A diterpene compound in cardamom, its pharmaceutical composition, preparation method and application thereof

Abstract

The present invention discloses a diterpenoid compound from cardamom, a pharmaceutical composition thereof, a preparation method, and an application thereof, and relates to the field of pharmaceutical technology. The present invention extracts a diterpenoid compound from the fruit of cardamom for the first time, and the structure of the diterpenoid compound is a new compound reported for the first time. The diterpenoid compound isolated from the fruit of cardamom has significant GLP-1 secretion-promoting and DPP-4 inhibitory activities. Experiments have shown that the diterpenoid compound extracted from the fruit of cardamom has blood sugar-lowering and weight-loss effects.

Description

Technical Field

[0001] The present invention relates to the technical field of medicines, and in particular to a diterpene compound in red cardamom, a pharmaceutical composition thereof, a preparation method thereof and an application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a complex metabolic disease characterized by insulin resistance or insufficient insulin secretion leading to increased blood sugar levels in the body. Type 2 diabetes often leads to serious complications such as retinopathy, nephropathy, and hypertension.

[0003] Currently, there are a variety of oral hypoglycemic drugs on the market, including biguanides, α-glucosidase inhibitors, insulin secretagogues, insulin sensitizers, glucagon-like peptide-1 (GLP-1) receptor agonists, dipeptidyl peptidase-4 (DPP4) inhibitors, and sodium-glucose cotransporter-2 (SGLT-2) inhibitors. However, the various side effects of these oral hypoglycemic drugs (such as acute hypoglycemia, weight gain, gastrointestinal discomfort, and hepatotoxicity) limit their application. Therefore, new antidiabetic drugs with high efficacy and low risk of hypoglycemia are still under development.

[0004] Glucagon-like peptide 1 (GLP-1) is an incretin hormone secreted by enteroendocrine epithelial L cells that stimulates insulin secretion and reduces glucagon secretion in a glucose-dependent manner. Currently, two types of GLP-1 receptor agonists are commercially available: GLP-1 receptor agonists and DPP-4 inhibitors. Most GLP-1 receptor agonists are peptide analogs, which have disadvantages such as high cost, injection, and gastrointestinal and pancreatic side effects. Furthermore, existing DPP4 inhibitors are synthetic compounds, resulting in low selectivity and poor safety. Therefore, the search for GLP-1 receptor agonists from natural products is of great significance.

[0005] Traditional Chinese medicine, used for thousands of years in China, is an important source for exploring new anti-diabetic drugs. Cardamom, a key herb, has been shown in relevant studies to have anti-gastric ulcer and protective effects against gastric mucosal damage. Studies have found that cardamom contains a variety of compounds, including volatile oils, flavonoids, sesquiterpenes, diterpenes, phenylpropanoids, glycosides, lignans, and organic acids, which have antioxidant, antibacterial, anti-tumor, analgesic, anti-ulcer, and anti-diarrhea properties.

[0006] In this regard, the inventors believe that how to extract effective active substances from red cardamom for the preparation of hypoglycemic or weight-loss drugs is a technical problem that urgently needs to be solved by technicians in this field.

[0007] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0008] In response to the above technical problems, the embodiments of the present invention provide a diterpene compound in cardamom, a pharmaceutical composition thereof, a preparation method and an application thereof, to solve the problems raised in the above background technology.

[0009] A diterpene compound having a structural formula as one of Formulas 1-17; the diterpene compound is named: alpigalangin;

[0010]

[0011] A use of the diterpene compound as described above in the preparation of a GLP-1 secretagogue drug.

[0012] A use of the diterpene compound as described above in the preparation of DPP-4 inhibitor drugs.

[0013] A use of the diterpene compound as described above in the preparation of a blood sugar lowering drug or a weight loss drug.

[0014] A use of the diterpene compound as described above in the preparation of a health food; the health food has the function of lowering blood sugar or losing weight.

[0015] A pharmaceutical composition, wherein the active ingredient comprises the diterpene compound as described above or a pharmaceutically acceptable salt thereof.

