/mL (figure 6).
The above results indicate that compound 1 is able to induce neurogenesis and axonal elongation in primary neurons.
Example 7 autophagy-inducing Activity of Compound 1 in YOM38 Yeast
The experimental method comprises the following steps:
after inoculating YOM38 yeast cells containing pRS316-GFP-ATG8 plasmid into glucose liquid medium (w/v, 2% glucose, 2% peptone and 1% yeast extract) and shake-culturing under dark conditions (180rpm, 28 ℃) for 24 hours, an appropriate amount of yeast cells were transferred to 20mL of synthetic medium to make the initial OD 600 The value was 0.1, and the sample was added after correcting the OD value. The samples were dissolved in ethanol and formulated to the desired concentrations with compound 1 final concentrations of 0.1,0.3, 1 μ M, resveratrol as a positive control and 300 μ M ethanol as a negative control. The sample solution or control was loaded at a volume of 20. Mu.L. The YOM38 yeast cells were co-incubated with the sample for 22 hours in the dark. After the effect time was reached, 500. Mu.L of the cell culture medium was washed three times with PBS per group, and then stained with DAPI at a final concentration of 20. Mu.g/mL for 12 minutes in the dark, and finally, extracellular DAPI was washed with PBS. The yeast cells were then resuspended in a 30% glycerol solution and observed with a two-photon confocal fluorescence microscope and fluorescence pictures taken. The percentage of cells containing free GFP was analyzed using GraphPad Prism Version 5.01.
In compound dose-related western blot analysis experiments, YOM38 yeast cells were treated with 300 μ M resveratrol or 0,0.1, 0.3 and 1 μ M compound 1 for 22 hours. In experiments with optimal dose and varying duration of action, YOM38 yeast was incubated with 0.3 μ M of compound 1 for 0, 8, 15 and 22 hours. After incubation for the corresponding time, cells were obtained by centrifugation (12,000 × g,3 min), washed three times with PBS and finally resuspended in 500 μ L PBS. The collected yeast cells were sonicated on ice for 5 minutes and the supernatant was obtained by centrifugation (12,000 Xg, 10 minutes) as a protein sample. The concentration of protein in the supernatant was determined using the BCA assay kit. First, 200 μ L BCA working solution (a: B = 50. Subsequently, the 96-well plate was incubated at 37 ℃ for 25 minutes, and immediately after completion of the incubation, the absorbance of the sample at 562nm was measured using a BioTek plate reader. And drawing a standard curve according to the obtained standard protein absorbance value, and further calculating the protein concentration of the sample by a linear equation. Then, 20. Mu.g of yeast protein, GFP-Atg8 and free GFP were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected in Western blot analysis using GFP-specific primary antibody and goat anti-rabbit IgG conjugated to horseradish peroxidase. The internal reference is detected by adopting beta-actin primary antibody and goat anti-mouse IgG connected with horseradish peroxidase.
The experimental results are as follows:
free GFP in YOM38 yeast cells after 22 hours of treatment with 0.1,0.3 and 1 μ M compound 1 was significantly increased compared to the control group and was comparable to 300 μ M resveratrol (figure 7). Western blot analysis of GFP-Atg8 and free GFP showed a significant increase in free GFP expression in compound 1 treated group, with 0.3 μ M being the most effective (fig. 8). Compared to the control group, 0.3. Mu.M Compound 1 significantly increased the expression of free GFP by 22 hours of action (FIG. 9).
The research result shows that the compound 1 can obviously improve the level of the yeast autophagy and achieve the optimal induction effect within 22 hours.
EXAMPLE 8 Compound 1 induces mitophagy in YOM38 Yeast
The experimental method comprises the following steps:
the yeast cell culture and loading steps in the mitophagy experiments were the same as those described for the autophagy experiments in example 3. The subsequent treatments were distinguished by staining the YoM38 yeast cells with 300nM Mito-Tracker Red CMXRos for 1h at 37 ℃ before staining with DAPI (20. Mu.g/mL). The percentage of cells containing free GFP co-localized with the mitochondrial dye Mito-Tracker Red CMXRos was finally analyzed using GraphPad Prism Version 5.01.
The YOM38 yeast cells were exposed to 300 μ M resveratrol or 0,0.1, 0.3 and 1 μ M compound 1 for 22 hours before centrifugation to harvest the cells and sonicate on ice, and the cell lysate was centrifuged twice at 5000 × g for 15 minutes each, and the supernatant was taken. The supernatant was then centrifuged (12,000 Xg, 30 min) to obtain a mitochondrial pellet, which was lysed with RAPI lysis buffer containing 1% protease inhibitor and incubated on ice for 20 min. After centrifugation (12,000 × g,15 min), the supernatant was subjected to ubiquitin western blot analysis as a protein sample. The concentration of protein in the supernatant was determined using the BCA assay kit as described in example 3. Then, 20. Mu.g of yeast protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and in Western blot analysis of ubiquitin specific primary antibody and horseradish peroxidase-linked goat anti-rabbit IgG was used for detection. The internal reference was detected using mitochondrial outer membrane protein porin 1 (VDAC 1) primary antibody together with horseradish peroxidase-linked goat anti-mouse IgG.
In the replicative life measurement, the K6001 strain stored at-30 ℃ was first placed in a 15mL centrifuge tube, washed with 5mL of Phosphate Buffer Solution (PBS) each time, washed 3 times in total, finally inoculated in 5mL of galactose liquid medium, and cultured for 24 hours under shaking (180rpm, 28 ℃). K6001 yeast cells that reached logarithmic growth phase were washed three times with PBS and 4000 cells were plated on glucose agar plates containing either compound 1 at a concentration of 0, 0.3. Mu.M or resveratrol at 10. Mu.M after counting. After culturing at 28 ℃ for 48 hours, 40 microcolonies formed on an agar plate were randomly selected and observed under an Olympus normal microscope, and the number of daughter cells generated by a single mother cell in each microcolony was counted. The determination of the replicative life time of the K6001 mutants Δ atg32, Δ atg2 was in accordance with the procedure described above.
