Microtoxane type sesquiterpenoids and application of microtoxane type sesquiterpenoids in preparation of anti-diabetic drugs

Abstract

The invention relates to a microtoxane type sesquiterpenoids compound and an application of the microtoxane type sesquiterpenoids compound in preparation of an anti-diabetic drug. According to the present invention, the microtoxane type sesquiterpenoids are extracted from Dendrobium maximowiczianum, comprise a compound 1, a compound 2 and a compound 3, have significant improvement effects on insulin resistance, and have anti-diabetic activity. The compounds are extracted from plants, have the advantages of high efficiency and low toxicity, and are expected to be developed into novel antidiabetic drugs.

Description

Technical Field

[0001] The present application relates to the technical field of plant chemistry, and in particular to a class of picrotoxane-type sesquiterpenoid compounds and their application in the preparation of anti-diabetic drugs. Background Art

[0002] Picrotoxane-type sesquiterpenes are a class of chemical components containing multiple chiral centers, mainly existing in the form of picrotoxane-type alkaloids and non-alkaloids. So far, more than 130 compounds of this type have been isolated and reported from plants such as Orchidaceae (Dendrobium nobile, Dendrobium macrobractefolium, Dendrobium clubroot, Dendrobium hook, Dendrobium candidum), Moraceae, and Menispermaceae. At present, the biological functional applications of this type of sesquiterpenes are mainly focused on the effects on the nervous system, the immune system, and anti-tumor effects.

[0003] Dendrobium plants have stems that nourish yin, clear heat, benefit the stomach and promote the production of body fluids, and have brightly colored flowers. They are perennial epiphytic herbs that combine medicinal and ornamental values. Their stems can be used as medicine, and their flowers are brightly colored, so they are often used as ornamental plants. Dendrobium plants are rich in sesquiterpenes and their alkaloids, phenylpropanoids, bibenzyls, flavonoids, phenanthrene, steroids, and other common types of compounds in Dendrobium plants. Among them, piccrotoxane-type sesquiterpenoid compounds are one of the representative chemical components of some plants in the Dendrobium genus (Dendrobium nobile, Dendrobium scalyx, Dendrobium scalyx, etc.). Related pharmacological activity studies have shown that the total alkaloid components in Dendrobium nobile and Dendrobium scalyx have certain anti-diabetic activity. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present application provides a class of picrotoxane-type sesquiterpenoid compounds, which have anti-diabetic activity.

[0005] The present application provides a class of picrotoxane-type sesquiterpenoid compounds, characterized in that they have one of the following structures:

[0006]

[0007] The present application also provides a method for preparing the above-mentioned picrotoxane-type sesquiterpenoid compound, comprising the following steps:

[0008] 1) crushing the dried stem of Dendrobium candidum, adding ethanol to extract, obtaining an ethanol extract, and concentrating to obtain an extract;

[0009] 2) separating the extract by normal phase silica gel column chromatography to obtain component Fr.10 and component Fr.12; the eluent used for the normal phase silica gel column chromatography separation is chloroform-acetone solvent;

[0010] 3) Component Fr.10 is separated by MCI column to obtain 5 components, namely component Fr.10.1, component Fr.10.2, component Fr.10.3, component Fr.10.4 and component Fr.10.5;

[0011] The eluent used for MCI column separation is methanol-water solvent, and the volume ratio of methanol to water in the methanol-water solvent is 40:60-80:20; component Fr.10.1 is separated into three components by Sephadex LH-20, and then recrystallized by reverse phase RP-18 column chromatography to obtain compound 1 and compound 3; the eluent used for Sephadex LH-20 is methanol;

[0012] 4) Component Fr.12 is separated by MCI column to obtain 4 components, namely, component Fr.12.1, component Fr.12.2, component Fr.12.3 and component Fr.12.4;

[0013] The eluent used for MCI column separation is methanol-water solvent, and the volume ratio of methanol to water in the methanol-water solvent is 30:70-80:20; component Fr.12.2 is separated by silica gel column chromatography, purified by Sephadex LH-20, and recrystallized in a mixed solvent of acetone and methanol to obtain compound 2.

