An isosporane diterpene compound, its pharmaceutical composition, its preparation method and application

Abstract

This invention discloses an isosporanoid diterpenoid compound, its pharmaceutical composition, its preparation method, and its application, relating to the field of pharmaceutical technology. This invention is the first to extract the isosporanoid diterpenoid compound 3-epi-kravanhin A from the stems and leaves of *Amomum villosum*, and the structure of this isosporanoid diterpenoid is a novel compound reported for the first time. The isosporanoid diterpenoid compound 3-epi-kravanhin A isolated from the stems and leaves of *Amomum villosum* exhibits significant GLP-1 secretion-promoting and DPP-4-inhibiting activities. Experiments show that compound 3-epi-kravanhin A has hypoglycemic and weight-loss effects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an isosporane diterpenoid compound, its pharmaceutical composition, its preparation method, and its application. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a chronic disease caused by insulin resistance or insufficient insulin secretion, which leads to elevated blood sugar levels. Due to long-term high blood sugar, type 2 diabetes often leads to serious complications such as retinopathy, nephropathy, and hypertension.

[0003] Currently, there are various oral hypoglycemic agents on the market, including biguanides, alpha-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 use of these oral hypoglycemic agents is limited by side effects such as acute hypoglycemia, weight gain, gastrointestinal discomfort, and hepatotoxicity.

[0004] Amomum tsao-ko is an important spice in my country, often used in traditional Chinese medicine to treat ailments such as abdominal distension, vomiting, and malaria. Previous studies have isolated various compounds from Amomum tsao-ko, including diarylheptanes, flavonoids, and monoterpenes. Some of these compounds possess biological activities such as antiemetic, antiasthmatic, anti-gastric ulcer, antiproliferative, antiviral, antioxidant, anti-inflammatory, and antibacterial properties.

[0005] Currently, there are no reports in existing research on the preparation of hypoglycemic or weight-loss drugs by extracting active substances from cardamom; therefore, how to extract and prepare hypoglycemic or weight-loss drugs or health foods from cardamom is a technical problem that urgently needs to be solved by those skilled in the art.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an isosporane diterpene compound, its pharmaceutical composition, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0008] An isosporane diterpene compound, characterized by having the structural formula shown in formula (I): This isosporane diterpene compound is named: 3- and -kravanhin A;

[0009] (I).

[0010] The use of an isosporane diterpene compound as described above in the preparation of GLP-1 secretagogue drugs.

[0011] The use of an isosporane diterpene compound as described above in the preparation of a DPP-4 inhibitor drug.

[0012] The application of an isosporane diterpene compound as described above in the preparation of hypoglycemic or weight-loss drugs.

[0013] A pharmaceutical composition wherein the active ingredient comprises an isosporanine diterpene compound as described in claim 1 or a pharmaceutically acceptable salt thereof.

[0014] Preferably, it also includes at least one of pharmaceutically acceptable carriers, excipients, excipients and mediators.

[0015] The use of a pharmaceutical composition as described above in the preparation of hypoglycemic drugs or weight-loss drugs.

[0016] A method for extracting isospongane diterpenoids as described above, wherein the isospongane diterpenoids are extracted from the stems and leaves of cardamom.

[0017] Preferably, the extraction method specifically includes the following steps:

[0018] The stems and leaves of the cardamom were crushed and extracted twice with 70% ethanol at room temperature, each time for 48 hours. The ethanol extracts were then combined.

[0019] The combined ethanol extract was recovered under reduced pressure to obtain an extract, which was then dispersed in water and extracted with ethyl acetate. The extract was then concentrated to obtain the ethyl acetate extract.

[0020] The ethyl acetate extract was subjected to silica gel column chromatography and eluted sequentially with acetone-petroleum ether at volume ratios of 0:100, 2:98, 5:95, 10:90, 20:80 and 100:0. The resulting primary fractions were eluted with ethyl acetate-petroleum ether at volume ratios of 10:90-80:20 to obtain secondary fractions. The secondary fractions were analyzed by TLC and fractions with the same characteristics were combined.

