Compound hibiscus odoratin and its preparation method and use

Abstract

The present invention belongs to the field of medicine, and specifically relates to a new compound, hibiscus odoratum, 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 18 O8, chemically named 8-(1"-(3"',4"'-dihydroxyphenyl)ethyl)-3,5,7,4'-tetrahydroxyflavone, and named hibiscus flower element, English name Mutabilfloin. 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 in particular relates to a new compound hibiscus odoratum and a preparation method and application 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 flowers. 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 18 O8.

[0006]

[0007] The chemical name of the compound of formula I of the present invention is 8-(1"-(3"',4"'-dihydroxyphenyl)ethyl)-3,5,7,4'-tetrahydroxyflavone, and is named as hibiscus flower element, and the English name is Mutabilfloin. The nuclear magnetic resonance data (600MHz, 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 in formula I of the present invention are: brown powder, easily soluble in methanol.

[0012] Spectral data of the compound described in formula I of the present invention: (c=0.05,MeOH),IR(KBr)v max 3458,1733,1682,1560;UVλ max 205nm; LC-MS: m / z 423.16; 1H NMR (600MHz, Methanol-d4) δ7.62(2H,d,J=8.2Hz,H-2',6'),6.78(2H,d,J=8.1Hz,H-3',5'),6.75(1H,d,J=2.1Hz,H-2"'),6.70(1H, d,J=8.3Hz,H-5”'),6.65(1H,dd,J=8.4,2.2Hz,H-6”’),6.29(1H,s,H-6),4.81(1H,q,J=7.2Hz,H-1”),1.66(3H,d,J=7.2Hz,2”-CH3); 13 C NMR(151MHz,MeOD)δ160.4(C-2),123.8(C-3),177.7(C-4),160.3(C-5),98.9(C-6),1 63.0(C-7),112.5(C-8),155.7(C-9),105.1(C-10),136.8(C-1'),131.1(C-2'),116.1 (C-3'),148.6(C-4'),116.1(C-5'),131.1(C-6'),33.0(C-1”),18.5(2”-CH3),138.2 (C-1"'),115.4(C-2"'),144.0(C-3"'),146.0(C-4"'),116.0(C-5"'),119.1(C-6"').

[0013] Optical rotation of the compound of formula I of the present invention (c 0.05, methanol), from the infrared spectrum, we can observe 3458cm -1 、1733cm -1 、1682cm -1 、1560cm -1 There is absorption at 205nm and 280nm, indicating that the compound contains hydroxyl, carbonyl and benzene rings. The UV spectrum shows that the compound has strong absorption at 205nm and 280nm. The LC-MS quasi-molecular ion peak is at m / z423.16[M+H] + The calculated value is 423.11, the degree of unsaturation is Ω=15, and the molecular formula is determined to be C 23 H 18 O8.

[0014] The compounds of formula I of the present invention 1 H NMR spectra and 1 H- 1The HCOSY spectrum (see Table 1) showed that δ7.62 (2H, J = 8.2 Hz, H-2', 6') and δ6.87 (2H, J = 8.1 Hz, H-3', 5') were characteristic signals of 4'-hydroxyflavonol, and the HSQC spectrum showed that δ H 7.62 and δ C 131.1 connected, δ H 6.87 and δ C 116.1. Compound *24 13 C data and kaempferol 13 C NMR data were compared to determine that the flavonoid core in the molecule was kaempferol. 1 H NMR spectrum and the coupling value to deduce δ H 6.75 (1H, d, J = 2.1 Hz), 6.70 (1H, d, J = 8.3 Hz), 6.65 (1H, dd, J = 8.4, 2.2 Hz) are hydrogen signals of the ABX system on the benzene ring. HSQC and HMBC spectra show that δ C 160.3(C-5) and δ C 163.0 (C-7) proton connected to hydroxyl, δ C 105.9 (C-4) is a tertiary carbon proton, δ C 98.9 (C-6) is a single peak (s), indicating that the C-8 position of the kaempferol core is substituted. 1 H NMR spectra and 1 H- 1 HCOSY spectrum shows δ H 4.81 is the methylene proton signal, with a quartet (q), J = 7.2 Hz, coupled with three protons, δ H The signal at 1.66 is a methyl proton signal, which is coupled with two protons, so it is judged that there is an ethyl group in compound *24. The HMBC spectrum shows that the tertiary carbon δ C 33.0 and δ C 18.5,112.5,115.4,119.1,138.2 are related, so the position of the benzene ring, ethyl group and kaempferol core structure of the ABX system can be determined, and it can be judged from the unsaturation Ω=15 that there is no ring structure between the kaempferol core part and the phenyl substituent part. In summary, the structure of the compound of formula I of the present invention is determined to be 8-(1"-(3"',4"'-dihydroxyphenyl)ethyl)-3,5,7,4'-tetrahydroxyflavone, and it is named Mutabilfloin, and its English name is Mutabilfloin.