[0014] Optionally, the mass fraction of ethanol in step 1) is 95%.

[0015] Optionally, in step 1), the volume ratio (kg:L) of the mass of the dried stem of Dendrobium candidum to ethanol is 1:20.

[0016] Optionally, in step 1), the ethanol extraction is performed twice, each extraction time is 2 hours, and the extraction temperature is 25°C.

[0017] The present application also provides the use of a picrotoxane sesquiterpenoid compound in the preparation of an antidiabetic drug, wherein the picrotoxane sesquiterpenoid compound is any one of the above-mentioned compounds 1-3 or compounds 4-16. The use of a picrotoxane sesquiterpenoid compound in the preparation of an antidiabetic drug is characterized in that the picrotoxane sesquiterpenoid compound is any one of the compounds 1-3 or compounds 4-16 described in claim 1; the structural formula of the compound 4-16 is:

[0018]

[0019] The technical solution provided by this application may have the following beneficial effects:

[0020] 1) The present application extracts sesquiterpenoid compounds from Dendrobium macrobractefolium, including Compound 1, Compound 2 and Compound 3. The sesquiterpenoid compounds have a significant improvement effect on insulin resistance and have anti-diabetic activity, and Compound 1 shows a significant dose-dependency.

[0021] 2) The present application extracts compounds 4, 5, 7, 10, 11-13 and 15 from Dendrobium candidum and Dendrobium schrenkiana, all of which have a significant improvement effect on insulin resistance. Compounds 3-5 and 11-15 can significantly increase glucose-stimulated insulin secretion. The insulin secretion-promoting effects of compound 5 at a concentration of 12.5 μM, compound 11 at a concentration of 12.5-50 μM, compound 12 at a concentration of 50 μM, and compound 15 at a concentration of 12.5 μM and 25 μM are all stronger than those of the positive control drug glibenclamide, and compounds 11-13 and compound 15 all show good dose dependence. It provides a new option for clinical screening and / or preparation of anti-diabetic drugs.

[0022] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0024] Figure 1This is a schematic diagram of the structural formula of the picrotoxane-type sesquiterpene compounds shown in the present application: dendrowardin K (1), dendrowardin L (2), dendrowardin O (3), dendrobiumane C (4), dendrowardin E (5), dendrowardin C (6), aduncin (7), dendrowardin P (8), dendroterpene D (9), amotin (10), crystallinin (11), (+)-(1R,2S,3R,4S,5R,6S,9R)-2,11,12-Trihydroxypicrotoxane-3 (15)-lactone (12), dendroside F (13), dendromoniliside D (14), 6-hydroxydendroxine (15), and dendrobine (16).

[0025] Figure 2 This is a schematic diagram showing the effect of picrotoxane-type sesquiterpenoid compounds on the survival rate of Hep-G2 cells shown in the present application;

[0026] Figure 3 This is a schematic diagram showing the effect of picrotoxane-type sesquiterpenoid compounds on glucose consumption in Hep-G2 cells that induce insulin resistance;

[0027] Figure 4 It is a schematic diagram showing the effect of picrotoxane-type sesquiterpenoid compounds on the survival rate of β-CT-116 cells shown in the present application;

[0028] Figure 5 This is a schematic diagram of the glucose-stimulated insulin secretion activity of picrotoxane-type sesquiterpenoid compounds shown in the present application;

[0029] Figure 6 The dendrowardin K shown in this application 1 H NMR spectrum;

[0030] Figure 7 The dendrowardin K shown in this application 13 C NMR and DEPT spectra;

[0031] Figure 8 The dendrowardin K shown in this application 1 H- 1 H COSY spectrum;

[0032] Fig. 9 is the HSQC spectrum of dendrowardin K shown in this application;

[0033] Fig.10 is the HMBC spectrum of dendrowardin K shown in the present application;

[0034] Fig.11 is the ROESY spectrum of dendrowardin K shown in the present application;

[0035] Fig.12 The dendrowardin L shown in this application 1 H NMR spectrum;

[0036] Fig.13 The dendrowardin L shown in this application 13 C NMR and DEPT spectra;

