Procyanidine tripolymer compound and application thereof
By isolating and preparing the proanthocyanidin trimer compound Psychotriflavanol A from *Symplocos rubra*, a plant in the Rubiaceae family, the problem of the lack of effective α-amylase inhibitors in the prior art has been solved, achieving a significant hypoglycemic effect and demonstrating good potential for drug development.
Patent Information
- Application Number
- CN202511223124.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-12-02
AI Technical Summary
Current technologies have not yet systematically studied the chemical composition and pharmacological activities of plants in the genus *Achyranthes*, especially *Achyranthes rubrum*. There is a lack of effective α-amylase inhibitors for the treatment of type 2 diabetes, and existing drugs have gastrointestinal adverse reactions and compliance issues.
Psychotriflavanol A, a proanthocyanidin trimer, was isolated and prepared from *Syngonium stenoptera*, a plant in the Rubiaceae family. It achieves a hypoglycemic effect by inhibiting α-amylase activity. The compound was obtained by multi-step column chromatography and purification techniques and applied to the preparation of various drug dosage forms.
The proanthocyanidin trimer compound Psychotriflavanol A significantly inhibits α-amylase activity, with an IC50 value of 81.54 ± 6.32 nM, which is superior to acarbose. It has good water solubility and safety, and is suitable for the preparation of hypoglycemic drugs, especially for the treatment of type 2 diabetes.
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Figure CN121045124A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a proanthocyanidin trimer compound and its applications. Background Technology
[0002] Nine-sectioned ( *Rubiaceae* is a genus of plants, comprising approximately 800-1500 species worldwide, widely distributed in tropical and subtropical regions, particularly abundant in the Americas. my country has 17 species and 1 variety of this genus, including *Symplocos rubra*. ), Manjiujie ( ), Meiguo Jiujie ( ), Nine-section bone fixation ( Four species have medicinal value and are used for clearing heat and detoxifying, reducing swelling and removing toxins, dispelling wind and dampness, relaxing muscles and tendons, and strengthening muscles and bones. In recent years, scholars at home and abroad have conducted extensive research on the chemical composition and pharmacological activity of plants in the genus *Nyctaginus*. The results show that plants in this genus contain novel alkaloids, including indole alkaloids, bisindole alkaloids, quinoline alkaloids, and polyalkaloids (South African Journal of Botany, 2019, 121: 494-499.), and have a wide range of pharmacological activities, including antitumor, antibacterial and antiviral effects, as well as regulation of central nervous system activity (such as hallucinogenic, sedative and analgesic effects) (Current Neuropharmacology, 2019, 17(2): 108-128; Natural Products and Bioprospecting, 2019, 9(4): 251-265.). In Guangdong, plants in the genus *Nyctaginus* ( The phenolic components isolated from *Gynostemma pentaphyllum* show anti-diabetic potential by inhibiting α-glucosidase activity, reducing intestinal glucose absorption, and enhancing liver glycogen synthesis; however, the specific mechanisms and clinical applications still require further verification. Currently, research on this genus in my country is still very limited, with existing work focusing only on *Gynostemma pentaphyllum* (Yunnan *Gynostemma pentaphyllum*). Mao Jiujie ( ) ), Manjiujie ( ) and Hainan Jiujie ( Preliminary isolation and identification of a few species, such as [list of species], have been achieved, but systematic research reports on the chemical composition and pharmacological activities of other species in this genus have not yet been found.
[0003] Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia, primarily caused by insufficient insulin secretion or impaired insulin action. Long-term hyperglycemia can lead to various complications, seriously threatening human health. In type 2 diabetes mellitus (T2DM), insulin resistance and postprandial hyperglycemia are the main pathophysiological features, and the rapid rise in postprandial blood glucose largely depends on the conversion of carbohydrates into absorbable monosaccharides in the digestive tract. α-Amylase, as a key enzyme in the hydrolysis of starch and glycogen, dominates the initial breakdown process of polysaccharides into oligosaccharides and glucose, thus becoming a core target for controlling postprandial glucose production and absorption. Inhibiting α-amylase activity can significantly slow down the rate of carbohydrate digestion, reduce postprandial blood glucose peaks, and alleviate insulin burden, which is directly related to the metabolic management and complication risk control of T2DM. Although existing clinical inhibitors (such as acarbose) are effective in reducing postprandial hyperglycemia, their gastrointestinal adverse reactions limit long-term adherence. In contrast, natural α-amylase inhibitors derived from plants show significant research and application potential due to their good biocompatibility and lower side effects. Therefore, developing efficient and safe natural inhibitors of α-amylase is of great clinical significance for the prevention and treatment of diabetes, especially postprandial hyperglycemia.
