A rice bran alkaloid with lipase inhibitory activity, a pharmaceutical composition, and a preparation method and application thereof

Abstract

The present invention discloses a rice bran alkaloid with lipase inhibitory activity, a pharmaceutical composition, a preparation method and an application thereof. The alkaloid is 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid, and its structural formula is shown as formula I: #imgabs0# (Formula I). The alkaloid is obtained by extraction from rice bran and is a natural product with characteristics such as low toxicity and side effects and good activity. It has been confirmed by in vitro pharmacological experiments that its IC 50 value is 187.1 μM, and it has excellent pancreatic lipase inhibitory activity, and is expected to be developed into a new type of pancreatic lipase inhibitor drug as a lead compound. The preparation method of the present invention can effectively extract the alkaloid in rice bran, the preparation process conditions are controllable, the extraction is convenient, environmentally friendly and has potential good economic benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicines, and particularly relates to a rice bran alkaloid with lipase inhibitory activity, a pharmaceutical composition, and a preparation method and application thereof. Background Art

[0002] Obesity is a chronic metabolic disease caused by the long-term imbalance between energy absorption and consumption in the human body, resulting in excessive body fat or abnormal distribution and weight gain.

[0003] Pancreatic lipase is a key enzyme involved in fat absorption and metabolism in the human body. After being secreted by the pancreas, it is released into the gastrointestinal system and is responsible for hydrolyzing about 50-70% of the fat in the human diet. Pancreatic lipase inhibitors can effectively reduce the decomposition and absorption of fat in the digestive system and play a role in improving obesity and hyperlipidemia, thus becoming a popular target for the treatment of obesity.

[0004] Orlistat is a commonly used lipase inhibitor in clinical practice at present, which can effectively reduce the absorption of fat in the human body and improve obesity. However, orlistat is accompanied by many gastrointestinal adverse reactions in clinical applications, such as flatulence, abdominal cramps, fatty stools, etc. Therefore, it is of great significance to find new lipase inhibitors. Natural products play an important role in drug discovery with their good activity and small toxic and side effects, and have always been the focus of attention of medicinal chemists, providing a source for natural product-type active substances with lipase inhibitory activity. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a rice bran alkaloid with lipase inhibitory activity, which comes from the plant rice bran, has the characteristics of stable properties and easy preservation, has good pancreatic lipase inhibitory activity, and is expected to be developed into a new lipase inhibitory drug as a lead compound.

[0006] The second object of the present invention is to provide a preparation method of the above-mentioned rice bran alkaloid with lipase inhibitory activity.

[0007] The third object of the present invention is to provide a medicinal salt or an esterified derivative of the above-mentioned rice bran alkaloid with lipase inhibitory activity.

[0008] The fourth object of the present invention is to provide a pharmaceutical composition.

[0009] The fifth object of the present invention is to provide an application of the rice bran alkaloid with lipase inhibitory activity.

[0010] One of the objects of the present invention can be achieved by adopting the following technical solutions:

[0011] A rice bran alkaloid with lipase inhibitory activity, wherein the rice bran alkaloid with lipase inhibitory activity is 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid, and the structure is shown in Formula I:

[0012]

[0013] The second object of the present invention can be achieved by adopting the following technical solutions:

[0014] A preparation method of a rice bran alkaloid with lipase inhibitory activity, comprising the following steps:

[0015] S1. After removing the oil from the rice bran, extract it with an aqueous solution of alcohol, and collect the residue after extraction;

[0016] S2. Extract the collected residue with an alkali solution, then adjust the solution to acidic to obtain an extract, extract the extract with an organic solvent, collect the organic phase, and obtain an extract after removing the solvent from the organic phase;

[0017] S3. Subject the extract to normal-phase silica gel column chromatography, and perform gradient elution with chloroform / methanol as the eluent, and collect the elution fraction with a chloroform / methanol volume ratio of (90:10)-(70:30);

[0018] S4. Subject the elution fraction collected in S3 to ODS-A medium-pressure column chromatography, and perform gradient elution with methanol / water as the eluent, and collect the elution fraction with a methanol / water volume ratio of (20:80)-(40:60);

[0019] S5. The fraction collected in step S4 is separated and purified by high-performance liquid chromatography to obtain 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

[0020] Further, in step S1, the removal of oil is achieved by subcritical extraction; the solvent for subcritical extraction is an alkane with C3-C6; the aqueous solution of alcohol is an aqueous solution of methanol or ethanol, and the volume of alcohol in the aqueous solution of alcohol is 75%-95%.