[0016] Preferably, it further comprises at least one of a pharmaceutically acceptable carrier, excipient, adjuvant and vehicle.

[0017] A use of the above-mentioned pharmaceutical composition in the preparation of a hypoglycemic drug or a weight-loss drug or a health food with hypoglycemic and weight-loss functions.

[0018] A method for extracting diterpenoid compounds as described above, wherein the diterpenoid compounds are extracted from the fruit of red cardamom; preferably, the fruit of red cardamom is a dried mature fruit.

[0019] Preferably, the extraction method specifically comprises the following steps:

[0020] The cardamom fruit was crushed and cold-extracted with 90% ethanol three times, and the ethanol extracts were combined;

[0021] The combined ethanol extracts are decompressed to recover ethanol to obtain an extract, the extract is dispersed in water, extracted with ethyl acetate, and concentrated to obtain an ethyl acetate extract;

[0022] The ethyl acetate extract was subjected to silica gel column chromatography, and gradient elution was performed using acetone-petroleum ether with volume ratios of 0:100, 5:95, 10:90, 20:80, 50:50 and 100:0 as eluents to obtain multiple primary fractions;

[0023] The multiple primary fractions were chromatographed on a silica gel column using ethyl acetate-petroleum ether in a volume ratio of 10:90 to 50:50 as eluent to obtain multiple secondary fractions;

[0024] The multiple secondary fractions were chromatographed on a silica gel column using methanol-water with a volume ratio of 50:50, 70:30, and 90:10 as eluent to obtain multiple tertiary fractions;

[0025] Multiple tertiary fractions were chromatographed on a silica gel column using petroleum ether-acetone and chloroform-methanol as eluents, and then chromatographed on a Sephadex LH-20 column using chloroform-methanol with a volume ratio of 1:1 as eluent. Finally, they were purified by semi-preparative high-performance liquid chromatography and qualitatively screened to obtain diterpenoid compounds.

[0026] The present invention provides a diterpene compound, a pharmaceutical composition thereof, a preparation method thereof and an application thereof, which have the following characteristics:

[0027] Beneficial effects:

[0028] 1. This invention extracts 17 diterpenoids from the fruit of red cardamom for the first time, and the structures of these 17 diterpenoids are new compounds reported for the first time;

[0029] 2. The diterpenoid compounds isolated from the fruit of red cardamom have significant GLP-1 secretion-promoting and DPP-4 inhibitory activities, indicating that these diterpenoid compounds have potential hypoglycemic effects;

[0030] 3. The preparation method of the present invention is simple and easy to operate, has a high yield, is environmentally friendly and safe, and has high feasibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the structural formula of compounds 1-17 of the present invention;

[0032] Figure 2 The promoting effect of compounds 1-17 of the present invention on GLP-1 secretion in NCl-H716 cells; cholic acid (CA) was used as a positive control, and the values ​​are expressed as mean ± standard deviation (n=3). DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0034] In response to the above technical problems, the embodiments of the present invention provide a diterpene compound in cardamom, a pharmaceutical composition thereof, a preparation method and an application thereof, to solve the problems raised in the above background technology.

[0035] Example 1:

[0036] Preparation of compounds 1–17:

[0037] The dried ripe fruits of red cardamom were crushed and cold-extracted with 90% ethanol for three times. The ethanol extracts were combined and the ethanol was recovered under reduced pressure to obtain an extract. The extract was dispersed in water and extracted with ethyl acetate. The extract was concentrated to obtain an ethyl acetate extract. The ethyl acetate extract was then subjected to silica gel column chromatography using acetone-petroleum ether (0:100, 5:95, 10:90, 20:80, 50:50 and 100:0, v / v) as the eluent for gradient elution to obtain 11 fractions from Fr.1 to Fr.11; Fr.5 was subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 10:90-50:50) to obtain 9 fractions from Fr.5-1 to Fr.5-9; Fr.5-7 was subjected to Rp-C 18 Column chromatography (methanol-water, 50:50, 70:30, and 90:10, v / v) yielded three fractions, Fr.5-7-1 through Fr.5-7-3. Fr.5-7-2 was purified by multiple silica gel column chromatography (petroleum ether-acetone; chloroform-methanol, etc.), Sephadex LH-20 column chromatography (chloroform-methanol = 1:1), and semi-preparative HPLC purification (acetonitrile-water, methanol-water) to afford compound 1-17.

