CN111595972A - Preparation method and quality detection method of lithocarpus polystachyus rehd total flavone extract - Google Patents
Preparation method and quality detection method of lithocarpus polystachyus rehd total flavone extract Download PDFInfo
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- CN111595972A CN111595972A CN202010475809.5A CN202010475809A CN111595972A CN 111595972 A CN111595972 A CN 111595972A CN 202010475809 A CN202010475809 A CN 202010475809A CN 111595972 A CN111595972 A CN 111595972A
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- Prior art keywords
- rehd
- lithocarpus polystachyus
- total
- phlorizin
- solution
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Abstract
The invention relates to a preparation method and a quality detection method of total flavonoids of lithocarpus polystachyus rehd, wherein the preparation method of the total flavonoids of lithocarpus polystachyus rehd comprises the following steps: dried leaves of lithocarpus polystachyus → crushing → methanol or ethanol heating reflux extraction → concentration → methanol or ethanol recovery → crude extract is added with water for suspension → macroporous absorption resin column chromatography → elution → concentration → drying, thus obtaining the total flavone of lithocarpus polystachyus. The invention researches the preparation process of the total flavone extract in lithocarpus polystachyus rehd, researches the quality standard of the total flavone extract of lithocarpus polystachyus rehd, and provides a reference basis for establishing the complete quality standard and developing and utilizing resources in the future. Pharmacological experiments prove that the total flavone extract of lithocarpus polystachyus rehd prepared by the invention has better blood sugar reducing effect and can be applied to preparing blood sugar reducing medicines.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine extract preparation, in particular to a preparation method and a quality detection method of a lithocarpus polystachyus rehd total flavone extract.
Background
Lithocarpus polystachyus (also known as Lithocarpus litseifolius, Lithocarpus polystachyus (Rehd.) Rehd, and Stevlae Rebaudiana (Rehd.) Rehd, A. RehdLithocarpus litseifolius（Hance）Chun.) The dried tender leaves of the tea are usually drunk as tea leaves in Guangxi folk, and have the effects of reducing blood sugar, resisting oxidation, reducing blood pressure, resisting bacteria, resisting allergy, resisting cancer and the like. The lithocarpus polystachyus rehd is not recorded in the herbal medicines of the past generations, is scattered in Chinese herbal medicine monographs and Chinese herbal medicine manuals or phytology monographs in southern regions, and has no legal quality standard at present, such as Chinese plant records, Chinese higher plant atlas, Guizhou plant records, Yunnan plant records, Guangxi medicinal plant famous records, national Chinese herbal medicine assembly and the like. Through research, the main active ingredients in the lithocarpus polystachyus rehd are phlorizin and trilobatin, and the lithocarpus polystachyus rehd has high content and can be cultivated in a large area in areas such as Guangxi province.
At present, the research on the flavonoid substances of lithocarpus polystachyus rehd in the existing literature mainly has the following aspects:
1. [ PROBLEM ] extraction and antibacterial effect research of active ingredients of Lithocarpus polystachyus (Li Sheng Hua, Wuxian, Zhaoxiang, Gnakai ] extraction and antibacterial effect research [ J ] food technology, 2010,35(3): 211-214) is that 5g of Lithocarpus polystachyus (Koehne) medicinal material powder is respectively decocted by 50%, 70% and 90% ethanol and extracted by microwave assistance, and the content of total flavone in an extracting solution is determined by adopting an ultraviolet spectrophotometry and taking rutin as a reference substance, so that the content of the total flavone is highest in the Lithocarpus polystachyus (Koehne) extract taking a 70% ethanol solution as an extracting solvent; in the aspect of bacteriostasis effect, the lithocarpus polystachyus rehd extract extracted by 70% ethanol solution has the best bacteriostasis effect.