[0021] Further, in step S2, the alkali solution is one or a combination of two of an aqueous solution of sodium hydroxide or potassium hydroxide; the concentration of the alkali solution is 0.5 mol / L-3 mol / L; the volume-mass ratio of the alkali solution to the residue is (10-30 mL):1 g; the extraction time with the alkali solution is 1-5 h; the extraction with the alkali solution is carried out in an inert gas environment.

[0022] Further, in step S2, adjusting the solution to acidic means adjusting the pH value of the solution to 1-2; adjusting the solution to acidic uses one or a combination of two of hydrochloric acid or sulfuric acid.

[0023] Further, in step S3, chloroform / methanol is used as the eluent for gradient elution, and the volume percentage of chloroform is gradually decreased by 10% from 100% until 60%;

[0024] In step S4, methanol / water is used as the eluent for gradient elution, and the volume percentage of methanol is gradually increased by 10% from 20% until 90%.

[0025] Further, in step S5, the addition for high performance liquid separation is as follows: an YMC-Pack ODS-A chromatographic column is used, the chromatographic column size is 20×250 mm, the particle size is 5 μm, the mobile phase is an acetonitrile aqueous solution with a volume percentage of 16%, and the flow rate is 5 mL / min.

[0026] The third object of the present invention can be achieved by adopting the following technical solutions:

[0027] A medicinal salt of a rice bran alkaloid with lipase inhibitory activity or an esterified derivative thereof.

[0028] The fourth object of the present invention can be achieved by adopting the following technical solutions:

[0029] A pharmaceutical composition comprising the above-mentioned rice bran alkaloid with lipase inhibitory activity or a medicinal salt of the above-mentioned rice bran alkaloid with lipase inhibitory activity or an esterified derivative thereof.

[0030] The fifth object of the present invention can be achieved by adopting the following technical solutions:

[0031] Use of a rice bran alkaloid with lipase inhibitory activity or a medicinal salt of a rice bran alkaloid with lipase inhibitory activity or an esterified derivative thereof in the preparation of a lipase inhibitor drug.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. The rice bran alkaloid with lipase inhibitory activity of the present invention is extracted from rice bran and is a natural product, which has the characteristics of small toxic and side effects and good activity; it is confirmed by in vitro pharmacological experiments that it has excellent pancreatic lipase inhibitory activity, and its half inhibitory concentration is 187.1 μM; the raw material source is wide and it can be utilized and produced on a large scale.

[0034] 2. A preparation method of rice bran alkaloids with lipase inhibitory activity according to the present invention. After degreasing and treating rice bran with an ethanol aqueous solution, extraction is carried out under an alkaline solution, acidified, extracted with an organic solvent, and then purified by chromatography and high performance liquid separation to obtain 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid. This method can effectively extract the alkaloids in rice bran, with controllable preparation process conditions, convenient extraction, environmental friendliness and potential good economic benefits.

[0035] 3. The present invention also discloses and protects a medicinal salt or its esterified derivative of rice bran alkaloids with lipase inhibitory activity, which is convenient for drug production and application.

[0036] 4. A pharmaceutical composition of the present invention contains an active ingredient of rice bran alkaloids with lipase inhibitory activity and realizes its application in the preparation of lipase inhibitor drugs. This pharmaceutical composition also has characteristics such as low toxicity and side effects and good activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the 1 1H NMR spectrum of the extract of the example;

[0038] Figure 2 is the 13 13C NMR spectrum of the extract of the example;

[0039] Figure 3 is the 1 H- 1 H COSY spectrum of the extract of the example;

[0040] Figure 4 is the HSQC spectrum of the extract of the example;

[0041] Figure 5 is the HMBC spectrum of the extract of the example;

[0042] Figure 6 is the main 1 H- 1 H COSY and HMBC correlation signals of the extract of the example;

[0043] Figure 7 is the HR-ESI-MS spectrum of the extract of the example. DETAILED DESCRIPTION OF THE INVENTION

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0045] Rice bran is a by-product generated during the processing of the plant Oryza sativa L. of the Gramineae family, mainly including pericarp, seed coat, aleurone layer and embryo. Rice bran contains a variety of active substances with rich contents. Therefore, the present invention extracts the rice bran alkaloid 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid with lipase inhibitory activity from rice bran, which has excellent pancreatic lipase inhibitory activity.

