A lignan compound containing a tannin structure isolated from Phyllanthus emblica and its hepatoprotective uses.

Abstract

This invention discloses lignan compounds containing tannin structures, as shown in Formula I, isolated from Phyllanthus emblica. The inventors used an automated biochemical analyzer and the IFCC method to determine the AST and ALT activities in the serum of mice with a CCl4-induced liver injury model, finding that both AST and ALT were significantly reduced, indicating that the compound possesses hepatoprotective activity. This invention also discloses the use of the aforementioned lignan compounds containing tannin structures in the preparation of hepatoprotective drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel lignan compound containing a tannin structure isolated from Phyllanthus emblica, as well as the preparation method and hepatoprotective uses of the compound. Background Technology

[0002] Phyllanthus emblica L. is the dried, ripe fruit of the deciduous shrub or small tree of the genus Phyllanthus in the family Euphorbiaceae. It is also known as Amla, Yunnan olive, and oil-licorice. As a medicinal and edible herb, Phyllanthus emblica has a history of over two thousand years in my country. Studies have shown that it contains a rich variety of chemical components, including phenolic acids, tannins, flavonoids, lignans, sterols, triterpenes, and alkaloids. Modern pharmacological research has found that Phyllanthus emblica has good hepatoprotective, antioxidant, antitumor, anti-inflammatory, anti-aging, hypoglycemic, hypolipidemic, and immunomodulatory effects. Since 1977, Phyllanthus emblica has been included in various editions of the Chinese Pharmacopoeia as a traditional Tibetan medicine. It has a sweet, sour, and astringent taste, and is cool in nature, possessing the effects of soothing the liver and gallbladder, clearing heat and cooling the blood, promoting digestion and strengthening the stomach, and relieving cough and saliva production. Tibetan medicine often uses Phyllanthus emblica to treat "blood diseases," "Tripa diseases," and "Peigen diseases," which share many similarities with the clinical manifestations of blood heat and blood stasis in traditional Chinese medicine and liver and gallbladder diseases in modern medicine. Therefore, it is necessary to further explore the potential medicinal value of Phyllanthus emblica. Summary of the Invention

[0003] The inventors selected Phyllanthus emblica, a plant used for both medicinal and edible purposes, as the research object. They employed a bioactivity-guided separation method to isolate and purify the hepatoprotective active fraction of Phyllanthus emblica, obtaining a novel lignan compound containing a tannin structure. Simultaneously, the inventors used an automated biochemical analyzer and the IFCC method to measure the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of mice with a CCl4 liver injury model. They found that both AST and ALT were significantly reduced, indicating that the new compound possesses hepatoprotective activity. The purpose of this invention is to provide a novel lignan compound containing a tannin structure isolated from Phyllanthus emblica, as well as its preparation method and hepatoprotective activity.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A novel lignan compound containing a tannin structure, with the structure shown in Formula I, isolated from Phyllanthus emblica, for medicinal use:

[0006]

[0007] Compound I: English name: 1-O-herpetol-3,6-digalloyl-O-β-D-glucoside; Chinese name: 1-O-herpetol-3,6-digalloyl-O-β-D-glucoside; Molecular formula: C 40 H 38 O 19 .

[0008] A method for preparing the lignan compound containing a tannin structure, comprising the following steps:

[0009] Step (1): Crush the dried amla fruit, soak it in 90-100% ethanol for 20-24 hours, heat and reflux to extract 3-5 times, 4-6 hours each time, combine the filtrates to obtain the total extract of amla fruit;

[0010] Step (2): The total extract of Phyllanthus emblica is concentrated under vacuum to obtain a total viscous paste of Phyllanthus emblica;

[0011] Step (3): The total viscous extract of Phyllanthus emblica is uniformly suspended in distilled water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether extract, ethyl acetate extract and n-butanol extract, respectively.

[0012] Step (4): The macroporous adsorption resin column of the n-butanol extraction section is eluted sequentially with 10% ethanol → 25% ethanol → 45% ethanol → 70% ethanol → 100% ethanol to obtain 5 main fractions: main fraction A, main fraction B, main fraction C, main fraction D, and main fraction E.

[0013] Step (5): Separate the main fraction B using a medium-pressure chromatography column to obtain three sub-fractions: sub-fraction B-1, sub-fraction B-2, and sub-fraction B-3.

[0014] Step (6): Load sub-fraction B-2 onto a Toyopearl HW-40C column and elute with dichloromethane-methanol volume ratio of 1:2 to obtain sub-fraction B-2-1 and sub-fraction B-2-2;

[0015] Step (7): Load sub-fraction B-2-2 onto a Sephadex LH-20 column and elute with 70-90% methanol to obtain fine fractions B-2-2-1 and B-2-2-2.

