A zygophyllin L, and a preparation method and use thereof

Abstract

The application provides a compound shown in formula I or a pharmaceutically acceptable salt thereof: wherein R4 is selected from a hydroxyl group, an O-beta-D Glc group, and an O-beta-D Glc-(2-1)-alpha-L-Rha group; and R8 is selected from a hydroxyl group, an O-beta-D Glc group, and an O-beta-D Glc-(2-1)-alpha-L-Rha group; and the application provides the use of the compound in the preparation of a blood sugar reducing drug and in the inhibition of alpha-glucosidase activity, and has the beneficial effect that the application provides the use of zeylasteroside D and zeylasteroside J in the preparation of a blood sugar reducing drug and in the inhibition of alpha-glucosidase activity, and discloses the compound structure of zeylasteroside L and the use of the zeylasteroside L in the preparation of a blood sugar reducing drug and in the inhibition of alpha-glucosidase activity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicines, and particularly relates to a zygophyllum saponin L and a preparation method and application thereof. BACKGROUND

[0002] Zygophyllum (Actinostemma lobatum Maxim) is an annual herbaceous plant of Cucurbitaceae Zygophyllum, which is first recorded in Bencao Shiyi. Zygophyllum grows on roadsides, mountain grasslands or water edges. Through medical research over the ages, zygophyllum is mainly used as a medicine with whole grass and seeds, and has the effects of clearing heat and resolving toxins, diuresis and detumescence, heat-clearing and dampness-removing. Zygophyllum is mainly used for treating nephritis edema, ascites edema, snakebite and initial malnutrition.

[0003] In the early 1980s, Japanese scholars Fujinobu Tadashi et al. took Cucurbitaceae plants as the research object, and isolated 19 kinds of saponin monomer compounds from the whole grass and seeds of various plants in the process of active ingredient screening, mainly being saponin compounds of tetracyclic triterpenes, including 6 kinds of dammarane type and 2 kinds of bakuchiol type zygophyllum saponins (actinostemmosides), which have similar structures to ginsenosides.

[0004] However, the zygophyllum saponin D and the zygophyllum saponin J disclosed in the existing literature do not indicate the application thereof in preparing hypoglycemic drugs and in inhibiting the activity of alpha-glucosidase, and no existing literature discloses the compound structure of the zygophyllum saponin L and the application thereof in preparing hypoglycemic drugs and in inhibiting the activity of alpha-glucosidase. SUMMARY

[0005] In view of the above problems,

[0006] In one aspect, the application provides a compound shown as formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008] In formula I, R4 is selected from a hydroxyl group, an O-beta-DGlc group and an O-beta-DGlc-(2-1)-alpha-L-Rha group, and R8 is selected from a hydroxyl group, an O-beta-DGlc group and an O-beta-DGlc-(2-1)-alpha-L-Rha group.

[0009] The compound shown as formula I is shown in Table 1, including zygophyllum saponin D and J.

[0010] Table 1. Zygophyllum saponin D, J.

[0011]

[0012] Further, the compound is shown as formula II.

[0013]

[0014] Another aspect of the present application provides a composition comprising two or more of the above-mentioned compounds or pharmaceutically acceptable salts thereof.

[0015] Another aspect of the present application provides an extract comprising two or more of the above-mentioned compounds or pharmaceutically acceptable salts thereof.

[0016] In another aspect, the present application also provides a preparation method of the compound or pharmaceutically acceptable salt thereof:

[0017] S1: Take 4.2 Kg of dried whole grass of Herba Eupatorium fortunei and immerse it in 70% ethanol solution for 2 days. Extract it twice with 10 times the amount of 70% ethanol solution, each time for 12 hours. Combine the two extraction solutions and concentrate them under reduced pressure using a rotary evaporator to obtain the ethanol extract solid extract. Dissolve the extract in a suitable amount of purified water and extract it three times with ethyl acetate (volume ratio of ethyl acetate to water is 1:1). Combine the water-soluble parts remaining after extraction and concentrate them under reduced pressure using a rotary evaporator to obtain 300 g of ethyl acetate extract extract and 785 g of water layer extract.