[0046] A rice bran alkaloid with lipase inhibitory activity, wherein the rice bran alkaloid with lipase inhibitory activity is 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid, and the structure is shown in Formula I:

[0047]

[0048] The rice bran alkaloid with lipase inhibitory activity is 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid, which has the main structure of 1,2,3,4-tetrahydroquinoline, and the substitution of dihydroxy and the substitution of carboxyl and one of the hydroxyl groups on the same carbon atom at the 4th carbon atom make the 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid have excellent pancreatic lipase inhibitory activity; and the 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid is extracted from rice bran and is obtained by extracting natural products, so it has small toxic and side effects, and is stable in nature and easy to store.

[0049] A preparation method of a rice bran alkaloid with lipase inhibitory activity, comprising the following steps:

[0050] S1. After removing the grease from the rice bran, it is extracted with an aqueous solution of alcohol, and the residue is collected after extraction;

[0051] S2. The collected residue is extracted with an alkaline solution, then the solution is adjusted to acidic to obtain an extract, the extract is extracted with an organic solvent, the organic phase is collected, and the organic phase is dried to obtain an extract after removing the solvent;

[0052] S3. The extract is subjected to normal-phase silica gel column chromatography, and gradient elution is carried out with chloroform / methanol as the eluent, and the elution fraction with a chloroform / methanol volume ratio of (90:10)-(70:30) is collected;

[0053] S4. Subject the elution components collected in S3 to medium-pressure column chromatography on ODS-A, and perform gradient elution using methanol / water as the eluent, and collect the elution components with a methanol / water volume ratio of (20:80)-(40:60);

[0054] S5. The components collected in step S4 are separated and purified by high-performance liquid chromatography to obtain 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

[0055] Rice bran contains parts such as seed coats and embryos, so it has more oil. The oil will dissolve in organic solvents and react with alkalis, so it will affect the extraction of alkaloids. Removing the ester from rice bran at the beginning of the extraction can reduce the subsequent extraction pressure. The target compound in the present invention is bound to rice bran fibers through chemical bonds. Therefore, after removing the ester, first extract with an aqueous solution of alcohol to remove other free components, and the target compound still binds to the rice bran fibers. Then, after collecting the extraction residue, the target compound is released by alkali hydrolysis for extraction; at the same time, the alkali solution can react with the carboxyl group of the target compound 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid to form carboxylate, or the hydroxyl group on the benzene ring forms phenolate, increasing its solubility in water; then when the solution is further adjusted to be acidic, the carboxylate in the solution is reduced to carboxylic acid again, and it can be separated from the aqueous solution by extraction with an organic solvent.

[0056] The purification steps sequentially select normal-phase silica gel column chromatography, medium-pressure column chromatography on ODS-A, and high-performance liquid separation, and the corresponding eluents are chloroform / methanol and methanol / water. Through gradient elution, components containing 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid at a specific eluent concentration can be obtained. Finally, pure 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid can be obtained through high-performance liquid separation.

[0057] In one of the embodiments, in step S1, removing the oil is achieved by subcritical extraction; the solvent for subcritical extraction is an alkane with C3-C6.

[0058] Subcritical extraction is carried out according to the principle of similar solubility of organic substances. Therefore, in the present invention, an alkane is selected as the extraction agent to remove the oil. In order to facilitate the separation of the extraction agent and the oil, an alkane with a lower number of carbon atoms is further selected in the present invention, and an alkane with C3-C6 is selected; preferably, butane is selected as the extraction agent in the present invention.

[0059] In one of the embodiments, in step S1, the aqueous solution of alcohol is an aqueous solution of methanol or ethanol, and the volume of alcohol in the aqueous solution of alcohol is 95%-75%.

[0060] An aqueous solution of alcohol is used for extraction. Both ethanol and methanol are good alcohol reagents, and the volume of alcohol in the aqueous solution of alcohol is 95%-75%; preferably, the volume of alcohol in the aqueous solution of alcohol is 95%-85%; more preferably, the volume of alcohol in the aqueous solution of alcohol is 90%-95%.

