Jasminoides W, extracts containing the compound, and uses of the same

Abstract

The present invention provides a novel compound, Jasminoides W, an extract containing the compound, and their use in the preparation of antidiabetic drugs. The Jasminoides W and extract containing the compound provided herein have an α-glucosidase inhibitory effect comparable to that of acarbose and can be used in the preparation of hypoglycemic drugs.

Description

Technical Field

[0001] The present invention belongs to the field of anti-diabetic drugs, and particularly relates to Jasminoides W, an extract containing the compound, and uses of the two in preparing anti-diabetic drugs. Background Art

[0002] Gardenia jasminoides (Gardenia jasminoides Ellis), a member of the Rubiaceae family, is harvested annually between June and July and used fresh or dried. It is one of the first medicinal herbs designated as both edible and medicinal by the National Health Commission of my country. Recorded in the Southern Yunnan Materia Medica, gardenia possesses a cold nature and bitter flavor, and is known to clear lung heat, relieve coughs caused by lung heat, stop bleeding, and eliminate phlegm.

[0003] Discovered as early as 1835, phlorizin has the effects of lowering blood sugar levels and improving insulin sensitivity. Phlorizin is mainly found in fruits and vegetables such as apples, strawberries, and pears. A large number of studies have shown that phlorizin has physiological functions such as antioxidant, antibacterial, tyrosinase inhibition, α-glucosidase inhibition, blood sugar reduction, cardiovascular protection, and anti-tumor. Not only that, phlorizin is also an effective inhibitor of sodium-glucose co-transporters (sodiumdependent glucose transporters, SGLT) 1 and 2 and glucose transporters (glucose transporter, GLUT) 1 and 2. However, phlorizin has obvious defects. It is easily broken down into phlorizin and glucose in the intestine. Its utilization in the human body is not high, and its anti-glycemic effect is not ideal. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention proposes a compound having a structure as shown in formula (1):

[0005]

[0006] Wherein, at least one of R1 and R2 is β-D-glucoside, preferably, both R1 and R2 are β-D-glucoside, and more preferably, the compound is Jasminoides W or Tetracentronside B.

[0007] The present invention also provides a gardenia flower extract for preparing an anti-diabetic drug, wherein the gardenia flower extract comprises the compound described in formula (1).

[0008] The present invention also provides a gardenia flower extract for preparing an anti-diabetic drug. The gardenia flower extract is a gardenia flower ethanol extract prepared by subjecting the gardenia flower to a 95% ethanol solvent reflux extraction step.

[0009] The present invention also provides a gardenia flower extract for preparing an anti-diabetic drug. The gardenia flower extract is a gardenia flower extract prepared by separating and purifying the gardenia flower extract by high performance liquid chromatography.

[0010] The present invention also provides a gardenia flower extract for preparing an anti-diabetic drug. The gardenia flower extract is prepared by subjecting the gardenia flower ethanol extract to polarity gradient extraction with organic solvents of different polarities.

[0011] The Jasminoides W and the extract containing the compound provided by the invention have the effect of inhibiting the activity of alpha-glucosidase and can be used for preparing blood sugar lowering drugs. DETAILED DESCRIPTION

[0012] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0013] Example 1

[0014] The ethanol extract of gardenia is prepared by extracting gardenia with 95% ethanol solvent under reflux. Specifically, 20 kg of dried gardenia is taken, soaked in 95% ethanol for 24 hours and extracted three times, and the combined extracts are concentrated under reduced pressure to obtain the ethanol extract of gardenia.

[0015] Example 2

[0016] The gardenia flower extract obtained in Example 1 was subjected to a polarity gradient extraction step using organic solvents of varying polarity to prepare a gardenia flower extract. Specifically, the gardenia flower ethanol extract obtained in Example 1 was dispersed in purified water and extracted sequentially with equal volumes of petroleum ether, ethyl acetate, and n-butanol to obtain 160 g of petroleum ether extract, 468 g of ethyl acetate extract, 228 g of n-butanol extract, and 2400 g of aqueous extract, respectively. The 2400 g aqueous extract was used as the gardenia flower extract in Example 3.