[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 ethyl acetate fraction and separate it by normal phase silica gel column chromatography, use petroleum ether-ethyl acetate as the elution system for gradient elution, collect the fractions, check and combine them by TLC, and obtain 7 components in sequence, named Fr.YA~Fr.YG, collect Fr.YC for later use;

[0020] D. Fr.YC obtained in step C was separated using an MCI medium pressure column, gradient eluted using a methanol-water solvent system, and the fractions were collected and detected and combined using TLC to obtain the following components, which were named Fr.YC1-13, and Fr.YC 6 was collected for later use;

[0021] E. Fr.YC 6 obtained in step D was purified by semi-preparative liquid phase using 62% by volume methanol solution as the mobile phase to obtain the compound of formula I of the present invention.

[0022] In the technical scheme of the above preparation method:

[0023] The ethanol in step A is 70-100% ethanol by volume; preferably, 90% ethanol by volume.

[0024] The extraction method in step A is reflux extraction.

[0025] 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.

[0026] The method for removing ethanol in step A adopts reduced pressure recovery, and the end point is the absence of alcohol taste.

[0027] In step B, the amount of water added to the crude extract of ethanol extract of Hibiscus mutabilis is 8-12 times the amount, preferably 10 times the amount.

[0028] The water in step B is ultrapure water.

[0029] In step B, extraction is performed using equal volumes of petroleum ether (60-90° C.), ethyl acetate, and n-butanol (saturated with water) in sequence.

[0030] 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.

[0031] In step C, the petroleum ether-ethyl acetate solvent system is eluted according to a gradient of 20:1, 8:1, 5:1, 2:1, 1:1, 1:2, 1:4, 1:10, and 0:100, and the mixture is collected.

[0032] In step D, the methanol-water solvent elution system is 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0, respectively. The fractions are collected, detected and combined by TLC to obtain the following components, respectively named Fr.YC1~13.

[0033] As in step C, 80g of the ethyl acetate fraction was separated by normal phase silica gel column chromatography, and petroleum ether-ethyl acetate was used as the elution system to elute according to the gradient of 20:1, 8:1, 5:1, 2:1, 1:1, 1:2, 1:4, 1:10, 0:100, and collected, and a total of 127 bottles of fractions were collected. Then the 127 bottles of fractions collected were checked and combined by TLC to obtain 7 components, named Fr.YA~Fr.YG, among which 1-15 is Fr.YA, 26-37 is Fr.YB, 38-52 is Fr.YC, 53-68 is Fr.YD, 69-90 is Fr.YE, 91-109 is Fr.YF, and 110-127 is Fr.YG. Fr.YC was collected for later use;

[0034] The Fr.YC obtained in step C corresponds to the portion eluted with petroleum ether:ethyl acetate = 2:1.