[0061] In one embodiment, in step S2, the lye is one or a combination of two of an aqueous solution of sodium hydroxide or potassium hydroxide; the concentration of the lye is 0.5 mol / L - 3 mol / L; the volume-mass ratio of the lye to the residue is (10 - 30 mL) : 1 g; the time for extracting with the lye is 1 - 5 h; the extraction with the lye is carried out in an inert gas environment.

[0062] The lye can make 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid form a salt or the hydroxyl group on the benzene ring form a phenolate, thereby promoting dissolution in water and promoting leaching. Therefore, sodium hydroxide and potassium hydroxide, which are lye that easily react with carboxylic acids, and the concentration is between 0.5 mol / L - 3 mol / L; preferably, the concentration of the lye is 1 mol / L - 2.5 mol / L; preferably, the concentration of the lye is 1.5 mol / L - 2 mol / L; more preferably, the concentration of the lye is 2 mol / L. The liquid-solid ratio of the extraction residue to the lye is 1 g : (10 - 30 mL); preferably, the liquid-solid ratio of the extraction residue to the lye is 1 g : 20 mL. The extraction time is 1 - 5 h; preferably, the extraction time is 2 - 4 h; more preferably, the extraction time is 3 - 4 h. The extraction with the lye is carried out in an inert gas environment, and the inert gas environment is preferably a nitrogen environment.

[0063] In one embodiment, in step S2, adjusting the solution to acidic means adjusting the pH value of the solution to 1 - 2; hydrochloric acid or sulfuric acid, or a combination of the two, is used to adjust the solution to acidic. Any acid stronger than carboxylic acid can be used to adjust the pH, and hydrochloric acid or sulfuric acid is used in the present invention. Preferably, hydrochloric acid is used in the present invention to adjust the pH.

[0064] In one embodiment, organic solvent extraction is used. The organic solvent refers to an organic reagent that is immiscible with water, preferably ethyl acetate. When carrying out extraction, the dosage of the organic solvent is not fixedly limited. The extraction of the acidic solution can be repeated 3 - 5 times, and then the organic phases are combined. Preferably, the dosage of the organic solvent for each extraction is the same as the volume of the acidic solution, and the organic phases are combined after repeating the extraction 3 - 5 times.

[0065] In one embodiment, in step S3, chloroform / methanol is used as the eluent for gradient elution, and the volume percentage of chloroform is gradually reduced by 10% from 100% until 60%.

[0066] In step S4, gradient elution is performed using methanol / water as the eluent, and the volume percentage of methanol is gradually increased by 10% from 20% until 90%.

[0067] The extract is chromatographically separated by normal-phase silica gel column chromatography. Chloroform / methanol is a good eluent, and gradient elution is performed with a volume ratio of chloroform / methanol from 100:0 to 60:40. The eluents can be set to 100:0, 90:10, 80:20, 70:30, 60:40 respectively; a substance containing 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid can be obtained in the eluent components of (90:10)-(70:30); preferably, the components of the 80:20 eluent are collected to obtain a substance containing 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

[0068] Further chromatographic separation is performed by ODS-A medium-pressure column chromatography. Methanol / water is a good eluent, and gradient elution is performed with a volume ratio of methanol / water from 20:80 to 90:10. The eluents can be set to 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 respectively; a substance containing 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid can be obtained in the eluent components of (20:80)-(40:60); preferably, the components of the 30:70 eluent are collected to obtain a substance containing 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

[0069] In one embodiment, in step S3, gradient elution is performed using chloroform / methanol as the eluent, and the volume percentage of chloroform is gradually decreased by 10% from 100% until 70%, and the components of the eluent with a chloroform / methanol volume ratio of (90:10)-(70:30) are collected; preferably, the volume percentage of chloroform is gradually decreased by 10% from 100% until 80%, and the components of the eluent with a chloroform / methanol volume ratio of 80:20 are collected.

[0070] In step S4, gradient elution is performed using methanol / water as the eluent, and the volume percentage of methanol is gradually increased by 10% from 20% until 40%. Just collect the components of the eluent with a methanol / water volume ratio of (20:80)-(40:60). Preferably, the volume percentage of methanol is gradually increased by 10% from 20% until 30%, and just collect the components of the eluent with a methanol / water volume ratio of 30:70.