[0038] It should be noted that the fraction Fr.5 obtained by gradient elution was subjected to silica gel column chromatography to obtain Fr.5-1-Fr.5-9, and then fraction Fr.5-7 was selected from Fr.5-1-Fr.5-9 for column chromatography to obtain Fr.5-7-1-Fr.5-7-3, and then fraction Fr.5-7-2 was selected from Fr.5-7-1-Fr.5-7-3 to be purified and eluted to obtain the target substance. This is the reverse process of the entire experimental method. After research, we found that the target molecule obtained from fraction Fr.5-7-2 has the target activity. Therefore, the compound in fraction Fr.5-7-2 was taken as the final target substance, the screening and extraction method was traced back, and the separation and acquisition method of the target substance was directly given.

[0039] Structural data of compounds 1–17:

[0040] Optical rotation was measured using a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan). Infrared spectra (IR) were measured using a Bio-Rad FTS-135 infrared spectrometer (Hercules, California, USA) using the KBr pellet method. Ultraviolet spectra were measured using a UV-2401PC spectrometer (Shimadzu, Kyoto, Japan). ECD spectra were measured using an Applied Photophysics circular dichroism spectrometer (Agilent, Santa Clara, United States); nuclear magnetic resonance spectra (1D and 2D NMR) were measured using an Avance III-600 superconducting nuclear magnetic resonance spectrometer (Bruker, Bremerhaven, Germany) using deuterated chloroform or deuterated acetone as solvents; high-resolution mass spectrometry (HRMS) was measured using an LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Thin-layer chromatography silica gel and column chromatography silica gel (200–300 mesh) were purchased from Qingdao Meigao and Qingdao Ocean Chemical Group Co., Ltd., and Sephadex LH-20 was purchased from Amersham Bioscience (Sweden). CHP20P MCI gel was purchased from Mitsubishi Chemical Corporation (Tokyo, Japan).

[0041]

[0042] alpigalangin A(1)

[0043] Molecular formula: C 20 H 32 O4

[0044] Molecular weight: 336

[0045] Appearance: Colorless needle crystal

[0046] HRESIMS m / z 337.2365([M+H] + , calcd.for C 20 H 32 O4, –0.8 mDa);

[0047] UV(MeOH)λ max (logε):228(2.86)nm;

[0048] IR(KBr)ν max:3432, 2924, 2868, 2846, 1730, 1670, 1640, 1214, 1062, 1032, 934, 736cm -1 ;

[0049] ECD(MeOH)λ max (Δε)229(+0.74), 266(–0.01)nm;

[0050] (c 0.1, MeOH);

[0051] 1 HNMR and 13 CNMR (DEPT) data are shown in Tables 1 and 2.

[0052]

[0053] alpigalangin B(2)

[0054] Molecular formula: C 20 H 30 O5

[0055] Molecular weight: 350

[0056] Appearance: white powder

[0057] HRESIMS m / z351.2160([M+H] + , calcd.for C 20 H 30 O5, –0.6 mDa);

[0058] UV(MeOH)λ max (logε):269(3.03)nm;

[0059] IR(KBr)ν max :3424, 2948, 2926, 2870, 2846, 1750, 1642, 1600, 1460, 1440, 1390, 1364, 1330, 1290, 958, 894cm -1 ;

[0060] ECD(MeOH)λ max (Δε)268(+1.66), 302(+0.22)nm;

[0061] (c 0.1, MeOH);

[0062] 1 H-NMR and 13C-NMR (DEPT) data are shown in Tables 1 and 2.

