Sesquiterpenoid caffeic acid ester compounds and their preparation method and application

Abstract

The present invention discloses a sesquiterpenoid caffeic acid ester compound and its preparation method and application. The present invention finds two new sesquiterpenoid caffeic acid ester compounds from the leguminous plant Bauhinia saddleleaf, which have an inhibitory activity IC of α-glucosidase. 50 The sesquiterpenoid caffeic acid ester compounds described in the present invention can be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions for the treatment of diabetes-related diseases and can also be used to prepare health products that lower blood sugar. The preparation method of the present invention is simple, easy to operate, and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of natural products, in particular to sesquiterpenoid caffeic acid ester compounds and preparation methods and applications thereof. Background Art

[0002] Diabetes is one of the three most serious diseases affecting human health, posing a serious threat to people's health and quality of life. Type 2 diabetes accounts for over 90% of cases. α-glucosidase, located in the brush border of intestinal epithelial cells, is a key enzyme that catalyzes the hydrolysis of oligosaccharides, increasing postprandial blood glucose levels. Increased α-glucosidase activity can easily lead to life-threatening hyperglycemia in diabetic patients. Therefore, inhibiting α-glucosidase activity is an effective treatment for type 2 diabetes. Currently, clinically used α-glucosidase inhibitors such as acarbose and miglitol have certain side effects. Therefore, the search for more active and less toxic drug molecules has always been a key focus of pharmaceutical research. Natural products have long been an important source of drug leads, offering advantages over chemical drugs such as fewer toxic side effects and longer-lasting efficacy. Therefore, the search for new glucose-lowering drugs from natural products holds great promise.

[0003] Bauhinia brychycarpa Wall.exBenth, a member of the genus Bauhinia in the Leguminosae family, is also known as large-leaved Bauhinia, saddle leaf, night gate, and saddle Bauhinia. It is an upright or climbing shrub primarily grown in Sichuan, Yunnan, Gansu, and Hubei. As a traditional Bai medicine, Bauhinia brychycarpa, primarily for its rhizomes, stems, and leaves, has wind-dispelling, meridian-opening, astringent, and detoxifying properties. It is primarily used to treat rheumatic pain, testicular swelling and pain, palpitations, insomnia, and traumatic injuries. Current research on this plant indicates that its chemical components include triterpenes, flavonoids, phenolic acids, and steroids, primarily flavonoids. Pharmacological studies have primarily focused on its analgesic activity, with no evidence of hypoglycemic activity. Summary of the Invention

[0004] The present invention separates and identifies two new sesquiterpenoid caffeic acid esters from the stems and leaves of Bauhinia saddleii. Bausesquitate A and bausesquitate B are esters formed by cadinane-type and myrrhanane-type sesquiterpenoid alcohols, respectively, and caffeic acid. They have strong inhibitory activity against α-glucosidase and have good prospects for preparing hypoglycemic drugs.

[0005] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0006] The present invention provides a sesquiterpenoid caffeic acid ester compound, wherein the compound is bausesquitate A or bausesquitate B, and the structural formulas are shown in Formula 1 and Formula 2 respectively;

[0007]

[0008] The present invention also provides application of the sesquiterpene alcohol caffeic acid ester compound in inhibiting α-glucosidase.

[0009] The present invention also provides application of the sesquiterpenoid caffeic acid ester compound in the preparation of blood sugar-lowering medicines or foods.

[0010] The present invention also provides use of the sesquiterpenoid caffeic acid ester compound in preparing a drug for preventing or treating diabetes.

[0011] As a further supplement to the above scheme: the compound uses the compound shown in the structural formula or its pharmaceutically acceptable salts, esters, and stereoisomers as the active pharmaceutical ingredient; the content of the active pharmaceutical ingredient is 0.1 to 99.0%.

[0012] As a further supplement to the above scheme: the compound is used in combination with a pharmaceutically acceptable carrier, adjuvant or excipient to prepare an injection, injection, powder, tablet, oral solution, capsule or granule.

[0013] The present invention also provides a method for preparing the sesquiterpene alcohol caffeic acid ester compound, and the method is performed as follows:

[0014] Step (1) taking the crushed stems and leaves of Bauhinia saddleii, extracting them with an organic solvent, and recovering the solvent to obtain a total extract;

[0015] Step (2) separating and purifying the total extract by various combinations of normal phase silica gel column, MCI chromatographic column, Sephadex LH-20 gel column and preparative or semi-preparative HPLC to obtain compounds bausesquitate A and bausesquitate B.