[0016] Step (8): The secondary fraction B-2-2-1 was separated and purified by preparative liquid chromatography to obtain compound I.

[0017] In step (1), the dried amla seeds are pulverized to 80-100 mesh.

[0018] In step (2), the total extract of Phyllanthus emblica is concentrated under vacuum at a temperature of 50-60℃ to obtain a total viscous paste of Phyllanthus emblica.

[0019] In step (3), the total viscous extract of Phyllanthus emblica is uniformly suspended in distilled water according to the volume ratio of Phyllanthus emblica to distilled water = 1:1.5.

[0020] The activity of different extraction fractions was screened using the IFCC method, and the n-butanol extraction fraction was identified as the hepatoprotective active fraction.

[0021] In step (4), the macroporous adsorption resin is AB-8 type macroporous adsorption resin.

[0022] The hepatoprotective activity of five main fractions was screened using the IFCC method, and the results showed that main fraction B had significant hepatoprotective activity.

[0023] In step (5), the medium-pressure chromatography column is a YMC-Pack ODS-A column (250×50mm, 5μm); gradient elution is performed using 15% methanol, 25% methanol and 35% methanol as the mobile phase, with an absorption wavelength of 205-230nm and a flow rate of 15-20mL / min.

[0024] In step (8), the chromatographic column for preparing the liquid chromatography is a YMC-Pack ODS-A column (250×20mm, 5μm); the mobile phase is 10-25% methanol, the absorption wavelength is 200-220nm, and the flow rate is 2-4mL / min.

[0025] The inventors used a fully automated biochemical analyzer and the IFCC method to determine the activities of ALT and AST in serum, and screened and evaluated the hepatoprotective activity of the new compound I. The results showed that the compound has significant hepatoprotective activity.

[0026] This invention provides the use of the lignan compounds containing tannin structures in the preparation of hepatoprotective drugs.

[0027] This invention provides the use of the lignan compounds containing tannin structures in the preparation of drugs for treating liver injury.

[0028] The present invention also provides a pharmaceutical composition with hepatoprotective activity, wherein the pharmaceutical composition comprises the lignan compound containing a tannin structure as the active ingredient.

[0029] The beneficial effects of this invention are:

[0030] This invention discloses a novel lignan compound containing a tannin structure, isolated from Phyllanthus emblica, a plant resource that is abundant and readily available. The isolation method for this compound is simple and easy to operate, and the compound exhibits a novel and unique structure with hepatoprotective activity, making it suitable for formulation into a hepatoprotective drug. Attached Figure Description

[0031] Figure 1 This is a flowchart of the preparation process of compound I. Detailed Implementation

[0032] The following specific embodiments further illustrate the substantive content of the present invention, but do not limit the scope of protection of the present invention.

[0033] Example 1

[0034] The preparation method of compound I is as follows: Figure 1 As shown, the steps are as follows:

[0035] Step (1): Crush 8.5 kg of dried amla into 90 mesh, soak in 90% ethanol for 22 hours, and heat and reflux to extract 4 times, 5 hours each time. Combine the filtrates to obtain the total extract of amla.

[0036] Step (2): The total extract of Phyllanthus emblica was concentrated under vacuum at 55°C to obtain a total thick paste of Phyllanthus emblica (1.1 kg);

[0037] Step (3): The total viscous extract of Phyllanthus emblica was uniformly suspended in distilled water (total viscous extract of Phyllanthus emblica and distilled water = 1:1.5, V / V), and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract (98.6 g), ethyl acetate extract (207.8 g) and n-butanol extract (554.6 g) respectively.

[0038] Step (4): The different extract fractions obtained in step (3) were screened for activity using the IFCC method to determine the n-butanol extract fraction as the hepatoprotective active fraction.

[0039] Step (5): The hepatoprotective active fraction obtained in step (4) was loaded onto an AB-8 macroporous adsorption resin column and eluted sequentially with 10% ethanol → 25% ethanol → 45% ethanol → 70% ethanol → 100% ethanol to obtain 5 main fractions: A (75.6 g), B (98.0 g), C (60.2 g), D (45.1 g), and E (36.7 g).

[0040] Step (6): The five main fractions obtained in step (5) were screened for hepatoprotective activity using the IFCC method. The results showed that main fraction B had significant hepatoprotective activity. Main fraction B was subjected to medium-pressure chromatography on a YMC-Pack ODS-A column (column length 250 × inner diameter 50 mm, particle size 5 μm). Gradient elution was performed using 15% methanol, 25% methanol, and 35% methanol as the mobile phase. The absorption wavelength was 205-230 nm, and the flow rate was 15-20 mL / min. Three secondary fractions were obtained: B-1 (35.7 g), B-2 (26.8 g), and B-3 (11.3 g).

