Indole compounds and preparation methods and uses thereof

Abstract

The present invention belongs to the field of medicine and health care products, and specifically relates to a new indole compound and its preparation method and use. The compound of the present invention is extracted and separated from Hibiscus mutabilis for the first time, and its structure is shown in Formula I, and its chemical formula is C 23 H 29 NO 12 , chemically named 2-O-[β-D-glucopyranosyl (1→6)]-[α-L-rhamnopyranosyl (1→6)]-3-indoleacrylic acid. The compound of the present invention has multi-purpose application prospects such as anti-inflammatory. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the field of medicine and health care products, and specifically relates to a new indole compound and a preparation method and use thereof. Background Art

[0002] Hibiscus mutabilis.L is a plant of the genus Hibiscus in the Malvaceae family. Both its flowers and leaves can be used as medicine. The record of its medicinal value began in the "Compendium of Materia Medica" of the Song Dynasty, "Hibiscus mutabilis grows in Dingzhou. It tastes spicy, flat and non-toxic. The flowers are used to treat malignant sores, and the leaves are used to apply swelling and pain. They are harvested in September." Hibiscus mutabilis is also known as birch, frost-rejecting, Xia Pai Bei (Zhuang language), and Ben Niang Zha (Miao name). It is planted in Sichuan, Zhejiang, Fujian, Yunnan, Taiwan and other places. Overseas planting resources are mostly distributed in South Korea, Japan, Nepal, Laos, Thailand and other places. After more than ten years of research, Chengdu Botanical Garden has successfully researched and cultivated 18 varieties of Hibiscus mutabilis. Hibiscus mutabilis leaves are included in the 2020 edition of the "Chinese Pharmacopoeia", and its flowers are included in the 2019 edition of the "Guangdong Provincial Traditional Chinese Medicine Standard Volume 3". It is pungent in taste and neutral in nature. It has the effects of detoxification, swelling, heat-clearing, cooling blood and pus discharge. It can be used externally to treat carbuncle, furuncle, back ulcer, burns, mastitis, traumatic injury, etc. It can be taken internally to treat lung heat cough, menstruation, excessive leucorrhea, etc. "Compendium of Materia Medica" records: "Hibiscus mutabilis flowers and leaves are neutral in nature, neither cold nor hot, slightly pungent in taste, slippery and sticky in nature, and its effect in treating carbuncle is very effective". Modern pharmacological research shows that it not only has anti-inflammatory, analgesic, antioxidant, anti-allergic, and blood sugar-lowering effects, but is also widely used in cosmetics and food raw materials or additives.

[0003] In terms of its main medicinal value, Hibiscus mutabilis has the effects of clearing away heat and detoxifying, reducing swelling and draining pus, cooling blood and stopping bleeding. It is used for lung heat cough, menorrhagia, and leucorrhea; it is used externally to treat carbuncle, sore, furuncle, mastitis, lymphadenitis, mumps, burns, scalds, poisonous snake bites, and traumatic injuries. Usage and dosage: 0.3-1 liang; for external use, mash fresh leaves and flowers and apply them to the affected area or grind dry leaves and flowers into powder and apply them with oil, vaseline, wine, vinegar or strong tea. The prescriptions reported are: 1. For vomiting blood, uterine bleeding, fire eyes, sores, and lung abscesses: 3 to 1 liang of hibiscus flowers, decocted and taken. (Common Chinese Herbal Medicines in Shanghai); 2. For carbuncle and swelling: Hibiscus mutabilis flowers, leaves, and moutan bark. Decoction and wash. (Hunan Pharmacy); 3. For snake head furuncle and snake venom: 2 liang of fresh Hibiscus mutabilis flowers and 5 qian of winter honey. Mash and apply, change two to three times a day. (Fujian "Practical Folk Herbs"); 4. Treat water burns: dry hibiscus flowers, grind them into powder, and apply with sesame oil. ("Hunan Pharmacy"); 5. Treat moxibustion sores that do not heal: grind hibiscus flowers into powder and apply. ("Wonderful Prescriptions"); 6. Treat consumptive cough: 2 to 4 liang of hibiscus flowers, 1 liang of deer bite grass, 2 liang of brown sugar, stew pig heart and lungs and take; if there is no sugar, salt can also be added. ("Chongqing Herbs"); 7. Treat continuous menstrual bleeding: divide frost-resistant flowers and lotus shells into equal parts. Grind them into powder, and take 2 qian each time with rice. ("Women's Prescriptions").

[0004] The chemical components of Hibiscus mutabilis are mainly flavonoids and their glycosides, organic acids and volatile components, among which flavonoids and their glycosides are the characteristic components and effective components of Hibiscus mutabilis. So far, 73 flavonoid compounds have been separated and identified from Hibiscus mutabilis, including 48 flavonoid components separated and identified from Hibiscus mutabilis leaves and 36 flavonoid components separated and identified from Hibiscus mutabilis flowers, mainly flavonoid glycoside compounds with kaempferol, quercetin, apigenin, etc. as the parent nucleus. In this application background, the inventor of the present invention discovered a new compound in the study of Hibiscus mutabilis. Summary of the invention

[0005] The first technical problem solved by the present invention is to provide a new compound having a structure as shown in Formula I and a chemical formula of C 23 H 29 NO 12 .

[0006]

[0007] The chemical name is 2-O-[β-D-glucopyranosyl (1→6)]-[α-L-rhamnopyranosyl (1→6)]-3-indoleacrylic acid, which belongs to indole compounds. The NMR data (600 MHz, CD3OH, J inHz) of the compound of formula I of the present invention are shown in Table 1.