2. [ PROBLEM ] extraction and separation technology study of Lithocarpus polystachyus (Rochebrun, Liujia, Liuxiong, Rana.) extraction and separation technology study of Lithocarpus polystachyus (J.) Kagaku (Kagaku) technical study of extraction and separation [ J ]. Guangzhou chemical industry, 2014,42(14): 69-71): respectively measuring 10mL of soaked AB-8 and D101 resins, transferring the resins into two glass columns, loading 70 mL of 0.4 mg/mL of the flocculated solution of lithocarpus polystachyus rehd at the flow rate of 1BV/h, collecting effluent, and measuring the absorbance of the effluent until the concentration of flavone in the effluent is the same as that of the initial solution. Washing the resin which is adsorbed and saturated by deionized water at a rate of 3BV/h to remove water-soluble impurities, performing gradient elution by using ethanol at a flow rate of 1BV/h, and collecting the effluent components, so that after the AB-8 column is separated and purified, the purity of phlorizin reaches 52.84 percent, and the recovery rate is 73.82 percent; after separation and purification by a D101 column, the purity of phlorizin is 44.68%, and the recovery rate reaches 72.36%.
3. [ PROBLEMS ] the main active ingredients and content changes of wild Lithocarpus polystachyus (Wangkun, Li Kaixiang, Chenjinyan, Huangjian, Malaying.) the main active ingredients and content changes of wild Lithocarpus polystachyus [ J ] the economic forest research, 2016,34(04):96-100+ 122.): control solution: precisely weighing phlorizin and trilobatin which are dried in vacuum to constant mass and 5.00mg, and respectively dissolving in 5mL of methanol to obtain the finished product. The sample solution is prepared by collecting Lithocarpus polystachyus powder 0.25g, placing in 10ml centrifuge tube, adding methanol 5ml, performing ultrasonic treatment for 15min, centrifuging for 15min, transferring the supernatant to 25ml volumetric flask with dropper, adding methanol to desired volume of 25ml, shaking, filtering with 0.45 μm microporous membrane, and collecting the filtrate and injecting into liquid chromatograph. Chromatographic conditions are as follows: the column was Athena C18-WP (100A, 2.1 mm. times.100 mm, 3 μm); the mobile phase was acetonitrile-0.04% formic acid (28: 72); the flow rate is 0.8 mL/min; the detection wavelength is 285 nm; the column temperature is room temperature; the sample amount is 5 mu L, and the number of theoretical plates is not less than 6000 calculated according to phlorizin peak. As a result, the method is proved to be accurate and reasonable by methodology, and can be used as a content determination method of phlorizin and trilobatin in lithocarpus polystachyus rehd.
4. [ PROBLEMS ] a response surface method is used for optimizing the extraction process of lithocarpus polystachyus rehd general flavone and the research on antioxidant activity (Tangjian people, Zhuchenghao, Korea plum, bear elegant orchid, Zhongrong, bear loyalty and Jiansheng; the response surface method is used for optimizing the extraction process of lithocarpus polystachyus rehd general flavone and the research on antioxidant activity [ J ] Anhui agricultural science, 2020,48(01):181 & 185.): the optimal extraction conditions of the lithocarpus polystachyus rehd total flavone are that the ultrasonic power is 328.8W, the ultrasonic frequency is 35 kHz, the ethanol concentration is 60%, the material-liquid ratio is 1: 41.2 (g: mL), the extraction temperature is 63.2 ℃, the extraction time is 37.8min, and the extraction rate of the total flavone is 19.18%. The antioxidant activity test results show that when the concentration of flavone is 2mg/mL, the maximum clearance rates of OH, DPPH and O2-are respectively 11.70%, 75.86% and 32.00%, and the flavone has good reducing capability.
5. [ PROBLEMS ] ultrasonic extraction-process study of sweetener in resin-purified Lithocarpus polysachyus leaves (Li Aimin, Li Sheng, Zhang Yuan, Liu Ying, Li bin, Wuxian further. ultrasonic extraction-process study of sweetener in resin-purified Lithocarpus polysachyus leaves [ J ]. wild plant resources in China, 2014,33(05): 1-7.): the optimum ultrasonic extraction conditions of the Lithocarpus polystachyus leaf and the optimum AB-8 resin purification process are determined by using phlorizin and neohesperidin dihydrochalcone (NHDC) purity as indexes and adopting single factor and L9(34) orthogonal design. As a result, the optimum ultrasonic extraction conditions are that the ethanol concentration is 70%, the solid-to-liquid ratio is 1:25, and the ultrasonic time is 35 min; the optimal purification process conditions of the AB-8 resin are as follows: the adsorption flow rate is 0.5 mL/min, the concentration of the elution ethanol is 90 percent, the elution volume is 1.875 BV, and the flow rate of the eluent is 1 mL/min. Under the condition of extraction and purification, the purity of the obtained phlorizin is 88.616%, and the purity of the neohesperidin dihydrochalcone is 71.823%.
