Aurone-lignan glycoside compound separated from Tibetan monascus and application of aurone-lignan glycoside compound in resisting non-alcoholic fatty liver
By isolating and preparing novel quercetin glycosides from saffron, the problem of the lack of effective anti-non-alcoholic fatty liver drugs in the prior art has been solved. The new compounds significantly reduce relevant serum indicators and increase beneficial indicators, demonstrating significant anti-non-alcoholic fatty liver activity.
Patent Information
- Application Number
- CN202610079219.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-21
- Publication Date
- 2026-05-05
AI Technical Summary
Current technologies have failed to effectively utilize the chemical components in saffron to develop significant anti-non-alcoholic fatty liver drugs, and there is a lack of effective treatments for non-alcoholic fatty liver disease.
A novel saffron lignan glycoside compound was isolated and prepared from saffron yeast. Its structure was analyzed using modern spectroscopic techniques, and its effect on non-alcoholic fatty liver disease was detected by biochemical methods. The compound was obtained by a multi-step extraction and purification method.
This compound significantly reduced the levels of ALP, AST, ALT, TG, TC, and LDL-C in the serum of golden hamsters, a model of non-alcoholic fatty liver disease, and significantly increased the levels of APN and HDL-C, demonstrating significant anti-non-alcoholic fatty liver activity.
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Figure CN121974967A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel saffron lignan glycoside compound isolated from saffron yeast, its preparation method, and its use in the preparation of drugs for treating non-alcoholic fatty liver disease. Technical Background
[0002] Saffron red yeast rice is a functional red yeast rice produced by solid-state fermentation of the fruit of barley (a member of the Poaceae family) after inoculation with Monascus purpureus. It possesses both medicinal and edible properties. As an important Tibetan medicine, saffron red yeast rice is recorded in both the *Compendium of Materia Medica* and the *Dictionary of Chinese Medicine*. It has various effects, including promoting blood circulation, invigorating blood and removing blood stasis, soothing the liver and regulating qi, aiding digestion, and strengthening the spleen and stomach. It is suitable for treating symptoms such as hypochondriac pain and chest tightness caused by liver qi stagnation, and abdominal distension, loss of appetite, and stomach pain caused by spleen and stomach qi stagnation. Previous studies have found that saffron red yeast rice contains abundant chemical components, including flavonoids, lignans, alkaloids, red yeast pigments, monacolins, lactones, polysaccharides, statins, sterols, and enzymes. Modern pharmacological studies have found that saffron red yeast rice has multiple effects, including anti-aging, anti-tumor, anti-inflammatory, anti-fatigue, anticonvulsant, liver protection, lowering blood lipids, lowering blood pressure, and lowering blood sugar. Its liver protection effects are particularly noteworthy. Therefore, saffron yeast has potential development value in the prevention and treatment of liver diseases. Summary of the Invention
[0003] To further explore the potential medicinal value of saffron red yeast rice in protecting the liver, this invention selected saffron red yeast rice as the research object, from which a novel hesperidin lignan glycoside compound was isolated, and its chemical structure was analyzed and identified using modern spectroscopic techniques (NMR, MS, IR, UV, etc.). Simultaneously, this invention used biochemical methods to detect blood lipid indicators (TG, TC, LDL-C, HDL-C) and liver function indicators (ALP, AST, ALT, APN) in golden hamsters to evaluate the anti-non-alcoholic fatty liver disease (NAFLD) activity of this compound. This compound significantly reduced the serum levels of ALP, AST, ALT, TG, TC, and LDL-C in golden hamsters with NAFLD, and significantly increased the levels of APN and HDL-C, demonstrating significant anti-NAFLD activity.
[0004] The present invention aims to provide a novel saffron lignan glycoside compound isolated from saffron, as well as a method for preparing the compound and its use in the preparation of a drug for treating non-alcoholic fatty liver disease.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A novel hesperidin lignan glycoside compound isolated from saffron (denoted as Compound I) or its pharmaceutically acceptable salt, with the structure shown in Formula I:
[0007]
[0008] Ⅰ
[0009] The chemical name of compound I is: (7E)-5-hydroxy-6-rutino-8-(12,13-dimethyl-11-carbonyl-pentaconitone)-9''-(3'',4''-dihydroxy-styryl)-9''-(4'-hydroxy-phenyl)-orange ketone.