[0004] Given that plants in the genus *Achyranthes* are rich in structurally diverse alkaloids and phenolic compounds with broad pharmacological activities, research on the chemical composition and activities of *Achyranthes bidentata* is still not systematic. Therefore, it is necessary to conduct component isolation and functional screening to explore potential natural enzyme inhibitors and provide new drug lead structures for metabolic diseases such as diabetes. Summary of the Invention
[0005] The first objective of this invention is to provide a proanthocyanidin trimer compound isolated from *Agropyron cristatum*, a plant of the genus *Agropyron*, which significantly inhibits α-amylase activity.
[0006] A second objective of this invention is to provide a method for preparing the proanthocyanidin trimer compound.
[0007] A third objective of this invention is to provide the application of the aforementioned proanthocyanidin trimer compound in the preparation of a hypoglycemic drug.
[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0009] In a first aspect, the present invention provides a proanthocyanidin trimer compound with the following chemical structure:
[0010] .
[0011] The proanthocyanidin trimer compound was isolated and prepared from *Achyranthes bidentata*, a plant belonging to the Rubiaceae family.
[0012] A second aspect of the present invention provides a method for preparing the aforementioned proanthocyanidin trimer compound, comprising the following steps:
[0013] The stems, leaves, or whole plant of the plant *Mexicana spp.* are dried (air-dried), pulverized to 20-30 mesh, and extracted with solvent at least three times, soaking for at least 24 hours after each extraction. The extracts are then combined and concentrated under reduced pressure to obtain an extract.
[0014] The extract was obtained by solid-phase extraction with ethyl acetate to obtain an ethyl acetate extract, which was dissolved in chloroform / acetone (1:1 v / v). The extract was then subjected to column chromatography (2002-300 mesh) with segmentation and elution using a gradient of chloroform / methanol (1:0-0:1 v / v) to obtain 8 main fractions. The chloroform / methanol fraction (5:5 v / v) was subjected to silica gel column chromatography (1:1 v / v chloroform and methanol), RP-18 column chromatography (3:1 v / v acetonitrile and water), and Sephadex LH-20 column chromatography (1:1 v / v methanol and water) to obtain the proanthocyanidin trimer compound Psychotriflavanol A.
[0015] The solvent is selected from at least one of ethyl acetate, acetone, methanol, ethanol (preferably 95% ethanol) or water.
[0016] A third aspect of the present invention provides the use of the aforementioned proanthocyanidin trimer compound or its pharmaceutical salt in the preparation of a hypoglycemic drug.
[0017] The proanthocyanidin trimer compound of the present invention can lower blood sugar by inhibiting α-amylase.
[0018] The blood glucose-lowering drugs refer to those drugs whose primary or clearly defined secondary therapeutic goals are to lower plasma glucose levels or improve glucose metabolism. Specifically, these include drugs used to regulate blood glucose, treat type 2 diabetes (T2DM), and drugs that have been clinically proven to significantly improve fasting blood glucose, postprandial blood glucose, or glycated hemoglobin (HbA1c) levels.
[0019] The blood sugar-lowering drug is composed of the proanthocyanidin trimer compound or its pharmaceutical salt as a single active ingredient, or in combination with a pharmaceutically acceptable carrier.
[0020] In a fourth aspect, the present invention provides a pharmaceutical preparation made from the said proanthocyanidin trimer compound or its pharmaceutical salt and medically acceptable excipients.
[0021] The dosage form of the pharmaceutical preparation is selected from tablets, ampoules, capsules, suspensions, granules, powders, emulsions, solutions, drop pills, injections, suppositories, enemas, aerosols, patches, or drops.
[0022] The drug formulation can be administered orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, sublingually, transdermally, via rectal suppository, via parenteral administration, via direct intracerebral administration, via nasal brain-targeted delivery, via genetic engineering technology, or via receptor-mediated transport.
[0023] In a fifth aspect, the present invention provides the use of the aforementioned proanthocyanidin trimer compound or a pharmaceutical salt thereof in the preparation of a medicament for treating diabetes.