[0008] Table 1

[0009]

[0010] a) The measurement frequency is 600MHz; b) The measurement frequency is 150MHz.

[0011] The physicochemical properties of the compound described by formula I of the present invention are: yellow-brown amorphous crystals, easily soluble in methanol.

[0012] Spectral data of the compound described in formula I of the present invention: 86.00 (c=0.05, MeOH); IR (KBr)v max 3442,2949,1746,1647;UVλ max 228nm; HR-ESI-MS: m / z 512.17487; 1H NMR (600MHz, Methanol-d4) δ8.40(1H,s,H-2),7.65(1H,d,J=7.7Hz,H-4),7.57(1H,s,H-10),7.35(1H,d,J=7.9Hz,H-7),7.12(1H,ddd,J=8.0,7. 1,1.3Hz,H-6),7.09(1H,ddd,J=8.2,7.0,1.2Hz,H-5),4.92(1H,d,J=7.8Hz,H-1'),4.61(1H,d,J=1.8Hz,H-1”),3.79(1H,dd,J=11.1,1.9Hz,H-6 'a),3.77(1H,dd,J=3.5,1.8Hz,H-3”),3.70(1H,dd,J=9.4,3.4Hz,H-2” ),3.54–3.57(1H,m,H-5”),3.51–3.54(1H,m,H-6’b),3.48–3.51(1H,m,H -2'),3.47(1H,d,J=9.3Hz,H-4'),3.43(1H,d,J=9.0Hz,H-3'),3.38(1H, d,J=9.5Hz,H-4”),3.32–3.36(1H,m,H-5’),1.06(3H,d,J=6.2Hz,H-6”); 13 C NMR(151MHz,MeOD)δ130.7(C-2),109.5(C-3),118.7(C-4),121.4(C-5),123.2(C -6),112.6(C-7),137.2(C-8),128.8(C-9),120.7(C-10),139.4(C-11),169.2(C- 12),105.2(C-1'),75.9(C-2'),78.3(C-3'),70.7(C-4'),77.0(C-5'),67.4(C-6' ),102.2(C-1”),72.5(C-2”),72.1(C-3”),74.2(C-4”),69.4(C-5”),17.9(C-6”).

[0013] Optical rotation of the compound of formula I of the present invention 86.00 (c 0.05, methanol), hydroxyl groups (3442 cm -1 ), carbonyl (1746cm -1 ). The UV spectrum shows that the compound has a maximum absorption at 228nm and a strong absorption at 330nm. The HR-ESI-MS quasi-molecular ion peak is at m / z 512.17487[M+H] +appears at (calculated value is 512.17625), unsaturation Ω = 10, and the molecular formula is determined to be C 23 H 29 NO 12 .

[0014] The compounds of formula I of the present invention 1 H NMR and COSY spectra (see Table 1) show a 3-substituted indole ring (table 1-1), δ H 7.65 (1H, d, J = 7.7 Hz), 7.35 (1H, d, J = 7.9 Hz), 7.12 (1H, ddd, J = 8.0, 7.1, 1.3 Hz), 7.09 (1H, ddd, J = 8.2, 7.0, 1.2 Hz) are proton signals in the aromatic region, 8.40 (s, 1H) is a proton signal on the nitrogen-containing pyrrole ring; a methine proton signal, 7.57 (1H, s); 7 proton signals of glucose, 4.92 (1H, d, J = 7.8 Hz, H-1'), 3.48-3.51 (1H, m, H-2'), 3.43 (1H, d, J = 9.0 Hz, H-3'), 3.47 (1H, d, J = 9.3 Hz, H-4'), 3.32-3.36 (1H, m, H-5'), 3.79 (1H, dd, J = 11.1, 1.9 Hz, H-6'), 3.51-3.54 (1H, m, H-6'); 6 proton signals of one rhamnose, 4.61 (1H, d, J = 1.8 Hz, H-1"), 3.70 (1H, dd, J = 9.4, 3.4 Hz, H-2"), 3.77 (1H, dd, J = 3.5, 1.8 Hz, H-3"), 3.38 (1H, d, J = 9.5 Hz, H-4"), 3.54-3.57 (1H, m, H-5"), 1.06 (3H, d, J = 6.2 Hz, H-6"). According to 13 C NMR data and HSQC spectrum show that one sugar signal δ C 169.2, 2 methine carbon signals δ C 130.7, 120.7, 6 aromatic carbon signals 118.7, 121.4, 123.2, 112.6, 137.2, 128.8; 6 carbon signals of glucose δ C 105.2, 75.9, 78.3, 70.7, 77.0, 67.4, and by δ H The proton signal coupling constant of 4.92 (1H, d, J = 7.8 Hz) can be used to analyze that the terminal carbon of glucose is in α configuration; the 6 carbon signals of rhamnose are δ C 102.2, 72.5, 72.1, 74.2, 69.4, 17.9, and by δ HThe proton signal coupling constant of 4.61 (1H, d, J = 1.8 Hz) can be analyzed that the terminal carbon of the rhamnose group is in β configuration. According to the HMBC spectrum, δ H 7.57 is related to C-2 (130.7), C-9 (128.8), C-11 (139.4), and C12 (169.2). The end proton of glucose δ H 4.92 is related to C-11 (139.4), the rhamnose proton δ H 4.61 is related to C-6'(67.4), so the position of the sugar group and the side chain can be determined. After acid hydrolysis and derivatization, the sugar components of the compound of formula I of the present invention are identified as D-glucose and L-rhamnose by HPLC. In summary, the structure of the compound is determined to be 2-O-[β-D-glucopyranoside(1→6)]-[α-L-rhamnopyranosyl(1→6)]-3-indoleacrylic acid, i.e., 2-O-[β-D-glucopyranoside(1→6)]-[α-L-rhamnopyranosyl(1→6)]-3-(1H-indol-3-yl)acrylic acid.