[0063]

[0064] alpigalangin C(3)

[0065] Molecular formula: C 19 H 32 O4

[0066] Molecular weight: 324

[0067] Appearance: white powder

[0068] HRESIMS m / z 347.2215([M+Na] + , calcd.for C 19 H 32 O4, +2.2 mDa);

[0069] UV(MeOH)λ max (logε):235(3.13)nm;

[0070] IR(KBr)ν max :3426, 2946, 2926, 2848, 1718, 1660, 1622, 1462, 1440, 1368, 1244, 1166, 1030, 890, 774, 562cm -1 ;

[0071] ECD(MeOH)λ max (Δε)207(+3.61), 234(–0.24)nm;

[0072] [α] 2 D 4 +17.9 (c 0.08, MeOH);

[0073] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 1 and 2.

[0074]

[0075] alpigalangin D(4)

[0076] Molecular formula: C 17 H 28 O4,

[0077] Molecular weight: 296

[0078] Appearance: white powder

[0079] HRESIMS m / z 319.1872([M+Na] + , calcd.for C 17 H 28 O4, –0.8 mDa);

[0080] UV(MeOH)λ max (logε):219(3.13)nm;

[0081] IR(KBr)ν max :3568, 3516, 3420, 2950, ​​2927, 2848, 2624, 1691, 1647, 1545, 1436, 1390, 1284, 1196, 998, 928, 770cm -1 ;

[0082] ECD(MeOH)λ max (Δε)196(+3.85), 234(+0.65)nm;

[0083] (c 0.1, MeOH);

[0084] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 1 and 2.

[0085]

[0086] alpigalangin E(5)

[0087] Molecular formula: C 21 H 34 O5

[0088] Molecular weight: 366

[0089] Appearance: white powder

[0090] HRESIMS m / z 389.2294([M+Na] + , calcd.for C 21 H 34 O5, –0.4 mDa);

[0091] UV(MeOH)λ max (logε):230(3.07)nm;

[0092] IR(KBr)ν max:3432, 2926, 2868, 1761, 1670, 1630, 1462, 1368, 1208, 1180, 1082, 1040, 982, 888, 560cm -1 ;

[0093] ECD(MeOH)λ max (Δε)224(+4.87), 263(–0.43)nm;

[0094] (c 0.1, MeOH);

[0095] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 1 and 3.

[0096]

[0097] alpigalangin F(6)

[0098] Molecular formula: C 22 H 36 O5,

[0099] Molecular weight: 380

[0100] Appearance: white powder

[0101] HRESIMS m / z 403.2464 ([M+Na] + , calcd.for C 22 H 36 O5, +0.9 mDa);

[0102] UV(MeOH)λ max (logε):228(3.14)nm;

[0103] IR(KBr)ν max :3324, 2982, 2932, 2872, 1748, 1674, 1642, 1450, 1220, 1188, 1048, 998, 956cm -1 ;

[0104] ECD(MeOH)λ max (Δε)201(+3.14), 246(+0.49)nm;

[0105] [α] 2 D 4 +56.5 (c 0.12, MeOH);

[0106] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 1 and 3.

[0107]

[0108] alpigalangin G(7)

[0109] Molecular formula: C 22 H 36 O4,

[0110] Molecular weight: 364

[0111] Appearance: white powder

[0112] HRESIMS m / z 363.2535([MH] - , calcd.for C 22 H 36 O4, -0.6 mDa);

[0113] UV(MeOH)λ max (logε):228(3.06)nm;

[0114] IR(KBr)ν max :3440, 2928, 2848, 1756, 1674, 1462, 1444, 1368, 1210, 1068, 1032, 966cm -1 ;

[0115] ECD(MeOH)λ max (Δε)209(+2.30), 237(–0.55)nm;

[0116] [α] 2 D 0 +13.6 (c 0.05, MeOH);

[0117] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 1 and 3.