[0037] Fig.14 The dendrowardin L shown in this application 1 H- 1 H COSY spectrum;

[0038] Fig.15 is the HSQC spectrum of dendrowardin L shown in this application;

[0039] Fig.16 is the HMBC spectrum of dendrowardin L shown in the present application;

[0040] Fig.17 is the ROESY spectrum of dendrowardin L shown in the present application;

[0041] Fig.18 The dendrowardin O shown in this application 1 H NMR spectrum;

[0042] Fig.19 The dendrowardin O shown in this application 13 C NMR and DEPT spectra;

[0043] Fig. 20 The dendrowardin O shown in this application 1 H- 1 H COSY spectrum;

[0044] Fig.21 is the HSQC spectrum of dendrowardin O shown in this application;

[0045] Fig. 22 is the HMBC spectrum of dendrowardin O shown in the present application;

[0046] Fig.23 This is the ROESY spectrum of dendrowardin O shown in this application. DETAILED DESCRIPTION

[0047] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0048] Example 1

[0049] The dried stems and leaves of Dendrobium candidum were crushed and extracted with 95% industrial ethanol (4×30L, once every two days) under reflux. The extraction times were 2 times, each extraction time was 2 hours, the extraction temperature was 25°C, the mass of the dried stems of Dendrobium candidum to the volume ratio of ethanol (kg:L) was 1:20, the ethanol was recovered under reduced pressure, concentrated to an extract, dissolved and filtered with methanol, mixed with silica gel, and roughly segmented by normal phase silica gel column chromatography (chloroform-acetone volume ratio of 15:1-0:1), and silica gel thin layer chromatography was used for detection and merging of parts with similar polarity to obtain 9 components Fr.5-Fr.13. Component Fr.10 (14 g) was eluted through an MCI column (methanol-water volume ratio of 40:60-80:20), detected by silica gel thin layer chromatography and combined with the parts with similar polarity to obtain 5 components, namely component Fr.10.1, component Fr.10.2, component Fr.10.3, component Fr.10.4 and component Fr.10.5. Component Fr.10 used chloroform and acetone in a volume ratio of 1:1; component Fr.12 used chloroform and acetone in a volume ratio of 1:0.

[0050] Component Fr.5 (15 g): Firstly separated by hydroxypropyl glucose gel (chloroform-methanol volume ratio of 1:1), detected by silica gel thin layer chromatography and combined with the parts with similar polarity to obtain three parts Fr.5.1-Fr.5.3, among which Fr.5.2 was purified by silica gel column chromatography to obtain two components Fr.5.2.1 and component Fr.5.2.2; component Fr.5.2.2 was purified by silica gel column chromatography (petroleum ether-acetone volume ratio of 30:1), and recrystallized to obtain dendrowardin P (compound 8, 100 mg).

[0051] Component Fr.6 (20 g) was firstly separated by Sephadex LH-20 (chloroform-methanol volume ratio of 1:1) to obtain 8 components Fr.6.1-Fr.6.8. Component Fr.6.4 was repeatedly separated by silica gel column chromatography and then by preparative high performance liquid chromatography (ZORBAX SB-C18 column, 75% methanol-water, 2.0 mL / min) and repeated recrystallization to obtain aduncin (compound 7, 6 g, t R =5.33min).

[0052] Component Fr.7 (8 g) was eluted by MCI column (methanol-water volume ratio of 60:40-100:0), detected by silica gel thin layer chromatography and combined with the parts of similar polarity to obtain three components, Fr.7.1-Fr.7.3. Component Fr.7.2 was separated into two components by Sephadex LH-20 (methanol), and then subjected to silica gel column chromatography to obtain dendroterpene D (compound 9, 8 mg).

[0053] Component Fr.10 (14 g) was eluted with MCI column (methanol-water volume ratio of 40:60-80:20), detected by silica gel thin layer chromatography and combined with the parts with similar polarity to obtain 5 components, Fr.10.1-Fr.10.5. Component Fr.10.1 was separated into 3 components by Sephadex LH-20 (methanol), and then chromatographed by reverse phase RP-18 column, and repeatedly recrystallized to obtain dendrowardin K (compound 1, 35 mg), dendrowardin O (compound 3, 15 mg), and dendrobiumane C (compound 4, 6 mg).