[0015] The second technical problem solved by the present invention is to provide a preparation method of the compound. The compound of formula I of the present invention is prepared by an extraction method, and the raw material used is dried hibiscus flowers, that is, the flowers that have just opened or the buds that are about to open are picked during the flowering period and dried in the sun or in the oven.

[0016] The preparation method of the compound of formula I of the present invention comprises the following steps:

[0017] A. crushing and drying hibiscus mutabilis flowers, extracting with ethanol, collecting the filtrate, and removing the ethanol to obtain a crude hibiscus mutabilis flower ethanol extract;

[0018] B. The crude extract of the ethanol extract of Hibiscus mutabilis obtained in step A is suspended in water, and extracted 3-5 times in sequence using petroleum ether (60-90° C.), ethyl acetate, and n-butanol (water saturated), respectively, and the extracts are collected and removed to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively, and the remainder is an aqueous phase fraction;

[0019] C. Take the n-butanol fraction and dissolve it in water. Use D101 macroporous adsorption resin as a carrier, wet load the sample, and use a 0:100→100:0 water-methanol solvent gradient elution to obtain several elution fractions. Collect the 70% methanol elution fraction;

[0020] D. Take the 70% methanol elution portion obtained in step C, and collect the fractions by C18 medium pressure column chromatography with a methanol-water gradient elution of 5:100→100:0;

[0021] E. The fractions obtained in step D were divided and combined by high performance liquid chromatography to obtain 16 parts Fr.ZD1-16, and Fr.ZD 3 was collected for later use;

[0022] F. Fr.ZD 3 obtained in step E is subjected to semi-preparative liquid phase separation using acetonitrile-formic acid water as mobile phase to obtain the compound of formula I of the present invention.

[0023] In the technical scheme of the above preparation method:

[0024] The ethanol in step A is 70-100% ethanol by volume; preferably, 90% ethanol by volume.

[0025] The extraction method in step A is reflux extraction.

[0026] The extraction conditions of the reflux extraction in the extraction method of step A are 1-3 times, each time for 0.5-3 hours; preferably, the extraction conditions are 2 times, each time for 2 hours.

[0027] The method for removing ethanol in step A adopts reduced pressure recovery, and the end point is the absence of alcohol taste.

[0028] In step B, the amount of water added to the crude extract of ethanol extract of Hibiscus mutabilis flower is 8-12 times the amount, preferably 10 times the amount.

[0029] The water in step B is ultrapure water.

[0030] Step B: extracting with equal volumes of petroleum ether (60-90° C.), ethyl acetate and n-butanol (saturated with water) in sequence.

[0031] In step B, petroleum ether (60-90° C.), ethyl acetate, and n-butanol (water-saturated) are sequentially used for extraction 1-5 times, preferably 3 times.

[0032] The n-butanol part in step C is dissolved in water, and the ratio of the n-butanol part to water is: 300-400 g of n-butanol part and 3-4 L of water; preferably 350 g of n-butanol part and 3.5 L of water.

[0033] Step C is performed by gradient elution with water, 25-35% methanol, 45-55% methanol, 65-75% methanol and 90-100% methanol in sequence, and the elution fraction with 65-75% methanol is collected.

[0034] In step C, preferably, gradient elution is performed using water, 30% methanol, 50% methanol, 70% methanol, and 95% methanol in sequence, and the 70% methanol elution portion is collected.

[0035] In step D, the gradient elution was performed with methanol-water of 5:100→100:0, with an increase of 5%-10%, and a certain number of fractions were collected for each gradient.

[0036] Preferably, in step D, gradient elution with methanol-water of 5:100→100:0 is performed with a solvent system in which the volume fractions of methanol-water are 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 50:50, 60:40, 70:30, 80:20, and 100:0, respectively, and the mixture is collected;

[0037] For example, 20 g of 70% methanol was used as the elution fraction and chromatographed on a C18 medium pressure column with a gradient of 5% or 10%, with each gradient being about 20 bottles, and a total of 146 bottles of fractions were collected. Then the collected 146 bottles of fractions were segmented and combined by HPLC to obtain 16 components, named Fr.ZD 1-16; among them, 1-6 is Fr.ZD 1, 7-14 is Fr.ZD 2, 15-18 is Fr.ZD 3, 19-24 is Fr.ZD 4, 25-30 is Fr.ZD 5, 31-36 is Fr.ZD 6, 37-42 is Fr.ZD 7, 42-52 is Fr.ZD 8, 53-68 is Fr.ZD 9, 69-90 is Fr.ZD 10, 71-101 is Fr.ZD 11, 102-112 is Fr.ZD 12, 113-116 is Fr.ZD 13, 117-120 is Fr.ZD 14, 121-126 are Fr.ZD 15, 127-146 are Fr.ZD 16; collect Fr.ZD 3 for future use.

[0038] The Fr.ZD 3 corresponds to the portion eluted with 5% methanol, i.e., a methanol-water 5:95 solvent system.

[0039] In the above technical solution, the high performance liquid chromatograph described in step E adopts DAD.

[0040] In step F, the mobile phase used for the semi-preparative liquid phase separation is: A: 0.025% formic acid water B: acetonitrile, A:B (5:100)→(100:0) as the mobile phase.