[0021] The secondary fractions, after being combined with the same fractions, were eluted with methanol-water at a volume ratio of 20:80-100:0 to obtain the tertiary fractions. The tertiary fractions were purified by HPLC and eluted with acetonitrile-water to obtain the quaternary fractions. The quaternary fractions were then qualitatively screened to obtain isosporane diterpenoid compounds.

[0022] The present invention provides an isosporane diterpene compound, its pharmaceutical composition, its preparation method, and its application, which have the following beneficial effects:

[0023] This invention is the first to extract isosporanine diterpenoid compound 3- from the stems and leaves of cardamom. and -kravanhin A, and the structure of this isospongine diterpene is a novel compound reported for the first time;

[0024] 2. This invention isolates isosporane diterpenoid compound 3- from the stems and leaves of *Amomum villosum*. and -kravanhin A exhibits significant GLP-1 secretion-promoting and DPP-4-inhibiting activities, indicating that compound 3- and -kravanhin A has the effects of lowering blood sugar and aiding weight loss;

[0025] 3. Compound 3- of the present invention and -kravanhin A possesses both GLP-1 secretion-promoting and DPP-4-inhibiting activities, making it a novel multi-target molecule with hypoglycemic and weight-loss activities.

[0026] 4. The preparation method of the present invention is simple and easy to implement, convenient to operate, uses waste cardamom stems and leaves as raw materials, has low cost, is environmentally friendly and safe, and has high feasibility. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structural formula of the compound 3-epi-kravanhin A of the present invention;

[0028] Figure 2 The dose-response relationship of the compound 3-epi-kravanhin A in this invention in promoting GLP-1 secretion;

[0029] Bile acid (CA) was used as a positive control;* P < 0.05, ** P < 0.01, *** P < 0.001 relative to the control group; values ​​are expressed as mean ± standard deviation. n = 3). Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] To address the aforementioned technical problems, this invention provides an isosporane diterpene compound, its pharmaceutical composition, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0032] Example 1

[0033] 1. Compound 3- and Preparation of -kravanhin A:

[0034] The stems and leaves of *Amomum tsao-ko* were pulverized and extracted twice with 70% ethanol at room temperature for 48 hours each time. 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 the ethyl acetate extract. The ethyl acetate extract was then subjected to silica gel column chromatography with acetone-petroleum ether (0:100, 2:98, 5:95, 10:90, 20:80, and 100:0). v / v The solution was eluted with a gradient of ethyl acetate-petroleum ether (10:90-80:20) to obtain 10 fractions. Fraction Fr.8 was subjected to silica gel column chromatography with ethyl acetate-petroleum ether (10:90-80:20) as the eluent. TLC analysis showed that the fractions were combined to obtain Fr.8-1-Fr.8-6. Fr.8-4 was subjected to MCI CHP20P (methanol-water, 20:80-100:0) column chromatography to obtain Fr.8-4-1-Fr.8-4-4. Fr.8-4-1 was purified by HPLC (Rp-C8 column) with acetonitrile-water (39:61) as the eluent to obtain compound 3- and -kravanhin A.

[0035] It should be noted that the process of reversing the experimental procedure, from obtaining fraction Fr.8 from gradient elution to Fr.8-1-Fr.8-6 via silica gel column chromatography, then selecting fraction Fr.8-4 from Fr.8-1-Fr.8-6 for column chromatography to obtain Fr.8-4-1-Fr.8-4-4, and finally selecting fraction Fr.8-4-1 from Fr.8-4-1-Fr.8-4-4 for purification and elution to obtain the target substance, is a process of reversing the experimental procedure. Through our research, we found that the target molecule obtained from fraction Fr.8-4-1 has the target activity. Therefore, we use fraction Fr.8-4-1 as the final target substance, and by reversing the screening and extraction method, we directly provide a method for separating and obtaining the target substance.

