Guanacastane type diterpene derivative candollins AL and its preparation method and application

Abstract

The present invention provides a Guanacastane diterpene derivative candollinsA-L and a preparation method and application thereof, and relates to the technical field of pharmaceutical chemical preparation. The Guanacastane diterpene derivative candollinsA-L is a secondary metabolite obtained by solid fermentation of yam agar using yam endophyte Psathyrella candolleana SY 7×1 with a preservation number of CCTCC NO: M 2024722 as a raw material. The compound candollinsA-L is a new compound with protection by MPP + Guanacastane-type diterpene derivatives that induce damaged nerve cells provide a research basis for the discovery of small molecule drugs that can treat or alleviate neurodegenerative diseases such as Parkinson's disease.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemical preparation, and in particular to a Guanacastane-type diterpene derivative candollins AL and a preparation method and application thereof. Background Art

[0002] Parkinson's disease (PD) is a neurodegenerative disease caused by protein aggregation, neuronal death and tension in the central nervous system. In recent years, the prevalence of Parkinson's disease has increased year by year, seriously threatening the life safety and quality of life of the middle-aged and elderly population. However, there is no effective therapeutic drug that can effectively cure or alleviate the pathological process of Parkinson's disease. Mitochondrial damage and dysfunction caused by the inhibition of electron transfer of complex I by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+) is an important pathogenic mechanism of Parkinson's disease. Research and exploration of drugs that can protect neuronal cells damaged by MPP+ is one of the important ways to discover neurodegenerative diseases such as Parkinson's disease. Plant endophyte secondary metabolites are one of the important ways to discover natural small molecule drugs with biological activity. They are characterized by simple and efficient preparation methods, low production costs, and easy mass production. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides a Guanacastane-type diterpene derivative candollins AL and a preparation method and application thereof, which can be obtained by solid fermentation of plant endophytes agar and has a novel protective effect by MPP. + Guanacastane-type diterpene derivatives that induce damaged nerve cells provide a research basis for the discovery of small molecule drugs that can treat or alleviate neurodegenerative diseases such as Parkinson's disease.

[0004] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0005] Guanacastane-type diterpene derivatives candollins AL, the chemical structures of the candollins AL are shown below:

[0006] Candollins A: Candollins B:

[0007] Candollins C: Candollins D:

[0008] Candollins E: Candollins F:

[0009] Candollins G: Candollins H:

[0010] Candollins I: candollins J:

[0011] Candollins K: Candollins L:

[0012] The preparation method of guanacastane-type diterpene derivative candollins AL comprises the following steps:

[0013] S1. Using the yam endophyte Psathyrella candolleana SY 7×1 with a preservation number of CCTCC NO: M 2024722 as a raw material, inoculating the yam endophyte Psathyrella candolleana SY 7×1 into a yam agar solid culture medium for solid fermentation culture, obtaining a solid fermentation product after the fermentation is completed, and extracting the solid fermentation product with ethyl acetate to obtain a yam solid fermentation crude extract of Psathyrella candolleana SY 7×1;

[0014] S2. The crude fermentation extract was repeatedly separated by normal phase chromatography and reverse phase chromatography to obtain the compound candollins AL.

[0015] Preferably, the formula of the yam agar culture medium in step S1 is 200g yam, 20g glucose, 15g agar, and 1L water; and the fermentation condition is static fermentation at room temperature for 2 months.

[0016] Preferably, the method for preparing the crude solid fermentation extract in step S1 is to mix and extract the solid fermentation product with equal volumes of ethyl acetate for 5 times, and then vacuum concentrate and combine to obtain the crude extract.

[0017] Preferably, the separation and purification method of normal phase chromatography and reverse phase chromatography in step S2 comprises the following steps:

[0018] S2-1, dissolving the crude extract in a dichloromethane-methanol mixed solution with a volume ratio of 1:1 to obtain a concentrated crude extract solution;

[0019] S2-2, the crude extract concentrated solution was mixed with silica gel of the same mass as the dry weight of the crude extract, placed in a constant temperature oven at 55°C to dry to remove the organic solvent, and loaded with 100-200 mesh silica gel column, and gradient eluted with the following solutions in sequence, and divided into seven fractions 1-7 according to polarity differences:

[0020] Petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 30:1, a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 20:1, a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1, dichloromethane, a mixed solution of dichloromethane and methanol in a volume ratio of 50:1, and a mixed solution of dichloromethane and methanol in a volume ratio of 10:1;

[0021] S2-3, using a normal phase column to perform gradient elution in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1, 30:1, 25:1, 20:1, 15:1 and 10:1, and dividing it into 7 parts Fr.4.1-4.7 according to TLC analysis;

[0022] S2-4, take Fr.4.3 segment and use gel chromatography with methanol as eluent, combine the parts with the same TLC analysis results to obtain three parts Fr.4.3.1-Fr.4.3.3; take Fr.4.3.1 and use silica gel chromatography to separate, using a mixed solution of dichloromethane:methanol with a volume ratio of 150:1 as eluent, to separate candollins L;

[0023] S2-5, Fr.4.3.2 was separated by silica gel chromatography, the eluent being a mixed solution of dichloromethane:methanol in a volume ratio of 200:1, and then candollins I was separated by gel chromatography using methanol as the eluent;

[0024] S2-6, take Fr.4.4 segment and use gel chromatography with methanol as eluent to elute, combine the parts with the same TLC analysis results to obtain 4 parts Fr.4.4.1-Fr.4.4.4, take Fr.4.4.1 and use silica gel chromatography to separate, the eluent is a mixed solution of dichloromethane: methanol in a volume ratio of 150:1, to obtain candollins A;

[0025] S2-7, take Fr.4.4.3 segment and separate it by silica gel chromatography, using the eluents as a mixed solution of petroleum ether:ethyl acetate in a volume ratio of 5:1 and dichloromethane:methanol in a volume ratio of 100:1, to obtain candollins D;

[0026] S2-8, take Fr.4.5 segment and use gel chromatography with methanol as eluent to elute, combine the parts with the same TLC analysis results to obtain 5 parts Fr.4.5.1-Fr.4.5.5, take Fr.4.5.2 segment and use silica gel chromatography to separate, the eluent is a mixed solution of dichloromethane: methanol with a volume ratio of 100:1 and petroleum ether: ethyl acetate with a volume ratio of 5:1, then use gel chromatography with methanol as eluent to elute to obtain candollins B;