[0041] Preferably, in step F, the mobile phase used for the semi-preparative liquid phase separation is: A: 0.025% formic acid water B: acetonitrile, with A:B being 75:25.

[0042] In step F, in the semi-preparative liquid phase separation, the elution time of the compound of formula I of the present invention is 13 minutes.

[0043] Most preferably, the preparation method of the compound of formula I of the present invention is as follows:

[0044] A. Grind the dried Hibiscus mutabilis flower into coarse powder, extract it with 90% ethanol by volume twice under reflux, each time for 2 hours, filter and combine the filtrates, and recover under reduced pressure until there is no alcohol taste, thereby obtaining a crude extract of ethanol extract of Hibiscus mutabilis flower;

[0045] B. Add 10 times the amount of ultrapure water suspension extract, extract three times with equal volumes of petroleum ether (60-90°C), ethyl acetate, and n-butanol (water saturated), respectively, combine the extracts, and recover under reduced pressure to obtain a petroleum ether portion, an ethyl acetate portion, and an n-butanol portion, and the remainder is an aqueous phase;

[0046] C. Dissolve the n-butanol fraction in water at a ratio of 100 g to 1 L of water, and apply the fraction to D101 macroporous adsorption resin, and then elute with water, 30% methanol, 50% methanol, 70% methanol, and 95% methanol in sequence (the volume of each gradient elution is 1 / 5 of the water volume);

[0047] D. Take the 70% methanol elution fraction and pass it through C18 medium pressure column chromatography, and use methanol-water (5-100%) gradient elution, increasing by 5%, and collect about 20 bottles of each gradient according to the ratio of 20g of the 70% methanol elution fraction, and collect 146 bottles of fractions in total;

[0048] E. The fragments were divided and merged by high performance liquid chromatography: 1-6 were Fr.ZD 1, 7-14 were Fr.ZD 2, 15-18 were Fr.ZD 3, 19-24 were Fr.ZD 4, 25-30 were Fr.ZD 5, 31-36 were Fr.ZD 6, 37-42 were Fr.ZD 7, 42-52 were Fr.ZD 8, 53-68 were Fr.ZD 9, 69-90 were Fr.ZD 10, 71-101 were Fr.ZD 11, 102-112 were Fr.ZD 12, 113-116 were Fr.ZD 13, 117-120 were Fr.ZD 14, 121-126 were Fr.ZD 15, 127-146 for Fr. ZD 16;

[0049] F. Fr.ZD3 was treated with acetonitrile-0.025% formic acid water as the mobile phase (25:75) for 13 min to obtain the compound of formula I.

[0050] By adopting the above preparation method, taking 20 kg of dried Hibiscus mutabilis flowers as an example, step A can obtain 3.34 kg of crude ethanol extract of Hibiscus mutabilis flowers; step B can obtain 93.6 g of petroleum ether, 89.7 g of ethyl acetate, 389 g of n-butanol, and the rest is the aqueous phase; step C takes 350 g of n-butanol and dissolves it in 3.5 L of water, and the volume of each gradient elution is 7 L; step D uses 20 g of 70% methanol elution part to pass through C18 medium pressure column chromatography, and uses methanol-water (5-100%) gradient elution, increasing by 5%, each gradient elution 320 mL is a bottle of eluent, and a total of 20 bottles are connected, about 6.5 L; step F 84.10 mg Fr.3 is eluted to obtain the compound of formula I of the present invention.

[0051] The third technical problem solved by the present invention is to provide at least one new use of the compound of formula I.

[0052] Pharmacological tests have shown that the compounds of formula I of the present invention have a new use in the preparation of anti-tumor drugs or health products; in particular, the compounds of formula I of the present invention have a new use in the preparation of anti-liver cancer drugs or health products. The compounds of formula I of the present invention also have a new use in the preparation of anti-inflammatory drugs or health products.

[0053] The fourth technical problem solved by the present invention is to provide a derivative product prepared using the compound of formula I as a raw material.

[0054] The medicine is prepared by using the compound of formula I of the present invention as raw material and adding pharmaceutically acceptable auxiliary ingredients.

[0055] The health care product is prepared by using the compound of formula I of the present invention as raw material and adding auxiliary ingredients acceptable to health care products.

[0056] The cosmetics are prepared by using the compound of formula I of the present invention as raw material and adding auxiliary ingredients acceptable to cosmetics.

[0057] The veterinary drug is prepared by using the compound of formula I of the present invention as raw material and adding auxiliary ingredients acceptable to veterinary drugs.

[0058] Beneficial effects of the present invention: The present invention obtains a new compound of formula I through extraction and separation technology, and confirms that it has anti-inflammatory and anti-tumor medical uses, providing the public with a new compound and its preparation method and multiple application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Flow chart of extraction, separation and purification.

[0060] Figure 2 Acid hydrolysis HPLC chromatogram of the compound of formula I of the present invention.

[0061] Figure 3 Effect of the compounds of formula I of the present invention on the release of TNF-α.

[0062] Note: Compared with the blank group ## P<0.01; compared with the model group * P<0.05, ** P<0.01, **** P<0.001

[0063] Figure 4 Effect of the compounds of formula I of the present invention on NO release.

[0064] Note: Compared with the blank group ## P<0.01; compared with the model group * P<0.05, ** P<0.01, *** P<0.001 DETAILED DESCRIPTION

[0065] The following describes specific embodiments of the present invention but does not limit the present invention.

[0066] The compound of formula I of the present invention has a structure as shown in formula I and a chemical formula of C 23 H 29 NO 12 .