[0041] Step (7): Load sub-fraction B-2 onto a Toyopearl HW-40C column and elute with dichloromethane-methanol = 1:2 (V / V) to obtain sub-fractions B-2-1 (9.6 g) and B-2-2 (13.5 g);

[0042] Step (8): Load sub-fraction B-2-2 onto a Sephadex LH-20 column and elute with 85% methanol to obtain fine fractions B-2-2-1 (4.3 g) and B-2-2-2 (3.1 g);

[0043] Step (9): The fine fraction B-2-2-1 was purified by preparative liquid chromatography using a YMC-Pack ODS-A column (column length 250 × inner diameter 20 mm, particle size 5 μm), mobile phase: 20% methanol, absorption wavelength: 210 nm, flow rate: 3 ml / min. After multiple preparations and purifications, the new compound I (12.25 mg) was finally obtained.

[0044] Compound I is a white solid, HR-ESI-MS m / z 844.1827 [M+Na] + Its molecular formula is C 40 H 38 O 19 (calcd.for C 40 H 37 O 19 Na, 844.1823). IRν of compound I max 3291, 1701, 1618, 1378cm -1 ,UV(MeOH)λ max :205, 210, 306, 320 nm. In compound I 1 H NMR and 13 In the C NMR spectrum, there are two typical galloyl group signals: δ H 7.08 (2H, s, H-2”' / 6”') and δc 122.5 (C-1”'), 109.1 (C-2”' / 6”'), 166.2 (C-7”'), 146.0 (C-3”' / 5”'), 138.9 (C-4”'). Based on the correlation between H-3 and C-7”' in the HMBC spectrum, the galloyl group is determined to be attached to the C-3 position of glucose. δ H7.04 (2H, s, H-2”” / 6””) and δc 122.1 (C-1””), 108.8 (C-2”” / 6””), 166.0 (C-7””), 145.0 (C-3”” / 5””), 138.7 (C-4””), based on the correlation between H-6 and C-7”” in the HMBC spectrum, it is determined that the galloyl group is linked to the C-6 position of glucose. Additionally, a typical ABX system signal: δ H 6.53 (1H,d,J=2.0Hz,H-2'), 6.50 (1H,d,J=8.0,2.0Hz,H-6'), 7.30 (1H,d,J=8.0Hz,H-5') and δc 159.7 (C-1'), 100.3 (C-2'), 160.3 (C-3'), 110.9 (C-4'), 127.5 (C-5'), 107.1 (C-6') are shown in compound I. 1 H NMR and 13 In the 1 / 2C NMR spectrum, the correlation between H-1 and C-1' in the HMBC spectrum indicates that the ABX system is linked at the C-1' position. Further analysis revealed a fragment signal of 1'-allyl-8'-hydroxymethyl-3”-methoxy-phenylpropanuran appearing in... 1 HNMR and 13 In the C NMR spectrum: δ H and δc 132.1 (C-1”), 105.3 (C-2”), 144.7 (C-3”), 148.7 (C-4”), 125.3 (C-5”), 108.9 (C-6”), 130.2 (C-7”), 127.5 (C-8”), 64.3 (C-9”), 151.1 (C-7’), 115.2 (C-8’), 50.0 (C-9’). Based on the correlation between H-5 and C-7’ in the HMBC spectrum, this fragment is attached at the C-4’ position. This compound… 1 HNMR (DMSO-d6, 600MHz) and 13 The C10 NMR (DMSO-d6, 150MHz) data are shown in Table 1. Based on the spectral data of compound I and HMBC, 1 H- 1 H COSY coupling information, through SciFinder search, compound I is a new lignan compound containing a tannin structure.

[0045]

[0046] Table 1. Compound I 1 H NMR, 13 C NMR and HMBC related data

[0047]

[0048]

[0049] Example 2

[0050] Hepatoprotective activity assay of compound I

[0051] 1.1 Laboratory animals, reagents and instruments

[0052] SPF-grade male Kunming mice; carbon tetrachloride, silymarin, edible peanut oil, aspartate aminotransferase (AST), alanine aminotransferase (ALT), CS-600B fully automated biochemical analyzer, HC-3618R high-speed low-temperature refrigerated centrifuge, ZT-14V2 biological tissue dehydrator, BX51 microscope, etc.

[0053] 1.2 Modeling and Drug Administration

[0054] SPF-grade male Kunming mice were randomly divided into 6 groups: model group, control group, positive control group, high-dose group of compound I, medium-dose group of compound I, and low-dose group of compound I. The model group and control group were administered 0.5% sodium carboxymethyl cellulose solution (10 mL / kg) by gavage daily; the positive control group was administered 0.0842 g / kg of silymarin (0.5% sodium carboxymethyl cellulose solution) by gavage; the high-, medium-, and low-dose groups were administered 2.38 g / kg, 1.19 g / kg, and 0.595 g / kg of compound I (0.5% sodium carboxymethyl cellulose solution) by gavage, respectively. Each group was administered the drug for 15 consecutive days. One hour after the last administration, except for the control group, all mice in the other groups were injected intraperitoneally with a 0.15% CCl4 peanut oil solution (dose 10 mL / kg). They were then fasted but allowed to drink water. After 16 hours, the eyeballs were removed, blood was collected from the vein, and the serum ALT and AST activities were measured by centrifugation at 5000 rpm for 15 min using an automated biochemical analyzer and the IFCC method.