[0035] In step D, based on 80 g of ethyl acetate, the methanol-water solvent elution system was 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, and a total of 90 bottles of fractions were connected. After detection and merging by TLC, the following components were obtained in sequence, named Fr.YC1~13, among which: 1-4 is Fr.YC 1, 5-9 is Fr.YC 2, 10-13 is Fr.YC 3, 14-19 is Fr.YC 4, 20-24 is Fr.YC 5, 25-28 is Fr.YC 6, 29-33 is Fr.YC 7, 34-38 is Fr.YC 8, 39-44 is Fr.YC 9, 45-48 is Fr.YC 10, 49-62 is Fr.YC 11, 63-79 is Fr.YC 12, and 80-90 is Fr.YC 13.

[0036] The Fr.YC 6 obtained in step D corresponds to the portion eluted with the methanol-water solvent system = 60:40, i.e., 60% methanol.

[0037] Step E was purified by semi-preparative liquid phase, with 62% methanol solution as the mobile phase, and the elution time was 33 min.

[0038] Most preferably, the preparation method of the compound of formula I of the present invention is as follows:

[0039] 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;

[0040] 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;

[0041] C. Take the ethyl acetate fraction and separate it by normal phase silica gel column chromatography, use petroleum ether-ethyl acetate as the elution system for gradient elution, collect the fractions, check and combine them by TLC, and obtain 7 components in sequence, named Fr.YA~Fr.YG, collect Fr.YC for later use;

[0042] D. Fr.YC obtained in step C was separated using an MCI medium pressure column, gradient eluted using a methanol-water solvent system, and the fractions were collected and detected and combined using TLC to obtain the following components, which were named Fr.YC1-13, and Fr.YC 6 was collected for later use;

[0043] E. Fr.YC 6 obtained in step D was purified by semi-preparative liquid phase with 62% methanol solution as mobile phase and the peak elution time was 33 min to obtain the compound of formula I of the present invention.

[0044] By adopting the above preparation method, taking 20 kg of dried hibiscus flower as an example, step A can obtain 3.34 kg of crude ethanol extract of hibiscus flower; step B can obtain 93.6 g of petroleum ether part, 89.7 g of ethyl acetate part, 389 g of n-butanol part, and the rest is an aqueous phase; step C takes 80 g of the ethyl acetate part, uses petroleum ether-ethyl acetate as an elution system, and elutes according to a gradient of 20:1, 8:1, 5:1, 2:1, 1:1, 1:2, 1:4, 1:10, and 0:100, and collects, and a total of 127 bottles of fractions are connected. Then the collected 127 bottles of fractions were checked and combined by TLC to obtain 7 components, named Fr.YA~Fr.YG, among which 1-15 were Fr.YA, 26-37 were Fr.YB, 38-52 were Fr.YC, 53-68 were Fr.YD, 69-90 were Fr.YE, 91-109 were Fr.YF, and 110-127 were Fr.YG. Fr.YC was collected for use; 14.97g of fraction Fr.YC was collected; then Fr.YC was separated by MCI medium pressure column, eluted by methanol-water solvent system (50:50, 60:40, 70:30, 80:20, 90:10, 100:0), and a total of 90 bottles of fractions were collected. 1-4 is Fr.YC 1, 5-9 is Fr.YC 2, 10-13 is Fr.YC 3, 14-19 is Fr.YC 4, 20-24 is Fr.YC 5, 25-28 is Fr.YC 6, 29-33 is Fr.YC 7, 34-38 is Fr.YC 8, 39-44 is Fr.YC 9, 45-48 is Fr.YC 10, 49-62 is Fr.YC 11, 63-79 is Fr.YC 12, 80-90 is Fr.YC 13. The collected Fr.YC 6 corresponds to the part eluted with the methanol-water solvent system = 60:40, i.e. 60% methanol, and is purified by semi-preparative liquid phase to obtain the compound of formula I of the present invention.

[0045] The third technical problem solved by the present invention is to provide at least one new use of the compound of formula I.