[0067]

[0068] The chemical name is 2-O-[β-D-glucopyranosyl (1→6)]-[α-L-rhamnopyranosyl (1→6)]-3-indoleacrylic acid.

[0069] The compound of formula I of the present invention is discovered for the first time. The inventor obtains the monomer compound by separating and purifying the chemical components from the ethanol extract of Hibiscus mutabilis flower.

[0070] The hibiscus flower samples in the present invention were collected from a hibiscus flower planting base in Sanhe Town, Chengdu City, Sichuan Province in mid-October 2019, naturally dried in the shade, and identified as dried flowers of Hibiscus mutabilis L.

[0071] 1. Extraction, separation and purification of the compound of formula I of the present invention

[0072] 1 Extraction, separation and purification

[0073] 1.1 Extraction

[0074] 20 kg of dried Hibiscus mutabilis flowers were crushed into coarse powder, extracted with 90% ethanol by volume for 2 times, each time for 2 hours, filtered and combined, and the filtrate was recovered under reduced pressure until there was no alcohol taste, to obtain a crude extract (3.34 kg) of ethanol extract of Hibiscus mutabilis flowers. 10 times the amount of ultrapure water suspension extract was added, and extracted with equal volumes of petroleum ether (60-90°C), ethyl acetate, and n-butanol (water saturated) for 3 times in sequence, and the extracts were combined and recovered under reduced pressure to obtain a petroleum ether portion (93.6 g), an ethyl acetate portion (89.7 g), and an n-butanol portion (389.0 g), and the rest was an aqueous phase.

[0075] 1.2 Separation and purification

[0076] 350.0 g of the extract from the n-butanol part was separated by D101 macroporous adsorption resin, and gradient elution was performed using methanol-water (5:100, 30:70, 50:50, 70:30, 100:0) as the elution system. The eluate was collected and evaporated to dryness under reduced pressure to obtain 5 components, named Fr.ZA~Fr.ZE. Fr.ZD (22.0 g) was separated by ODS C 18 The medium pressure column was used for separation, and gradient elution was performed with a methanol-water (5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 50:50, 60:40, 70:30, 80:20, 100:0) solvent system. The fractions were collected and tested and combined by HPLC to obtain components Fr.ZD 1 to 16. Among them, Fr.ZD 3 was purified by semi-preparative liquid phase, with a volume fraction of 25% acetonitrile solution as the mobile phase to obtain the compound of formula I of the present invention (6.3 mg); the extraction separation and purification flow chart is as follows Figure 1 shown.

[0077] The compound obtained by extraction and purification is yellow-brown amorphous crystal, easily soluble in methanol, with an optical rotation of [α]2 D 5 86.00 (c0.05, methanol), and 3442 cm-1 was observed from the infrared spectrum. -1 、2949cm -1 、1746cm -1 、1647cm -1 There is absorption at 228nm and 330nm, indicating the presence of hydroxyl, carbonyl and indole rings in the structure. The UV spectrum shows that the compound has strong absorption at 228nm and 330nm. The HR-ESI-MS quasi-molecular ion peak is at m / z 512.17487[M+H] + appears at (calculated value is 512.17625), unsaturation Ω = 10, and the molecular formula is determined to be C 23 H 29 NO 12 . 1H NMR and COSY spectra (see Table 1) show a 3-substituted indole ring, δ H 7.65 (1H, d, J = 7.7 Hz), 7.35 (1H, d, J = 7.9 Hz), 7.12 (1H, ddd, J = 8.0, 7.1, 1.3 Hz), 7.09 (1H, ddd, J = 8.2, 7.0, 1.2 Hz) are proton signals in the aromatic region, 8.40 (1H, s) is a proton signal on the nitrogen-containing pyrrole ring; a methine proton signal, 7.57 (1H, s); 7 proton signals of glucose, 4.92 (1H, d, J = 7.8 Hz, H-1'), 3.48-3.51 (1H, m, H-2'), 3.43 (1H, d, J = 9.0 Hz, H-3'), 3.47 (1H, d, J = 9.3 Hz, H-4'), 3.32–3.36 (1H, m, H-5'), 3.79 (1H, dd, J=11.1, 1.9 Hz, H-6'), 3.51–3.54 (1H, m, H-6'); six proton signals of rhamnose, 4.61 (1H, d, J=1.8 Hz, H-1”), 3.70 (1H, dd, J=9.4, 3.4 Hz, H-2”), 3.77 (1H, dd, J=3.5, 1.8 Hz, H-3”), 3.38 (1H, d, J=9.5 Hz, H-4”), 3.54–3.57 (1H, m, H-5”), 1.06 (3H, d, J=6.2 Hz, H-6”). According to 13 C NMR data and HSQC spectrum show that one sugar signal δ C 169.2, 2 methine carbon signals δ C 130.7, 120.7, 6 aromatic carbon signals 118.7, 121.4, 123.2, 112.6, 137.2, 128.8; 6 carbon signals of glucose δ C 105.2, 75.9, 78.3, 70.7, 77.0, 67.4, and by δ H The proton signal coupling constant of 4.92 (1H, d, J = 7.8 Hz) can be used to analyze that the terminal carbon of glucose is in α configuration; the 6 carbon signals of rhamnose are δ C 102.2, 72.5, 72.1, 74.2, 69.4, 17.9, and by δ H The proton signal coupling constant of 4.61 (1H, d, J = 1.8 Hz) can be analyzed that the terminal carbon of the rhamnose group is in β configuration. According to the HMBC spectrum, δ H 7.57 is related to C-2 (130.7), C-9 (128.8), C-11 (139.4), and C-12 (169.2). The end proton of glucose δH 4.92 is related to C-11 (139.4), the rhamnose proton δ H 4.61 is related to C-6'(67.4), so the position of the sugar group and the side chain can be determined. After acid hydrolysis and derivatization, the sugar components of the compound of formula I of the present invention are identified as D-glucose and L-rhamnose by HPLC. In summary, the structure of the compound of formula I of the present invention is determined to be 2-O-[β-D-glucopyranoside(1→6)]-[α-L-rhamnopyranosyl(1→6)]-3-indoleacrylic acid, i.e., 2-O-[β-D-glucopyranoside(1→6)]-[α-L-rhamnopyranosyl(1→6)]-3-(1H-indol-3-yl)acrylic acid.