[0036] 2. Compound 3- and -Kravanhin A's structural data:

[0037] Optical rotation was measured using a Jasco Model 1020 polarimeter (Horiba, Tokyo, Japan); infrared (IR) spectra were measured using the KBr pellet method with a Bio-Rad FTS-135 infrared spectrometer (Hercules, California, USA); and ultraviolet (UV) spectra were measured using a UV-2401PC UV spectrometer (Shimadzu, Kyoto, Japan). ECD spectra were determined using an Applied Photophysics circular dichroism chromatograph (Agilent, Santa Clara, United States); nuclear magnetic resonance (1D and 2D NMR) spectra were determined using an AV800 or Avance III-600 superconducting NMR spectrometer (Bruker, Bremerhaven, Germany) with deuterated chloroform as the solvent; high-resolution mass spectrometry (HRMS) was performed 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.; dextran gel LH-20 (Sephadex LH-20) was purchased from Amersham Bioscience (Sweden); CHP20P MCI gel was purchased from Mitsubishi Chemical Corporation (Tokyo, Japan).

[0038] The structural data for Deoxycalyxin A are as follows: It is an isosporan diterpene compound, named 3- and -kravanhinA; Molecular formula: C 20 H 28 O4; Molecular weight: 332; Appearance: White needle-like crystals; HRESIMS m / z 355.1880 ([M + Na] + ,calcd. for C 20 H 28 O4, 355.1880, 0.0 mDa); ‒175.2 ( c 0.3, MeOH); UV (MeOH) λ max (log ε ): 217 (3.02) nm; 1 H NMR and 13 The C NMR (DEPT) data are shown in Table 1.

[0039] Table 1. Compound 3- and-kravanhin A 13 C (150 MHz) and 1 H NMR (600 MHz, J (in Hz) data, the test solvent was CDCl3 ( δ in ppm, J (in Hz).

[0040] Example 2

[0041] Compound's GLP-1 secretion-promoting activity and DPP-4 inhibitory activity

[0042] 1. Materials and Methods

[0043] 1.1. Materials

[0044] STC-1 cells were purchased from the ATCC cell bank in the United States; DMEM and RPMI-1640 media were purchased from Vivacell in Italy; F12 and F12K media were purchased from Thermo Fisher Scientific in the United States; the protease inhibitor mixture was purchased from Beyotime Biotechnology Co., Ltd. in Shanghai, China; fatty acid-free bovine serum albumin was purchased from Shanghai Liji Biotechnology Co., Ltd.; Matrigel was purchased from Becton, Dickinson and Company in the United States; and the mouse GLP-1 kit was purchased from Wuhan Huamei Biotechnology Co., Ltd.

[0045] 1.2. Instruments

[0046] The Flex Station 3 benchtop multi-functional microplate reader was purchased from Bio-Rad Laboratories; the FlexA-200 full-wavelength microplate reader was purchased from Hangzhou Ausen Instruments Co., Ltd.; the ME104E analytical balance was purchased from Mettler Toledo Instruments (Shanghai) Co., Ltd.; the DHP-9082 electric thermostatic incubator was purchased from Shanghai Yiheng Technology Co., Ltd.; the cell incubator was purchased from esco Technology Co., Ltd. (Singapore); the inverted fluorescence microscope was purchased from Olympus Corporation (Japan); and the fluorescence and chemiluminescence imaging analysis system was purchased from Beijing Saizhi Venture Technology Co., Ltd. (China).

[0047] 1.3. Experimental Procedure

[0048] GLP-1 secretion assay: STC-1 cells were cultured in DMEM medium containing 10% FBS and 1% PS in an incubator at 37°C and 5% CO2 saturated humidity; when the cells grew to 80%-90%, the diluted cell suspension was seeded into 24-well plates (5×10⁻⁶ cells / wells). 5 (1 sample per well), add KRBH to each well for 1 hour of starvation; add KRBH working solution containing the compound to each well for 2 hours of starvation, and collect the supernatant; detect the GLP-1 content in the supernatant using a mouse-derived GLP-1 ELISA kit.