[0016] Preferably, the organic solvent in step (1) is selected from one or more of ethyl acetate, acetone, n-butanol, ethanol, methanol, aqueous n-butanol, aqueous acetone, aqueous ethanol and aqueous methanol; and the mass volume ratio of the Bauhinia saddleii leaves to the organic solvent is 1:1-15.

[0017] Preferably, the eluent for the normal phase silica gel column chromatography in step (2) is a combination of one of petroleum ether, n-hexane, chloroform and dichloromethane and one of ethyl acetate, acetone and methanol, and the elution is performed in a gradient of 100:0 to 0:100 by volume.

[0018] Preferably, the MCI column chromatography uses a gradient elution of methanol, ethanol, or acetone to water in a volume ratio from 0:100 to 100:0;

[0019] Preferably, the Sephadex LH-20 gel column chromatography is eluted using one of methanol, acetone, methanol and chloroform, methanol and water, and acetone and water;

[0020] Preferably, the preparative or semi-preparative HPLC uses a combination of methanol or acetonitrile and water as a solvent, and performs gradient or isocratic elution according to a volume ratio.

[0021] Preferably, the method further comprises extracting the total extract, wherein the extraction operation requires dispersing the total extract in water, and the solvent used for the extraction comprises at least one of petroleum ether, ethyl acetate, chloroform, ethyl acetate and n-butanol.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the two new sesquiterpenoid caffeic acid ester compounds bausesquitate A and bausesquitate B (compounds 1 and 2) provided by the present invention have an inhibitory activity IC of α-glucosidase. 50 It is significantly stronger than the existing clinical α-glucosidase inhibitor acarbose and can be used to treat hyperglycemia, diabetes and other related diseases.

[0023] In addition, the present invention provides a method for extracting and purifying new compounds 1 and 2. The method has readily available raw materials, is simple to operate, and is suitable for industrial operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a flow chart of the preparation process of bausesquitate A and bausesquitate B;

[0025] Figure 2 For the compound bausesquitate A 1 HNMR spectrum;

[0026] Figure 3 For the compound bausesquitate A 13 C NMR spectrum;

[0027] Figure 4 HR-ESI-MS spectrum of compound bausesquitate A;

[0028] Figure 5 For compound bausesquitate B 1 HNMR spectrum;

[0029] Figure 6 For compound bausesquitate B 13 C NMR spectrum;

[0030] Figure 7 HR-ESI-MS spectrum of compound bausesquitate B;

[0031] Figure 8 This is the molecular docking result of bausesquitate A and α-glucosidase protein;

[0032] Figure 9 This is the molecular docking result of bausesquitate B and α-glucosidase protein. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these solutions is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise indicated, the technical and scientific terms used in this invention have the same meanings as those commonly understood by those skilled in the art to which this invention belongs. The terms used in this specification are only for the purpose of describing specific embodiments and are not intended to limit the scope of the invention in any form. As used herein, the term "and / or" includes any and all combinations of one or more of the relevant listed items.

[0035] Example 1 Preparation Method of Bausesquitate A and Bausesquitate B

[0036] The dried stems and leaves of Bauhinia saddleii were crushed and cold-extracted with 8 times the volume (m / v) of 95% ethanol for 3 times, each time for 3 days. The extracts were combined and the solvent was recovered under reduced pressure to obtain the total extract.

[0037] The total extract was dispersed in water and extracted with ethyl acetate. After extraction, the extract was concentrated under reduced pressure and then applied to a normal phase silica gel column. Gradient elution was performed using petroleum ether / ethyl acetate as the eluent (volume ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 0:100, 3 to 5 column volumes for each gradient). Under thin layer chromatography detection (developing solvent: petroleum ether / ethyl acetate volume ratio of 8:2), 7 component segments Fr.A to Fr.G were obtained by combination.