[0046] Pharmacological experiments have shown that the compound of formula I of the present invention also has a new use in the preparation of anti-inflammatory drugs and cosmetics.

[0047] 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.

[0048] The medicine is prepared by using the compound of formula I of the present invention as raw material and adding pharmaceutically acceptable auxiliary ingredients.

[0049] 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.

[0050] 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.

[0051] 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 medical uses, providing the public with a new compound and its preparation method and multiple application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Flow chart of extraction, separation and purification.

[0053] Figure 2 Effects of the compounds of formula I of the present invention on the proliferation of RAW264.7 cells.

[0054] Figure 3 Effect of the compounds of formula I of the present invention on the release of TNF-α.

[0055] Note: Compared with the blank group ## P<0.01; compared with the model group * P<0.05, ** P<0.01, **** P<0.001

[0056] Figure 4 Effect of the compounds of formula I of the present invention on NO release.

[0057] Note: Compared with the blank group ## P<0.01; compared with the model group ** P<0.01, *** P<0.001 DETAILED DESCRIPTION

[0058] The following is a description of the specific embodiments of the present invention, but does not limit the present invention. The compound of the present invention has a structure as shown in Formula I, and a chemical formula of C 23 H 18 O8.

[0059]

[0060] The chemical name of the compound of the present invention is 8-(1"-(3"',4"'-dihydroxyphenyl)ethyl)-3,5,7,4'-tetrahydroxyflavone, and is also named as mutabilfloin, and its English name is Mutabilfloin.

[0061] 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.

[0062] 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.

[0063] 1. Extraction, separation and purification of the compound of formula I of the present invention

[0064] 1 Extraction, separation and purification

[0065] 1.1 Extraction

[0066] 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.

[0067] 1.2 Separation and purification

[0068] Take 80.0g of the ethyl acetate extract and separate it by normal phase silica gel column chromatography, use petroleum ether-ethyl acetate as the elution system for gradient elution, collect the fractions and use TLC to check and merge, and get 7 components, named Fr.YA~Fr.YG. Fr.YA (10.97g) and YC (14.97g) segments are separated by MCI medium pressure column, and use methanol-water solvent system for gradient elution, collect the fractions and use TLC to check and merge, and get components Fr.YA1~11, Fr.YC1~13. Among them, Fr.YC 6 is semi-preparative liquid phase purified, and 62% methanol solution by volume is used as the mobile phase to obtain the compound of formula I of the present invention. The extraction separation and purification flow chart is as follows Figure 1 shown.

[0069] The compound of formula I obtained by the extraction and purification method is a brown powder, which is easily soluble in methanol. (c 0.05, methanol), from the infrared spectrum, we can observe 3458cm -1 、1733cm -1 、1682cm -1 、1560cm -1There is absorption at 205nm and 280nm, indicating that the compound contains hydroxyl, carbonyl and benzene rings. The UV spectrum shows that the compound has strong absorption at 205nm and 280nm. The LC-MS quasi-molecular ion peak is at m / z423.16[M+H] + The calculated value is 423.11, the degree of unsaturation is Ω=15, and the molecular formula is determined to be C 23 H 18 O8. 1 H NMR spectra and 1 H- 1 The HCOSY spectrum (see Table 1) showed that δ7.62 (2H, J = 8.2 Hz, H-2', 6') and δ6.87 (2H, J = 8.1 Hz, H-3', 5') were characteristic signals of 4'-hydroxyflavonol, and the HSQC spectrum showed that δ H 7.62 and δ C 131.1 connected, δ H 6.87 and δ C 116.1. Compound *24 13 C data and kaempferol 13 C NMR data were compared to determine that the flavonoid core in the molecule was kaempferol. 1 H NMR spectrum and the coupling value to deduce δ H 6.75 (1H, d, J = 2.1 Hz), 6.70 (1H, d, J = 8.3 Hz), 6.65 (1H, dd, J = 8.4, 2.2 Hz) are hydrogen signals of the ABX system on the benzene ring. HSQC and HMBC spectra show that δ C 160.3(C-5) and δ C 163.0 (C-7) proton connected to hydroxyl, δ C 105.9 (C-4) is a tertiary carbon proton, δ C 98.9 (C-6) is a single peak (s), indicating that the C-8 position of the kaempferol core is substituted. 1 HNMR spectra and 1 H- 1 HCOSY spectrum shows δ H 4.81 is the methylene proton signal, with a quartet (q), J = 7.2 Hz, coupled with three protons, δ H The signal at 1.66 is a methyl proton signal, which is coupled with two protons, so it is judged that there is an ethyl group in compound *24. The HMBC spectrum shows that the tertiary carbon δ C 33.0 and δ C18.5,112.5,115.4,119.1,138.2 are related, so the position of the ABX system benzene ring, ethyl and kaempferol core structure can be determined, and the unsaturation Ω=15 shows that there is no ring structure between the kaempferol core part and the phenyl substituent part. In summary, the structure of the compound of formula I of the present invention is determined to be 1"'-methoxy-3,5,7,4',3",4"-hexahydroxyflavone, that is, 1"'-methoxy-3,5,7,4',3",4"-hexahydroxyflavone, and is named Mutabilfloin.