[0049] DPP-4 activity screening: The working buffer (Tris-HCl) was prepared by dissolving 605.6 mg of Tris and 28.5 mL of 0.118 mol / L hydrochloric acid solution, adjusting the pH to 8.0, in 100 mL of ultrapure water. Glycylproline p-nitroaniline (GLY-PRO-PNA 25 mg) was dissolved in 7.6 mL of Tris-HCl as a substrate. 420 mg of NaHCO3 was dissolved in 50 mL of ultrapure water as the NaHCO3 stop solution. 70... μ L Tris-HCl, 10 μ L of the test sample dissolved in DMSO and 10 μ LPTP1B enzyme (5 mg / L) was added sequentially to 96-well plates, incubated at 37°C for 10 min, and then diluted with 10 mg / L of LPTP1B enzyme. μ The reaction was initiated with L substrate and then incubated for 30 min. 100 μL of the substrate was added to the reaction mixture. μ The reaction was terminated with L Na2CO3 solution, and the absorbance was measured at 405 nm using an ELISA reader. The results were recorded. DMSO was used instead of the test solution for the negative control, and sitagliptin was used for the positive control; other methods were the same. The DPP-4 inhibition rate was calculated as: Inhibition rate (%) = (Δenzyme – Δsample / Δenzyme – Δnegative) × 100%. The experimental results were analyzed using Graphpadprism 8.0 software.

[0050] 2. Results:

[0051] 2.1. GLP-1 secretory activity:

[0052] The isosporane diterpenoid compound disclosed in this invention is the first to be isolated from the stems and leaves of *Amomum villosum*, compound 3- and -kravanhin A was acted on STC-1 cells to evaluate its effect on GLP-1 secretion. As shown in Table 2, compound 3- and -kravanhin A at 60 μ M significantly promotes GLP-1 secretion, with a secretion rate of 109.7%.

[0053] Further research is needed on its range of 5-160. μ The dose-response relationship at the concentration of M (see) Figure 2 ), compound 3- and -kravanhin A can dose-dependently promote GLP-1 secretion without affecting cell survival, indicating that 3- and -kravanhin A has significant GLP-1 secretion-promoting activity.

[0054] Table 2. Compound 3- and -Kravanhin A promoting effect on GLP-1 secretion in STC-1 cells (60 μM) a .

[0055] a Numerical values ​​are expressed as mean ± standard deviation. n = 3); b The secretion rate represents the percentage increase in GLP-1 content compared to the control group; c Bile acid was used as a positive control.

[0056] 2.2. DPP-4 inhibitory activity:

[0057] Endogenous GLP-1 is unstable in vivo and is rapidly degraded by the DPP-4 enzyme. Therefore, DPP-4 inhibitors are effective drugs for increasing endogenous GLP-1 levels in the human body. This study further evaluated compound 3- and -Inhibitory effect of kravanhin A on DPP-4 enzyme (see Table 3). Compound 3- and -kravanhin A at concentrations of 200 and 400 μ At M, it has a moderate inhibitory effect on DPP-4 enzyme, with inhibition rates of 31.0% and 46.5%, respectively.

[0058] Table 3. Compound 3- and -Kravanhin A's inhibitory activity against DPP-4 a .

[0059] a The inhibitory effect is expressed as mean ± standard deviation ( ). n = 3); Sitagliptin was used as a positive control.

[0060] It should be noted that GLP-1 and DPP-4 are biomolecules related to insulin regulation and blood glucose control: GLP-1 is an insulin secretin before insulin secretion, which can promote insulin secretion, improve insulin sensitivity, reduce insulin breakdown and inhibit glucagon secretion, and can also act on the central nervous system to reduce appetite and increase satiety.

[0061] DPP-4 is an enzyme found in various cell types. Its 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.