[0118]

[0119] alpigalangin H(8)

[0120] Molecular formula: C 22 H 34 O5,

[0121] Molecular weight: 398

[0122] Appearance: white powder

[0123] HRESIMS m / z 401.2294 ([M+Na] + , calcd.for C 22 H 34 O5, -0.4 mDa);

[0124] UV(MeOH)λ max (logε):228(3.13)nm;

[0125] IR(KBr)ν max :3496, 3032, 2924, 2868, 2846, 1750, 1728, 1678, 1442, 1380, 1236, 1184, 1128, 1032, 1008, 890, 816, 684, 568cm -1 ;

[0126] ECD(MeOH)λ max (Δε)207(+0.89), 228(+0.14), 245(+0.97)nm;

[0127] [α] 2 D 0 +2.6 (c 0.1, MeOH);

[0128] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 1 and 3.

[0129]

[0130] alpigalangin I(9)

[0131] Molecular formula: C 20 H 30 O4,

[0132] Molecular weight: 334

[0133] Appearance: white powder

[0134] HRESIMS m / z 357.2024([M+Na] + , calcd.for C 20 H 30 O4, -1.2 mDa);

[0135] UV(MeOH)λ max (logε):224(3.15)nm;

[0136] IR(KBr)ν max :3390, 2944, 2928, 2844, 1728, 1674, 1642, 1388, 1296, 1224, 1050, 982, 890, 834cm -1 ;

[0137] ECD(MeOH)λ max (Δε)231(+8.06), 263(+0.13)nm;

[0138] (c 0.1, MeOH);

[0139] 1 H-NMR and 13 The C-NMR (DEPT) data are shown in Tables 4 and 5.

[0140]

[0141] alpigalangin J(10)

[0142] Molecular formula: C 21 H 32 O4,

[0143] Molecular weight: 348

[0144] Appearance: white powder

[0145] HRESIMS m / z 371.2187([M+Na] + , calcd.for C 21 H 32 O4, –0.6 mDa);

[0146] UV(MeOH)λ max (logε):226(3.14)nm;

[0147] IR(KBr)ν max :3432, 2928, 2866, 2848, 1764, 1678, 1630, 1444, 1366, 1342, 1184, 1016, 942, 856, 716, 634cm -1 ;

[0148] ECD(MeOH)λ max (Δε)234(+16.94), 295(+1.18)nm;

[0149] (c 0.11, MeOH);

[0150] 1 H-NMR and 13 The C-NMR (DEPT) data are shown in Tables 4 and 5.

[0151]

[0152] alpigalangin K(11)

[0153] Molecular formula: C 21 H 32 O4,

[0154] Molecular weight: 348

[0155] Appearance: white powder

[0156] HRESIMS m / z 371.2197([M+Na] + , calcd.for C 21 H 32 O4, +0.4 mDa);

[0157] UV(MeOH)λ max (logε):226(2.91)nm;

[0158] IR(KBr)ν max :3432, 2996, 2928, 2849, 1756, 1678, 1636, 1336, 1218, 1020, 930, 836, 634cm -1 ;

[0159] ECD(MeOH)λ max (Δε)235(+4.36), 268(+0.34)nm;

[0160] (c 0.12, MeOH);

[0161] 1 H-NMR and 13 The C-NMR (DEPT) data are shown in Tables 4 and 5.

[0162]

[0163] alpigalangin L(12)

[0164] Molecular formula: C 21 H 34 O4,

[0165] Molecular weight: 350

[0166] Appearance: white powder

[0167] HRESIMS m / z 373.2343 ([M+Na] + , calcd.for C 21 H 34 O4, –0.6 mDa);

[0168] UV(MeOH)λ max (logε):221(3.24)nm;

[0169] IR(KBr)ν max :3394, 2946, 2922, 2866, 2846, 1728, 1678, 1648, 1220, 1048, 980, 908, 872cm -1 ;

[0170] ECD(MeOH)λ max (Δε)213(+8.03), 248(–3.12)nm;

[0171] [α] 2 D 0 +3.9 (c 0.08, MeOH);

[0172] 1 H-NMR and 13 The C-NMR (DEPT) data are shown in Tables 4 and 5.