[0010] The molecular formula of compound I is C 41 H 42 O 18 .
[0011] Another object of the present invention is to provide a method for preparing the aforementioned orange ketone lignan glycoside compound, comprising the following steps:
[0012] Step (1): Extract saffron yeast with 90-100% ethanol by heating and reflux for 3-5 times, 3-5 hours each time, to obtain an extract. Extract the residue with 60-80% ethanol by heating and reflux for 3-5 times, 3-5 hours each time, to obtain an extract. Combine the extracts to obtain the total saffron yeast extract.
[0013] Step (2): The total extract of saffron yeast is concentrated and dried under reduced pressure to obtain a dry total paste;
[0014] Step (3): The total saffron koji paste is uniformly suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively.
[0015] Step (4): The n-butanol extract was loaded onto a macroporous adsorption resin column and eluted sequentially with 15%, 30%, 50%, and 100% ethanol to obtain four fractions: A, B, C, and D.
[0016] Step (5): Fraction B is subjected to normal phase silica gel column chromatography with gradient elution using dichloromethane and methanol in a volume ratio of 20:1 to 1:1 to obtain four subfractions: subfraction B-1, subfraction B-2, subfraction B-3, and subfraction B-4.
[0017] Step (6): Load subfraction B-3 onto a Sephadex LH-20 gel column and elute with 80%→100% methanol to obtain three subfractions: subfraction B-3-1, subfraction B-3-2, and subfraction B-3-3.
[0018] Step (7): Load fraction B-3-2 onto a Toyopearl HW-40C gel column and elute with 85%→95% methanol to obtain three small fractions: fraction B-3-2-1, fraction B-3-2-2, and fraction B-3-2-3.
[0019] Step (8): The small fraction B-3-2-2 is separated by preparative liquid chromatography to obtain the orange ketone lignan glycoside compound.
[0020] In step (1), when saffron is extracted by heating and reflux with 90-100% ethanol, the ratio of saffron to 90-100% ethanol is 1:1-1:2 kg / L for each extraction; when the residue is extracted by heating and reflux with 60-80% ethanol, the ratio of residue (calculated as saffron) to 60-80% ethanol is 1:1-1:2 kg / L for each extraction.
[0021] Preferably, the dried saffron is extracted by heating and refluxing with 95% ethanol four times for 4 hours each time, according to a material-to-liquid ratio of 1:1 kg / L, to obtain an extract; the residue is then extracted by heating and refluxing with 70% ethanol four times for 4 hours each time, according to a material-to-liquid ratio of 1:1 kg / L, to obtain an extract; the extracts are combined to obtain the total saffron extract.
[0022] In step (2), the temperature of the vacuum concentration and drying is 45-55℃, and the pressure is under vacuum conditions.
[0023] In step (3), the total saffron slurry is evenly suspended in water according to the volume ratio of saffron slurry to water of 1:1-1:3.
[0024] In step (4), the macroporous adsorption resin is D101 type macroporous adsorption resin.
[0025] In step (5), the normal phase silica gel column chromatography uses 200-300 mesh silica gel.
[0026] In step (8), the conditions for preparing liquid chromatography are: YMC-Pack ODS-A column (250 × 20 mm, 5 μm), mobile phase: 15-40% methanol, absorption wavelength: 150-400 nm, flow rate: 2-4 mL / min.
[0027] Another object of the present invention is to provide the use of the aforementioned novel quercetin glycoside compounds in the preparation of lipid-lowering drugs.
[0028] Another object of the present invention is to provide the use of the aforementioned novel quercetin glycosides in the preparation of a medicament for treating non-alcoholic fatty liver disease.
[0029] Another object of the present invention is to provide a pharmaceutical composition comprising the aforementioned neo-orange lignan glycoside compound as the active ingredient, and formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.