[0024] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:
[0025] This invention systematically studied *Symplocos rubrum*, a plant belonging to the Rubiaceae family and the *Symplocos* genus, and discovered a proanthocyanidin trimer compound, Psychotriflavanol A. Its hypoglycemic potential was evaluated through in vitro enzyme inhibition experiments. The results showed that this compound exhibited extremely strong inhibitory activity against α-amylase, with an IC50 value of [missing information]. 50 The value was 81.54 ± 6.32 nM; compared with the positive control drug acarbose (IC50). 50 Compared to acarbose (1.38±0.21 μM), this compound exhibits significantly greater activity in inhibiting α-amylase, with an inhibitory capacity 16.9 times that of acarbose. This compound possesses good water solubility, is derived from natural plant resources, has a well-defined structure, and demonstrates good safety, showing great promise for the development of novel naturally derived hypoglycemic drugs.
[0026] The proanthocyanidin trimer compound Psychotriflavanol A provided by this invention is derived from *Symplocos rubra*, a plant in the Rubiaceae family. This plant is abundant, extraction is feasible, and the resource is sustainable. Chemically, this compound is a proanthocyanidin flavanol derivative with a well-defined trimer structure, exhibiting typical characteristics of polyphenolic compounds. It is easily separated and purified using modern chromatographic techniques and also possesses potential for synthetic modification. Functionally, Psychotriflavanol A significantly inhibits α-amylase activity even at extremely low concentrations, suggesting that it may regulate blood glucose levels by delaying carbohydrate digestion and absorption. Therefore, this compound holds promise as a lead structure for treating metabolic diseases such as type 2 diabetes and has significant value in drug development. This invention expands the pharmaceutical applications of proanthocyanidin trimers from *Symplocos rubra*. Attached Figure Description
[0027] Figure 1 This is a schematic diagram showing the inhibition rate of α-amylase by the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention.
[0028] Figure 2 The IC50 of the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention against α-amylase 50 Schematic diagram of the value measurement results. Detailed Implementation
[0029] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.
[0030] Example 1
[0031] Psychotriflavanol A, a proanthocyanidin trimer
[0032] 1. Sample Source
[0033] Rubiaceae family, genus *Nine-joint* plant, *Nine-joint* fruit ( The samples were provided by Kunming Zhifen Biotechnology Co., Ltd., and the original collection site was Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province.
[0034] 2. Methods for extraction, separation, and purification of chemical components
[0035] The stems and leaves of 10.02 kg of the plant *Agrostis pilosa* were dried in the shade, pulverized to 20-30 mesh, and extracted three times with 50 L of 95% ethanol at room temperature each time. The extracts were then soaked for 24 h and extracted again. The extracts were combined and concentrated under reduced pressure to obtain 1600 g of extract.
[0036] 1600 g of extract was subjected to solid-phase extraction with 24 L of ethyl acetate to obtain 660 g of ethyl acetate extract. The extract was dissolved in chloroform / acetone (1:1 volume ratio of chloroform to acetone), mixed with 80-100 mesh silica gel, and then subjected to column chromatography with 200-300 mesh silica gel for fractionation. Gradient elution with chloroform / methanol at a volume ratio of 1:0 to 0:1 yielded 8 main fractions. The chloroform / methanol fraction with a volume ratio of 5:5 was subjected to silica gel column chromatography (1:1 volume ratio of chloroform and methanol), RP-18 column chromatography (3:1 volume ratio of acetonitrile and water), and Sephadex LH-20 column chromatography (1:1 volume ratio of methanol and water) to obtain 0.1 g of the proanthocyanidin trimer compound Psychotriflavanol A.
[0037] 3. The structure of the proanthocyanidin trimer compound Psychotriflavanol A was determined.
[0038] use 1 H-NMR, 13 Multiple spectral techniques, including C-NMR, CD, OR, and HR-ESI-MS, were used to obtain the NMR and molecular weight information of the compound, and the structure of the compound was determined as shown below:
[0039]
[0040]
[0041] Example 2
[0042] Detection of α-amylase resistance in Psychotriflavanol A, a proanthocyanidin trimer compound prepared in this invention:
[0043] 1. Experimental materials
[0044] α-Amylase (from Aspergillus oryzae), soluble starch (starch substrate), DNS reagent (3,5-dinitrosalicylic acid, used for reducing sugar detection), PBS phosphate buffer (pH 7.0) (used to prepare enzyme and substrate solutions), and acarbose (positive control) were all purchased from Shanghai Shiga Biotechnology Co., Ltd.