[0070] Spectral data of the compound described in formula I of the present invention: (c=0.05,MeOH),IR(KBr)v max 3458,1733,1682,1560;UVλ max 205nm; LC-MS: m / z 423.16; 1 H NMR (600MHz, Methanol-d4) δ7.62(2H,d,J=8.2Hz,H-2',6'),6.78(2H,d,J=8.1Hz,H-3',5'),6.75(1H,d,J=2.1Hz,H-2"'),6.70(1H, d,J=8.3Hz,H-5”'),6.65(1H,dd,J=8.4,2.2Hz,H-6”’),6.29(1H,s,H-6),4.81(1H,q,J=7.2Hz,H-1”),1.66(3H,d,J=7.2Hz,2”-CH3); 13 C NMR(151MHz,MeOD)δ160.4(C-2),123.8(C-3),177.7(C-4),160.3(C-5),98.9(C-6),1 63.0(C-7),112.5(C-8),155.7(C-9),105.1(C-10),136.8(C-1'),131.1(C-2'),116.1 (C-3'),148.6(C-4'),116.1(C-5'),131.1(C-6'),33.0(C-1”),18.5(2”-CH3),138.2 (C-1"'),115.4(C-2"'),144.0(C-3"'),146.0(C-4"'),116.0(C-5"'),119.1(C-6"').

[0071] II. Use of the compounds of formula I of the present invention

[0072] Mouse macrophage cell line RAW264.7 was placed in DMEM complete medium containing antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin) and 10% fetal bovine serum (FBS) and cultured in an incubator at 37° C. and 5% CO 2 .

[0073] Experimental Example 1: Effect on the proliferation of RAW264.7 cells

[0074] 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 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 DEME solution containing different concentrations (3.125, 6.25, 12.5, 25, 50μmol / L) of the compound of formula I of the present invention was added to the experimental group, and 100ug / ml of dexamethasone was added to the positive control group. The plates were cultured in a 37°C, 5% CO2 incubator for 24h. The original culture medium in the 96-well plate was discarded, and 100μL of serum-free culture medium and 10μL of CCK-8 solution were added under light-proof conditions. The duplicate wells without cells were set as blank groups. The cell well plates were placed in a cell incubator and incubated in the dark for 3h. The absorbance (A450) values ​​were measured three times in parallel at 450nm by an enzyme reader to calculate the relative survival rate of the cells. Figure 2 The study found that the compound of formula I of the present invention can significantly inhibit the proliferation of RAW264.7 macrophages within a certain molar concentration range (25-50 μmol / L) (P<0.05), and is concentration-dependent, and has a significant inhibitory effect on the proliferation of RAW264.7 cells.