[0054] Component Fr.11 (18 g) was gradient eluted through an MCI column (methanol-water volume ratio of 30:70-80:20), detected by silica gel thin layer chromatography and combined with the parts with similar polarity to obtain three components Fr.11.1-Fr.11.3. Component Fr.11.1 was purified by Sephadex LH-20 (methanol) and then prepared by preparative high performance liquid chromatography (ZORBAX SB-C18 column, 70% methanol-water, 2.0 mL / min) and repeated recrystallization to obtain amotin (compound 10, 7 g, t R =5.12min).

[0055] Component Fr.12 (30 g) was gradient eluted by MCI column (methanol-water volume ratio of 30:70-80:20), detected by silica gel thin layer chromatography and combined with similar polarity parts to obtain four components Fr.12.1-Fr.12.4. Component Fr.12.1 was purified by Sephadex LH-20 (methanol) several times, and finally recrystallized in methanol to obtain dendrowardin E (compound 5, 400 mg). Component Fr.12.2 was purified by silica gel column chromatography and Sephadex LH-20 (methanol) several times, and recrystallized in a mixed solvent of acetone and methanol to obtain dendrowardin L (compound 2, 234 mg). Component Fr.12-3 was purified by Sephadex LH-20 (methanol) and then by normal phase silica gel column chromatography (petroleum ether-acetone volume ratio of 15:1-0:1) gradient elution, and Sephadex LH-20 (methanol-chloroform volume ratio of 1:1) column chromatography to obtain dendrowardin C (compound 6, 36 mg).

[0056] Take the naturally shade-dried stems of Dendrobium officinale, crush them and extract them with 90% ethanol by reflux for four times (40L×4, 2d). Recover the ethanol under reduced pressure, concentrate it into a water suspension, and then extract it with ethyl acetate (5L×3) and n-butanol (5L×3) respectively. The n-butanol part (250g) is eluted with chloroform-methanol solvent (the volume ratio of chloroform and methanol in chloroform-methanol solvent is 95:5, 90:10, 80:20, 60:40 respectively), and detect it with silica gel thin layer chromatography and merge the parts with similar polarity into 5 components, namely component F, component G, component H, component I and component J.

[0057] Component G (45 g) was eluted through an MCI column (the eluent was methanol-water solvent, and the volume ratios of methanol-water in the methanol-water solvent were 10:90, 30:70, 60:40, and 80:20, respectively), detected by silica gel thin layer chromatography, and the parts with similar polarity were combined to obtain 4 components, namely, component G1, component G2, component G3, and component G4.

[0058] Component G2 (8 g) was eluted by silica gel column chromatography (eluent was ethyl acetate-methanol solvent, the volume ratio of ethyl acetate to methanol in ethyl acetate-methanol solvent was 15:1, 10:1, 4:1), and further purified by RP-18 (eluent was methanol-water solvent, the volume ratio of methanol to water in methanol-water solvent was 20:80, 40:60, 60:40) to obtain 6-hydroxydendroxine (compound 15, 3 mg) and dendrobine (compound 16, 3 mg);

[0059] Component G3 (8 g) was eluted by normal phase silica gel column chromatography (eluent was ethyl acetate-methanol solvent, the volume ratio of ethyl acetate to methanol in ethyl acetate-methanol solvent was 10:1, 4:1) and RP-18 (eluent was methanol-water solvent, the volume ratio of methanol to water in methanol-water solvent was 20:80, 40:60, 60:40) to obtain crystallinin (compound 11, 2 mg) and dendromoniliside D (compound 14, 2 mg).