The lithocarpus polystachyus rehd contains abundant flavonoid substances, the content of the flavonoid substances can reach 9.8 percent, and the flavonoid substances are dihydrochalcone glycosides in a small amount in other plants. The research on the extraction process and the quality detection method of the high-purity lithocarpus polystachyus rehd flavone has important significance for further researching the properties, developing and utilizing the lithocarpus polystachyus rehd flavone.
Disclosure of Invention
The invention explores the preparation process of the total flavone extract in lithocarpus polystachyus rehd, and researches the quality standard of the total flavone extract of lithocarpus polystachyus rehd. The thin-layer chromatography is established to identify kaempferol, quercetin and phlorizin in the lithocarpus polystachyus rehd total flavone extract, and the ultraviolet spectrophotometry and the ultra-high performance liquid chromatography are used to measure the content of the lithocarpus polystachyus rehd total flavone, phlorizin and trilobatin, so that a reference basis is provided for establishing a complete quality standard and developing and utilizing resources in the future.
The invention aims to provide a preparation method and a quality detection method of total flavonoids of lithocarpus polystachyus rehd, wherein the preparation method of the total flavonoids of lithocarpus polystachyus rehd comprises the following steps: dried leaves of lithocarpus polystachyus (rehd.) rehd → pulverization → methanol or ethanol heating reflux extraction → concentration → methanol or ethanol recovery → crude extract is added with water for suspension → macroporous absorption resin column chromatography → elution → concentration → vacuum drying or freeze drying, thus obtaining the total flavone of lithocarpus polystachyus (rehd.) rehd. Pharmacological experiments prove that the total flavone extract of lithocarpus polystachyus rehd prepared by the invention has better blood sugar reducing effect and can be applied to preparing blood sugar reducing medicines.
The invention is realized by the following technical scheme:
a method for preparing total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd comprises the following steps:
(1) heating and refluxing a methanol or ethanol solution:
firstly, drying leaves of lithocarpus polystachyus rehd, crushing, and adding 5-20 times of methanol or ethanol with the volume concentration of 50-90% by weight;
heating, refluxing and extracting for 2-4 times at the temperature of 60-90 ℃, each time for 0.5-2.5 hours, filtering, and combining the filtrates;
③ recovering methanol or ethanol from the filtrate, concentrating the filtrate until no alcohol smell exists, adding water to dilute the filtrate until the concentration of the extract of lithocarpus polystachyus rehd is 0.05-0.06 g crude drug amount/ml, and putting the extract on a column for use;
(2) separating and purifying by macroporous adsorption resin:
selecting medium polarity or non-polarity macroporous adsorption resin;
adsorption: the concentration of the lithocarpus polystachyus rehd extract on the upper column is 0.05-0.06 g crude drug amount/ml, the volume of the lithocarpus polystachyus rehd extract on the upper column is 8-16 times of the volume of the filled macroporous adsorption resin, and the flow rate on the upper column is 1-4 BV/h;
flushing: the eluent is water, the dosage of the eluent is 1-3 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 1-3 BV/h, and the eluent is discarded;
ethanol solution elution: and the eluent is ethanol with the volume concentration of 30-90%, the dosage of the eluent is 10-20 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 1-3 BV/h, the eluent is collected, the ethanol is recovered under reduced pressure, the concentrated solution is dried, and then the vacuum drying or freeze drying is carried out, so that the lithocarpus polystachyus rehd total flavone extract is obtained.