[0017] Example 3

[0018] The gardenia flower extract is prepared by subjecting the gardenia flower extract to a high performance liquid chromatography separation and purification step. Specifically, 2400 g of the aqueous layer of the gardenia flower is initially separated using D-101 macroporous adsorption resin, and gradient eluted with 10%, 30%, 60%, and 100% ethanol, respectively. The eluted fractions are concentrated and combined to obtain four fractions (fractions 1 to 4) according to the different ethanol volume fractions.

[0019] Fraction 3 (53.7 g) was mixed with 110 g of ODS and loaded onto a column using a dry-phase elution gradient of methanol:water (30:70, 60:40, 80:20, and 100:0). Identical fractions were combined by TLC to yield 10 fractions (fractions 3.1 to 3.10). Fraction 3.3 (12.2 g) was mixed with 26 g of silica gel (100-200 mesh) and loaded onto a column using a wet-phase elution gradient of dichloromethane:methanol (15:1, 10:1, 5:1, and 1:1). Identical fractions were combined by TLC to yield 5 fractions (fractions 3.3.1 to 3.3.5). Fraction 3.3.4 (1.1 g) was purified by Sephadex LH-20 gel column chromatography with methanol as eluent and TLC identification and combined identical fractions to obtain seven subfractions (fraction 3.3.4.1-fraction 3.3.4.7). Fraction 3.3.4.1 (0.1 g) was separated and purified by preparative HPLC chromatography [methanol: water (50:50)] to give compound 20 (11 mg, tR = 50 min).

[0020] The chemical structure of the Gardenia jasminoides extract, compound 1, was determined.

[0021] Compound 1 is a brown solid that is readily soluble in methanol. The Molish reaction is positive, suggesting it is a glycoside. Hydrolysis with a 10% H2SO4-CH3CH2OH solution yields Glc. HR-ESI-MS yields: m / z 683.2596 [M+H] + Quasi-molecular ion peak (C 32 H 43 O 16 + , calculated value is 683.2551), combined with NMR data, it can be inferred that its molecular formula is C 32 H 42 O 16 ,

[0022] 1H-NMR (400MHz, Pyridine-d5) δ: 6.99 (2H, d, J = 4.43Hz, H-2, 2'), 6.85 (4H, q, J = 4.91Hz, H-5, 6, 5', 6') , 5.93 (4H, d, J = 4.93Hz, 3, 4-OCH2O- / 3', 4'-OCH2O-), 5.14 (1H, dd, J = 8.01, 4.69Hz, H-1"'), 4.77-4.8 8(2H, m, H-1", 9'a), 4.52 (1H, m, H-6"'a), 2.97 (4H, m, H-7, 7'), 2.49 (1H, m, H-8), 2.16 (1H, q, J = 6.44, 5.89Hz, H-8'); 13C-NMR (101MHz, Pyridine-d5) δ: 148.5 (C-3), 148.5 (C-3'), 146.6 (C-4), 146.6 (C-4 '), 136.1(C-1'), 135.8(C-1), 123.2(C-6), 123.1(C-6'), 110.7(C-2), 110.7(C-2'), 108.9(C-5), 10 8.8(C-5'), 105.9(C-1"'), 105.3(C-1"), 101.6(C-3,4-OCH2O-), 101.6(C-3',4'-OCH2O-), 79.1(C-3 ”'), 78.9(C-3”), 78.9(C-5”’), 77.8(C-5”), 75.7(C-2”), 75.6(C-2”’), 72.2(C-4”), 72.1(C-4”’), 7 0.7(C-9'), 70.4(C-9), 63.2(C-6"'), 61.8(C-6"), 44.5(C-8'), 42.4(C-8), 35.8(C-7'), 35.7(C-7).

[0023] through 13 C-NMR data analysis revealed compounds similar to those reported in the literature:

[0024] The data for (8R,8'R)-9-O-(6'-O-α-L-arabinofuranosyl)-β-D-glucopyranosyldihydrocubebin and TetracentronsideB can be compared.