[0038] The Fr.B fraction was separated on an MCI column (small-pore resin gel column, polystyrene-based reverse-phase resin packing) using a methanol / water gradient elution (volume ratios of 60:40, 80:20, and 100:0, 3-5 column volumes per gradient). Five fractions, Fr.B-1 to Fr.B-5, were obtained by thin-layer chromatography (developing solvent: petroleum ether / ethyl acetate, volume ratio 8:2).

[0039] Fr.B-3 was separated by Sephadex LH-20 gel column (hydroxypropyl dextran gel column) chromatography (methanol elution) and then purified by semi-preparative HPLC using a methanol:water volume ratio of 90:10 as the mobile phase to obtain compounds bausesquitate A and bausesquitate B.

[0040] Preparation process of bausesquitate A and bausesquitate B: According to the experimental summary, the preparation process of bausesquitate A and bausesquitate B is summarized as follows Figure 1 .

[0041] Example 2 Preparation Method 2 of Bausesquitate A and Bausesquitate B

[0042] The dried stems and leaves of Bauhinia saddleii were crushed and extracted with 6 times (m / v) ethyl acetate as solvent at room temperature for 3 times, each time for 24 hours. The extracts were combined and the solvent was recovered under reduced pressure to obtain the total extract.

[0043] The total extract was applied to a normal phase silica gel column and gradient elution was performed with dichloromethane / methanol as the eluent (volume ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 0:100, 3 to 5 column volumes for each gradient). Under detection by thin layer chromatography (developing solvent: petroleum ether / ethyl acetate, volume ratio of 8:2), 8 component segments Fr.A to Fr.H were combined.

[0044] The Fr.C fraction was separated on an MCI column using a methanol / water gradient elution (volume ratios of 0:100, 30:70, 50:50, 70:30, and 100:0, with each gradient lasting 3 to 5 column volumes). Six fractions, Fr.C-1 to Fr.C-6, were obtained by thin-layer chromatography (developing solvent: petroleum ether / acetone, volume ratio 8:2).

[0045] Fr.C-4 was separated by Sephadex LH-20 gel column chromatography (chloroform / methanol, 1:1, v / v) and then purified by semi-preparative HPLC using acetonitrile:water (volume ratio: 80:20) as the mobile phase to obtain compounds bausesquitate A and bausesquitate B.

[0046] Example 3 Structural Characterization of Bausesquitate A and Bausesquitate B

[0047] Physical constants and spectral data of bausesquitate A: colorless oil; UV(MeOH)λ max (logε)244(2.13),299(2.20),329(2.29)nm; IR(KBr)ν max 3432,2930,1631,1281,and 1110cm -1 ; HR-ESI-MS m / z 383.2228[MH] - (Calculated value: C 24 H 31 O4,383.2232). 1 H NMR and 13 C NMR data are shown in Table 1. 1 H NMR spectrum is shown in Figure 2 ; Compound bausesquitate A 13 C NMR spectrum is shown in Figure 3 ; HRESIMS spectrum of compound bausesquitate A is shown in Figure 4 .

[0048] Physical constants and spectral data of bausesquitate B: colorless oil; UV(MeOH)λ max (logε)216(3.10),245(2.93),303(3.04),328(3.16)nm; IR(KBr)ν max 3399,2927,1671,1604,1280,and 1169cm -1 ;HR-ESI-MS m / z407.2190[M+Na] + (Calculated value: C 24 H 32 O4Na,407.2193). 1 HNMR and 13C NMR data are shown in Table 1. 1 H NMR spectrum is shown in Figure 5 ; Compound bausesquitate B 13 C NMR spectrum is shown in Figure 6 ; The HR-ESI-MS spectrum of compound bausesquitate B is shown in Figure 7 .

[0049] Table 1 Bausesquitate A and Bausesquitate B 1 H and 13 C NMR data

[0050]

[0051]

[0052] Note: a The data were measured using CDCl3 as solvent ( 1 HNMR 600 MHz, 13 CNMR 150MHz).