Specifically, the macroporous adsorption resin in the step (2) is moderate-polarity XAD-18 or nonpolar XAD-1600N, D101 macroporous adsorption resin.
According to the preparation method of the total flavonoids of the lithocarpus polystachyus rehd, the total flavonoids of the total flavonoids extract prepared by the method account for more than 50 percent of the total weight of the extract in terms of phlorizin, the main components of the total flavonoids extract comprise phlorizin and trilobatin with a dihydrochalcone structure mother nucleus and two unknown compounds with similar structures of the flavone mother nucleus and the phlorizin (the chemical structures of the two unknown compounds can not be measured), the four compounds account for more than 50 percent of the total flavonoids of the lithocarpus polystachyus rehd, and the phlorizin and the trilobatin account for more than 30 percent of the total flavonoids.
FIG. 1 is a flow chart of the preparation process of total flavonoids of Lithocarpus polystachyus Rehd. Through detection, the dried leaves, rhizomes and fruits of lithocarpus polystachyus rehd all contain total flavonoids, and all the total flavonoids can be extracted and prepared by the method.
The invention relates to a method for detecting the quality of total flavone extract of lithocarpus polystachyus rehd, which comprises the following steps:
the invention relates to a method for detecting the quality of total flavone extract of lithocarpus polystachyus rehd, wherein the content of total flavone of lithocarpus polystachyus rehd can be measured by adopting an ultraviolet spectrophotometry, and the detection steps are as follows:
(1) preparation of control solutions: precisely weighing appropriate amount of phlorizin reference substance, and adding methanol to obtain solution containing 0.7mg per 1 ml;
(2) preparation of a standard curve: precisely sucking 1ml, 2ml, 3ml, 4ml, 5ml, 6ml and 7ml of phlorizin reference substance solution, respectively placing the phlorizin reference substance solution in a 100ml measuring flask, adding methanol to dilute the solution to a scale, shaking the solution uniformly, measuring the absorbance at 284 +/-2 nm by using an ultraviolet-visible spectrophotometry with the methanol as a blank, drawing a standard curve by using the concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and obtaining a regression equation of Y =0.0376X +0.0252 and r = 0.9998;
(3) the determination method comprises the following steps: precisely weighing 25mg of total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd, placing in a 25ml measuring flask, adding appropriate amount of methanol to dissolve and dilute to scale, shaking, precisely weighing 1ml, placing in a 50ml measuring flask, adding appropriate amount of methanol to dissolve and dilute to scale, shaking, measuring absorbance at 284 + -2 nm with methanol as blank, wherein the total flavone content is not less than 50.0% calculated as phlorizin.
The invention relates to a method for detecting the quality of total flavone extract of lithocarpus polystachyus rehd, wherein the content of the total flavone of the lithocarpus polystachyus rehd can be measured by adopting a high performance liquid chromatography, and the detection steps are as follows:
(1) chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking 30: 70-90: 10 methanol A-0.1% formic acid aqueous solution B as a mobile phase, and carrying out gradient elution, wherein the elution gradient is as follows: 0-2 min, 25% A → 35% A; 2-20 min, 35% A → 90% A; the flow rate is 0.2-1 ml/min, and the detection wavelength is 284 nm; the number of theoretical plates is not less than 3000 calculated by phlorizin and trilobatin;
(2) preparation of a test solution: precisely weighing 16 mg of total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd, placing in a 100ml volumetric flask, adding 50% methanol solution, performing ultrasonic treatment for 10min to dissolve, treating at 320W and 80KHz frequency, adding 50% methanol solution to scale, shaking, filtering, and collecting filtrate;
(3) preparation of control solutions: accurately weighing appropriate amount of phlorizin and trilobatin reference substances, and adding methanol to obtain mixed reference substance solution containing phlorizin 35 μ g and trilobatin 16 μ g per 1 ml;
(4) the determination method comprises the following steps: respectively sucking mixed reference solution and sample solution 10 μ l each, injecting into liquid chromatograph, measuring, and calculating total amount of phlorizin and trilobatin not less than 30.0% by external standard method with phlorizin and trilobatin as reference.