[0071] In one embodiment, the addition in the high performance liquid separation in step S5 is as follows: an YMC-Pack ODS-A chromatographic column is used, the size of the chromatographic column is 20×250 mm, the particle size is 5 μm, the mobile phase is an acetonitrile aqueous solution with a volume percentage of 16%, and the flow rate is 5 mL / min.

[0072] High performance liquid chromatography separation is a method that can further completely separate the extract. High performance liquid chromatography under various different conditions in the art can achieve the separation of the extract to obtain 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid. Preferably, in this application, an YMC-Pack ODS-A chromatographic column is used, the size of the chromatographic column is 20×250 mm, the particle size is 5 μm, the mobile phase is an acetonitrile aqueous solution with a volume percentage of 16%, and the separation is carried out at a flow rate of 5 mL / min. The substance obtained when the retention time tR is 62 min is 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

[0073] The present invention also discloses a medicinal salt or an esterified derivative of a rice bran alkaloid having lipase inhibitory activity, which is a medicinal salt or an esterified derivative of 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

[0074] The medicinal salt of an active compound is one of the conventional utilization methods in the art. The 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid in this application has dihydroxy and carboxyl groups, so its esterified derivative is also one of the utilization methods in the art.

[0075] The present invention discloses a pharmaceutical composition, which comprises the above-mentioned rice bran alkaloid having lipase inhibitory activity or a medicinal salt or an esterified derivative of the above-mentioned rice bran alkaloid having lipase inhibitory activity. The rice bran alkaloid having lipase inhibitory activity in this application or a medicinal salt or an esterified derivative of the above-mentioned rice bran alkaloid having lipase inhibitory activity can be combined with excipients or carriers permitted by the preparation or drug to prepare a pharmaceutical composition having lipase inhibitory activity and can be used for the treatment of obesity.

[0076] In one embodiment, the drug or pharmaceutical composition can be in dosage forms such as wettable powders, tablets, granules, capsules, oral liquids, dripping pills, injections, aerosols, etc.; controlled release or sustained release dosage forms or nano-formulations well known in the modern pharmaceutical industry can also be used.

[0077] The present invention discloses the use of a rice bran alkaloid having lipase inhibitory activity or a medicinal salt or an esterified derivative of a rice bran alkaloid having lipase inhibitory activity in the preparation of a lipase inhibitor drug.

[0078] The following is a further description with specific examples. The rice bran samples for extraction are prepared by milling self-purchased brown rice, and the variety of brown rice used is Suijing 18 (Oryza sativa subsp. japonica); other reagents are purchased unless otherwise specified.

[0079] Example 1:

[0080] The rice bran is extracted with subcritical butane to remove oil, and then extracted with an aqueous solution of 95% ethanol. The extract is separated to collect the residue. 2M NaOH solution is added to the residue at a mass-to-volume ratio of 1g:20mL, and then extracted for 4 hours under a nitrogen environment. Subsequently, the pH is adjusted to 1 with 6M HCl, and then extracted 5 times with ethyl acetate of the same volume as the extract. The ethyl acetate phase is collected, and the organic solvent is removed by reduced pressure concentration to obtain the extract.

[0081] The extract is subjected to normal-phase silica gel column chromatography and eluted with chloroform / methanol in a gradient from a volume ratio of 100:0 to 60:40. The fraction eluted with a chloroform / methanol volume ratio of 80:20 is collected. After the solvent is evaporated, the residue is subjected to ODS-A medium-pressure column chromatography and eluted with methanol / water in a gradient from a volume ratio of 20:80 to 90:10. The fraction eluted with a methanol / water volume ratio of 30:70 is collected and then separated by high-performance liquid chromatography. The separation conditions are as follows: a YMC-Pack ODS-A chromatographic column with a column size of 20×250mm and a particle size of 5μm is used, and an aqueous solution of acetonitrile with a volume fraction of 16% is used as the mobile phase. Separation is carried out at a flow rate of 5mL / min, and the substance with a retention time of 62min is collected to obtain the 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

[0082] Example 2:

[0083] The rice bran is extracted with subcritical butane to remove oil, and then extracted with an aqueous solution of 90% ethanol. The extract is separated to collect the residue. 3M NaOH solution is added to the residue at a mass-to-volume ratio of 1g:10mL, and then extracted for 1 hour under a nitrogen environment. Subsequently, the pH is adjusted to 2 with 3M HCl, and then extracted 5 times with ethyl acetate of the same volume as the extract. The ethyl acetate phase is collected, and the organic solvent is removed by reduced pressure concentration to obtain the extract.