[0078] Spectral data of the compound of formula I of the present invention: 86.00 (c=0.05, MeOH); IR (KBr)v max 3442,2949,1746,1647;UVλ max 228nm; HR-ESI-MS: m / z 512.17487; 1 H NMR (600MHz, Methanol-d4) δ8.40(1H,s,H-2),7.65(1H,d,J=7.7Hz,H-4),7.57(1H,s,H-10),7.35(1H,d,J=7.9Hz,H-7),7.12(1H,ddd,J=8.0,7. 1,1.3Hz,H-6),7.09(1H,ddd,J=8.2,7.0,1.2Hz,H-5),4.92(1H,d,J=7.8Hz,H-1'),4.61(1H,d,J=1.8Hz,H-1”),3.79(1H,dd,J=11.1,1.9Hz,H-6 'a),3.77(1H,dd,J=3.5,1.8Hz,H-3”),3.70(1H,dd,J=9.4,3.4Hz,H-2” ),3.54–3.57(1H,m,H-5”),3.51–3.54(1H,m,H-6’b),3.48–3.51(1H,m,H -2'),3.47(1H,d,J=9.3Hz,H-4'),3.43(1H,d,J=9.0Hz,H-3'),3.38(1H, d,J=9.5Hz,H-4”),3.32–3.36(1H,m,H-5’),1.06(3H,d,J=6.2Hz,H-6”); 13C NMR(151MHz,MeOD)δ130.7(C-2),109.5(C-3),118.7(C-4),121.4(C-5),123.2(C -6),112.6(C-7),137.2(C-8),128.8(C-9),120.7(C-10),139.4(C-11),169.2(C- 12), 105.2 (C-1'), 75.9 (C-2'), 78.3 (C-3'), 70.7 (C-4'), 77.0 (C-5'), 67.4 (C-6'), 7102.2 (C-1"), 2.5 (C-2"), 72.1 (C-3"), 74.2 (C-4"), 69.4 (C-5"), 17.9 (C-6"). The NMR data of the compound of formula I of the present invention are shown in Table 1.

[0079] Acid hydrolysis of the compound of formula I of the present invention: 1.5 mg of the compound is added with 2 mL of a newly prepared 2 mol / L trifluoroacetic acid solution, and the mixture is hydrolyzed at 100°C for 6 h. 2 mL of methanol is added, and the solvent is evaporated under reduced pressure, and the process is repeated 4-6 times to remove all the trifluoroacetic acid, and 2 mL of ultrapure water is added to dissolve the evaporated sample, and 2 mL of ethyl acetate is added for extraction, and the process is repeated 3 times. The water layer is dried under reduced pressure to obtain the hydrolyzed monosaccharide. The monosaccharide obtained by hydrolysis is added with 0.5 mL of pyridine and 1 mg of L-cysteine ​​methyl ester hydrochloride as the experimental group, and 1.5 mg of glucose standard and 1.5 mg of rhamnose standard are added with 0.5 mL of pyridine and 1 mg of L-cysteine ​​methyl ester hydrochloride as the glucose control group and the rhamnose control group, respectively, and reacted at 60°C for 1 h. Then, 0.5 mL of phenyl isothiocyanate is added to the three groups respectively, and reacted at 60°C for 1 h. The reaction solution is analyzed on an Ultimated 3000 high performance liquid chromatograph. Chromatographic conditions: chromatographic column: Inertsil ODS-3 (4.6×250mm, 5um); mobile phase: acetonitrile (A)-0.05% phosphoric acid water (B); flow rate: 1mL / min; detection wavelength: 250nm; column temperature: 30°C; injection volume: 10uL, 25% acetonitrile isocratic elution for 45min. Compared with the retention time of monosaccharide derivatization, D-glucose is 11min, L-rhamnose is 14min, and the high performance liquid chromatogram of the compound of formula I of the present invention is shown in Figure 2 .

[0080] II. Use of the compounds of formula I of the present invention

[0081] Among them, the mouse macrophage cell line RAW264.7 used in Experimental Example 2-3 was placed in DMEM complete culture medium containing antibiotics (100U / mL penicillin, 100μg / mL streptomycin) and 10% fetal bovine serum (FBS), and cultured in an incubator at 37°C and 5% CO2.

[0082] Experimental Example 1: Cytotoxicity to hepG2 liver cancer cells

[0083] HepG2 cells in the logarithmic growth phase were taken and 3×10 5 / mL density, 100μL per well inoculated in 96-well plate, sample group added 50umol / L DMSO solution containing the compound of formula I of the present invention, control added equal volume of DMSO, each group of 5 duplicate wells, positive control group added 60umol / L 5-fluorouracil, cultured in 37℃, 5% CO2 incubator for 24h and 48h respectively. Discard the original culture medium in the 96-well plate, add 100μL serum-free culture medium and 20μL CCK-8 solution under light-proof conditions, set the cell-free secondary well as blank control group. Place the cell well plate in a cell culture incubator and incubate for 3h in the dark, and measure the absorbance (A450) value three times at 450nm in parallel with an enzyme reader. The study found that the inhibition rate of HepG2 cells treated with 50umol / L of the compound of formula I of the present invention for 24h was -37.73%, and there was no obvious inhibitory effect on HepG2 cells. The inhibition rate of 48h treatment was 593.3%, with obvious inhibitory effect.