The invention relates to a method for detecting the quality of total flavone extract of lithocarpus polystachyus rehd, wherein the percentage content of the total flavone of the lithocarpus polystachyus rehd is measured by adopting a chromatographic area normalization method, and the detection steps are as follows:
(1) determination of each chromatographic peak in the liquid chromatogram of the test solution: injecting 10 μ l of the mixed reference solution and test solution into a liquid chromatograph, measuring chromatogram and 3D spectrogram, and comparing relative retention time with 3D spectrogram to determine that 3 and 4 chromatographic peaks in the test solution are phlorizin and trilobatin respectively; comparing with 3D spectrogram, wherein the spectral characteristics and maximum absorption values of No. 1 and No. 2 chromatographic peaks in the sample solution are substantially consistent with those of No. 3 and No. 4 chromatographic peaks, and are flavonoids; 1. the No. 2 chromatographic peak is basically consistent with the ultraviolet absorption spectrogram of phlorizin, and the No. 1 and No. 2 chromatographic peaks are determined to be flavonoid substances;
(2) the determination method comprises the following steps: recording the chromatogram; measuring the area of the No. 1-4 peak and the total chromatographic peak area except the solvent peak on the chromatogram according to a chromatographic area normalization method, and calculating the percentage of the sum of the No. 1-4 peak areas in the total peak area; the relative percentage content of total flavone of Lithocarpus polystachyus (Rehd.) Rehd is not less than 50.0% based on the sum of peak areas of No. 1-4 chromatographic peaks.
The quality detection method of the lithocarpus polystachyus rehd total flavone extract detects kaempferol and quercetin, wherein the thin-layer chromatography identification of kaempferol and quercetin in the lithocarpus polystachyus rehd total flavone comprises the following steps:
(1) preparation of a test solution: adding methanol into the total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd to obtain a sample solution containing about 3mg of total flavone per 1 ml;
(2) preparation of control solutions: adding methanol into kaempferol reference substance to obtain kaempferol reference substance solution containing kaempferol 0.05mg per 1 ml; adding methanol into quercetin control to obtain quercetin control solution containing quercetin 0.06mg per 1 ml;
(3) the determination method comprises the following steps: taking the above test solution and control solution, dropping 5 μ l each on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate-formic acid (10: 9: 0.5) as developing agent, spraying with 3% aluminum trichloride ethanol solution, and observing under ultraviolet light 365nm to obtain the same fluorescent spot on the corresponding position of the sample chromatogram and the control chromatogram.
The method for detecting the quality of the total flavone extract of lithocarpus polystachyus rehd detects phlorizin, wherein the thin-layer chromatography identification of phlorizin in the total flavone of lithocarpus polystachyus rehd comprises the following steps:
(1) preparation of a test solution: adding methanol into the total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd to obtain a sample solution containing about 3mg of total flavone per 1 ml;
(2) preparation of control solutions: adding methanol into phlorizin reference substance to obtain phlorizin reference substance solution containing 0.6mg of phlorizin per 1 ml;
(3) the determination method comprises the following steps: taking the above test solution and control solution, dropping 5 μ l each on the same silica gel G thin layer plate, developing with ethyl acetate-formic acid-water (7: 0.5: 0.5) as developing agent, spraying with 3% aluminum trichloride ethanol solution, and observing under ultraviolet light 365nm to obtain the same fluorescent spot at the corresponding position of the sample chromatogram and the control chromatogram.
The total flavone extract of lithocarpus polystachyus rehd prepared by the method has the pharmacological action of reducing blood sugar through mouse experiments, and can be applied to the preparation of hypoglycemic drugs.