[0045] 2. Experimental Methods
[0046] The inhibitory activity of the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention against α-amylase was evaluated in vitro in 96-well plates using the p-p-nitrophenyl substrate colorimetric method.
[0047] The experimental procedure is as follows: In the pre-reaction system, 50 μL of the test compound solution (final concentration 50 μM) and 50 μL of α-amylase solution (2 U / mL) were added to each well sequentially, and incubated at 37℃ for 10 min. Then, 100 μL of soluble starch solution (2%, w / v) was added as substrate, and the reaction was continued at 37℃ for 15 min. To terminate the reaction, 50 μL of DNS chromogenic reagent was added, and the mixture was heated in a boiling water bath for 10 min. After cooling, 100 μL of deionized water was added for dilution. The absorbance of each well was measured at 540 nm using a microplate reader. Each group had three replicates, with acarbose (final concentration 10 μM) as a positive control. The α-amylase inhibition rate was calculated using the following formula:
[0048] Inhibition rate (%) = [1 - (Absorbance of sample group - Absorbance of negative control group) / (Absorbance of blank group - Absorbance of blank control group)] × 100%
[0049] 3. Experimental Results
[0050] The activity data are shown in Table 1:
[0051] Table 1. Anti-α-amylase activity data of the proanthocyanidin trimer compound Psychotriflavanol A
[0052] (mean ± s, n = 3)
[0053]
[0054] In an in vitro α-amylase inhibition assay, the compound Psychotriflavanol A (50 μM) of this invention showed an inhibition rate of 98.92%, which was higher than the 95.96% inhibition rate of the control drug acarbose under the same conditions. This data indicates that the compound of this invention has a significant advantage in inhibiting amylase hydrolytic activity, and therefore can serve as a candidate active ingredient or lead compound for controlling postprandial blood glucose, for further drug development or the preparation of functional formulations (including but not limited to hypoglycemic drug compositions, functional food components, etc.).
[0055] Example 3
[0056] The IC50 of the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention against α-amylase... 50 Value determination:
[0057] Preliminary screening of α-amylase inhibitory activity at a concentration of 50 μM showed that the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention exhibited an inhibition rate of over 80% against α-amylase, demonstrating strong inhibitory potential.
[0058] Based on this, its half-maximal inhibitory concentration (IC50) was further determined. 50 Inhibition experiments on α-amylase were conducted using six concentration gradients: 1.00 μM, 0.50 μM, 0.25 μM, 0.125 μM, 0.0625 μM, and 0.0313 μM. The inhibition rate was calculated and dose-response curves were plotted.
[0059] The results are as follows Figure 1 and Figure 2 As shown, Figure 1 This is a schematic diagram showing the inhibition rate of the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention against α-amylase. Figure 2 The IC50 of the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention against α-amylase 50 A schematic diagram of the value measurement results. From... Figure 1 As can be seen from the results, Psychotriflavanol A exhibited inhibitory activity comparable to or slightly superior to that of the clinically commonly used inhibitor acarbose in in vitro α-amylase inhibition experiments (inhibition rate of 98.92% vs. acarbose 95.96% at 50 μM), indicating its promising development potential as a potent α-amylase inhibitor from a natural source. Figure 2 As can be seen from the results, the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention exhibits an IC50 response to α-amylase. 50 The value was 81.54±6.32 nM, showing extremely strong α-amylase inhibitory activity, which was 16.9 times that of the positive control drug acarbose.
[0060] Example 4
[0061] The proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention was formulated into tablets:
[0062] The ingredients included Psychotriflavanol A (a proanthocyanidin trimer) 10 mg, lactose 180 mg, starch 55 mg, and magnesium stearate 5 mg.
[0063] Tablet preparation method: Psychotriflavanol A, a proanthocyanidin trimer, is mixed evenly with lactose and starch. An appropriate amount of propylene glycol is added as a wetting agent, and after thorough wetting, the mixture is granulated through a sieve. The resulting wet granules are dried at low temperature, then granulated again through a sieve, magnesium stearate is added, and the mixture is mixed thoroughly before being compressed into tablets. The resulting tablets have a smooth appearance and uniform color, with each tablet weighing 250 mg, containing 10 mg of the active ingredient, Psychotriflavanol A.