[0053] The final chemical structures of bausesquitate A (1) and bausesquitate B (2) are as follows:

[0054]

[0055] Example 4 α-glucosidase inhibitory activity of bausesquitate A and bausesquitate B

[0056] 1) Experimental Principle

[0057] α-glucosidase belongs to the class of oligosaccharide hydrolases. Its inhibitors competitively inhibit the action of glycosidases on the villi of the small intestinal epithelium, reducing carbohydrate degradation and delaying carbohydrate digestion and absorption. This effectively reduces peak postprandial blood glucose concentrations in diabetic patients, achieving glycemic control. α-glucosidase inhibitors (such as acarbose) are currently widely used as oral hypoglycemic drugs in clinical practice. α-glucosidase inhibitor activity can be screened by conducting an in vitro enzymatic reaction between the enzyme and its substrate, 4-Nitrophenylα-D-glucopyranoside (PNPG, a maltose analog). When α-glucosidase is added to the substrate, the enzyme catalyzes its decomposition into p-nitrophenol (PNP) and glucose. PNP is a colored substance with a maximum absorption at 405 nm. It can be measured using a microplate reader, and the inhibitory activity of the sample is calculated based on the OD value.

[0058] 2) Experimental methods

[0059] α-glucosidase and PNPG were diluted to 0.25 unit / mL and 2.5 mM, respectively, using PBS. At the same time, the sample to be tested was diluted to an appropriate concentration using PBS. 60 μL PBS, 10 μL test sample, 10 μL α-glucosidase and 20 μL PNPG were added to a 96-well plate in sequence. Three replicates were set for each sample, incubated in a 37 ° C incubator for 50 min, and 40 μL Na2CO3 (0.1 M) was added to quench the reaction. The absorbance of the reaction mixture was recorded at 405 nm and the α-glucosidase inhibitory activity (%) relative to the blank was calculated.

[0060] Inhibition rate (%) = [1-(As-Ab) / (Ac-Ab)] × 100%)

[0061] As--sample detection absorbance; Ac--blank detection absorbance; Ab--background detection absorbance.

[0062] Acarbose was used as a positive control in the experiment.

[0063] 3) Experimental results

[0064] Table 2 α-glucosidase inhibitory activity of bausesquitate A, bausesquitate B and acarbose

[0065]

[0066] As shown in Table 2, bausesquitate A and bausesquitate B exhibit significantly higher α-glucosidase inhibitory activity than acarbose, a first-line clinical α-glucosidase inhibitor. Both compounds exhibit stronger α-glucosidase inhibitory activity and are potential drugs for lowering blood sugar or treating diabetes.

[0067] 4) Compound molecular docking results

[0068] Molecular docking results of bausesquitate A and α-glucosidase protein ( Figure 8 ), and found that the oxygen atom of the ester group of bausesquitate A was in contact with the glutamine at position 279 of the enzyme protein peptide chain (GLN279: ) forms a hydrogen bond, the hydroxyl hydrogen at position 3 forms a hydrogen bond with the hydroxyl oxygen atom at position 4 and the serine at position 241 (SER241: ) forms a hydrogen bond, and the hydroxyl oxygen atom at position 3 forms a hydrogen bond with aspartic acid at position 242 (ASP242: ) forms a hydrogen bond, the hydroxyl hydrogen atom at position 4 forms a hydrogen bond with lysine at position 156 (LYS156: ) formed hydrogen bonds. This indicated that bausesquitate A interacted with α-glucosidase protein.

[0069] Molecular docking results of bausesquitate B and α-glucosidase protein ( Figure 9 ) and found that the hydrogen atom of the hydroxyl group at position 4 of bausesquitate B interacted with the lysine at position 156 of the enzyme protein peptide chain (LYS156: ) formed hydrogen bonds. This indicated that bausesquitate B interacted with α-glucosidase protein.

[0070] In summary, the sesquiterpenoid caffeic acid ester compounds provided by the present invention can be used to inhibit α-glucosidase; and further can be used to prepare hypoglycemic drugs or foods; or to prepare drugs for preventing or treating diabetes.

[0071] The present invention also provides a pharmaceutical composition or a hypoglycemic food composition containing the compound bausesquitate A and / or bausesquitate B. The compound bausesquitate A and / or bausesquitate B is prepared into different dosage forms with a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier, adjuvant, additive or excipient.

[0072] Example 5 Preparation of injection

[0073] Compound bausesquitate A and / or bausesquitate B is added with injection water according to conventional methods, finely filtered, and sterilized by filling to prepare an injection solution.