[0030] The dosage forms mentioned are tablets, capsules, granules, pills, oral liquids, and suspensions.
[0031] Another object of the present invention is to provide the application of the aforementioned novel quercetin glycoside compounds in the preparation of health foods that help maintain healthy blood lipid (cholesterol / triglyceride) levels.
[0032] The beneficial effects of this invention are:
[0033] This invention selects saffron as the research object, as this medicinal material is abundant and readily available. This invention employs modern separation and purification methods and spectroscopic techniques to obtain a novel hesperidin lignan glycoside compound from the extract of saffron. This compound has a novel structure, and its preparation method is simple, rapid, and easy to operate.
[0034] The novel citrione lignan glycoside compound of this invention can significantly reduce the levels of ALP, AST, ALT, TG, TC, and LDL-C in the serum of golden hamsters, a model of non-alcoholic fatty liver disease, and can significantly increase the levels of APN and HDL-C. This invention discloses for the first time the use of this compound in the preparation of anti-non-alcoholic fatty liver drugs. Attached Figure Description
[0035] Figure 1 This is a flowchart illustrating the preparation process of the novel compound I of this invention.
[0036] Figure 2 HMBC of the novel compound I of this invention and 1 H- 1 H COSY coupling correlation diagram.
[0037] Figure 3 This is a NOESY correlation diagram of the novel compound I of this invention. Detailed Implementation
[0038] 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.
[0039] Example 1
[0040] like Figure 1 As shown, a new citronellol glycoside compound was isolated from saffron yeast, and the preparation method is as follows:
[0041] Step (1): Extract 10.0 kg of dried saffron yeast with 95% ethanol by reflux 4 times, using 10 L of 95% ethanol for 4 hours each time, to obtain extract T1; extract the residue with 70% ethanol by reflux 4 times, using 10 L of 70% ethanol for 4 hours each time, to obtain extract T2; combine extract T1 and extract T2 to obtain total saffron yeast extract.
[0042] Step (2): Using a rotary evaporator, the total extract of saffron was concentrated and dried under reduced pressure at low temperature (55 °C) and vacuum conditions to obtain dried total saffron paste (1.5 kg).
[0043] Step (3): The total saffron spore paste was uniformly suspended in water (the volume ratio of total saffron spore paste to water = 1:2, V / V), and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract (189.6 g), ethyl acetate extract (363.7 g) and n-butanol extract (588.6 g), respectively.
[0044] Step (4): The n-butanol extract was loaded onto a macroporous adsorption resin (D101 type) column and eluted sequentially with 15%, 30%, 50%, and 100% ethanol to obtain four fractions, which were denoted as fraction A (89.4 g), fraction B (130.5 g), fraction C (180.3 g), and fraction D (68.6 g).
[0045] Step (5): Fraction B was subjected to normal-phase silica gel (200-300 mesh) column chromatography with gradient elution using dichloromethane and methanol = 20:1→10:1→5:1→1:1 (V / V) to obtain 4 subfractions: subfraction B-1 (26.7 g), subfraction B-2 (38.3 g), subfraction B-3 (40.6 g), and subfraction B-4 (19.9 g);
[0046] Step (6): Load subfraction B-3 onto a Sephadex LH-20 gel column and elute with 80%→90%→100% methanol to obtain three subfractions: subfraction B-3-1 (11.3 g), subfraction B-3-2 (15.6 g), and subfraction B-3-3 (9.8 g).
[0047] Step (7): Load fraction B-3-2 onto a Toyopearl HW-40C gel column and elute with 85%→90%→95% methanol to obtain three small fractions: fraction B-3-2-1 (4.5 g), fraction B-3-2-2 (6.5 g), and fraction B-3-2-3 (3.6 g).
[0048] Step (8): The small fraction B-3-2-2 was subjected to preparative liquid phase separation (YMC-Pack ODS-A column: length 250 × inner diameter 20 mm, particle size 5 μm; mobile phase: 28% methanol, absorption wavelength: 210 nm, flow rate: 3.5 mL / min). After multiple preparations, the new compound I (8.96 mg) was obtained.