[0018] S2: Take the water-extracted extract and dissolve it in a suitable amount of methanol solution. Weigh about 200 g of ODS (120-50 mesh) and place it in an evaporating dish. Place the evaporating dish in a water bath and heat it while adding the sample solution to the ODS and stirring. Weigh ODS (120-50 mesh) (about 3 times the weight of the water fraction sample) and dry pack it into a column (8.5*65 cm). Add a certain amount of methanol reagent to the column and flush it several times to remove air bubbles. Add the stirred ODS to the chromatographic column and load it three times, and elute it with a methanol-water gradient system. The gradient system is methanol: water = 20:80, 60:40, 100% methanol. Concentrate the eluted fractions under reduced pressure and develop them by thin layer chromatography. Combine the eluted samples with similar spots and the same Rf value. Analyze and detect the eluted samples by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). Combine the eluted samples with the same absorption peak to obtain Fr.1, Fr.2 and Fr.3.

[0019] S3: Fr.2 was separated by Sephadex LH-20 gel column chromatography, eluted with dichloromethane:methanol = 1:1, and the sample was spotted on TLC plate to give four fractions, Fr.2.1, Fr.2.2, Fr.2.3 and Fr.2.4, respectively; Fr.2.2 was separated by semi-preparative HPLC gradient elution (C18, acetonitrile-water, 26:74-56:44, 30 min, 10 mL / min) to give three fractions, Fr.2.2.1, Fr.2.2.2 and Fr.2.2.3, respectively; Fr.2.2.2 was separated by semi-preparative HPLC (C18, acetonitrile-water, 28:72, 10 mL / min) to give six fractions, Fr.2.2.2.1, Fr.2.2.2.2, Fr.2.2.2.3, Fr.2.2.2.3 was separated by semi-preparative HPLC (C18, acetonitrile-water, 26:74, 10 mL / min) to give three fractions, Fr.2.2.2.3.1, Fr.2.2.2.3.2 and Fr.2.2.2.3.3, respectively; Fr.2.2.2.3.3 was separated by semi-preparative HPLC (C18, acetonitrile-water, 22:78, 10 mL / min) to give three fractions, Fr.2.2.2.3.3.1, Fr.2.2.2.3.3.2 and Fr.2.2.2.3.3.3, respectively; Fr.2.2.2.3.3.2 was separated by semi-preparative HPLC (C18, acetonitrile-water, 22:78, 10 mL / min), filtered, recrystallized to give saponin L (tR= 54.6 min, 23.8 mg).

[0020] In another aspect, the present application also provides the use of the compound of formula I or its pharmaceutically acceptable salt in the preparation of a hypoglycemic drug, preferably the hypoglycemic drug is an α-glucosidase inhibitor,

[0021]

[0022] wherein R4 is selected from hydroxyl, O-β-DGlc group, O-β-DGlc-(2-1)-α-L-Rha group, and R8 is selected from hydroxyl, O-β-DGlc group, O-β-DGlc-(2-1)-α-L-Rha group.

[0023] Further, the use of the compound of formula I or its pharmaceutically acceptable salt in the preparation of a hypoglycemic drug, wherein R4 is selected from O-β-DGlc-(2-1)-α-L-Rha group and R8 is selected from hydroxyl.

[0024] Further, the use of the compound of formula I or its pharmaceutically acceptable salt in the preparation of a hypoglycemic drug, R4 is selected from hydroxyl, and R8 is selected from O-β-DGlc-(2-1)-α-L-Rha group.

[0025] Further, the application of the compound shown in formula I or its pharmaceutically acceptable salt in preparing the medicine for reducing blood sugar, wherein R4 is selected from O-β-DGlc group, and R8 is selected from O-β-DGlc group.

[0026] The application has the advantages that the application provides the application of zizyphus jujuba cv. jujuba var. spinosa bunge saponin D and zizyphus jujuba cv. jujuba var. spinosa bunge saponin J in preparing the medicine for reducing blood sugar and the application in α-glucosidase inhibition activity, and discloses the compound structure of zizyphus jujuba cv. jujuba var. spinosa bunge saponin L and the application in preparing the medicine for reducing blood sugar and the application in α-glucosidase inhibition activity. DETAILED DESCRIPTION

[0027] The following examples further illustrate the present application but should not be construed as limiting the application. Modifications or variations of the method, steps or conditions of the application can be made by those skilled in the art without departing from the spirit and scope of the application.

[0028] Example 4: Water layer extract

[0029] Take 4.2 Kg of dried whole grass of zizyphus jujuba cv. jujuba var. spinosa bunge and immerse in 70% ethanol solution for 2 days, and extract twice with 10 times the amount of 70% ethanol solution, each time for 12 hours, combine the two extraction solutions, and then concentrate under reduced pressure by using a rotary evaporator to obtain the solid extract of the alcohol extract. Dissolve the extract in a suitable amount of purified water, and extract three times with ethyl acetate (the volume ratio of ethyl acetate to water is 1:1), combine the water-soluble parts remaining after extraction, and then concentrate under reduced pressure by using a rotary evaporator to obtain 300 g of ethyl acetate extract and 785 g of water layer extract.