[0075] Experimental Example 2: Elisa method to detect the release of inflammatory factor TNF-α in RAW264.7 cells

[0076] 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 (25μmol / 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 3It 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 (25 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.

[0077] Experimental Example 3: Determination of NO Release by Griess Method

[0078] RAW264.7 cells in the logarithmic growth phase were selected and cultured at 3×10 5 / mL density, 100μL per well were inoculated in 96-well plates, and cultured in an incubator at 37°C and 5% CO2, and NO standard curves were 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 (6.25, 12.5μmol / 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%.

[0079] like Figure 4 As shown, LPS can increase the NO release in the supernatant of macrophages, with a significant difference (P<0.05). After adding the compound of formula I of the present invention (6.25, 12.5 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 (6.25, 12.5 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.

[0080] 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 cytotoxicity of the compounds of Formula I of the present invention at different concentrations was detected by CCK-8 method, and it was found that the compounds of Formula I of the present invention could inhibit the proliferation of inflammatory cells in a certain concentration range (25-50 μmol / L) and it was concentration-dependent. Then, the release of TNF-α inflammatory factor was detected by Elisa method, and it was found that 25 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 release of NO was detected by Griess method, and it was found that 6.25 and 12.5 μmol / L of the compounds of Formula I of the present invention could reduce the release of NO of 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.

[0081] In summary, the compound of the present invention has a structure as shown in Formula I and a chemical formula of C 23 H 18 O8, chemically named 8-(1”-(3”',4”'-dihydroxyphenyl)ethyl)-3,5,7,4'-tetrahydroxyflavone, and named Mutabilfloin. It was first extracted, separated and purified from the extract of Hibiscus mutabilfloin and has a clear anti-inflammatory effect.

Claims

1. A compound, characterized in that: The structure is as formula Ⅰ, the chemical formula is C 23 H 18 O8:

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 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 ethyl acetate fraction and separate it by normal phase silica gel column chromatography, use petroleum ether-ethyl acetate as the elution system for gradient elution, collect the fractions, check and combine them by TLC, and obtain 7 components in sequence, named Fr.YA~Fr.YG, collect Fr.YC for later use; D. Fr.YC obtained in step C was separated using an MCI medium pressure column, gradient eluted using a methanol-water solvent system, and the fractions were collected and detected and combined using TLC to obtain the following components, which were named Fr.YC1-13, and Fr.YC 6 was collected for later use; E. Fr.YC 6 obtained in step D was purified by semi-preparative liquid phase purification using 62% by volume methanol solution as the mobile phase to obtain the compound of formula I.

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 ethanol in step A is 70-100% ethanol by volume; 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; Step B is to extract with 60-90° C. petroleum ether, ethyl acetate, and water-saturated n-butanol in sequence for 1-5 times; Step C: the petroleum ether-ethyl acetate solvent system is eluted according to the gradient of 20:1, 8:1, 5:1, 2:1, 1:1, 1:2, 1:4, 1:10, 0:100, and collected; The Fr.YC obtained in step C corresponds to the fraction eluted with petroleum ether:ethyl acetate = 2:1; Step D: The methanol-water solvent elution system is 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, and the fractions are collected and detected and combined by TLC to obtain the following components, which are named Fr.YC1-13 respectively; The Fr.YC 6 obtained in step D corresponds to the portion eluted with the methanol-water solvent system = 60:40, i.e., 60% methanol.