[0084] The subsequent chromatography and high-performance liquid chromatography separation conditions are the same as those in Example 1.

[0085] Example 3:

[0086] The rice bran was extracted with subcritical n-pentane to remove oil, and then extracted with 75% ethanol aqueous solution. The extract was separated and the residue was collected. 0.5 M NaOH solution was added to the residue at a mass-to-volume ratio of 1 g:30 mL, and then extracted for 5 hours under a nitrogen atmosphere. Subsequently, the pH was adjusted to 1 with 2 M HCl, and then extracted 5 times with ethyl acetate in an equal volume to the extract. The ethyl acetate phase was collected and concentrated under reduced pressure to remove the organic solvent to obtain the extract.

[0087] The subsequent chromatography and high performance liquid chromatography separation conditions were the same as those in Example 1.

[0088] Compound characterization:

[0089] The final extracts obtained in Examples 1-3 were subjected to one-dimensional and two-dimensional nuclear magnetic resonance and mass spectrometry detection, and the results are as Figures 1-6 shown, where Figure 1 is 1 the 1H NMR spectrum; Figure 2 is 13 the 13C NMR spectrum; Figure 3 is 1 the H- 1 H COSY spectrum; Figure 4 is the HSQC spectrum; Figure 5 is the HMBC spectrum; Figure 7 is the HR-ESI-MS spectrum.

[0090] The final extracts obtained in Examples 1-3 were dissolved in methanol for ultraviolet absorption spectrum analysis, and the ultraviolet absorption spectrum absorption peaks were: UV(MeOH) λmax(logε): 205 nm(3.89), 261 nm(3.47) and 306 nm(2.89).

[0091] HR-ESI-MS m / z: 224.0554[M+H]+(theoretical calculation C 10 H 10 NO 5 + , 224.0553);

[0092] 246.0373[M+Na]+(theoretical calculation C 10 H 9 NNaO 5 + 246.0373).

[0093] 1 1H(500 MHz) and 13 13C(125 MHz) nuclear magnetic resonance data are shown in Table 1:

[0094] Table 1 NMR data of the extracts in the examples (in CD3 OD)

[0095]

[0096] According to HR-ESI-MS: m / z 224.0554 [M+H]+ (theoretical calculation C 10 H 10 NO 5 + , 224.0553); 246.0373 [M+Na]+ (theoretical calculation C 10 H 9 NNaO 5 + 246.0373). The molecular weight was determined to be 223, and the molecular formula was C 10 H 9 NO 5 , with an unsaturation degree of 7. The 1H NMR spectrum (Table 1) showed a signal of an ABX-coupled aromatic ring at δ 6.92 (1H, d, J = 2.6 Hz, H-5), 6.78 (1H, d, J = 8.5 Hz, H-8), 6.72 (1H, dd, J = 8.5, 2.6 Hz, H-7); a methylene signal at δ 3.02 (1H, d, J = 16.1 Hz, H2-3a) and 2.78 (1H, d, J = 16.1 Hz, H2-3b). The 13C NMR spectrum showed (Table 1) 6 aromatic ring carbon signals (δ 154.7 - 113.8), 1 methylene carbon signal at δ 42.9 (C-3), 2 carbonyl carbon signals at δ 175.9 (C-11), 170.6 (C-2), and 1 oxygen-linked quaternary carbon signal at δ 74.8. Combining the presence of a nitrogen atom in the molecular formula, it was inferred that there was a 2-quinolone nucleus in the structure. According to the 1H-1H COSY and HSQC spectra, the carbon-hydrogen signals were assigned. As Figure 6 shown, in the HMBC spectrum, H2-3 was correlated with δ 74.8 (C-4), 127.3 (C-10), 170.6 (C-2), and 175.9 (C-11); H-5 was correlated with δ 74.8 (C-4), 117.3 (C-7), 130.4 (C-9), and 154.7 (C-6); H-7 was correlated with δ 113.8 (C-5), 130.4 (C-9), 154.7 (C-6); H-8 was correlated with δ 74.8 (C-4), 127.3 (C-10), and 154.7 (C-6). Based on the above information, it was determined that there was a hydroxyl substitution on the benzene ring at the 6-position of the 2-quinolone nucleus structure of the extract, and the extraction products of Examples 1 - 3 were resolved to have the structure shown in Formula I, which was named 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid according to the compound naming rules:

[0097]

[0098] Compound activity evaluation:

[0099] (1) Instruments and materials

[0100] The substrate 4-MUO 75164 (4-Methylumbelliferyl oleate) and porcine pancreatic lipase (EC3.1.1.3, L3126) were purchased from Sigma. Phosphate buffer PBS was purchased from Life (Thermo Fisher Scientific, China). DMSO and MeOH were both of analytical grade and purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; citric acid and sodium citrate were purchased from Tianjin Fuchen Chemical Reagent Factory. Microplate reader Tecan GENios (Tecan Group Ltd., Switzerland); 96-well plates (Corning Costar, Cambridge, MA, USA).

[0101] (2) Evaluation of lipase inhibitory activity

[0102] The colorimetric method (Ercan et al, 2016) was used to test the inhibition rate of the compound obtained by extracting in Example 1 on porcine pancreatic lipase through a 96-well cell culture plate. The substrate 4-MUO generates fluorescent 4-methylumbelliferone under the action of porcine pancreatic lipase, and the inhibitory activity of the compound on lipase was judged by measuring the fluorescence intensity of each well after the reaction. The test steps are as follows:

[0103] a) Prepare 0.1M citric acid-sodium citrate buffer (pH = 4.2) with ultrapure water; prepare 1mg / mL porcine pancreatic lipase solution, 0.1mM 4-MUO solution, and 0.2M Na 2 CO 3 solution with PBS (pH = 7.4).

[0104] b) Take the prepared 1mg / ml porcine pancreatic lipase solution and heat it in a 90°C water bath for 20 minutes to inactivate the enzyme.

[0105] c) Dilute the extract of Example 1 and the positive control (orlistat) with DMSO in equal ratios to prepare test solution samples with initial concentrations of 10, 5, 2.5, 1.25, 0.625, and 0.3125 mM.

[0106] d) Add samples according to the following reaction system (the DMSO concentration does not exceed 5% of the whole system):

[0107] Sample group: 1 μL sample + 24 μL PBS + 25 μL enzyme;

[0108] Sample blank group: 1 μL sample + 24 μL PBS + 25 μL inactivated enzyme;

[0109] Negative group: 1 μL DMSO + 24 μL PBS + 25 μL enzyme;

[0110] Negative blank group: 1 μL DMSO + 24 μL PBS + 25 μL inactivated enzyme;

[0111] After adding the samples and mixing well, add 50 μL of 0.1 mM reaction substrate 4-MUO to each well. After reacting for 20 min in a constant temperature incubator at 25 °C, add 100 μL of citric acid - sodium citrate solution (0.1 M, pH = 4.2) to each sample well to terminate the reaction. Measure the fluorescence absorbance value of each well with an enzyme-linked immunosorbent assay (ELISA) reader. The excitation wavelength is 320 nm, and the detection wavelength is 450 nm. Each sample is repeated at least 3 times, and the inhibition rate is calculated using the following formula:

[0112] Inhibition rate (%) = [1 - (A_sample – A_sample blank) / (A_negative – A_negative blank)]

[0113] × 100;

[0114] Use SPSS 19.0 statistical analysis software to calculate the half-maximal inhibitory concentration IC 50 value.

[0115] (3) Results of pancreatic lipase inhibitory activity:

[0116] The inhibitory results of the compound extracted in Example 1 on porcine pancreatic lipase are shown in Table 2:

[0117] Table 2 Pancreatic lipase inhibitory activity of the extracts in the examples

[0118]

[0119] As can be seen from the results in Table 2, the half-maximal inhibitory concentration IC 50 value of the rice bran alkaloid 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid with lipase inhibitory activity extracted in the examples of the present invention on porcine pancreatic lipase is 187.1 μM, while the half-maximal inhibitory concentration IC 50The value is 117 nM. Therefore, it can be known that the rice bran alkaloid 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid with lipase inhibitory activity provided by the present invention has good pancreatic lipase inhibitory activity, and is expected to be developed for the preparation of drugs for pancreatic lipase inhibitors, with wide application potential and good prospects.