[0030] Take the water layer extract

[0031] Example 1: Zizyphus jujuba cv. jujuba var. spinosa bunge saponin L

[0032] Take the water layer extract, and add a suitable amount of methanol solution to dissolve it. Take about 200 g of ODS (120-50 mesh) and place it in an evaporating dish, and then place the evaporating dish in a water bath and heat it, while adding the sample solution to the ODS and stirring the sample. Take ODS (120-50 mesh) (about 3 times the amount of the water part sample) and dry pack the column (8.5*65 cm), and then add a certain amount of methanol reagent to the column and flush the column several times to remove air bubbles. Add the stirred ODS to the chromatographic column, and then add the sample in three portions, and use the methanol-water gradient system to elute it in sequence. The gradient system is methanol: water = 20:80, 60:40, and 100% methanol. Concentrate the eluted fractions under reduced pressure, and then develop them by thin layer chromatography, and combine the eluted samples with similar spots and the same Rf value. Analyze and detect the eluted samples by using an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) instrument, and then combine the eluted samples with the same absorption peak to obtain the fractions Fr.1, Fr.2, and Fr.3.

[0033] Fr.2 was separated by Sephadex LH-20 gel column chromatography, eluted with dichloromethane:methanol = 1:1, and the sample spotted on thin layer chromatography (TLC) was divided into four parts, Fr.2.1, Fr.2.2, Fr.2.3 and Fr.2.4, respectively; Fr.2.2 was separated by semi-preparative HPLC gradient elution (C18, acetonitrile-water, 26:74-56:44, 30 min, 10 mL / min) to obtain three parts, Fr.2.2.1, Fr.2.2.2 and Fr.2.2.3, respectively; Fr.2.2.2 was separated by semi-preparative HPLC (C18, acetonitrile-water, 28:72, 10 mL / min) to obtain six parts, Fr.2.2.2.1, Fr.2.2.2.2, Fr.2.2.2.3, Fr.2.2.2.3 was separated by semi-preparative HPLC (C18, acetonitrile-water, 26:74, 10 mL / min) to obtain three parts, Fr.2.2.2.3.1, Fr.2.2.2.3.2 and Fr.2.2.2.3.3, respectively; Fr.2.2.2.3.3 was separated by semi-preparative HPLC (C18, acetonitrile-water, 22:78, 10 mL / min) to obtain three parts, Fr.2.2.2.3.3.1, Fr.2.2.2.3.3.2 and Fr.2.2.2.3.3.3, respectively; Fr.2.2.2.3.3.2 was separated by semi-preparative HPLC (C18, acetonitrile-water, 22:78, 10 mL / min), filtered, recrystallized to obtain saponin L (t R = 54.6 min, 23.8 mg).

[0034] Compound structure determination:

[0035] White powder solid. 10% H2SO4-CH3CH2OH solution showed purple red, L-B reaction showed positive, Molish reaction showed positive, according to the above reaction results, it was speculated to be a triterpenoid saponin. 10% H2SO4-CH3CH2OH solution hydrolysis result detected Glc. HR-ESI-MS gave: m / z 801.5010 [M+H] + molecular ion peak (C 42 H 73 O 14 + , the calculated value is 801.5000), combined with NMR data, it was speculated that the molecular formula was C 42 H 72 O 14 .

[0036] The 1H-NMR (400 MHz, C5D5N) spectrum of actinostemmoside L gave the following signals: δ 0.90 (3H, s), 0.95 (3H, s), 0.99 (3H, s), 1.35 (3H, s), 1.40 (3H, s), 1.89 (3H, s), 2.01 (3H, s), 5.42 (1H, t, J = 6.8 Hz), 5.12 (1H, d, J = 5.2 Hz), 4.98 (1H, d, J = 7.3 Hz). The 13C-NMR (100 MHz, C5D5N) spectrum of actinostemmoside L gave the following signals: δ 135.8 (C-24), 127.4 (C-25), 106.9, 98.3, 89.4 (C-3), 81.7, 78.8, 78.5, 78.1, 77.7, 75.6, 75.3 (C-20), 72.0, 71.6, 67.4 (C-6), 63.0, 62.8, 61.6, 60.7 (C-27), 50.4, 49.9, 47.8, 47.3, 41.9, 41.5, 40.3, 38.9, 38.7, 38.4, 31.25 (C-28), 30.9, 27.17, 26.4 (C-21), 24.9, 22.2, 21.8, 21.7, 21.5 (C-26), 17.3 (C-19), 17.2 (C-18), 16.8 (C-30), 16.4 (C-29).