The invention examines the content determination methodology of the total flavone extract in lithocarpus polystachyus rehd as follows:
content determination (ultraviolet spectrophotometry) of total flavonoids of Lithocarpus polystachyus Rehd
(1) Preparation of a standard curve: 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml and 8ml of a control solution containing 720 mu g/ml phlorizin are precisely measured, respectively placed in a 100ml measuring flask, diluted to scales by adding methanol, shaken up, and subjected to ultraviolet-visible spectrophotometry (0401 in the four-part general rule of Chinese pharmacopoeia 2015) at 284nm to measure absorbance, and a standard curve is drawn by taking the concentration as an abscissa and the absorbance as an ordinate. The regression equation Y = 0.037X-0.025 and r =0.999 shows that the linear relation of phlorizin is good when the regression equation Y = 7.037X-0.025 and r = 57.6 mu g/mL.
(2) And (3) precision test: taking the total flavone extract test solution with the concentration of 1.1mg/ml, continuously measuring for 6 times, recording the absorbance, and calculating the RSD of the result of 6 times to be 0.09%.
(3) And (3) stability test: and (3) taking the same test solution, respectively measuring for 0 hour, 2 hours, 4 hours, 8 hours, 12 hours, 20 hours and 24 hours, recording the absorbance, and judging that the RSD of the results of 7 times is 1.27 percent, which indicates that the test solution is stable within 24 hours.
(4) Recovery rate test: respectively and precisely adding a proper amount of phlorizin reference substances into the total flavone extract of lithocarpus polystachyus rehd with the known content of 57%, measuring the content according to a proposed method, and calculating the recovery rate, wherein the average recovery rate is 99.6%, and the RSD is 1.09%.
The results of the methodology tests show that: the method is accurate, stable and simple to operate, and can ensure the quality of products.
Content determination (high performance liquid chromatography and area normalization method) of total flavonoids of Lithocarpus polystachyus Rehd
1. Content determination of total flavonoids of Lithocarpus polystachyus (high performance liquid chromatography)
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking 30: 70-90: 10 methanol A-0.1% formic acid aqueous solution B as a mobile phase, and carrying out gradient elution, wherein the elution gradient is as follows: 0-2 min, 25% A → 35% A; 2-20 min, 35% A → 90% A; the flow rate is 0.2-1 ml/min, and the detection wavelength is 284 nm; the number of theoretical plates is not less than 3000 calculated by phlorizin and trilobatin;
(2) preparation of a test solution: precisely weighing 16 mg of total flavones of Lithocarpus polystachyus (Rehd.) Rehd, placing in a 100ml volumetric flask, adding 50% methanol solution, performing ultrasonic treatment (power 320W, frequency 80 KHz) for 10min for dissolving, adding 50% methanol solution to scale, shaking, filtering, and collecting the filtrate;
(3) preparation of control solutions: accurately weighing appropriate amount of phlorizin and trilobatin reference substances, and adding methanol to obtain mixed reference substance solution containing phlorizin 35 μ g and trilobatin 16 μ g per 1 ml;
(4) the determination method comprises the following steps: respectively sucking mixed reference solution and sample solution 10 μ l each, injecting into liquid chromatograph, measuring, taking phlorizin and trilobatin as reference, and respectively calculating total amount of phlorizin and trilobatin not less than 30.0% by external standard method.
2. Content determination of total flavonoids of Lithocarpus polystachyus (area normalization method)
(1) Determination of each chromatographic peak in the liquid chromatogram of the test solution: precisely sucking 10 μ l of phlorizin, trilobatin reference solution and test solution respectively, injecting into a liquid chromatograph, measuring chromatogram and 3D spectrogram thereof according to the liquid phase method, and comparing relative retention time with the 3D spectrogram to determine that 3 and 4 chromatographic peaks in the test solution are phlorizin and trilobatin respectively; comparing with 3D spectrogram, the spectral characteristics and maximum absorption values of No. 1 and No. 2 chromatographic peaks in the sample solution are substantially consistent with those of No. 3 and No. 4 chromatographic peaks, and are flavonoid compounds with dihydrochalcone structure; 1. the No. 2 chromatographic peak is basically consistent with the ultraviolet absorption spectrogram of phlorizin, and the No. 1 and No. 2 chromatographic peaks are confirmed to be flavonoid substances with dihydrochalcone structures;
(2) the determination method comprises the following steps: precisely absorbing 10 mu l of test solution, injecting into a liquid chromatograph, and recording a chromatogram; measuring the area of the No. 1-4 peak and the total chromatographic peak area except the solvent peak on the chromatogram according to a chromatographic area normalization method, and calculating the percentage of the sum of the No. 1-4 peak areas in the total peak area; the relative percentage content of total flavone of Lithocarpus polystachyus (Rehd.) Rehd is not less than 50.0% based on the sum of peak areas of No. 1-4 chromatographic peaks.