[0060] Component H (50 g) was eluted by normal phase silica gel column chromatography (eluent was ethyl acetate-methanol solvent, and the volume ratio of ethyl acetate to methanol solvent in ethyl acetate-methanol solvent was 15:1, 9:1, 2:1), detected by silica gel thin layer chromatography and combined with the parts with similar polarity to obtain 4 components, namely component H1, component H2, component H3 and component H4. Component H1 was decolorized by MCI and further purified by RP-18 (eluent was methanol-water solvent, and the volume ratio of methanol to water in methanol-water solvent was 20:80, 40:60, 60:40) to obtain (+)-(1R,2S,3R,4S,5R,6S,9R)-2,11,12-Trihydroxypicrotoxane-3(15)-lactone (compound 12, 3 mg) and dendroside F (compound 13, 4 mg).

[0061] The spectral data of compound 1, compound 2, compound 3 and compound 8 are as follows:

[0062] Dendrowardin K (Compound 1): Colorless needle-shaped crystals (methanol); molecular formula: C 15 H 20 O 5 ,mp:227–228℃; IR(KBr)νmax 3434.58,2963.58,2931.79,2875.48,1770.80,1629.67,1669.91cm -1 ;(-)HRESIMS m / z279.1235[MH] - ,(C 15 H 19 O 5 , calculated value is 279.1238);

[0063] Dendrowardin L (Compound 2): Colorless needle-shaped crystals (methanol); molecular formula: C 16 H 24 O 5 ,mp:160–162℃; CD(MeOH)λmax(Δε)227(+17.0)nm,210(+1.6); UV(MeOH)λmax(logε)202(1.74)nm; IR(KBr)ν max 3433.05,29657.76,2925.89,2873.19,285676,1714.72,1633.00cm -1 ;(-)HRESIMS m / z 341.1605[MH] + ,(C 15 H 23 O 4 , calculated value is 341.1606);

[0064] Dendrowardin O (compound 3): colorless needle crystals (methanol); molecular formula is C 15 H 24 O 4 ; UV(MeOH)λmax(logε)202(1.42)nm; CD(MeOH)λmax(Δε)220,(+17.5)nm; IR(KBr)ν max 3400.36,2959.58,2916.50,2879.10,1771.21,1716.19 1442.29cm -1 ;(+)HR ESIMS m / z 341.1605[M+COOH] + ,(C 16 H 24 O 6 , calculated value is 341.1605);

[0065] Dendrowardin Q (Compound 8): Colorless needle-shaped crystals (methanol); molecular formula: C 15 H 18 O 5 ; mp: 290–291°C; UV(MeOH)λmax(logε)220(2.67),260(1.59); CD(MeOH)λmax(Δε)229(+0.07)nm; IR(KBr)ν max 3440.06,2967.62,2872.55,1795.30,1778.62,1828.56,1454.09,734.36cm -1 ; (+)HRESI-MS m / z301.1048([C 15 H18 O 5 ] + Na) + , the calculated value is 301.1046.

[0066] Table 0 Compound 1, Compound 2, Compound 3 and Compound 8 1 H NMR data

[0067]

[0068]

[0069] a 500MHz in CD 3 OD; b 500MHz in CDCl 3

[0070] Table 3 Compound 1, Compound 2, Compound 3 and Compound 8 13 C NMR data

[0071]

[0072] ( a 125 MHz in CD 3 OD; b 125MHz in CDCl 3 )

[0073] The structures of compound 1 to compound 16 are as follows Figure 1 shown.

[0074] Example 2

[0075] 1. Experimental study on the activity of picrotoxane-type sesquiterpenoids in improving insulin resistance based on the IR-Hep-G2 model

[0076] Experimental instruments and materials

[0077]

[0078] 1. Effects of picrotoxane-type sesquiterpenoids on the survival rate of Hep-G2 cells

[0079] Experimental methods:

[0080] The toxicity of 16 sesquiterpenoid compounds to Hep-G2 cells was tested in order to screen out compounds that had no effect on the survival rate of Hep-G2 cells for the next step of the experiment. First, logarithmically growing Hep-G2 cells were selected and plated at a density of 10 5Hep-G2 cell fluid at a concentration of 100 μg / mL was inoculated into a 24-well plate. After culturing for 24 hours, the supernatant was discarded and the plate was washed once with PBS. Serum-free culture medium containing 12.5, 25, and 50 μM sesquiterpene samples was added to each well. Each group had 3 replicate wells. The samples were treated for 0, 24, and 48 hours. The cell viability of Hep-G2 was detected by MTS method.