4. The method for preparing the compound according to claim 2, 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 the reflux extraction in the extraction method of step A are as follows: the extraction conditions 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; In step C, 80 g of the ethyl acetate fraction was separated by normal phase silica gel column chromatography, and petroleum ether-ethyl acetate was used as the elution system to perform gradient elution according to 20:1, 8:1, 5:1, 2:1, 1:1, 1:2, 1:4, 1:10, and 0:100, and the fractions were collected and 127 bottles of fractions were connected; then the collected 127 bottles of fractions were inspected and combined by TLC to obtain 7 components, named Fr.YA to Fr.YG, among which 1-15 was Fr.YA, 26-37 was Fr.YB, 38-52 was Fr.YC, 53-68 was Fr.YD, 69-90 was Fr.YE, 91-109 was Fr.YF, and 110-127 was Fr.YG; Fr.YC was collected for later use, and the obtained Fr.YC corresponded to the part eluted with petroleum ether: ethyl acetate = 2:1; In step D, 80 g of the ethyl acetate fraction was taken according to step C, and the methanol-water solvent elution system was 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, and 90 bottles of fractions were connected; TLC was used to detect and merge, and the following components were obtained in sequence, which were named Fr.YC1-13, respectively, wherein: 1-4 is Fr.YC 1, 5-9 is Fr.YC 2, 10-13 is Fr.YC 3, 14-19 is Fr.YC4, 20-24 is Fr.YC 5, 25-28 is Fr.YC 6, 29-33 is Fr.YC 7, 34-38 is Fr.YC 8, 39-44 is Fr.YC 9, 45-48 is Fr.YC 10, and 49-62 is Fr.YC 11, 63-79 are Fr.YC 12, 80-90 are Fr.YC 13; the obtained Fr.YC 6 corresponds to the part eluted with the methanol-water solvent system = 60:40, i.e. 60% methanol; Step E was purified by semi-preparative liquid phase, with 62% methanol solution as the mobile phase, and the elution time was 33 min.

5. The method for preparing the compound according to claim 2, characterized in that: The steps include: 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. Take the ethyl acetate fraction and separate it by normal phase silica gel column chromatography, use petroleum ether-ethyl acetate = 20:1, 8:1, 5:1, 2:1, 1:1, 1:2, 1:4, 1:10, 0:100 as the elution system for gradient elution, collect the fractions, connect 127 bottles of fractions in total, check and merge them by TLC, and get 7 components in turn, named Fr.YA~Fr.YG, among which 1-15 is Fr.YA, 26-37 is Fr.YB, 38-52 is Fr.YC, 53-68 is Fr.YD, 69- 90 is Fr.YE, 91-109 are Fr.YF, 110-127 are Fr.YG, and Fr.YC is collected for later use; D. Fr.YC obtained in step C is separated by MCI medium pressure column, and gradient elution is performed with methanol-water = 50:50, 60:40, 70:30, 80:20, 90:10, 100:0 as the solvent system, and the fractions are collected, and a total of 90 fractions are connected to the bottles, and the fractions are detected and combined by TLC to obtain the following components, which are named Fr.YC1-13 respectively, among which: 1-4 is Fr.YC 1, 5-9 are Fr.YC 2, 10-13 are Fr.YC 3, 14-19 are Fr.YC 4, 20-24 are Fr.YC 5, 25-28 are Fr.YC 6, 29-33 are Fr.YC 7, 34-38 are Fr.YC 8, 39-44 are Fr.YC 9, 45-48 are Fr.YC 10, 49-62 are Fr.YC 11, 63-79 are Fr.YC 12, 80-90 are Fr.YC 13, collect Fr.YC 6 for backup; E. Fr.YC 6 obtained in step D was purified by semi-preparative liquid phase purification using 62% methanol solution as the mobile phase and the elution time was 33 min to obtain the compound of formula I.

6. The compound according to claim 1 is used in the preparation of anti-inflammatory drugs, cosmetics and veterinary drugs.

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

8. The cosmetic prepared from the compound of claim 1, characterized in that: The cosmetics are prepared by using the compound of formula I as raw material and adding auxiliary ingredients acceptable to cosmetics.

9. The veterinary drug prepared from the compound according to claim 1, characterized in that: The veterinary drug is prepared by using the compound of formula I as raw material and adding auxiliary ingredients acceptable to veterinary drugs.

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