[0084] Experimental Example 2: Elisa method to detect the release of inflammatory factor TNF-α in RAW264.7 cells

[0085] RAW264.7 cells in the logarithmic growth phase were selected and cultured at 3×10 5 / mL density, 100μL per well was inoculated in a 96-well plate, and cultured in a 37°C, 5% CO2 incubator for 24h, the supernatant was removed, 1mL of DEME solution was added to the blank group, 1mL of DEME solution containing LPS (1μg / mL) was added to the model group and the experimental group, and 1mL of DEME solution containing the compound (50μmol / L) was added to the experimental group after 0.5h, and cultured in a 37°C, 5% CO2 incubator for 24h, and the inflammatory factor TNF-α level was detected by Elisa kit, and its content was calculated according to the standard curve. Figure 3 It can be seen that compared with the blank group, LPS can increase the release of TNF-α in RAW264.7 macrophages, with a significant difference (P<0.05). After adding the compound of formula I of the present invention (50 μmol / L) for treatment, the ability of RAW264.7 cells to secrete TNF-α inflammatory factors can be effectively reduced, indicating that the compound of formula I of the present invention exhibits anti-inflammatory activity by reducing the release of TNF-α.

[0086] Experimental Example 3: Determination of NO Release by Griess Method

[0087] RAW264.7 cells in the logarithmic growth phase were selected and cultured at 3×10 5 / mL density, 100μL per well was inoculated in a 96-well plate, and cultured in an incubator at 37°C and 5% CO2, and a NO standard curve was prepared according to the instructions of the kit. 1mL of DEME solution was added to the blank group, 1mL of DEME solution containing LPS (1μg / mL) was added to the model group and the experimental group, and 1mL of DEME solution containing the compound of formula I of the present invention (25, 50μmol / L) was added to the experimental group after 0.5h, and 24h later, centrifuged (1000g, 15min), 50μL of culture supernatant was drawn to a 96-well cell culture plate, 50μL of Griess reagent A was added to each well and mixed, and the mixture was kept at room temperature and away from light for 5min, and 50μL of Griess reagent B was added to each well and mixed, and the mixture was kept at room temperature and away from light for 10min, and the absorbance (A540) was detected by an enzyme marker at 540nm, and the concentration of NO in each experimental group was determined by the calibration curve, and each group was repeated for 6 wells. The inhibitory effect on nitric oxide (NO) was evaluated. The NO inhibition rate was calculated according to the following formula: NO inhibition rate = (A model group - A drug group) / (A model group - A blank group) × 100%.

[0088] like Figure 4 As shown, LPS can increase the NO release in the supernatant of macrophages, with significant difference (P<0.05). After adding the compound of formula I of the present invention (25, 50 μmol / L) for treatment, the NO content in the supernatant of RAW264.7 macrophages treated with the compound of formula I of the present invention (50 μmol / L) was significantly reduced (P<0.05), and it was concentration-dependent, indicating that the compound of formula I of the present invention can show anti-inflammatory activity by reducing the NO release.

[0089] LPS was used as an inducer to stimulate RAW264.7 macrophages to establish an inflammatory model, and the isolated compounds of Formula I of the present invention were evaluated for in vitro anti-inflammatory activity. First, the CCK-8 method was used to detect the cytotoxicity of the compounds of Formula I of the present invention at different concentrations, and it was found that the compounds of Formula I of the present invention had no effect on the proliferation of inflammatory cells within this concentration range. Then, the release of TNF-α inflammatory factors was detected by Elisa method, and it was found that 50 μmol / L of the compounds of Formula I of the present invention could reduce the release of TNF-α compared with the model group; the Griess method was used to detect the release of NO, and it was found that 50 μmol / L of the compounds of Formula I of the present invention could reduce the release of NO in RAW264.7 macrophages compared with the model group. The experimental results showed that the anti-inflammatory effect of the compounds of Formula I of the present invention was mainly related to the inhibition of the release of inflammatory factors TNF-α and NO.

[0090] In summary, the indole compound of the present invention has a structure as shown in Formula I, and a chemical formula of C 23 H 29 NO 12, chemically named 2-O-[β-D-glucopyranosyl (1→6)]-[α-L-rhamnopyranosyl (1→6)]-3-indoleacrylic acid. It was first extracted, separated and purified from Hibiscus mutabilis flower extract and has clear anti-inflammatory and anti-tumor effects, especially against liver cancer.