[0049] Compound I is a yellow solid, HR-ESI-MS m / z 845.2267 [M+Na] + Its molecular formula is C 41 H 42 O 18 (calcd. for C) 41 H 42 O 18 Na, 845.2269). UV (MeOH)λ of compound I. max : 210, 255, 270, 395 nm; IRν max : 3408, 1676, 1604, 1385 cm -1 In compound I 1 In the H NMR spectrum, there is a typical set of AA'BB' system signal peaks: δ H 7.15 (2H, d, J = 8.5 Hz, H-2' / 6'), 6.71 (2H, d, J = 8.5 Hz, H-3' / 5'), combined with its 13 C NMR spectral data δ C 130.5 (C-1'), 128.5 (C-2' / 6'), 116.4 (C-3' / 5'), 157.4 (C-4') (Table 1). Based on the correlations between H-2' / 6' and C-9'', and H-8'' and C-1' in the HMBC correlation spectrum of compound I, it is inferred that the AA'BB' system fragment is connected at the C-9'' position (Figure 2). Simultaneously, there is an ABX system signal peak: δ H 6.65 (1H, d, J = 2.0 Hz, H-2''), 6.75 (1H, dd, J = 8.0, 2.0 Hz, H-6''), 6.52 (1H, d, J = 8.0 Hz, H-5''), combined with its 13 C NMR spectral data δ C129.6 (C-1''), 115.7 (C-2''), 145.1 (C-3''), 144.3 (C-4''), 118.5 (C-5''), 120.8 (C-6'') (Table 1). Based on the correlations between H-7'' and C-2'' / 6'', and H-8'' and C-1'' in the HMBC correlation spectrum of this compound, it is inferred that the AA'BB' system fragment is connected at the C-7'' position (Figure 2). Additionally, a typical 12,13-dimethyl-11-carbonyl-pentacolone ring is present: δ H 7.85 (1H, s, H-9), 1.50 (6H, s, 12 / 13-CH3), combined with its 13 C NMR spectral data δ C 131.2 (C-8), 137.6 (C-9), 78.6 (C-10), 166.1 (C-11), 25.3 (12 / 13-CH3) (Table 1). Based on H-9 and C-7 / 11 in the HMBC correlation spectrum of this compound, it is inferred that the five-membered lactone ring is attached at the C-7 position (Figure 2). In compound I... 1 H NMR and 13 In the mid-field plot of C NMR, there is a set of typical rutin-based characteristic signals: δ H 5.81 (1H, d, J = 7.8 Hz, H-1'''), 3.87 (1H, m, H-2'''), 3.56 (1H, m, H-3'''), 3.42 (1H, m, H-4'''), 3.98 (1H, m, H-5'''), 3.52 (1H, dd, J = 14.4, 7.2 Hz, H-6'''a), 3.90 (1H, J = 7.2, 3.6 Hz, H-6'''b) and δ C 102.0 (C-1'''), 72.0 (C-2'''), 69.3 (C-3'''), 67.7 (C-4'''), 70.3 (C-5'''), 63.8 (C-6''); 4.99 (1H, d, J = 1.8 Hz, H-1''''), 3.67 (1H, m, H-2''''), 3.55 (1H, m, H-3''''), 3.34 (1H, m, H-4''''), 3.79 (1H, m, H-5''''), 1.22 (1H, d, J = 7.2 Hz, H-6'''') and δ C99.5 (C-1''''), 71.7 (C-2''''), 68.6 (C-3''''), 72.9 (C-4''''), 64.0 (C-5''''), 15.2 (C-6''''') (Table 1). According to the HMBC correlation diagram of the rutin base: H-1''' is correlated with C-6 / 3''', H-6''' is correlated with C-4''' / 1'''', H-1'''' is correlated with C-6''' / 3'''', H-4'''' is correlated with C-6'''', it is determined that the rutin base is connected at the C-6 position (Figure 2). In addition, the other fragment linkage positions of this compound can be determined based on its HMBC correlation spectrum: H-4 and C-3 / 6, H-3' / 5' and C-1', H-8'' and C-2'', H-2'' / 6'' and C-4'' (Figure 2); 1 H- 1 H COSY correlation spectra: H-2' and H-3', H-5' and H-6', H-5'' and H-6'', H-7'' and H-8'' (Figure 2); NOESY correlation spectra: H-2'' and H-7'', H-6'' and H-8'', H-4'''' and 6''''-CH3, H-9 and 13-CH3 (Figure 3), etc., were verified. Combined with compound I... 1 H NMR, 13 C NMR data and HMBC, 1 H- 1 H COSY and NOESY spectra coupled with relevant information, and a SciFinder search, identified compound I as a novel orange ketone lignan glycoside. The chemical name of compound I is: (7E)-5-hydroxy-6-rutinosyl-8-(12,13-dimethyl-11-carbonyl-pentaconitone)-9''-(3'',4''-dihydroxy-styryl)-9''-(4'-hydroxy-phenyl)-orange ketone.