[0074] Example 6 Injection Preparation

[0075] Take compound bausesquitate A and / or bausesquitate B, dissolve or suspend them in sterile water for injection, stir evenly, filter with a sterile suction funnel, and then sterile fine filter, divide into ampoules, freeze-dry at low temperature, and aseptically seal them to obtain injections.

[0076] Example 7 Powder Preparation

[0077] Compound bausesquitate A and / or bausesquitate B are added to the excipient at a weight ratio of 9:1 to prepare a powder.

[0078] Example 8 Tablet Preparation

[0079] Compound bausesquitate A and / or bausesquitate B are added to the excipients at a weight ratio of 1:5 to 1:10, and the mixture is granulated and tableted.

[0080] Example 9 Oral Liquid Preparation

[0081] Take compound bausesquitate A and / or bausesquitate B and prepare oral liquid according to conventional oral liquid preparation method.

[0082] Example 10 Preparation of capsules, granules or granules

[0083] Take compound bausesquitate A and / or bausesquitate B, add it to the excipient at a weight ratio of 3-5:1 to prepare capsules, granules or granules.

[0084] Example 11 Food Preparation

[0085] The compound bausesquitate A and / or bausesquitate B is prepared by adding conventional food auxiliary materials.

[0086] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention extends to all other methods and applications with the same function.

Claims

1. A sesquiterpenoid caffeic acid ester compound, characterized in that: The compound is bausesquitate A or bausesquitate B, and the structural formulas are shown in Formula 1 and Formula 2 respectively; 。 2. Use of the sesquiterpene alcohol caffeic acid ester compound according to claim 1 in the preparation of drugs for inhibiting α-glucosidase.

3. Use of the sesquiterpene alcohol caffeic acid ester compound according to claim 1 in the preparation of hypoglycemic drugs.

4. Use of the sesquiterpene alcohol caffeic acid ester compound according to claim 1 in preparing a drug for preventing or treating diabetes.

5. The use according to any one of claims 2 to 4, characterized in that: The compound uses the compound shown in the structural formula or a pharmaceutically acceptable salt thereof as a pharmaceutically active component; the content of the pharmaceutically active component is 0.1-99.0%.

6. The use according to claim 5, characterized in that The compound is used in combination with a pharmaceutically acceptable carrier, adjuvant or excipient to prepare an injection, injection, powder, tablet, oral solution, capsule or granule.

7. The method for preparing the sesquiterpene alcohol caffeic acid ester compound according to claim 1, wherein The method operates as follows: Step (1) taking the crushed stems and leaves of Bauhinia saddleii, extracting them with an organic solvent, and recovering the solvent to obtain a total extract; In step (2), the total extract is separated and purified by a normal phase silica gel column, an MCI chromatographic column, a Sephadex LH-20 gel column, and various combinations of preparative or semi-preparative HPLC to obtain compounds bausesquitate A and bausesquitate B.

8. The method for preparing the sesquiterpene alcohol caffeic acid ester compound according to claim 7, wherein: The organic solvent in step (1) is selected from one or more of ethyl acetate, acetone, n-butanol, ethanol, methanol, aqueous n-butanol, aqueous acetone, aqueous ethanol and aqueous methanol; and the mass volume ratio of the Bauhinia saddleii leaves to the organic solvent is 1:1-15.

9. The method for preparing the sesquiterpene alcohol caffeic acid ester compound according to claim 7, wherein: The eluent for the normal phase silica gel column chromatography in step (2) is selected from: a combination of one of petroleum ether, n-hexane, chloroform and dichloromethane and one of ethyl acetate, acetone and methanol, and eluted in a gradient of 100:0 to 0:100 by volume; The MCI column chromatography uses methanol or ethanol or acetone and water in a volume ratio of 0:100 to 100:0 for gradient elution; The Sephadex LH-20 gel column chromatography is eluted using one of methanol, acetone, methanol and chloroform, methanol and water, and acetone and water; The preparative or semi-preparative HPLC uses a combination solvent of methanol or acetonitrile and water, and performs gradient or isocratic elution according to a volume ratio.

10. The method for preparing the sesquiterpene alcohol caffeic acid ester compound according to claim 7, characterized in that: The method further comprises extracting the total extract, wherein the extraction operation requires dispersing the total extract in water, and the solvent used for the extraction comprises at least one of petroleum ether, ethyl acetate, chloroform, ethyl acetate and n-butanol.

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