[0027] S2-9, take Fr.4.6 segment and use gel chromatography with methanol as eluent to elute, combine the parts with the same TLC analysis results to obtain 4 parts Fr.4.6.1-Fr.4.6.4; take Fr.4.6.2 segment and use silica gel chromatography to separate, the eluent is a mixed solution of petroleum ether: ethyl acetate with a volume ratio of 5:1 and dichloromethane: methanol with a volume ratio of 150:1, to obtain candollins E and candollins F;

[0028] S2-10, take Fr.4.6.3 segment and separate it by silica gel chromatography, the eluent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1, and then use gel chromatography with methanol as the eluent to elute, to obtain candollins G;

[0029] S2-11, take Fr.4.6.4 segment and separate it by silica gel chromatography, the eluent is a mixed solution of petroleum ether:acetone with a volume ratio of 5:1 and dichloromethane:methanol with a volume ratio of 100:1, and then use gel chromatography with methanol as the eluent to elute, to obtain candollins C and candollins K;

[0030] S2-12. Take segment Fr.4.7 and use gel chromatography with methanol as the eluent to elute it and divide it into 5 parts Fr.4.7.1-4.7.5 according to the TLC analysis results; take segment Fr.4.7.2 and use silica gel chromatography to separate it. The eluent is a mixed solution of dichloromethane: methanol in a volume ratio of 150:1 and petroleum ether: ethyl acetate in a volume ratio of 6:1, to obtain candollins H and candollins J.

[0031] The 12 guanacastane diterpene derivatives candollins AL or the solid fermentation products of Pleurotus eryngii agar or the ethyl acetate extraction products thereof provided by the present invention, which have the function of protecting PC12 cells from MPP+-induced damage, can be specifically used in the preparation of protective drugs for MPP+-induced nervous system damage.

[0032] The present invention provides a Guanacastane-type diterpene derivative candollins AL and a preparation method and application thereof, which have the following advantages over the prior art:

[0033] The compounds candollins AL provided by the present invention are all guanacastane-type diterpene derivatives with novel structures. Screening tests on the neuron protection activity of the series of compounds show that all the compounds have a protective effect on PC12 cells induced by MPP+ damage; and the compound candollins AL is a secondary metabolite of yam agar solid fermentation of white yellow brittle mushroom Psathyrella candolleana SY 7×1. The compound candollins AL obtained by the method has a short overall fermentation cycle and mild conditions, and the extraction and separation process of the secondary metabolites is simple to operate, has strong stereoselectivity, is low in cost, is easy to realize industrialization, and can achieve MPP+ damage. + The protective effect of induced nerve cell damage provides a research basis for the development of small molecule drugs to treat or alleviate neurodegenerative diseases such as Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a morphological diagram of the white and yellow brittle mushroom (Psathyrella candolleana) SY 7×1 of the present invention;

[0035] Figure 2 The compound candollin A of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0036] Figure 3 The compound candollin A of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0037] Figure 4 The HMBC spectrum of candollin A (CDCl 3 );

[0038] Figure 5 The HSQC spectrum of candollin A (CDCl 3 );

[0039] Figure 6 The compound candollin A of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0040] Figure 7 The NOESY spectrum of candollin A (CDCl 3 );

[0041] Figure 8 is the HR-ESI-MS spectrum of candollin A, a compound of the present invention;

[0042] Fig. 9 is the ECD spectrum of the compound candollin A of the present invention;

[0043] Fig.10 The compound candollin B of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0044] Fig.11 The compound candollin B of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0045] Fig.12 The HMBC spectrum of candollin B (CDCl 3 );

[0046] Fig.13 The HSQC spectrum of candollin B (CDCl 3 );

[0047] Fig.14 The compound candollin B of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0048] Fig.15 NOESY spectrum of candollin B (CDCl 3 );

[0049] Fig.16 is the HR-ESI-MS spectrum of candollin B, a compound of the present invention;

[0050] Fig.17 is the ECD spectrum of the compound candollin B of the present invention;

[0051] Fig.18 The compound candollin C of the present invention 1H-NMR spectrum (400MHz, CDCl 3 );

[0052] Fig.19 The compound candollin C of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0053] Fig. 20 The HMBC spectrum of candollin C of the present invention (CDCl 3 );

[0054] Fig.21 The HSQC spectrum of candollin C of the present invention (CDCl 3 );

[0055] Fig. 22 The compound candollin C of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0056] Fig.23 The NOESY spectrum of candollin C of the present invention (CDCl 3 );

[0057] Fig.24 is the HR-ESI-MS spectrum of the compound candollin C of the present invention;

[0058] Fig.25 is the ECD spectrum of the compound candollin C of the present invention;

[0059] Fig.26 The compound candollin D of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0060] Fig. 27 The compound candollin D of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0061] Fig.28 The HMBC spectrum of candollin D (CDCl 3 );

[0062] Fig.29The HSQC spectrum of candollin D (CDCl 3 );

[0063] Fig.30 The compound candollin D of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0064] Fig.31 The NOESY spectrum of candollin D (CDCl 3 );

[0065] Fig.32 is the HR-ESI-MS spectrum of candollin D, a compound of the present invention;

[0066] Fig.33 is the ECD spectrum of the compound candollin D of the present invention;

[0067] Fig.34 The compound candollin E of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0068] Fig.35 The compound candollin E of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0069] Fig.36 The HMBC spectrum of candollin E of the compound of the present invention (CDCl 3 );

[0070] Fig.37 The HSQC spectrum of candollin E of the present invention (CDCl 3 );

[0071] Fig.38 The compound candollin E of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0072] Fig.39 The NOESY spectrum of candollin E of the present invention (CDCl 3 );

[0073] Fig.40is the HR-ESI-MS spectrum of candollin E, a compound of the present invention;

[0074] Fig.41 is the ECD spectrum of the compound candollin E of the present invention;

[0075] Fig.42 The compound candollin F of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0076] Fig.43 The compound candollin F of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0077] Fig.44 The HMBC spectrum of candollin F, a compound of the present invention (CDCl 3 );

[0078] Fig.45 The HSQC spectrum of candollin F, a compound of the present invention (CDCl 3 );

[0079] Fig.46 The compound candollin F of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0080] Fig.47 The NOESY spectrum of candollin F, a compound of the present invention (CDCl 3 );

[0081] Fig.48 is the HR-ESI-MS spectrum of the compound candollin F of the present invention;

[0082] Fig.49 is the ECD spectrum of the compound candollin F of the present invention;

[0083] Fig.50 The compound candollin G of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0084] Fig.51 The compound candollin G of the present invention 13 C-NMR spectrum (100 MHz, CDCl3 );

[0085] Fig.52 The HMBC spectrum of candollin G, a compound of the present invention (CDCl 3 );

[0086] Fig.53 The HSQC spectrum of candollin G, a compound of the present invention (CDCl 3 );