[0050] Table 1. Compound I 1 H NMR, 13 C NMR and HMBC related data
[0051]
[0052] Note: 1 H NMR (600 MHz, CD3OD), 13 C NMR (125 MHz, CD3OD).
[0053] Example 2
[0054] Assay of Compound I's Anti-Non-Alcoholic Fatty Liver Activity
[0055] 1. Animals, reagents and instruments
[0056] SPF-grade golden hamsters (180-220 g, male), regular feed, high-fat feed, simvastatin, chloric acid hydrate, fully automated biochemical analyzer, centrifuge, enzyme-linked immunosorbent assay (ELISA) reader, etc.
[0057] 2. Animal grouping, model establishment, and drug administration
[0058] Sixty golden hamsters were randomly divided into six groups of ten each. After one week of normal feeding, the control group was fed a normal diet, while the other five groups were fed a high-fat diet to establish the model. Feeding continued for six weeks, with weekly weight measurements. Starting from week four, the four drug treatment groups were administered drugs via gavage: simvastatin (7.5 mg / kg daily), low-dose compound I (0.024 g / kg daily), medium-dose compound I (0.12 g / kg daily), and high-dose compound I (0.6 g / kg daily). Simvastatin and compound I were both prepared using 0.5% CMC-Na solution before gavage. The control and model groups were administered the same amount of distilled water via gavage. The hamsters were fasted but allowed water the night before the experiment ended.
[0059] 3. Serum Sample Preparation and Measurement
[0060] Golden hamsters were anesthetized with 10% chloral hydrate (0.3 mL / 100g), and blood was collected from the abdominal aorta. Serum was then separated (3000 r, 15 min). Serum biochemical indicators (ALP, AST, ALT, TG, TC, LDL-C, HDL-C) were detected using a fully automated biochemical analyzer. APN (adiponectin) levels were detected using a rat adiponectin kit ELISA.
[0061] 4. Screening results of the anti-non-alcoholic fatty liver activity of compound I
[0062] The results are shown in Tables 2 and 3. Compared with the normal control group, the model group showed significantly increased levels of ALP, AST, ALT, TG, TC, and LDL-C (P < 0.01), while significantly decreased levels of APN and HDL-C (P < 0.01). Compared with the model group, simvastatin significantly reduced serum levels of ALP, AST, ALT, TG, TC, and LDL-C (P < 0.05 or P < 0.01) and significantly increased levels of APN and HDL-C (P < 0.05 or P < 0.01). Compared with the model group, low, medium, and high doses of compound I significantly reduced serum levels of ALP, AST, ALT, TG, TC, and LDL-C (P < 0.05 or P < 0.01) and significantly increased levels of APN and HDL-C (P < 0.05 or P < 0.01), which were statistically significant.
[0063] Table 2. Effects of compound I on liver function indicators in rats of each group (mean ± standard deviation)
[0064]
[0065] Note: Compared with the normal control group: ## P < 0.01; Compared with the model group: * P < 0.05 ** P < 0.01.