[0173]

[0174] alpigalangin M(13)

[0175] Molecular formula: C 20 H 30 O3,

[0176] Molecular weight: 318

[0177] Appearance: white powder

[0178] HRESIMS m / z 341.2105([M+Na] + , calcd.for C 20 H 30 O3, +1.8 mDa);

[0179] UV(MeOH)λ max (logε):229(3.26)nm;

[0180] IR(KBr)νmax :3478, 2962, 2924, 2860, 1742, 1678, 1436, 1206, 1014, 964, 826, 776, 562cm -1 ;

[0181] ECD(MeOH)λ max (Δε)227(+6.19), 268(+0.46)nm;

[0182] (c 0.1, MeOH);

[0183] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 4 and 6.

[0184]

[0185] alpigalanginN(14)

[0186] Molecular formula: C 22 H 34 O4,

[0187] Molecular weight: 362

[0188] Appearance: white powder

[0189] HRESIMS m / z 385.2350([M+Na] + , calcd.for C 22 H 34 O4, +0.1 mDa);

[0190] UV(MeOH)λ max (logε):228(3.10)nm;

[0191] IR(KBr)ν max :3440, 2924, 2868, 2848, 1758, 1674, 1636, 1458, 1440, 1366, 1344, 1204, 1182, 1118, 1042, 1022, 946, 844cm -1 ;

[0192] ECD(MeOH)λ max (Δε)227(+2.79), 263(–0.01)nm;

[0193] (c 0.08, MeOH);

[0194] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 4 and 6.

[0195]

[0196] alpigalangin O(15)

[0197] Molecular formula: C 20 H 28 O4,

[0198] Molecular weight: 332

[0199] Appearance: white powder

[0200] HRESIMS m / z 331.1908([MH] - , calcd.for C 20 H 28 O4, -0.7 mDa);

[0201] UV(MeOH)λ max (logε):259(2.82)nm;

[0202] IR(KBr)ν max :3434, 2926, 2866, 2850, 1722, 1654, 1458, 1442, 1388, 1366, 1274, 1208, 1164, 1094, 1054, 1000, 958, 874cm -1 ;

[0203] ECD(MeOH)λ max (Δε)253(+1.69), 283(–0.20)nm;

[0204] (c 0.17, MeOH);

[0205] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 4 and 6.

[0206]

[0207] alpigalangin P(16)

[0208] Molecular formula: C 20 H 29 NO3,

[0209] Molecular weight: 331

[0210] Appearance: white powder

[0211] HRESIMS m / z 354.2042 ([M+Na] + , calcd.for C 20 H 29 NO3, +0.2mDa);

[0212] UV(MeOH)λ max (logε):214(2.68)nm;

[0213] IR(KBr)ν max :3440, 2992, 2926, 2850, 1702, 1648, 1456, 1436, 1388, 1294, 1214, 1128, 1092, 1078, 1052, 1026, 966, 870cm -1 ;

[0214] ECD(MeOH)λ max (Δε)203(+12.74), 240(+0.53)nm;

[0215]

[0216] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 4 and 6.

[0217]

[0218] alpigalangin Q(17)

[0219] Molecular formula: C 20 H 27 NO3,

[0220] Molecular weight: 329

[0221] Appearance: white powder

[0222] HRESIMS m / z 328.1934([MH] - , calcd.for C 20 H 27 NO3, +1.6mDa);

[0223] UV(MeOH)λ max (logε):220(3.16)nm;

[0224] IR(KBr)ν max:3424, 3282, 2952, 2926, 2846, 1772, 1718, 1638, 1460, 1444, 1346, 1270, 1254, 1108, 1088, 1072, 1006, 872cm -1 ;

[0225] ECD(MeOH)λ max (Δε)219(+4.93), 289(–3.62)nm;

[0226] [α] 2 D 0 -131.1 (c 0.09, MeOH);

[0227] 1 H-NMR and 13 C-NMR (DEPT) data are shown in Tables 4 and 6.