The experimental results are as follows:
mitochondria were localized with the Mito-Tracker Red CMXRos dye, and the co-localization of free GFP and Mito-Tracker Red CMXRos dye in the YOM38 yeast cells after 22 hours of treatment with 0.1,0.3 and 1 μ M of Compound 1 was significantly increased compared to the control group (FIG. 10). Western blot analysis of ubiquitin showed that the ubiquitin expression levels of 0.1 μ M and 1 μ M in the compound 1-treated group were significantly increased and were comparable to 300 μ M resveratrol, where the protein band was too shallow in the 0.3 μ M-treated group or due to incomplete mitochondrial purification (fig. 11). Compound 1 at 0.3 μ M was able to significantly extend the average replicative life span of K6001 compared to the control, but did not change the average replicative life span of Δ atg32, Δ atg2 (fig. 12).
The research results show that the compound 1 can induce mitophagy by regulating the genes ATG32 and ATG2 of the yeast and enhancing the expression of ubiquitin in mitochondria.
EXAMPLE 9 Effect of Compound 1 on autophagosome formation in PC12 cells
The experimental method comprises the following steps:
40X 10 cells were inoculated into 100mm dishes containing 10mL of DMEM medium (10% horse serum, 7.5% fetal bovine serum, 1% double antibody) 4 Two days later, the culture medium was replaced for each PC12 cell, and three days later, subculture was performed. Washing the cells twice with 5mL PBS, adding 10mL PBS to the culture dish, at 37 deg.C, 5% 2 The cells were cultured in a carbon dioxide incubator for 10 minutes, gently tapped, transferred to a 15mL disposable centrifuge tube, centrifuged at 800rpm for 3 minutes, and then counted on a hemocytometer. Each well of 24-well cell culture plate is previously added with 1mL of DMEM medium containing serum and double antibody and circular slide glass with the diameter of 1cm, and each well is connected with 8 multiplied by 10 4 Individual cell, CO 2 The incubator was ready for activity testing after 24 hours of incubation.
DMSO was used as a negative control at 0.1% and rapamycin (500 nM) + chloroquine (10 μ M) was used as a positive control, and compound 1 was dissolved in DMSO and formulated to the desired concentration. The 24-well cell plates were replaced per well of the original medium with 1mL of DMEM solution (serum-free) containing 0.1% DMSO, rapamycin (500 nM) + chloroquine (10. Mu.M) and 0.01, 0.03. Mu.M Compound 1, placed at 37 ℃ and 5% 2 Was cultured in an incubator for 18 hours. After the culture time is reached, according to CYTO-
Figure BDA0003367124350000111
Staining was performed according to the instructions of the Autophary Detection Kit (Enzo Life Sciences, inc. New York, USA), after which the circular slide was taken out, inverted on the slide, and mounted with mounting solution. Observation under an upright two-photon confocal microscopeEach autophagosome formation group was statistically analyzed by selecting at least 3 pictures from each group.
The experimental results are as follows:
fluorescence picture results show that the autophagosome formation rate in PC12 cells was significantly improved after 0.01 μ M,0.03 μ M compound 1 treatment compared to the control group, and the effect of 0.03 μ M compound 1 in combination with rapamycin (500 nM) + chloroquine (10 μ M) was comparable (fig. 13).
The above results indicate that compound 1 can achieve the same level of autophagy-inducing effect as rapamycin in PC12 cells at lower concentrations.

Claims (6)

1. A method for preparing a ketosteroid compound, wherein the ketosteroid compound has the following structure:
Figure DEST_PATH_IMAGE002
the method is characterized by comprising the following steps:
(1) Obtaining a crude extract: extracting dried root powder of Gentiana rigescens Bunge with methanol, filtering, spin-drying the crude extract, dissolving in water again, and distributing with n-hexane, ethyl acetate and n-butanol solvent to obtain n-hexane layer sample, ethyl acetate layer sample, n-butanol layer sample and water layer sample;
(2) Separation and purification: carrying out first separation on the water layer sample through a reversed-phase ODS open column, and combining the obtained components to obtain five samples I-1-I-5; collecting and combining methanol, namely performing secondary separation on an elution part with water of 7 as a sample I-4 by using a silica gel open column, combining the obtained components to obtain six samples II-1-II-6, collecting and combining ethyl acetate, namely performing HPLC purification on the elution part with methanol of 8 as a sample II-3, collecting one component every five minutes, and if an obvious peak appears within five minutes, separately collecting the peak, and separating for 64min to obtain a target compound 1;
wherein in the step (2), the first separation conditions are methanol; the second separation conditions were ethyl acetate-methanol =10, 0,9, 1,8, 7; the HPLC purification conditions were: cosmosil C18-AR-II packed column, Φ 10/250mm, flow rate: 3mL/min, detection wavelength: 210 And nm, 30-40% of methanol aqueous solution for 70 min.
2. Use of a ketosteroid compound obtained by the method of claim 1 for the preparation of an autophagy inducing agent.
3. The use of a ketosteroid compound obtained according to the method of claim 1 for the preparation of a medicament for anti-aging and preventing neurodegenerative diseases.
4. The use of claim 2, wherein the autophagy inducing agent is administered enterally and parenterally.
5. The use of claim 3, wherein the route of entry of said drug into the human body is enteral or parenteral.
6. The use according to claim 5, wherein the medicament is in the form of a solid or liquid formulation.
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