[0037] By analyzing the hydrogen nuclear magnetic resonance spectrum data, it was found that the data of actinostemmoside J reported in the literature (Study on the Chemical Constituents of Actinostemma Succosum) by Liu Qin could be compared, and it was found that the parent nucleus data was basically the same,

[0038] Therefore, it can be inferred that the aglycone parent nucleus of actinostemmoside L is: 3β, 6α, 20, 27-tetrahydroxy-(20S)-dammar-24-ene, and according to the glycosylation displacement rule, it can be inferred that the sugar group is substituted at the 3 and 20 positions of the parent nucleus. Among them, the 10 oxygen-containing carbon signals: δ 81.7, 78.8, 78.5, 78.1, 77.7, 75.6, 72.0, 71.6, 63.0, 62.8 and 2 double oxygen-containing carbon signals: δ 106.9, 98.3 constitute 12 sugar group carbon signals. Combined with the sugar end group proton signal in the hydrogen nuclear magnetic resonance spectrum and the hydrolysis result in 10% H2SO4-CH3CH2OH solution, it can be proved that there are 2 Glc in the compound.

[0039] In the heteronuclear multiple carbon correlation spectrum of 1H, the signal δ5.12 (1H, d, J=5.2 Hz, Glc H-1) in the end group proton signal of glucose and the signal δ75.32 (C-20) on the 20th position of the dammarane-type parent nucleus structure had a remote correlation, proving that glucose was connected to the 20th position of the dammarane-type parent nucleus; the signal δ4.98 (1H, d, J=7.3 Hz, Glc H-1) in the end group proton signal of glucose and the signal δ89.36 (C-3) on the 3rd position of the dammarane-type parent nucleus structure had a remote correlation, proving that glucose was connected to the 3rd position of the dammarane-type parent nucleus.

[0040] The absolute configuration of the sugar can be determined by gas chromatography analysis. The compound is subjected to acid hydrolysis and derivatization, respectively, and its gas chromatography is compared with the chromatography data of standard sugar derivatives. Research proves that the sugar therein is D-glucose. The chemical shift value and the coupling constant of the end group hydrogen are combined and compared, and the structure is identified as β-D-glucose.

[0041] In summary, the structure of the separated compound 2 is identified as zygophyllin L, which is a new compound not reported in the literature.

[0042] Comparative Example 1: Using acarbose as a positive control

[0043] Test Example 1

[0044] α-Glucosidase inhibitory activity experiment

[0045] α-Glucosidase widely exists in animals, plants and microorganisms, and is concentrated on the small intestinal brush border membrane epithelial cells in the human body, and is a key enzyme for hydrolyzing carbohydrates. It can hydrolyze the α-1,4-glucoside bond of oligosaccharides and polysaccharides from the non-reducing end, thereby releasing glucose, which is then absorbed into the blood through the small intestine, resulting in an increase in blood glucose. Therefore, inhibiting the activity of α-glucosidase can slow down the absorption of glucose by the human body and inhibit hyperglycemia.

[0046] In this experiment, p-nitrophenol-α-D-glucoside (PNPG) is selected as the reaction substrate to screen the compounds with α-glucosidase inhibitory activity. PNPG can be hydrolyzed into glucose and p-nitrophenol in the presence of α-glucosidase, and p-nitrophenol has a maximum absorbance value at 405 nm. When there is a substance that inhibits the activity of α-glucosidase in the reaction system, the p-nitrophenol produced by the hydrolysis of PNPG will decrease, and the absorbance value will correspondingly decrease.

[0047] Table 2 Main experimental materials and reagents

[0048]

[0049] Main instruments and equipment

[0050]

[0051] Solution preparation:

[0052] (1) 0.1M hydrochloric acid solution: take 9mL concentrated hydrochloric acid and add water to 1L;

[0053] (2) 0.1M PBS buffer solution: accurately weigh 3.5510g of anhydrous Na2HPO4 and 3.9019g of anhydrous NaH2PO4 into two beakers respectively, add 250mL of distilled water respectively to obtain 0.1M Na2HPO4 solution and 0.1M NaH2PO4 solution respectively, then mix them according to the volume ratio (61:39), and adjust the pH to 7 using 0.1M hydrochloric acid solution to obtain 0.1M PBS buffer solution for standby;

[0054] (3) Substrate PNPG solution: accurately weigh 7.5mg of PNPG solid powder into a 10mL volumetric flask, add 0.1M PBS buffer solution to make up the volume to obtain a 2.5mM PNPG solution, which is prepared and used immediately;

[0055] (4) α-glucosidase solution: take freeze-dried enzyme powder (enzyme activity is 69.6U / mg), first prepare 10U / mL α-glucosidase stock solution using 0.1M PBS buffer solution, then dilute it to 0.5U / mL α-glucosidase solution using the buffer solution, which is prepared and used immediately.