[0066] Table 3. Effects of Compound I on blood lipid levels in rats of each group (mean ± standard deviation)
[0067]
[0068] Note: Compared with the normal control group: ## P < 0.01; Compared with the model group: * P < 0.05 ** P < 0.01.
[0069] In summary, low, medium, and high doses of compound I significantly reduced serum ALP, AST, ALT, TG, TC, and LDL-C levels in golden hamsters with a non-alcoholic fatty liver disease (NAFLD) model (P < 0.05 or P < 0.01), and significantly increased APN and HDL-C levels (P < 0.05 or P < 0.01), showing statistically significant differences. Therefore, compound I exhibits significant anti-NAFLD activity, which is of important scientific significance for the development of novel anti-NAFLD drugs from saffron.
Claims
1. Orange ketone lignan glycosides with structures as shown in Formula I, or pharmaceutically acceptable salts thereof: ； Ⅰ。 2. A method for preparing the orange ketone lignan glycoside compound according to claim 1, characterized in that: Includes the following steps: Step (1): Extract saffron yeast with 90-100% ethanol by heating and reflux for 3-5 times, 3-5 hours each time, to obtain an extract. Extract the residue with 60-80% ethanol by heating and reflux for 3-5 times, 3-5 hours each time, to obtain an extract. Combine the extracts to obtain the total saffron yeast extract. Step (2): The total extract of saffron yeast is concentrated and dried under reduced pressure to obtain a dry total paste; Step (3): The total saffron koji paste is uniformly suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively. Step (4): The n-butanol extract was loaded onto a macroporous adsorption resin column and eluted sequentially with 15%, 30%, 50%, and 100% ethanol to obtain four fractions: A, B, C, and D. Step (5): Fraction B is subjected to normal phase silica gel column chromatography with gradient elution using dichloromethane and methanol in a volume ratio of 20:1 to 1:1 to obtain four subfractions: subfraction B-1, subfraction B-2, subfraction B-3, and subfraction B-4. Step (6): Load subfraction B-3 onto a Sephadex LH-20 gel column and elute with 80%→100% methanol to obtain three subfractions: subfraction B-3-1, subfraction B-3-2, and subfraction B-3-3. Step (7): Load fraction B-3-2 onto a Toyopearl HW-40C gel column and elute with 85%→95% methanol to obtain three small fractions: fraction B-3-2-1, fraction B-3-2-2, and fraction B-3-2-3. Step (8): The small fraction B-3-2-2 is separated by preparative liquid chromatography to obtain the orange ketone lignan glycoside compound.
3. The method for preparing the orange ketone lignan glycoside compound according to claim 2, characterized in that: In step (2), the temperature of the vacuum concentration and drying is 45-55℃.
4. The method for preparing the orange ketone lignan glycoside compound according to claim 2, characterized in that: In step (4), the macroporous adsorption resin is D101 type macroporous adsorption resin.
5. The method for preparing the orange ketone lignan glycoside compound according to claim 2, characterized in that: In step (8), the conditions for preparing liquid chromatography are: YMC-Pack ODS-A column (250 × 20 mm, 5 μm), mobile phase: 15-40% methanol, absorption wavelength: 150-400 nm, flow rate: 2-4 mL / min.
6. The use of the orange ketone lignan glycoside compound according to claim 1 in the preparation of lipid-lowering drugs.
7. The use of the orange ketone lignan glycoside compound according to claim 1 in the preparation of a drug for treating non-alcoholic fatty liver disease.
8. A pharmaceutical composition, characterized in that: The pharmaceutical composition uses the orange lignan glycoside compound of claim 1 as the active ingredient and is formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.
9. The pharmaceutical composition according to claim 8, characterized in that: The dosage forms mentioned are tablets, capsules, granules, pills, oral liquids, and suspensions.
10. The use of the orange ketone lignan glycoside compound of claim 1 in the preparation of health foods that help maintain healthy blood lipid (cholesterol / triglyceride) levels.