[0087] Fig.54 The compound candollin G of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0088] Fig.55 The NOESY spectrum of candollin G, a compound of the present invention (CDCl 3 );

[0089] Fig.56 is the HR-ESI-MS spectrum of candollin G, a compound of the present invention;

[0090] Fig.57 is the ECD spectrum of candollin G, a compound of the present invention;

[0091] Fig.58 The compound candollin H of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0092] Fig.59 The compound candollin H of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0093] Fig.60 The HMBC spectrum of candollin H of the compound of the present invention (CDCl 3 );

[0094] Fig.61 The HSQC spectrum of candollin H, a compound of the present invention (CDCl 3 );

[0095] Fig.62 The compound candollin H of the present invention 1 H- 1 H COSY spectrum (CDCl3 );

[0096] Fig.63 The NOESY spectrum of candollin H (CDCl 3 );

[0097] Fig.64 is the HR-ESI-MS spectrum of candollin H, a compound of the present invention;

[0098] Fig.65 is the ECD spectrum of the compound candollin H of the present invention;

[0099] Fig.66 The compound candollin I of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0100] Fig.67 The compound candollin I of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0101] Fig.68 The HMBC spectrum of candollin I (CDCl 3 );

[0102] Fig.69 The HSQC spectrum of candollin I of the present invention (CDCl 3 );

[0103] Fig.70 The compound candollin I of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0104] Fig.71 The NOESY spectrum of candollin I (CDCl 3 );

[0105] Fig.72 is the HR-ESI-MS spectrum of the compound candollin I of the present invention;

[0106] Fig.73 is the ECD spectrum of the compound candollin I of the present invention;

[0107] Fig.74 The compound candollin J of the present invention1 H-NMR spectrum (400MHz, CDCl 3 );

[0108] Fig.75 The compound candollin J of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0109] Fig.76 The HMBC spectrum of candollin J, a compound of the present invention (CDCl 3 );

[0110] Fig.77 The HSQC spectrum of candollin J of the present invention (CDCl 3 );

[0111] Fig.78 The compound candollin J of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0112] Fig.79 The NOESY spectrum of candollin J of the present invention (CDCl 3 );

[0113] Fig.80 is the HR-ESI-MS spectrum of candollin J, a compound of the present invention;

[0114] Fig.81 is the ECD spectrum of the compound candollin J of the present invention;

[0115] Fig.82 The compound candollin K of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0116] Fig.83 The compound candollin K of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0117] Fig.84 The HMBC spectrum of candollin K of the present invention (CDCl 3 );

[0118] Fig.85The HSQC spectrum of candollin K of the present invention (CDCl 3 );

[0119] Fig.86 The compound candollin K of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0120] Fig.87 The NOESY spectrum of candollin K (CDCl 3 );

[0121] Fig.88 is the HR-ESI-MS spectrum of candollin K, a compound of the present invention;

[0122] Fig.89 is the ECD spectrum of candollin K, a compound of the present invention;

[0123] Fig.90 The compound candollin L of the present invention 1 H-NMR spectrum (400MHz, CDCl 3 );

[0124] Fig.91 The compound candollin L of the present invention 13 C-NMR spectrum (100 MHz, CDCl 3 );

[0125] Fig.92 The HMBC spectrum of candollin L (CDCl 3 );

[0126] Fig.93 The HSQC spectrum of candollin L (CDCl 3 );

[0127] Fig.94 The compound candollin L of the present invention 1 H- 1 H COSY spectrum (CDCl 3 );

[0128] Fig.95 The NOESY spectrum of candollin L (CDCl 3 );

[0129] Fig.96is the HR-ESI-MS spectrum of candollin L, a compound of the present invention;

[0130] Fig.97 is the ECD spectrum of the compound candollin L of the present invention;

[0131] Fig.98 The absolute configuration of compound 6 of the present invention is calculated by ECD spectrum;

[0132] Fig.99 Compounds 1-12 of the present invention are MPP + Schematic diagram of the protective activity of PC12 cells induced with injury. DETAILED DESCRIPTION

[0133] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0134] In the present invention: Psathyrella candolleana SY 7×1, the preservation name is Psathyrella candolleana SY 7×1, the preservation number is CCTCC NO: M 2024722, the preservation address is China. Wuhan. Wuhan University. China Type Culture Collection, the culture was received by the preservation center on April 19, 2024, and registered. According to the request, it will be preserved for thirty years from that date, and will be preserved for another five years after receiving a request for providing a sample of the culture before the expiration. The viability of the culture was tested by the preservation center on April 26, 2024, and the result was survival.

[0135] Embodiment 1:

[0136] Isolation and identification of strain Psathyrella candolleana SY 7×1:

[0137] The collected yam was sterilized with 75% ethanol solution for 20 seconds, washed with sterile water for 6 times, and the excess ethanol solution was removed. Then the washed yam tissue was cut into 2 mm 3Use fire-sterilized tweezers to transfer the cut tissue to a plate (PDA, Bengal red culture medium), with 5 tissues per plate, and 20 plates for each culture medium. All the above operations are performed in a sterile clean bench. Seal the culture dish with sealing film and put it in a constant temperature incubator with a temperature set to 28°C. During the culture process, observe the morphology and growth of the fungi every other day until they grow to a suitable size for separation, then take them out for numbering. Inoculate them on PDA culture medium, and after a period of growth, observe whether they are single colonies, screen out the same strains, and inoculate the remaining strains after sifting out the weights on the slant of the test tube at 4°C for later use.

[0138] The strain SY7×1 was cultured on PDA medium, and the colony morphology was as follows Figure 1 As shown: The mycelium grows slowly, and after 5 days of cultivation at 28℃, it grows into a circular colony with a diameter of about 3 cm. The front of the colony is white and yellow flocculent, and the center of the colony on the back is black and the color gradually fades from the center to the periphery. The mycelium is dense and grows radially. No exudate is observed in the colony, and there is no obvious odor.

[0139] The ITS gene sequence of strain SY7×1 was obtained by sequencing and then spliced ​​to determine its credible interval sequence as shown in the following SQEID No.1:

[0140] SQE ID No.1:

[0141] ;

[0142] Combined with morphological characteristics and molecular identification, specifically by analyzing the rRNA internal transcribed spacer (ITS) region (mycelium samples were sent to Qingke Biotechnology Co., Ltd. for extraction, amplification and sequencing), the sequencing results were entered into GenBank (www.ncbi.nlm.nih.gov), and using the homologous sequences in GenBank, the results showed that the similarity between the endophyte SY7×1 and the Psathyrella candolleana EU520251.1 in NCBI reached 99.26%. The isolated endophyte was determined to be an endophytic fungus Psathyrella candolleana SY7×1 of the genus Psoralea of ​​the Coprinus family, and it was named and biologically preserved with the preservation number CCTCC NO: M 2024722.