[0081] Experimental results:

[0082] like Figure 2 As shown, 16 sesquiterpene samples in the concentration range of 12.5 to 50 μM had no significant effect on the survival rate of Hep-G2 cells after 0, 24, and 48 hours of treatment, indicating that the sesquiterpene samples at these concentrations are safe and non-toxic to Hep-G2 cells.

[0083] 2. Effects of picrotoxane-type sesquiterpenoids on glucose consumption in insulin-resistant Hep-G2 cells

[0084] Experimental methods:

[0085] The optimal modeling condition for insulin resistance was based on the previous research results of our research group (i.e., when the insulin concentration was 10 -5 mol / L, the action time is 24 hours, and the insulin resistance effect can last up to 48 hours).

[0086] First, add Hep-G2 cell suspension (density 10 5 / mL), the supernatant was discarded after culturing for 24 hours, the cells were washed once with PBS, and then cultured with HBSS buffer for 2 hours, the supernatant was discarded, and the cells were washed once with PBS. The cells were divided into blank group, model group, and sample group. Serum-free medium was added to the blank group, and a final concentration of 10 -5 mol / L insulin culture medium for 24 hours; discard the supernatant, wash once with PBS, then add serum-free culture medium to the blank group and model group, add serum-free culture medium containing 12.5, 25, and 50 μM sesquiterpene samples to the sample group, respectively. After culturing for 24 hours, collect the supernatant, centrifuge at 4℃ and 2500 rpm / min for 15 minutes, use a glucose detection kit to detect the glucose content in the cell supernatant, and calculate the glucose consumption of the cells.

[0087] Experimental results:

[0088] The glucose content in Hep-G2 cells after drug treatment was detected. The results are as follows Figure 3 As shown in the figure, it can be observed that after adding 10 -5After 24 hours of treatment with 1.5 mol / L insulin, the glucose consumption of the model group (Model) was significantly lower than that of the blank group (Control), indicating that Hep-G2 cells had developed insulin resistance at this time. The results showed that compared with the model group, the tested compounds in the range of 12.5 to 50 μM, compound 1, 2, 4-6, 8, 12, P-1 to P-3, P-5, P-6 groups all had a significant improvement on insulin resistance, and compound 1, 5, P-3, P-6 groups showed a significant dose-dependency.

[0089] 2. Experimental study on the insulin secretion-promoting activity of picrotoxane-type sesquiterpenoid compounds

[0090] Experimental instruments and materials

[0091]

[0092]

[0093] 1. Effect of sesquiterpenes on the survival rate of β-CT-116 cells

[0094] Experimental methods:

[0095] The toxicity of 16 sesquiterpenoid compounds to β-CT-116 cells was detected in order to screen out compounds that had no effect on the survival rate of β-CT-116 cells for the next experiment. First, logarithmically growing β-CT-116 cells were selected, and Hep-G2 cell fluid with a density of 105 / mL was inoculated into a 24-well plate. After culturing for 24 hours, the supernatant was discarded, and the plate was washed with PBS. Serum-free culture medium containing 12.5, 25, and 50 μM sesquiterpenoid samples was added to each well. There were 3 replicates in each group, and the samples were treated for 0, 24, and 48 hours. The cell viability of β-CT-116 was detected by MTS method.

[0096] Experimental results:

[0097] like Figure 4 As shown, 16 sesquiterpene samples in the concentration range of 12.5 to 50 μM had no significant effect on the survival rate of β-CT-116 cells after 0, 24, and 48 hours of treatment, indicating that sesquiterpene samples at these concentrations are safe for β-CT-116 cells and can be used for subsequent experiments.