[0228] Table 1. Compounds 1–8 13 C NMR (150 MHz) data

[0229]

[0230]

[0231] a acetone-d6; b CDCl3.

[0232] Table 2. Compounds 1-4 1 H NMR (600 MHz, δ in ppm, Jin Hz) data

[0233]

[0234]

[0235] a acetone-d6; b CDCl3.

[0236] Table 3. Compounds 5-8 1 H NMR (600 MHz, δ in ppm, Jin Hz) data

[0237]

[0238]

[0239] a acetone-d6;b CDCl3.

[0240] Table 4. Compounds 9-17 13 C NMR (150 MHz) data

[0241]

[0242] a acetone-d6; b CDCl3.

[0243] Table 5. Compounds 9-12 1 H NMR (600 MHz, δ in ppm, Jin Hz) data

[0244]

[0245]

[0246] a acetone-d6; b CDCl3.

[0247] Table 6. Compounds 13-17 1 H NMR (600 MHz, δ in ppm, Jin Hz) data

[0248]

[0249]

[0250] a acetone-d6; b CDCl3.

[0251] Example 2:

[0252] The compound has GLP-1 secretion promoting activity.

[0253] 1 Materials and Methods

[0254] 1.1 Materials

[0255] NCl-H716 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai); bovine serum was purchased from Shanghai Longtian Biotechnology Co., Ltd. (Shanghai); and the human GLP-1 ELISA kit was purchased from SAB (Maryland, USA).

[0256] 1.2 Instruments

[0257] Flex Station 3 desktop multifunctional microplate reader (Bio-RAD 680, USA); FlexA-200 full-wavelength microplate analyzer was purchased from Hangzhou Aosheng Instrument Co., Ltd.; analytical balance ME104E was purchased from Mettler-Toledo Instrument (Shanghai) Co., Ltd.; electric constant temperature incubator (DHP-9082) was purchased from Shanghai Yiheng Technology Co., Ltd.

[0258] 1.3 Experimental Procedure

[0259] GLP-1 secretion assay: NCl-H716 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% PS at 37°C in a 5% CO2 saturated humidity incubator. When the cells grew to 80%-90% of their growth rate, the diluted cell suspension was inoculated into 24-well plates (5×10 5 Each well was starved for 1 hour after adding KRBH working solution III; each well was starved for 2 hours after adding KRBH working solution IV containing the compound, and the supernatant was collected. The GLP-1 content in the supernatant was then detected using a human GLP-1 ELISA kit.

[0260] 2. Results:

[0261] The effect of compound 1-17 on GLP-1 secretion in NCI-H716 cells was evaluated at a concentration of 50 μM. Compared with the control group, compound 1-17 significantly increased GLP-1 secretion ( Figure 2 and Table 7). In particular, compounds 3, 4, 12, 14-17 promoted GLP-1 secretion by 246.0-413.8%; compounds 5, 6, 10, 11, and 13 promoted GLP-1 secretion by 82.6-115.0%. This suggests that diterpenoids from the traditional Chinese medicine cardamom have potential hypoglycemic effects.

[0262] Table 7. Effects of compounds on promoting GLP-1 secretion in NCI-H716 cells a

[0263]

[0264] a The secretion promotion rate was expressed as the percentage increase of GLP-1 content compared with the control group; b Cholic acid (CA) was used as a positive control.

[0265] 3. Conclusion

[0266] The present invention isolates 17 new diterpenoid compounds from red cardamom. At a concentration of 50 μM, compounds 3, 4, 12, 14-17 promote GLP-1 secretion by 246.0-413.8%, while compounds 5, 6, 10, 11, and 13 promote GLP-1 secretion by 82.6-115.0%. The present invention demonstrates that the diterpenoid compounds in red cardamom have a hypoglycemic effect.