[0064] The tablets prepared by this invention are simple to manufacture, use conventional excipients, and are suitable for large-scale production. Preliminary tests show that the tablets have good disintegration properties and physical stability, and can be used for subsequent in vitro and in vivo efficacy or pharmacokinetic studies.
[0065] Example 5
[0066] The proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention was formulated into an ampoule:
[0067] Preparation method of ampoules: 2 mg of the proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention was dissolved in 3 mL of propylene glycol. The solution was sterilized by filtration through a 0.22 μm microporous membrane. The filtrate was aseptically dispensed into sterilized ampoules, 3 mL per ampoule, and sealed to obtain the ampoules. The ampoules are colorless, clear liquids without visible impurities.
[0068] Propylene glycol, as a solvent, exhibits excellent solubility and biocompatibility, making it suitable for non-aqueous injectable formulations. The ampoule preparation process adheres to aseptic procedures and can serve as the basis for subsequent efficacy or toxicology studies.
[0069] Example 6
[0070] The proanthocyanidin trimer compound Psychotriflavanol A prepared in this invention was formulated into capsules:
[0071] The present invention prepares a proanthocyanidin trimer compound Psychotriflavanol A 10 mg, lactose 187 mg, and magnesium stearate 3 mg.
[0072] Preparation method of capsules: Psychotriflavanol A, a proanthocyanidin trimer, is mixed with lactose and magnesium stearate, sieved through an 80-mesh sieve, and then mixed again. The resulting powder is dispensed into No. 0 hard gelatin capsules, each capsule having a total mass of 200 mg and containing 10 mg of the active ingredient Psychotriflavanol A.
[0073] Capsules have advantages such as being easy to carry, convenient to take, and having a good taste-masking effect, making them suitable for preliminary drug efficacy screening and animal experiments. They can also be considered as a candidate form for oral formulations.
[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A proanthocyanidin trimer compound, characterized in that, The chemical structure is shown below: 。 2. A method for preparing the proanthocyanidin trimer compound according to claim 1, characterized in that, Includes the following steps: The stems, leaves, or whole plant of the plant *Mexicana spp.* are dried, pulverized to 20-30 mesh, and extracted with solvent at least three times, with each extraction followed by soaking for at least 24 hours. The extracts are then combined and concentrated under reduced pressure to obtain an extract. The extract was obtained by solid-phase extraction with ethyl acetate, which was dissolved in chloroform / acetone and fractionated by silica gel column chromatography. Gradient elution with chloroform / methanol at a volume ratio of 1:0 to 0:1 yielded 8 main fractions. The chloroform / methanol fraction with a volume ratio of 5:5 was subjected to silica gel column chromatography, RP-18 column chromatography, and Sephadex LH-20 column chromatography to obtain the proanthocyanidin trimer compound Psychotriflavanol A.
3. The method for preparing the proanthocyanidin trimer compound according to claim 2, characterized in that, The solvent is selected from at least one of ethyl acetate, acetone, methanol, ethanol, or water.
4. The use of the proanthocyanidin trimer compound of claim 1 or its pharmaceutical salt in the preparation of a hypoglycemic drug.
5. The application according to claim 4, characterized in that, The proanthocyanidin trimer compound can lower blood sugar by inhibiting α-amylase.
6. The application according to claim 4, characterized in that, The blood sugar-lowering drug is composed of the proanthocyanidin trimer compound or its pharmaceutical salt as a single active ingredient, or in combination with a pharmaceutically acceptable carrier.
7. A pharmaceutical preparation, characterized in that, It is made from the proanthocyanidin trimer compound or its pharmaceutical salt and medically acceptable excipients.
8. The pharmaceutical preparation according to claim 7, characterized in that, The dosage form of the pharmaceutical preparation is selected from tablets, ampoules, capsules, suspensions, granules, powders, emulsions, solutions, drop pills, injections, suppositories, enemas, aerosols, patches, or drops.
9. The pharmaceutical preparation according to claim 7, characterized in that, The drug formulation can be administered orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, sublingually, transdermally, via rectal suppository, via parenteral administration, via direct intracerebral administration, via nasal brain-targeted delivery, via genetic engineering technology, or via receptor-mediated transport.
10. The use of the proanthocyanidin trimer compound of claim 1 or its pharmaceutical salt in the preparation of a medicament for treating diabetes.