[0098] 2. Insulin content detection

[0099] Experimental methods:

[0100] The β-TC-6 cells grown to 80-90% were prepared with β-TC-6 medium to prepare a cell suspension (C = 400,000 / mL), and 24-well plates were used to add 500μL of cell suspension to each well. Incubate at 37°C in an incubator for 24h. Sample solutions with concentration gradients (12.5, 25, 50μM) were prepared using HBSS medium. The cells were starved, the cells were removed, the supernatant was discarded, and the cells were washed twice with PBS. 500μL of HBSS medium was added to each well, and the cells were incubated at 37°C in an incubator for 30min. The cells were induced with high glucose, the supernatant was discarded, the cells were washed twice with PBS, and 500μL of HG medium (D-Glu = 5.6mg / mL) was added to each well, and the cells were incubated at 37°C in an incubator for 60min. The cells were divided into a control group, a drug-added group, and a positive control group. The control group was HBSS, and the drug-added group and the positive control group were added with samples prepared with HBSS, and the samples were incubated in an incubator for 2.5h. The supernatant was collected into an EP tube and centrifuged at 2500 rpm / min and 4°C for 5 min. Finally, the insulin content in the cell supernatant was detected using a mouse insulin (INS) ELISA kit.

[0101] Experimental results:

[0102] The glucose-stimulated insulin secretion activity of 16 picrotoxane-type sesquiterpenes was evaluated in β-TC-6 cells. After high glucose induction treatment, the experimental results were as follows: Figure 5 As shown, Glibenclamide refers to the positive control group. Compared with the blank control group, the drug-added group compounds 4-6, P-1 to P-5 showed significant differences at concentrations of 12.5 to 50 μM; the secretion-promoting effects of compound 6 at a concentration of 12.5 μM, P-1 at a concentration of 12.5-50 μM, P-2 at a concentration of 50 μM, and P-5 at a concentration of 12.5 μM and 25 μM were all stronger than those of the positive control drug glibenclamide, among which compound 4, P-1 to P-3, P-5, and glibenclamide all showed good dose dependence.

[0103] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A picrotoxane-type sesquiterpenoid compound, characterized in that: Has one of the following structures:

2. A method for preparing a picrotoxane-type sesquiterpenoid compound as claimed in claim 1, characterized in that: The following steps are involved: 1) crushing the dried stem of Dendrobium candidum, adding ethanol to extract, obtaining an ethanol extract, and concentrating to obtain an extract; 2) separating the extract by normal phase silica gel column chromatography to obtain component Fr.10 and component Fr.12; the eluent used in the normal phase silica gel column chromatography separation is chloroform-acetone solvent; 3) The component Fr.10 is separated by an MCI column to obtain five components, namely, component Fr.10.1, component Fr.10.2, component Fr.10.3, component Fr.10.4 and component Fr.10.5; The eluent used for the MCI column separation is a methanol-water solvent, wherein the volume ratio of methanol to water in the methanol-water solvent is 40:60-80:20; the component Fr.10.1 is separated into three components by Sephadex LH-20, and then recrystallized by reverse phase RP-18 column chromatography to obtain compound 1 and compound 3; the eluent used for the Sephadex LH-20 is methanol; 4) The component Fr.12 is separated by an MCI column to obtain four components, namely, component Fr.12.1, component Fr.12.2, component Fr.12.3 and component Fr.12.4; The eluent used for the MCI column separation is a methanol-water solvent, in which the volume ratio of methanol to water is 30:70-80:20; the component Fr.12.2 is separated by silica gel column chromatography, purified by Sephadex LH-20, and recrystallized in a mixed solvent of acetone and methanol to obtain compound 2.

3. The preparation method according to claim 2, characterized in that: The mass fraction of ethanol in step 1) is 95%.

4. The preparation method according to claim 2, characterized in that: In the step 1), the volume ratio (kg:L) of the mass of the dried stems of Dendrobium candidum to ethanol is 1:

20.

5. The preparation method according to claim 2, characterized in that: In the step 1), the ethanol extraction is performed twice, each extraction time is 2 hours, and the extraction temperature is 25°C.

6. Use of picrotoxane-type sesquiterpenoid compounds in the preparation of antidiabetic drugs, characterized in that: The picrotoxane-type sesquiterpenoid compound is any one of the compounds 1-3 or compounds 4-16 described in claim 1; the structural formula of the compound 4-16 is:

Images