[0062] 3. Conclusion:

[0063] This invention provides the first isolation of a novel isospongine diterpenoid 3- from the stems and leaves of *Amomum villosum*. and -kravanhin A significantly promotes GLP-1 secretion at 60 μM in a dose-dependent manner, while exhibiting moderate inhibitory activity against DPP-4 enzyme at concentrations of 200 and 400 μM. Therefore, 3- and -kravanhin A possesses both GLP-1 secretion-promoting and DPP4-inhibiting activities, giving it a significant advantage over existing single-target drugs on the market (such as GLP-1 agonists or DPP4 inhibitors), and it has great potential to be developed into a hypoglycemic and weight-loss drug.

[0064] Example 3:

[0065] Formulation Examples:

[0066] Take compound 3- and -kravanhin A, dissolved in a small amount of DMSO, is then mixed with water for injection as usual, filtered, filled, and sterilized to prepare an injection solution;

[0067] Take compound 3- and -kravanhin A, dissolved in a small amount of DMSO, is then dissolved in sterile water for injection. The solution is stirred until dissolved, filtered through a sterile suction funnel, then aseptically filtered, dispensed into ampoules, freeze-dried at low temperature, and then aseptically sealed to obtain a powder for injection.

[0068] Take compound 3- and -kravanhin A, added to the excipients at a weight ratio of 9:1, to form a powder;

[0069] Take compound 3- and -kravanhin A, add excipients at a weight ratio of 5:1, granulate and compress;

[0070] Take compound 3- and -kravanhin A, prepared into an oral liquid using conventional oral liquid preparation methods;

[0071] Take compound 3- and -kravanhin A, added to excipients at a weight ratio of 5:1, to form capsules;

[0072] Take compound 3- and -kravanhin A, added to excipients at a weight ratio of 3:1, to form capsules;

[0073] Take compound 3- and -kravanhin A, added to the excipients at a weight ratio of 5:1, is used to make granules.

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An iso-spongiane diterpene compound, characterized in that, The structural formula is shown as formula (I): （I）。 2. Use of the isolated discodermide compound of claim 1 in the preparation of a GLP-1 secretagogue drug.

3. Use of the isolated discodermide compound of claim 1 in the preparation of a DPP-4 inhibitor drug.

4. Use of the isolated discodermide compound of claim 1 in the preparation of a hypoglycemic drug or a weight loss drug.

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

6. The pharmaceutical composition of claim 5, wherein, At least one of a pharmaceutically acceptable carrier, excipient, adjuvant and vehicle is further included.

7. Use of the pharmaceutical composition of claim 5 or 6 in the preparation of a hypoglycemic drug or a weight loss drug.

8. The extraction method of the iso-dispersine diterpene compound according to claim 1, characterized by, The isolated discodermide compound is extracted from Amomum villosum stem leaves.

9. The extraction method of the iso-dispersine diterpene compound according to claim 8, characterized by, The extraction method includes the following steps: Amomum villosum stem leaves are crushed, and extracted twice with 70% ethanol at room temperature by reflux extraction, each time for 48 hours, and the ethanol extracts are combined; The combined ethanol extracts are recovered under reduced pressure to obtain an extract, which is dispersed in water and extracted with ethyl acetate, and concentrated to obtain an ethyl acetate extract part; The ethyl acetate extract part is subjected to silica gel column chromatography, and gradient elution is performed with acetone-petroleum ether in the volume ratio of 0:100, 2:98, 5:95, 10:90, 20:80 and 100:0, respectively, and the eluted primary fractions are eluted with ethyl acetate-petroleum ether in the volume ratio of 10:90-80:20, respectively, to obtain secondary fractions, which are subjected to TLC detection and combined with the same fractions; The secondary fractions combined with the same fractions are eluted with methanol-water in the volume ratio of 20:80-100:0 to obtain tertiary fractions; the tertiary fractions obtained are purified by HPLC, eluted with acetonitrile-water to obtain quaternary fractions, and the quaternary fractions are subjected to qualitative screening to obtain the isolated discodermide compound.

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