[0120] The above embodiments are only the preferred embodiments of the present invention, and cannot be used to limit the scope of protection of the present invention. Any non-substantive changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of protection required by the present invention.

Claims

1. A rice bran alkaloid with lipase inhibitory activity, characterized in that, the rice bran alkaloid with lipase inhibitory activity is 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid, and its structure is shown in Formula I:

2. A preparation method of the rice bran alkaloid with lipase inhibitory activity according to Claim 1, characterized in that, it includes the following steps: S1. After removing the grease from the rice bran, extract it with an aqueous solution of alcohol, and collect the residue after extraction; S2. Extract the collected residue with an alkali solution, then adjust the pH of the extracted solution to acidic to obtain an extract, extract the extract with an organic solvent, collect the organic phase, and obtain an extract after removing the organic solvent from the organic phase; S3. Subject the extract to normal-phase silica gel column chromatography, and perform gradient elution with chloroform / methanol as the eluent, and collect the elution fractions with a chloroform / methanol volume ratio of (90:10)-(70:30); S4. Subject the elution fractions collected in S3 to ODS-A medium-pressure column chromatography, and perform gradient elution with methanol / water as the eluent, and collect the elution fractions with a methanol / water volume ratio of (20:80)-(40:60); S5. The elution fractions collected in step S4 are separated and purified by high-performance liquid chromatography to obtain 4,6-dihydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid.

3. The preparation method of the rice bran alkaloid with lipase inhibitory activity according to Claim 2, characterized in that, in step S1, the removal of grease is achieved by subcritical extraction; the solvent for subcritical extraction is an alkane with 3-6 carbon atoms; the aqueous solution of alcohol is an aqueous solution of methanol or ethanol, and the volume of alcohol in the aqueous solution of alcohol is 75%-95%.

4. The preparation method of the rice bran alkaloid with lipase inhibitory activity according to Claim 2, characterized in that, in step S2, the alkali solution is one or a combination of two of an aqueous solution of sodium hydroxide or potassium hydroxide; the concentration of the alkali solution is 0.5 mol / L - 3 mol / L; the volume-mass ratio of the alkali solution to the residue is (10 - 30 mL):1 g; the extraction time with the alkali solution is 1 - 5 h; the extraction with the alkali solution is carried out in an inert gas environment.

5. The preparation method of the rice bran alkaloid with lipase inhibitory activity according to Claim 2, characterized in that, in step S2, adjusting the solution to acidic means adjusting the pH value of the solution to 1 - 2; adjusting the solution to acidic uses one or a combination of two of hydrochloric acid or sulfuric acid.

6. The preparation method of the rice bran alkaloid with lipase inhibitory activity according to Claim 2, characterized in that, in step S3, gradient elution is performed with chloroform / methanol as the eluent, and the volume percentage of chloroform is gradually reduced by 10% from 100% until 60%; in step S4, gradient elution is performed with methanol / water as the eluent, and the volume percentage of methanol is gradually increased by 10% from 20% until 90%.

7. The preparation method of the rice bran alkaloid with lipase inhibitory activity according to Claim 2, characterized in that, The conditions for high performance liquid separation in step S5 are as follows: use a YMC-Pack ODS-A chromatographic column with a column size of 20×250 mm and a particle size of 5 μm. The mobile phase is an aqueous acetonitrile solution with a volume percentage of 16%, and the flow rate is 5 mL / min.

8. A medicinal salt of a rice bran alkaloid having lipase inhibitory activity as claimed in claim 1.

9. A pharmaceutical composition comprising a rice bran alkaloid having lipase inhibitory activity as claimed in claim 1 or a medicinal salt of a rice bran alkaloid having lipase inhibitory activity as claimed in claim 8.

10. Use of a rice bran alkaloid having lipase inhibitory activity as claimed in claim 1 or a medicinal salt of a rice bran alkaloid having lipase inhibitory activity as claimed in claim 8 in the preparation of a lipase inhibitor drug.

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