[0143] Embodiment 2:

[0144] Preparation of compound 1-12:

[0145] 1. Bacterial activation: Psathyrella candolleana SY7×1 isolated in Example 1 was inoculated into a PDA slant culture medium and placed in a 28° C. constant temperature incubator for 3 days to complete activation. The activated strain was placed in a 4° C. refrigerator for standby use.

[0146] 2. Preparation of yam agar solid medium: Wash and peel fresh yam and cut into 1cm 3 Weigh 200g of chopped yam and add it to 1L of water and boil for 20min. Filter through gauze to get the water extract of yam. Add 20g of glucose and 15g of agar successively and stir to dissolve evenly. Dispense into 1L conical bottles, 300ml of culture medium per bottle. Sterilize at 121℃ for 30min, then take out and cool naturally to solidify.

[0147] 3. Fermentation process: inoculate the activated strain into the prepared yam agar solid culture medium and ferment it at room temperature for 2 months.

[0148] 4. After the fermentation is completed, the solid fermentation medium is extracted with ethyl acetate, and the volume ratio of the medium to ethyl acetate is 1:1. The ethyl acetate extraction is performed 5 times, and the crude extract is obtained after vacuum concentration.

[0149] 5. Dissolve the obtained crude extract in a dichloromethane-methanol mixed solution with a volume ratio of 1:1 to obtain a concentrated solution of the crude extract;

[0150] The obtained crude extract is dissolved in an appropriate amount of a dichloromethane-methanol mixed solution with a volume ratio of 1:1 to obtain a crude extract concentrated solution; the above crude extract concentrated solution is mixed with silica gel of an equal mass to the dry weight of the crude extract, placed in a constant temperature oven at 55°C to dry to remove the organic solvent, and a 100-200 mesh silica gel column is used for column loading, and a mixed solution of petroleum ether, petroleum ether: ethyl acetate (v:v) 30:1, petroleum ether: ethyl acetate (v:v) 20:1, petroleum ether: ethyl acetate (v:v) 10:1, dichloromethane, dichloromethane: methanol (v:v) 50:1, and dichloromethane: methanol (v:v) 10:1 is used in sequence for gradient elution, thereby dividing the crude extract into seven fractions 1-7 according to polarity differences.

[0151] The petroleum ether: ethyl acetate (v:v) 10:1 segment (Fraction 4) was used with a normal phase column, and gradient elution was performed with a mixed solution system of petroleum ether: ethyl acetate (v:v) 30:1, petroleum ether: ethyl acetate (v:v) 25:1, petroleum ether: ethyl acetate (v:v) 20:1, petroleum ether: ethyl acetate (v:v) 15:1 and petroleum ether: ethyl acetate (v:v) 10:1 and divided into 7 fractions (Fr. 4.1-4.7) according to TLC analysis.

[0152] Fr.4.3 was subjected to gel chromatography using methanol as the eluent, and the parts with the same TLC analysis results were combined to obtain three parts Fr.4.3.1-Fr.4.3.3. Fr.4.3.1 was separated by silica gel chromatography using a mixed solution of dichloromethane:methanol (v:v) 150:1 as the eluent to obtain compound 12.

[0153] Fr.4.3.2 was separated by silica gel chromatography using a mixed solution of dichloromethane:methanol (v:v) 200:1 as the eluent, and then separated by gel chromatography using methanol as the eluent to obtain compound 9.

[0154] Take segment Fr.4.4 and use gel chromatography with methanol as eluent to elute, combine the parts with the same TLC analysis results to obtain 4 parts Fr.4.4.1-Fr.4.4.4, take Fr.4.4.1 and use silica gel chromatography to separate, the eluent is a mixed solution of dichloromethane:methanol (v:v) 150:1, to obtain compound 1.

[0155] Fr.4.4.3 segment was separated by silica gel chromatography using a mixed solution of petroleum ether:ethyl acetate (v:v) 5:1 and dichloromethane:methanol (v:v) 100:1 as the eluent to obtain compound 4.

[0156] Segment Fr.4.5 was separated by gel chromatography using methanol as the eluent, and the portions with the same TLC analysis results were combined to obtain five portions Fr.4.5.1-Fr.4.5.5. Segment Fr.4.5.2 was separated by silica gel chromatography using a mixed solution of dichloromethane:methanol (v:v) 100:1 and petroleum ether:ethyl acetate (v:v) 5:1 as the eluent, and then gel chromatography was performed using methanol as the eluent to obtain compound 2.

[0157] Fr.4.6 was separated by gel chromatography using methanol as the eluent, and the parts with the same TLC analysis results were combined to obtain four parts Fr.4.6.1-Fr.4.6.4. Fr.4.6.2 was separated by silica gel chromatography using a mixed solution of petroleum ether: ethyl acetate (v:v) 5:1 and dichloromethane: methanol (v:v) 150:1 as the eluent to obtain compounds 5 and 6.

[0158] Fr.4.6.3 segment was separated by silica gel chromatography using a mixed solution of petroleum ether and ethyl acetate (v:v) of 5:1 as the eluent, and then eluted by gel chromatography using methanol as the eluent to obtain compound 7.

[0159] Fr.4.6.4 segment was separated by silica gel chromatography, with the eluent being petroleum ether:acetone (v:v) 5:1 and dichloromethane:methanol (v:v) 100:1, and then gel chromatography was performed with methanol as the eluent to obtain compound 3 and compound 11.

[0160] Fr.4.7 was separated by gel chromatography using methanol as the eluent and divided into five parts (Fr.4.7.1-4.7.5) according to the TLC analysis results. Fr.4.7.2 was separated by silica gel chromatography using a mixed solution of dichloromethane:methanol (v:v) 150:1 and petroleum ether:ethyl acetate (v:v) 6:1 as the eluent to obtain Compound 8 and Compound 10.

[0161] Embodiment 3:

[0162] Structural identification of compounds 1-12:

[0163] The structure of candollins AL(1-12) was identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry).

[0164] Depend on 1 H-NMR and 13 C-NMR, combined with HSQC-DEPT spectrum, can obtain the chemical shifts corresponding to the C and H phases of the compound.

[0165] 1. For compound 1:

[0166] Compound 1 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 317.1750 [M+H] + ,calcd.for C 19 H 25 O 4 , 317.1753; indicating that its molecular formula is C 19 H 24 O 4 ; has 8 degrees of unsaturation.