[0025] Compound 1 exhibits an additional set of sugar signals at 105.9, 79.1, 78.9, 75.6, 72.1, and 63.2 compared to tetracentronside B. Combined with the sugar terminal proton signal at 5.14 ppm (1H, dd, J = 8.01, 4.69 Hz, H-1") in the 1H-NMR spectrum and the hydrolysis results with 10% H2SO4-CH3CH2OH solution, compound 1 demonstrates the presence of a β-D-glucose moiety. HBMC data analysis reveals a long-range correlation between the sugar terminal proton signal at 5.14 ppm (1H, dd, J = 8.01, 4.69 Hz, H-1") and the signal at the 9' position (C-9') on the parent nucleus at δ70.5 ppm, suggesting that the glucose moiety is attached to C-9'. Furthermore, a 9' position shift of 8 ppm upfield was observed in compound 20, consistent with the principle of glycosidation shift.

[0026] The chemical structure of the Gardenia jasminoides extract is as shown in formula (2):

[0027]

[0028] Name: Jasminoides W.

[0029] Comparative Example 1

[0030] Positive control example: Acarbose.

[0031] Comparative Example 2

[0032] The natural compound phlorizin, whose structure is shown in formula (3), is a natural glycoside derivative mainly derived from apple peel and pear trees. It was first isolated by a French chemist in 1835. Phlorizin can prevent the kidneys from reabsorbing glucose by inhibiting SGLT2 and can promote the kidneys to excrete sugar through urine in the long term.

[0033]

[0034] Comparative Example 3

[0035] Patent CN113461747B discloses a compound with hypoglycemic activity extracted from Rosa serrata fruit, the chemical name of which is 5-ethoxy-2-methoxymethyl-6-(1-methyl-butoxy)-tetrahydropyran-3,4-diol.

[0036] Comparative Example 4

[0037] The natural compound luteolin, whose structure is shown in formula (4), is a natural flavonoid found in a variety of plants. It exhibits a variety of pharmacological activities, including anti-inflammatory, anti-allergic, uric acid-lowering, anti-tumor, antibacterial, and antiviral. It is primarily used clinically for cough relief, expectoration, anti-inflammatory, uric acid-lowering, and treatment of cardiovascular disease, amyotrophic lateral sclerosis, SARS, and hepatitis. Luteolin can inhibit the absorption of glucose in the small intestine by inhibiting α-glucosidase.

[0038]

[0039] Test Example 1

[0040] Experimental materials and instruments:

[0041] Table 1. Main experimental materials and reagents

[0042]

[0043] Table 2. Main instruments and equipment used

[0044]

[0045]

[0046] Solution preparation:

[0047] (1) 0.1M hydrochloric acid solution: Take 9 mL of concentrated hydrochloric acid and add water to 1 L;

[0048] (2) 0.1 M phosphate buffer solution: Accurately weigh 3.5510 g of anhydrous Na2HPO4 and 3.9019 g of anhydrous NaH2PO4 and place them in two beakers, respectively. Add 250 mL of distilled water to each beaker to obtain 0.1 M Na2HPO4 solution and 0.1 M NaH2PO4 solution, respectively. Then, mix the two in a volume ratio of (61:39) and adjust the pH to 7 with 0.1 M hydrochloric acid solution to obtain 0.1 M phosphate buffer solution for later use.

[0049] (3) Substrate PNPG solution: Accurately weigh 7.5 mg of PNPG solid powder into a 10 mL volumetric flask and add 0.1 M phosphate buffer solution to make up to a concentration of 2.5 mM PNPG solution, which is prepared and used immediately;

[0050] (4) α-glucosidase solution: Take freeze-dried enzyme powder (enzyme activity is 69.6 U / mg), first use 0.1 M phosphate buffer solution to prepare 10 U / mL α-glucosidase stock solution, and then use buffer solution to dilute it to 0.5 U / mL α-glucosidase solution, ready for use.

[0051] (5) Acarbose (Comparative Example 1) solution: Accurately weigh 1.0 mg of acarbose solid, dissolve it in 1 mL of DMSO, and shake well to obtain the stock solution for later use;

[0052] (6) Stop solution: Accurately weigh 2.1201 g of anhydrous Na2CO3 solid into a 100 mL volumetric flask and distilled water to volume to obtain a 0.2 M Na2CO3 solution;

[0053] (7) Monomeric compound sample solutions. Some compounds were diluted.