[0267] It should be noted that GLP-1 and DPP-4 are biological molecules related to insulin regulation and blood sugar control: GLP-1 is an insulin secretogen before insulin secretion, which can promote insulin secretion, improve insulin sensitivity, reduce insulin breakdown and inhibit glucagon secretion. It can also act on the central nervous system to reduce appetite and increase satiety.

[0268] DPP-4 is an enzyme present in multiple cell types whose main function is to degrade glucagon and GLP-1; by degrading GLP-1, DPP-4 can reduce the biological activity and half-life of GLP-1, thereby regulating insulin secretion and blood glucose levels.

[0269] Example 3:

[0270] Preparation Example:

[0271] 1. Take any one of compounds 1-17 or any combination thereof, dissolve it in a small amount of DMSO, add water for injection as usual, filter finely, and sterilize by filling to prepare an injection solution.

[0272] 2. Take any one of compounds 1-17 or any combination thereof, dissolve it in a small amount of DMSO, dissolve it in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, and then sterile fine filter, package it into ampoules, freeze-dry it at low temperature, and aseptically seal it to obtain a powder injection.

[0273] 3. Take any one of compounds 1-17 or any combination thereof, add excipients at a weight ratio of 9:1 to the excipients, and prepare a powder.

[0274] 4. Take any one of compounds 1-17 or any combination thereof, add excipients at a weight ratio of 5:1, and granulate and tablet.

[0275] 5. Take any one of compounds 1-17 or any combination thereof and prepare an oral solution according to conventional oral solution preparation method.

[0276] 6. Take any one of compounds 1-17 or any combination thereof, add excipients at a weight ratio of 5:1 to the excipients, and prepare capsules.

[0277] 7. Take any one of compounds 1-17 or any combination thereof, add excipients at a weight ratio of 3:1 to the excipients, and prepare capsules.

[0278] 8. Take any one of compounds 1-17 or any combination thereof, add excipients at a weight ratio of 5:1 to the excipients to prepare granules.

[0279] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A diterpene compound, characterized in that The structural formula is one of formulas 2-7 and 10-17: 。 2. Use of the diterpene compound according to claim 1 in the preparation of a GLP-1 secretagogue.

3. Use of the diterpene compound according to claim 1 in the preparation of DPP-4 inhibitor drugs.

4. Use of the diterpene compound according to claim 1 in the preparation of a hypoglycemic drug or a weight-loss drug.

5. A pharmaceutical composition, characterized in that The active ingredient in the pharmaceutical composition includes the diterpene compound or a pharmaceutically acceptable salt thereof as claimed in claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that It also includes at least one of a pharmaceutically acceptable carrier, excipient, adjuvant and vehicle.

7. A method for extracting diterpenoid compounds as claimed in claim 1, characterized in that: The diterpenoid compound is extracted from the fruit of red cardamom; the extraction method comprises the following steps: The cardamom fruit was crushed and extracted with 90% ethanol for three times, and the ethanol extracts were combined; The combined ethanol extracts are decompressed to recover ethanol to obtain an extract, the extract is dispersed in water, extracted with ethyl acetate, and concentrated to obtain an ethyl acetate extract; The ethyl acetate extract was subjected to silica gel column chromatography, and gradient elution was performed using acetone-petroleum ether with volume ratios of 0:100, 5:95, 10:90, 20:80, 50:50 and 100:0 as eluents to obtain multiple primary fractions; The multiple primary fractions were chromatographed on a silica gel column using ethyl acetate-petroleum ether in a volume ratio of 10:90 to 50:50 as eluent to obtain multiple secondary fractions; The multiple secondary fractions were chromatographed on a silica gel column using methanol-water with a volume ratio of 50:50, 70:30, and 90:10 as eluent to obtain multiple tertiary fractions; Multiple tertiary fractions were chromatographed on a silica gel column using petroleum ether-acetone and chloroform-methanol as eluents, and then chromatographed on a Sephadex LH-20 column using chloroform-methanol with a volume ratio of 1:1 as eluent. Finally, they were purified by semi-preparative high performance liquid chromatography and qualitatively screened to obtain diterpene compounds.

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