[0167] according to 1 H-NMR and 13 C-NMR spectrum data showed that compound 1 contained 4 methyl groups [δ H1.73(3H,s,H-15),δ H 1.02(3H,s,H-16),δ H 1.03(3H,s,H-17),δ H 1.17(3H,t,H-15);δ C 11.1(C-15),δ C 26.0(C-16),δ C 19.6(C-17),δ C 11.2 (C-19)];

[0168] 4 methylene groups [δ H 1.90(1H,m,H-7β),2.13(1H,dd,J=13.0Hz,5.5Hz,H-7α); 1.64(1H,m,H-9β),2.44(1H,t d,J=14.1Hz,4.4Hz,H-9α);1.67(1H,m,H-10β),1.85(1H,m,H-10α);16.9(2H,m,H-18);δ C 45.5(C-7),33.6(C-9),29.6(C-10),16.9(C-18)];

[0169] 1 oxygen-containing methine [δ H 4.43(1H,dd,J=14.1Hz,5.6Hz,H-6),δ C 67.5(C-6)];

[0170] 1 olefin methine [δ H 6.99(1H,s,H-2)δ C 127.0(C-2)], 7 quaternary carbons [δ C 140.0(C-1),157.1(C-3),129.0(C-4),37.0(C-8),43.0(C-11),151.4(C-12),148.8(C-13)];

[0171] 2 carbonyl carbons [δ C 198.7 (C-5), 186.8 (C-14)];

[0172] From the NMR data of the compound, it can be seen that its structure is similar to the guanacastane diterpene psathin A reported in the literature [TDZhao, XQYang, J.Zhou, YBYang, ZTDing, Antibiotic GuanacastaneDiterpenoids with Two New Skeletons from Psathyrella candolleana Uncovered by Semisolid and Liquid Media, J.Agric.Food Chem., 2023, 71, 2006-2013], except that the compound lacks an oxygen-containing methylene at C-19 and a hydroxyl substitution at C-6. The correlation of H-18 with C-12 in the HMBC spectrum; H-19 with C-12 and C-18, and the correlation of H-18 with H-19 in the COSY spectrum determined that C-18 and C-19 are an ethyl group.

[0173] The planar structure of compound 1 was determined by HMBC correlation of H-7 with C-3, C-5, C-6 and C-8; H-15 with C-3, C-4 and C-5; H-16 with C-3, C-7, C-8 and C-9; H-17 with C-1, C-10, C-11 and C-12, and 1H-1H COSY correlation of H-6 / H-7 and H-9 / H-10. In order to determine the stereo configuration of the compound, the relative configuration of compound 1 was determined by NOESY correlation of H-6 / H-9α; H-16 / H-9β and H-17 / H-10α. The absolute configuration of compound 1 was determined by comparing its experimental CD spectrum with that of psathin A and its biosource. It was named candollin A with the structural formula:

[0174]

[0175] 2. For compound 2:

[0176] Compound 2 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 329.1390 ​​[M+H] + , calcd.for C 19 H 21 O 5 ,329.1389. This indicates that its molecular formula is C 19 H 20 O 5 . It has 10 degrees of unsaturation. 1 H-NMR and 13C-NMR spectral data showed that its structure was similar to candollins A, except that this compound lacked a methylene group at C-18, an oxygen-containing methine group at C-6, and a methylene group at C-7. An acetyl group was identified at C-18 and C-19 by correlation of H-19 with C-12 and C-18 in the HMBC spectrum. The planar structure of compound 2 was determined by HMBC correlation of H-2 with C-1 and C-14; H-7 with C-3, C-5, C-6, and C-8; H-15 with C-3, C-4, and C-5; H-16 with C-3, C-7, C-8, and C-9; and H-17 with C-1, C-10, C-11, and C-12, as well as 1H-1H COSY correlation of H-9 / H-10. The relative configuration of compound 2 was determined by NOESY correlation of H-16 / H-9β and H-17 / H-10α. The absolute configuration of compound 2 was determined by comparing its experimental CD spectrum and biosource with that of compound 1, and it was named candollin B with the structural formula:

[0177]

[0178] 3. For compound 3:

[0179] Compound 3 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 315.1592 [M+H] + calcd.for C 19 H 23 O 4 ,315.1596. This indicates that its molecular formula is C 19 H 22 O 4 . It has 9 degrees of unsaturation. 1 H-NMR and 13 C-NMR spectral data showed that its structure was similar to that of compounds 1 and 2. The structure of the five-membered ether ring was determined by HMBC correlation of H-2 with C-1, C-3 and C-14; H-15 with C-2, C-3 and C-4. The structure of the five-membered ether ring was determined by HMBC correlation of H-6 with C-5 and C-7; H-16 with C-7 and C-8; H-17 with C-1, C-10, C-11 and C-12; H-18 with C-12 and C-13; H-19 with C-12, C-18 and C-19, and the HMBC correlation of H-6 / H-7, H-9 / H-10 and H-18 / H-19 in the COSY spectrum. 1 H- 1The planar structure of compound 3 was determined by HCOSY correlation. The relative configuration of compound 3 was determined by NOESY correlation of H-2 / H-16, H-16 / H-9β and H-17 / H-10α. The absolute configuration of compound 3 was determined by comparing its experimental CD spectra and biosource with those of compounds 1 and 2. It was named candollin C with the structural formula:

[0180]

[0181] 4. For compound 4:

[0182] Compound 4 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 289.1433 [M+H] + calcd.for C 17 H 21 O 4 ,289.1440. This indicates that its molecular formula is C 17 H 20 O 4 . It has 8 degrees of unsaturation. According to 1H-NMR and 13C-NMR spectral data, its structure is similar to that of compound 1, except for the ethyl groups at C-18 and C-19. The structure of the five-membered ring was determined by HMBC correlation of H-12 with C-1, C-11, C-14 and C-17; H-17 with C-1 and C-11. The structure of the five-membered ring was determined by HMBC correlation of H-2 with C-1 and C-14; H-6 with C-5 and C-7; H-7 with C-3 and C-8; H-15 with C-3, C-4 and C-5; H-16 with C-3, C-8 and C-9; H-17 with C-10, as well as H-6 / H-7 and H-9 / H-10. 1 H- 1 The planar structure of compound 4 was determined by H COSY correlation. The relative configuration of compound 4 was determined by NOESY correlation of H-6 / H-9α, H-16 / H-9 and H-17 / H-10α in the spectrum. The absolute configuration of compound 4 was determined by comparing its experimental CD spectrum with that of compound 1 and its biosource. It was named candollin D and its structural formula is:

[0183]

[0184] 5. For compound 5:

[0185] Compound 5 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 317.1749 [M+H] + calcd.for C 19 H 25 O4 ,317.1753. This indicates that its molecular formula is C 19 H 24 O 4 . It has 8 degrees of unsaturation. 1 H-NMR and 13 C-NMR spectral data showed that its structure was similar to compound 1, except that the methyl group at C-15 and the ethyl groups at C-18 and C-19 disappeared. The HMBC correlations of H-4 with C-2, C-6 and C-8; H-6 with C-5; H-7 with C-6 and C-8 and the H-6 / H-7 1 H- 1 The structure of the six-membered ring was determined by HCOSY correlation. The isopropyl structures of C-18, C-19 and C-20 were determined by HMBC correlation of H-17 with C-12 and C-1; H-18 with C-12 and C-17; H-19 with C-12 and C-17 and 1H-1H COSY correlation of H-19 / H-18 / H-20. The planar structure of compound 5 was determined by HMBC correlation of H-2 with C-1, C-11 and C-14; H-6 with C-5; H-7 with C-6 and C-8; H-15 with C-3, C-7, C-8 and C-9; H-16 with C-1, C-10, C-11 and C-12 and 1H-1H COSY correlation of H-6 / H-7 and H-9 / H-10. The relative configuration of compound 5 was determined by NOESY correlation of H-6 / H-9α, H-16 / H-9 and H-17 / H-10α in the spectrum. The absolute configuration of compound 5 was determined by comparing its experimental CD spectrum with compound 1 and its biosource, and it was named candollin E, and its structural formula is:

[0186]

[0187] 6. For compound 6:

[0188] Compound 6 is a yellow oil, and the quasi-molecular ion peak measured by HR-ESI-MS is m / z 315.1588 [M+H] + calcd. for C19H23O4, 315.1596. This indicates that its molecular formula is C19H22O4. It has 9 unsaturations. According to 1H-NMR and 13C-NMR spectral data, its structure is similar to that of compound 5, except for the methyl group at C-4 and the methyl group at C-15. The structure of the benzene ring was determined by HMBC correlation of H-2 with C-3 and C-8; H-5 with C-6, C-7 and C-8; H-9 with C-8; H-10 with C-8 and H-15 with C-3, C-4 and C-5. The planar structure of compound 6 was determined by HMBC correlation of H-2 with C-1, C-11 and C-14; H-10 with C-11; H-16 with C-1, C-10, C-11 and C-12; H-17 with C-12 and C-13; H-18 with C-12 and C-17 and H-19 with C-12 and C-17, and 1H-1H COSY correlation of H-9 / H-10 and H-18 / H-17 / H-19. The relative configuration of compound 6 was determined by NOESY correlation of H-6 / H-9α, H-16 / H-9β and H-17 / H-10α. The absolute configuration of compound 6 was determined by ECD calculation spectrum (such as Fig.98 As shown in the figure, the source of the product was determined and named candollin F, and its structural formula is:

[0189]

[0190] 7. For compound 7:

[0191] Compound 7 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 403.2110 [M+H] + calcd.for C 23 H 31 O 6 , 403.2121. This indicates that its molecular formula is C 23 H 30 O 6 . It has 9 degrees of unsaturation. 1 H-NMR and 13 C-NMR spectral data showed that its structure was similar to compound 5, except for the methyl group at C-4. The HMBC correlations of H-15 with C-3, C-4, C-5 and C-17; H-16 with C-4, C-15 with C-17; H-18 with C-17 and the H-15 / H-16 1 H- 1The substituent at C-4 was determined to be methyl propionate by H COSY correlation. The planar structure of compound 7 was determined by HMBC correlation of H-2 with C-1, C-11 and C-14; H-6 with C-5 and C-7; H-7 with C-4, C-5 and C-8; H-19 with C-3, C-7, C-8 and C-9; H-20 with C-1, C-10, C-11 and C-12; H-21 with C-12 and C-13; H-22 with C-12 and C-21 and H-23 with C-12 and C-21, and 1H-1H COSY correlation of H-6 / H-7, H-9 / H-10 and H-22 / H-21 / H-23. The relative configuration of compound 7 was determined by NOESY correlation of H-6 / H-9α, H-16 and H-9β, and H-17 and H-10α. The absolute configuration of compound 7 was determined by comparing its experimental CD spectrum with that of compound 5 and its biosource, and it was named candollin G, and its structural formula is:

[0192]

[0193] 8. For compound 8:

[0194] Compound 8 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 347.1849 [M+H] + calcd.for C 20 H 27 O 5 ,347.1858. This indicates that its molecular formula is C 20 H 26 O 5 . It has 8 degrees of unsaturation. 1 H-NMR and 13C-NMR spectrum data showed that its structure was similar to that of psathin E in the literature [TDZhao, XQYang, J.Zhou, YBYang, ZTDing, Antibiotic Guanacastane Diterpenoids with Two New Skeletonsfrom Psathyrella candolleana Uncovered by Semisolid and Liquid Media, J.Agric.Food Chem., 2023, 71, 2006-2013], except for the oxygen-containing methine at the C-13 position and the olefin methine at the C-2 position. The planar structure of compound 8 was finally determined by HMBC correlation of H-1 with C-2, C-11 and C-14; H-7 with C-3, C-5, C-6 and C-8; H-12 with C-13 and C-18; H-13 with C-11, C-12, C-14 and C-18; H-15 with C-2, C-3 and C-4; H-16 with C-3, C-8 and C-9; H-17 with C-1, C-10 and C-11; H-18 with C-12; H-19 with C-18 and H-20 with C-18, as well as 1H-1H COSY correlation of H-1 / H-2, H-9 / H-10, H-13 / H-12 / H-18 and H-19 / H-18 / H-20. The relative configuration of compound 8 was determined by NOESY correlation of H-1 / H-17, H-2 / H-16, H-17 / H-10α, H-6 / H-9α, H-16 / H-9β, H-17 / H-10 and H-12 / H-13. The absolute configuration of compound 8 was determined by comparing its experimental CD spectrum and biosource with that of compound psathin E. It was named candollin H and its structural formula is as follows:

[0195]

[0196] 9. For compound 9:

[0197] Compound 9 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 343.1540 [M+H] + calcd.for C 20 H 23 O 5 ,343.1545. This indicates that its molecular formula is C 20 H 22 O 5 . It has 10 degrees of unsaturation. 1 H-NMR and 13C-NMR spectrum data showed that its structure was similar to that of psathin F in the literature [TDZhao, XQYang, J.Zhou, YBYang, ZTDing, Antibiotic Guanacastane Diterpenoids with Two NewSkeletons from Psathyrella candolleana Uncovered by Semisolid and LiquidMedia, J.Agric.Food Chem., 2023, 71, 2006-2013], except for the methylene groups at C-6 and C-7, the oxygen-containing methine at C-13, the carbonyl group at C-14 and the oxygen-containing methine at C-15. The planar structure of compound 9 was finally determined by HMBC correlation of H-7 with C-3, C-5, C-6 and C-8; H-12 with C-11, C-13 and C-18; H-15 with C-2, C-3 and C-4; H-16 with C-3, C-8 and C-9; H-17 with C-1, C-10 and C-11; H-19 with C-12 and C-18 and H-20 with C-12 and C-18, as well as 1H-1H COSY correlation of H-9 / H-10, H-12 / H-18 and H-19 / H-18 / H-20. The relative configuration of compound 9 was determined by NOESY correlation of H-16 / H-9β, H-10α / H-17 / H-18. The absolute configuration of compound 9 was determined by comparing its experimental CD spectra and biosource with those of compound psathin F, and it was named candollin I, and its structural formula is as follows:

[0198]

[0199] 10. For compound 10:

[0200] Compound 10 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 361.1639 [M+H] + calcd.for C 20 H 25 O 6 ,361.1651. This indicates that its molecular formula is C 20 H 24 O 6 . It has 10 degrees of unsaturation. 1 H-NMR and 13C-NMR spectrum data showed that its structure was similar to heptemerones E in the literature [M. Kettering, C. Valdivia, O. Sterner, H. Anke, E. Thines, Heptemerones A~G, Seven Novel Diterpenoids from Coprinus heptemerus: Producing Organism, Fermentation, Isolation and Biological Activities, J. Antibio, 2005, 58, 390-396]. The planar structure of compound 10 was finally determined by HMBC correlation of H-7 with C-3, C-5, C-6 and C-8; H-12 with C-11 and C-18; H-13 with C11, C-12 and C-14; H-15 with C-2, C-3 and C-4; H-16 with C-3, C-8 and C-9; H-17 with C-1, C-10 and C-11; H-19 with C-12 and C-18 and H-20 with C-12 and C-18, as well as 1H-1H COSY correlation of H-9 / H-10, H-13 / H-12 / H-18 and H-19 / H-18 / H-20. The relative configuration of compound 10 was determined by NOESY correlation of H-12 / H-1, H-16 / H-9β, H-10α / H-17 / H-18. The absolute configuration of compound 10 was determined by the biosource of heptemerones E. It was named candollin J and its structural formula is as follows:

[0201]

[0202] 11. For compound 11:

[0203] Compound 11 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 347.1854 [M+H] + calcd.for C 20 H 27 O 5 ,347.1858. This indicates that its molecular formula is C 20 H 26 O 5 . It has 10 degrees of unsaturation. 1 H-NMR and 13C-NMR spectral data showed that its structure was similar to that of compound 9. The planar structure of compound 11 was finally determined by HMBC correlation of H-2 with C-1 and C-3; H-6 with C-5; H-7 with C-3, C-5, C-6 and C-8; H-12 with C-11 and C-13; H-15 with C-2, C-3 and C-4; H-16 with C-3, C-8 and C-9; H-17 with C-1, C-10 and C-11; H-19 with C-12 and C-18 and H-20 with C-12 and C-18, and 1H-1H COSY correlation of H-6 / H-7, H-9 / H-10 and H-19 / H-18 / H-20. The relative configuration of compound 11 was determined by NOESY correlation of H-2 / H-16, H-6 / H-9α, H-16 / H-9β, H-10α / H-17 / H-18. The absolute configuration of compound 11 was determined by comparing the experimental CD spectrum and biosource of compound 9. It was named candollin K and its structural formula is as follows:

[0204]

[0205] 12. For compound 12:

[0206] Compound 12 is a yellow oil. The quasi-molecular ion peak of the compound measured by HR-ESI-MS is m / z 329.1749 [M+H] + calcd.for C 20 H 25 O 4 ,329.1753. This indicates that its molecular formula is C 20 H 24 O 4 . It has 9 degrees of unsaturation. 1 H-NMR and 13 C-NMR spectral data showed that its structure was similar to that of compound 10. The planar structure of compound 12 was finally determined by HMBC correlation of H-2 with C-1 and C-14; H-7 with C-3, C-5, C-6 and C-8; H-15 with C-3 and C-4; H-16 with C-3, C-8 and C-9; H-17 with C-1, C-10 and C-11; H-19 with C-12 and C-1 and H-20 with C-18, as well as 1H-1H COSY correlation of H-9 / H-10 and H-19 / H-18 / H-20. The relative configuration of compound 12 was determined by NOESY correlation of H-16 / H-9β, H-17 / H-10α. The absolute configuration of compound 12 was determined by comparing the experimental CD spectrum and biosource of compound 10. It was named candollinL, and its structural formula is as follows:

[0207]

[0208] Embodiment 4:

[0209] Effect of guanacastane-type diterpene derivatives candollins AL (compounds 1-12) on MPP + Induced protective activity against PC12 cell damage:

[0210] Select MPP + The Parkinson's disease model (PD) was established by treating dopaminergic cells PC12.

[0211] The specific experimental steps are as follows:

[0212] 1. PC12 poorly differentiated cells were cultured in DMEM high glucose + 10% FBS + 100U / mL double antibody culture medium, incubator temperature 37°C, 5% CO 2 ; When the PC12 poorly differentiated cells grew to a suitable number, they were digested with trypsin to prepare a cell suspension;

[0213] 2. Pipette the cell suspension into a 15 mL centrifuge tube and centrifuge at 800 rpm for 5 min. After centrifugation, discard the supernatant, add 5 mL of new complete culture medium and pipette into a uniform cell suspension;

[0214] 3. Take 0.02 ml of cell suspension and count using a cell counting plate to adjust the cell concentration to 1×10 5 cells / ml, add 96-well plate, 100 μL per well, and culture in a cell culture incubator;

[0215] 4. After 23 hours, the original culture medium was aspirated, and then new culture medium (same as the formula in 1) was added, and the sample to be tested was added (the final concentration of the compound in the system was 20 μM). After 1 hour, MPP was added (the final concentration of MPP in the system was 750 μM);

[0216] 5. Experimental design: Each group was designed with 3 replicates.

[0217] Blank group: only culture medium;

[0218] MPP group: medium plus MPP at a final concentration of 750 μM;

[0219] Compound group: The medium was supplemented with the compound at a final concentration of 20 μM, and then with MPP at a final concentration of 750 μM.

[0220] 6. After adding MPP for 24 hours, add MTS and read the value after 2 hours.