[0054] Seven groups of test samples, namely, Jasminoides W obtained by separation and purification in Example 1, Example 2, and Example 3, and Control Examples 2, 3, and 4, were taken and reacted in a 96-well cell culture plate. According to Table 3, phosphate buffer solution, α-glucosidase solution, sample solvent (DMSO) and test sample solutions of different concentrations were accurately pipetted, mixed, and incubated at 37°C for 10 minutes. Then, substrate (PNPG solution) was added, mixed thoroughly, and incubated at 37°C for 20 minutes. Finally, stop solution was added to terminate the reaction. The absorbance value was measured at a wavelength of 405 nm using an enzyme marker. The α-glucosidase inhibition rate formula (4) was used for calculation, and the obtained data was processed using Microsoft Excel 2016. A scatter plot was drawn using Origin software to calculate the IC 50 value.

[0055] Table 3. Amount of each reactant added

[0056]

[0057] Calculation formula for α-glucosidase inhibition rate:

[0058] Inhibition rate (%) = [(Ak-Akb)-(As-Asb)] / (Ak-Akb)×100% Formula (4)

[0059] Note: Ak: absorbance of blank group; Akb: absorbance of blank background group; As: absorbance of experimental group; Asb: absorbance of experimental background group.

[0060] Take control example 1 (positive control substance acarbose stock solution), first dilute it 1000 times with DMSO, and then dilute it using the two-fold dilution method to obtain six concentrations, namely 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 μg / mL. According to the above experimental method, the inhibition rate corresponding to each concentration was calculated, and the inhibition curve was drawn with acarbose concentration as the X-axis and α-glucosidase inhibition rate as the Y-axis, and the half-maximal inhibitory concentration (IC) of acarbose was calculated. 50 It is 0.17μg / mL.

[0061] The α-glucosidase inhibitory activity of each test sample was determined according to the above experimental procedures. The results are shown in Table 4.

[0062] Table 4. Inhibition rate of α-glucosidase

[0063]

[0064] Discussion: Gardenia jasminoides ethanol extract and gardenia jasminoides extract both had a certain inhibitory effect on α-glucosidase activity.

[0065] Jasminoides W has a significant inhibitory effect on α-glucosidase activity, and its effect is comparable to that of the positive control acarbose.

[0066] The inhibition rate of Jasminoides W at a concentration of 50 μg / mL reached 88.6%. Compared with luteolin, the inhibitory effect of Jasminoides W was more obvious. Jasminoides W was more efficient than Control Example 4, whose inhibition rate at a concentration of 50 μg / mL reached 48%, far lower than the 88.6% of Jasminoides W.

[0067] Unexpectedly, the direct attachment of the glucoside group of phlorizin in Control Example 2 to an aromatic group failed to achieve the same effect as Jasminoides W and Tetracentronside B, and the attachment of the glucoside group to a short-chain alkane in Control Example 3 also failed to achieve comparable effects as Jasminoides W and Tetracentronside B. This demonstrates that the basic structure of formula (1) is relevant to enhancing the inhibitory effect of α-glucosidase activity.

[0068] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A compound of formula (1) or a pharmaceutically acceptable salt thereof: in, R1 and R2 are both β-D-glucoside groups.

2. A Gardenia jasminoides extract for preparing an anti-diabetic drug, characterized in that: The gardenia extract comprises the compound as claimed in claim 1.

3. A Gardenia jasminoides extract for preparing an anti-diabetic drug, characterized in that: The gardenia extract is a gardenia ethanol extract prepared by subjecting gardenia to reflux extraction with a 95% ethanol solvent; the gardenia ethanol extract comprises the compound according to claim 1.

4. A Gardenia jasminoides extract for preparing an anti-diabetic drug, characterized in that: The gardenia extract is a gardenia extract prepared by subjecting the gardenia ethanol extract according to claim 3 to polarity gradient extraction using organic solvents of different polarities, and the gardenia extract includes the compound according to claim 1.

5. A preparation for treating diabetes, characterized in that: The preparation comprises the compound according to claim 1 or any one of the gardenia flower extracts according to claims 2 to 4 and pharmaceutical excipients.

6. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antidiabetic drug.

7. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting α-glucosidase activity.

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