[0056] (5) Stop solution: accurately weigh 2.1201g of anhydrous Na2CO3 solid into a 100mL volumetric flask, add distilled water to make up the volume to obtain a 0.2M Na2CO3 solution;

[0057] (6) Example 1 solution: accurately weigh 1.0mg of zygophyllin L compound, dissolve it in 1mL DMSO, shake well to obtain a stock solution for standby;

[0058] (7) Example 2-4 solution: refer to the stock solution of Example 1 for standby;

[0059] (8) Control Example 1 solution: accurately weigh 1.0mg of acarbose solid, dissolve it in 1mL DMSO, shake well to obtain a stock solution for standby;

[0060] Experimental method:

[0061] The experiment sets 4 groups, carries out the reaction in 96-hole cell culture plate, according to the data in table 3, accurately removes PBS buffer solution, α-glucosidase solution, sample solvent (DMSO) and different concentrations of positive control solution (or sample solution) first, mixes well, incubates at 37℃ for 10 min, then adds the substrate (PNPG solution), mixes well, incubates at 37℃ for 20 min, finally, adds the stop solution to stop the reaction. The absorbance value is measured at 405 nm wavelength by using the enzyme marker instrument. According to the α-glucosidase inhibition rate formula (3-2), the data obtained is processed by using Microsoft Excel 2016, and the IC 50 value is calculated.

[0062] Table 4: The amount of reactants added (μL)

[0063]

[0064] It should be noted that the sample solution in table 4 includes examples 1-4, and the positive control solution includes control examples 1-3.

[0065] The α-glucosidase inhibition rate calculation formula is:

[0066] Inhibition rate (%) = [(A k -A kb )-(A s -A sb )] / (A k -A kb )*100%

[0067] A k : Blank group absorbance; A kb : Blank background group absorbance; A s : Experimental group (control group) absorbance; A sb : Experimental background group absorbance.

[0068] Test 1: Take the experimental group Jujuba saponin L mother liquor, dilute 1000 times first using DMSO, then dilute to get six concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 μg / mL using the double dilution method. According to the above experimental method, the inhibition rate corresponding to each concentration is calculated, and the Jujuba saponin L concentration is taken as the X axis and the α-glucosidase inhibition rate as the Y axis to draw the inhibition curve, and the half maximal inhibitory concentration IC 50 of test 1 is calculated as 0.36 μg / mL.

[0069] Test 2: Different from test 1, Jujuba saponin D mother liquor is taken.

[0070] Test 3: Different from test 1, Jujuba saponin J mother liquor is taken.

[0071] Test 4: Take the solution of Example 4, first dilute 1000 times with DMSO, then dilute to six concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 μg / mL by using the double dilution method. Calculate the inhibition rate corresponding to each concentration according to the above experimental method, and calculate the half-inhibitory concentration IC50 of the positive control 50 0.17 μg / mL.

[0072] Test 7: Take the solution of Control Example 1 (acarbose mother liquor), first dilute 1000 times with DMSO, then dilute to six concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 μg / mL by using the double dilution method. Calculate the inhibition rate corresponding to each concentration according to the above experimental method, and calculate the half-inhibitory concentration IC50 of the positive control 50 0.17 μg.

[0073] Table 5 Inhibition rate and IC50 of the compounds on α-glucosidase 50

[0074]

[0075] As shown in Table 5, Examples 1-4 all have inhibitory effect on α-glucosidase, and the inhibition rate of Example 1 on α-glucosidase is the highest. The inhibition rate of the zygophyllin of Example 1, Example 2 and Example 3 on α-glucosidase is higher than that of other examples, which indicates that the structure-activity relationship of R4, R8 substituents and the parent ring affects the inhibition rate of α-glucosidase. Although the inhibition rate of acarbose of Control 1 on α-glucosidase is not much different from that of the present application, the present application provides a new compound and its application in reducing blood sugar.

[0076] Although the present application has been described in detail in the foregoing general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application.

Claims

1. Use of a compound of formula II or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for lowering blood glucose.

2. Use according to claim 1 wherein the medicament for lowering blood glucose is an α-glucosidase inhibitor.

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