[0221] The specific results are shown in the following table:

[0222]

[0223]

[0224] The experimental results showed that the screened compounds 1, 2, 8, 11, and 12 had an effect on MPP + induced neuronal damage (p<0.01); compounds 3-7, 9, and 10 had a significant protective effect on MPP + It had a very significant protective effect on the induced neuronal damage (p<0.001).

[0225] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Guanacastane-type diterpene derivative candollins AL, characterized in that The chemical structures of the candollins AL are shown below: candollins A: ;candollins B: ; candollins C: ;candollins D: ; candollins E: ;candollins F: ; candollins G: ;candollins H: ; candollins I: ;candollins J: ; candollins K: ;candollins L: 。 2. A method for preparing the Guanacastane-type diterpene derivative candollins AL as claimed in claim 1, characterized in that: The preparation method comprises the following steps: S1, using the yam endophyte Agaricus leucoderma with the preservation number of CCTCC NO: M 2024722 ( Psathyrella candolleana )SY 7´1 as a raw material, inoculating the yam endophyte Pleurotus eryngii SY 7´1 into a yam agar solid medium for solid fermentation, obtaining a solid fermentation product after the fermentation, and extracting the solid fermentation product with ethyl acetate to obtain a yam solid fermentation crude extract of Pleurotus eryngii SY 7´1; S2, repeatedly using normal phase chromatography and reverse phase chromatography to separate the fermentation crude extract to obtain compound candollins AL; The separation and purification method of normal phase chromatography and reverse phase chromatography comprises the following steps: S2-1, dissolving the crude extract in a dichloromethane-methanol mixed solution with a volume ratio of 1:1 to obtain a concentrated crude extract solution; S2-2, the crude extract concentrated solution was mixed with silica gel of the same mass as the dry weight of the crude extract, placed in a constant temperature oven at 55°C to dry to remove the organic solvent, and loaded with 100-200 mesh silica gel column, and gradient eluted with the following solutions in sequence, and divided into seven fractions 1-7 according to polarity differences: Petroleum ether, a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 30:1, a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 20:1, a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1, dichloromethane, a mixed solution of dichloromethane and methanol in a volume ratio of 50:1, and a mixed solution of dichloromethane and methanol in a volume ratio of 10:1; S2-3, using a normal phase column to perform gradient elution in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1, 30:1, 25:1, 20:1, 15:1 and 10:1, and dividing it into 7 parts Fr.4.1-4.7 according to TLC analysis; S2-4, take Fr.4.3 segment and use gel chromatography with methanol as eluent, combine the parts with the same TLC analysis results to obtain three parts Fr. 4.3.1-Fr. 4.3.3; take Fr. 4.3.1 and use silica gel chromatography with a mixed solution of dichloromethane:methanol in a volume ratio of 150:1 as eluent to separate candollins L; S2-5, Fr. 4.3.2 was separated by silica gel chromatography, the eluent being a mixed solution of dichloromethane:methanol in a volume ratio of 200:1, and then candollins I was separated by gel chromatography using methanol as the eluent; S2-6, take Fr. 4.4 and use gel chromatography with methanol as eluent to elute, combine the parts with the same TLC analysis results to obtain 4 parts Fr. 4.4.1-Fr. 4.4.4, take Fr. 4.4.1 and use silica gel chromatography to separate, using a mixed solution of dichloromethane: methanol in a volume ratio of 150:1 as eluent to obtain candollins A; S2-7, take Fr. 4.4.3 segment and separate by silica gel chromatography, using the eluents as a mixed solution of petroleum ether:ethyl acetate in a volume ratio of 5:1 and dichloromethane:methanol in a volume ratio of 100:1, to obtain candollins D; S2-8, take Fr. 4.5 segment and use gel chromatography with methanol as eluent to elute, combine the parts with the same TLC analysis results to obtain 5 parts Fr. 4.5.1-Fr.4.5.5, take Fr. 4.5.2 segment and use silica gel chromatography to separate, the eluent is a mixed solution of dichloromethane: methanol in a volume ratio of 100:1 and petroleum ether: ethyl acetate in a volume ratio of 5:1, then use gel chromatography with methanol as eluent to elute to obtain candollins B; S2-9, take Fr. 4.6 segment and use gel chromatography with methanol as eluent to elute, combine the parts with the same TLC analysis results to obtain 4 parts Fr. 4.6.1-Fr. 4.6.4; take Fr. 4.6.2 segment and use silica gel chromatography to separate, the eluent is a mixed solution of petroleum ether: ethyl acetate in a volume ratio of 5:1 and dichloromethane: methanol in a volume ratio of 150:1, to obtain candollins E and candollins F; S2-10, take Fr. 4.6.3 segment and separate it by silica gel chromatography, the eluent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1, and then use gel chromatography with methanol as the eluent to elute, to obtain candollins G; S2-11, take Fr. 4.6.4 and separate it by silica gel chromatography, using a mixture of petroleum ether:acetone in a volume ratio of 5:1 and dichloromethane:methanol in a volume ratio of 100:1 as the eluent, and then use gel chromatography with methanol as the eluent to obtain candollins C and candollins K; S2-12. Take segment Fr. 4.7 and use gel chromatography with methanol as the eluent to elute it and divide it into 5 parts Fr.4.7.1-4.7.5 according to the TLC analysis results; take segment Fr. 4.7.2 and use silica gel chromatography to separate it. The eluent is a mixed solution of dichloromethane: methanol in a volume ratio of 150:1 and petroleum ether: ethyl acetate in a volume ratio of 6:1, to obtain candollins H and candollins J.

3. The preparation method according to claim 2, characterized in that: The formula of the yam agar culture medium in step S1 is 200g yam, 20g glucose, 15g agar, and 1L water; the fermentation condition is static fermentation at room temperature for 2 months.

4. The preparation method according to claim 2, characterized in that: The method for preparing the solid fermentation crude extract in step S1 is to mix the solid fermentation product and ethyl acetate in equal volumes for extraction 5 times, and then vacuum concentrate and combine to obtain the crude extract.

5. Use of the Guanacastane-type diterpene derivative candollins AL as claimed in claim 1 or the solid fermentation product or crude extract prepared by any preparation method of claims 2 to 4 in the preparation of a protective agent for brain and nervous system damage.

6. The use according to claim 5, characterized in that: The brain nervous system damage is caused by the death of nerve cells induced by MPP+.

7. A neuroprotective drug, characterized in that: The drug comprises any one or more of the Guanacastane-type diterpene derivative candollins AL according to claim 1 or the solid fermentation product according to any one of claims 2-4, and the crude extract according to any one of claims 2-4.

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