[0055] 1.3 Effects of Compound I on ALT and AST Activities in Mouse Serum

[0056] The experimental results are shown in Table 2. The serum ALT and AST activities in the model group mice were significantly higher than those in the control group (P<0.01), indicating the successful establishment of the CCl4-induced liver injury mouse model. Compared with the model group, the serum ALT and AST activities in the positive control group (silymarin) and the three dosage groups of Compound I were all decreased; and compared with the model group, the serum ALT and AST activities in the three dosage groups of Compound I were all significantly decreased (P<0.05), indicating that the new Compound I has a certain protective effect against liver injury in mice. Compound I exhibits significant hepatoprotective activity, which has important scientific value for the development of novel hepatoprotective drugs from Phyllanthus emblica.

[0057] Table 2. Effects of compound I on serum AST and ALT in mice with CCl4-induced liver injury

[0058] Group Number of animals (individuals) Dosage (g / kg) AST(U / L) AST(U / L) Model group 12 - 241.84±35.46 230.54±41.57 control group 12 - 114.26±23.89** 89.53±16.52** Positive drug group 12 0.0842 202.05±56.17* 170.32±70.36* High-dose group 12 2.38 194.28±61.24** 160±50.52** medium dose group 12 1.19 197.54±42.43* 163.57±61.19* low-dose group 12 0.595 199.76±53.14* 166.37±56.27*

Claims

1. Lignans containing tannin structures, as shown in Formula I:

2. The method for preparing the lignan compound containing a tannin structure according to claim 1, characterized in that: Includes the following steps: Step (1): Crush the dried amla fruit, soak it in 90-100% ethanol for 20-24 hours, heat and reflux to extract 3-5 times, 4-6 hours each time, combine the filtrates to obtain the total extract of amla fruit; Step (2): The total extract of Phyllanthus emblica is concentrated under vacuum to obtain a total viscous paste of Phyllanthus emblica; Step (3): The total viscous extract of Phyllanthus emblica is uniformly suspended in distilled water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the petroleum ether extract, ethyl acetate extract and n-butanol extract, respectively. Step (4): The macroporous adsorption resin column of the n-butanol extraction section is eluted sequentially with 10% ethanol → 25% ethanol → 45% ethanol → 70% ethanol → 100% ethanol to obtain 5 main fractions: main fraction A, main fraction B, main fraction C, main fraction D, and main fraction E. Step (5): Separate the main fraction B using a medium-pressure chromatography column to obtain three sub-fractions: sub-fraction B-1, sub-fraction B-2, and sub-fraction B-3; the medium-pressure chromatography column is a YMC-Pack ODS-A column: 250×50mm, 5μm; gradient elution is performed using 15% methanol, 25% methanol, and 35% methanol as the mobile phase, with an absorption wavelength of 205-230nm and a flow rate of 15-20mL / min; Step (6): Load sub-fraction B-2 onto a Toyopearl HW-40C column and elute with dichloromethane-methanol volume ratio of 1:2 to obtain sub-fraction B-2-1 and sub-fraction B-2-2; Step (7): Load sub-fraction B-2-2 onto a Sephadex LH-20 column and elute with 70-90% methanol to obtain fine fractions B-2-2-1 and B-2-2-2. Step (8): The secondary fraction B-2-2-1 was separated and purified by preparative liquid chromatography to obtain compound I; the chromatographic column for preparative liquid chromatography was a YMC-Pack ODS-A column: 250×20mm, 5μm; Mobile phase: 10-25% methanol, absorption wavelength: 200-220nm, flow rate: 2-4mL / min.

3. The method for preparing lignan compounds containing tannin structures according to claim 2, characterized in that: In step (1), the dried amla seeds are pulverized to 80-100 mesh.

4. The method for preparing lignan compounds containing tannin structures according to claim 2, characterized in that: In step (4), the macroporous adsorption resin is AB-8 type macroporous adsorption resin.

5. Use of the lignan compound containing a tannin structure as described in claim 1 in the preparation of hepatoprotective drugs.

6. Use of the lignan compound containing a tannin structure as described in claim 1 in the preparation of a drug for treating liver injury.

7. A pharmaceutical composition with hepatoprotective activity, characterized in that: The pharmaceutical composition described herein uses the lignan compound containing a tannin structure as the active ingredient as described in claim 1.

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