Claims

1. A compound, characterized in that: The structure is as formula Ⅰ, the chemical formula is C 23 H 29 NO 12 :

2. The method for preparing the compound according to claim 1, characterized in that: The steps include: A. crushing and drying hibiscus mutabilis flowers, extracting with 70-100% by volume ethanol, collecting the filtrate, and removing the ethanol to obtain a crude hibiscus mutabilis flower ethanol extract; B. The crude extract of the ethanol extract of Hibiscus mutabilis obtained in step A is suspended in water, and extracted 3-5 times in sequence using petroleum ether, ethyl acetate, and water-saturated n-butanol at 60-90° C., respectively, and the extracts are collected and removed to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively, and the remainder is an aqueous phase fraction; C. Take the n-butanol fraction and dissolve it in water. Use D101 macroporous adsorption resin as a carrier, wet load the sample, and use a 0:100→100:0 water-methanol solvent gradient elution to obtain several elution fractions. Collect the 70% methanol elution fraction; D. Take the 70% methanol elution portion obtained in step C, and perform chromatography on a C18 medium pressure column, eluting with a methanol-water gradient of 5:95→100:0, and collect the fractions; E. The fractions obtained in step D were divided and combined by high performance liquid chromatography to obtain 16 parts Fr.ZD1-16, and Fr.ZD 3 was collected for later use; F. Fr.ZD 3 obtained in step E is subjected to semi-preparative liquid phase separation using acetonitrile-formic acid water as the mobile phase to obtain the compound of formula I; in, In step D, the fraction was eluted with a methanol-water gradient of 5:95→100:0, and eluted with 20 g of 70% methanol, and 146 bottles of fractions were collected in 5% increments by C18 medium pressure column chromatography; In step E, 146 vials of fractions obtained in step D were segmented and combined by HPLC to obtain 16 components, named Fr.ZD 1 to 16, wherein 1-6 are Fr.ZD 1, 7-14 are Fr.ZD 2, 15-18 are Fr.ZD 3, 19-24 are Fr.ZD 4, 25-30 are Fr.ZD 5, 31-36 are Fr.ZD 6, 37-42 are Fr.ZD 7, 43-52 are Fr.ZD 8, 53-68 are Fr.ZD 9, 69-90 are Fr.ZD 10, 91-101 are Fr.ZD 11, 102-112 are Fr.ZD 12, and 113-116 are Fr.ZD 13, 117-120 are Fr.ZD14, 121-126 are Fr.ZD 15, 127-146 are Fr.ZD 16; collect Fr.ZD 3 for future use.

3. The method for preparing the compound according to claim 2, characterized in that: At least one of the following is met: The dried hibiscus flowers are obtained by picking the flowers that have just opened or the buds that are about to open during the flowering period and drying them in the sun or in an oven; The extraction method in step A is reflux extraction; The extraction conditions of reflux extraction in the extraction method of step A are 1-3 times, each time for 0.5-3 hours; The method for removing ethanol in step A adopts reduced pressure recovery, and the end point is the absence of alcohol taste; The amount of water added to the crude extract of ethanol extract of Hibiscus mutabilis in step B is 8-12 times the amount; The water described in step B is ultrapure water; Step B: extracting with equal volumes of 60-90° C. petroleum ether, ethyl acetate, and water-saturated n-butanol in sequence; The n-butanol part of step C is dissolved in water, and the ratio of the n-butanol part to water is: 300-400 g of n-butanol part and 3-4 L of water; Step C: gradient elution with water, 25-35% methanol, 45-55% methanol, 65-75% methanol, and 90-100% methanol in sequence, and collecting the 65-75% methanol elution fraction; The high performance liquid chromatograph in step E adopts DAD; In step F, the mobile phase used for the semi-preparative liquid phase separation is: A: 0.025% formic acid water B: acetonitrile, A: B5:100→100:0 as the mobile phase; In step F, the semi-preparative liquid phase separation, the peak time of the compound of formula I is 13 minutes.

4. The method for preparing the compound according to claim 3, characterized in that: At least one of the following is met: The ethanol in step A is 90% ethanol by volume; The extraction conditions of reflux extraction in the extraction method of step A are 2 times, each time for 2 hours; The amount of water added to the crude extract of ethanol extract of Hibiscus mutabilis in step B is 10 times the amount; Step B is extracted three times with petroleum ether, ethyl acetate and water-saturated n-butanol at 60-90°C in sequence; The ratio of the n-butanol part to water in step C is: 350 g of n-butanol part and 3.5 L of water; Step C: gradient elution with water, 30% methanol, 50% methanol, 70% methanol, and 95% methanol in sequence, and collecting the 70% methanol elution fraction; In step F, the mobile phase used for the semi-preparative liquid phase separation is: A: 0.025% formic acid water B: acetonitrile, with A:B being 75:

25.

5. The preparation method according to claim 2, characterized in that: A. Grind the dried Hibiscus mutabilis flower into coarse powder, extract it with 90% ethanol by volume twice under reflux, each time for 2 hours, filter and combine the filtrates, and recover under reduced pressure until there is no alcohol taste, thereby obtaining a crude extract of ethanol extract of Hibiscus mutabilis flower; B. Add 10 times the amount of ultrapure water suspension extract, extract three times with equal volumes of 60-90°C petroleum ether, ethyl acetate, and water-saturated n-butanol, respectively, combine the extracts, and recover under reduced pressure to obtain a petroleum ether portion, an ethyl acetate portion, and an n-butanol portion, and the remainder is an aqueous phase; C. Dissolve the n-butanol fraction in water at a ratio of 100 g to 1 L of water, and apply the fraction to D101 macroporous adsorption resin in batches, eluting with water, 30% methanol, 50% methanol, 70% methanol, and 95% methanol in sequence; the volume of each gradient elution is 1 / 5 of the water volume; F. The compound of formula I was prepared by using Fr.ZD 3 with acetonitrile-0.025% formic acid water = 25:75 as the mobile phase for 13 min. The compound according to claim 1 is used in the preparation of anti-liver cancer drugs.

7. The compound according to claim 1 has use in preparing anti-inflammatory drugs.

8. The drug prepared from the compound of claim 1, characterized in that: The medicine is prepared by using the compound of formula I as raw material and adding pharmaceutically acceptable auxiliary ingredients.

Images