3. Methodology the following was investigated:
(1) linear range:
preparation of a standard curve mixed reference substance solutions with phlorizin 0.151mg/ml and trilobatin 0.109mg/ml are precisely sucked into measuring bottles with the volume of 1, 2, 3, 4 and 5ml to 25ml, and 50 percent methanol is added to the measuring bottles to fix the volume to the scale, so that the mixed reference substance solutions with the mass concentration of I-V are obtained.
Precisely sucking 10 μ l of mixed reference substance solution with mass concentration of I-V, and performing regression treatment on peak area integral value with sample amount (μ g) to obtain phlorizin and trilobatin regression equations of Y =2972999X +15190(r is 0.9999); y =898472X +3444(r ═ 0.9999);
(2) and (3) precision test: precisely sucking the same test solution, continuously injecting sample for 6 times under the chromatographic condition, and recording the peak areas of phlorizin and trilobatin, wherein the RSD% of phlorizin and trilobatin is 0.19% and 0.54% respectively after 6 times.
(3) And (3) stability test: and taking the same sample solution, injecting samples once every two hours within 0-24 hours, measuring, and recording the peak area. The RSD of the root bark glycoside peak area and the trefoil glycoside peak area are respectively 0.39% and 2.33%. Indicating that the test solution was stable within 24 hours.
(4) And (3) repeatability test: 6 parts of sample solution to be tested is prepared from the same batch of samples, and the average content of phlorizin and trilobatin is 240 mg/g and 120 mg/g respectively, and the RSD is 1.82 percent and 0.05 percent respectively according to the determination of a proposed method.
(5) Recovery rate test: precisely adding a certain amount of reference substance mixed solution into 9 parts of Lithocarpus polystachyus rehd extract with known content, preparing sample according to sample solution treatment method, measuring, and calculating sample recovery rate, wherein the average recovery rates of phlorizin and trilobatin are 100% and 103%, respectively, and RSD are 2.33% and 1.62%, respectively
As a result: by adopting the preparation and quality detection method of the total flavone extract of lithocarpus polystachyus rehd, proved by an empirical test, the total flavone content of the lithocarpus polystachyus rehd in the prepared total flavone extract of lithocarpus polystachyus rehd can reach more than 30 percent by the total amount of phlorizin and trilobatin, and the total flavone content can reach more than 50 percent by the calculation of an area normalization method.
The results of the methodology tests show that: the method is simple, accurate, and rapid, and can ensure the quality of total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd.
Wherein, FIG. 2 is the ultraviolet absorption spectrum of the control product of total flavonoids of Lithocarpus polystachyus (rehd.) Rehd and phlorizin of the invention, and it can be seen from FIG. 2 that the phlorizin and the total flavonoids of Lithocarpus polystachyus rehd both have the maximum absorption at 284 + -2 nm, and the absorption spectra are basically consistent. FIG. 3 is a high performance liquid chromatogram of total flavonoids of Lithocarpus polystachyus Rehd, wherein the chromatogram peak components are respectively: 1. unknown flavone, 2 unknown flavone, 3, phlorizin, 4, trilobatin. FIG. 4 is a high performance liquid chromatogram of the mixed reference solution, wherein the peak components of each chromatogram are 1, phlorizin, 2, trilobatin. FIG. 5 is a 3D spectrum of total flavonoids of Lithocarpus polystachyus Rehd, and it can be seen from FIG. 5 that the spectral features of No. 1-4 chromatographic peaks are consistent and are the same flavonoids compounds with a dihydrochalcone structure parent nucleus. FIG. 6 is the ultraviolet absorption spectrum from peak 1 to peak 4 in the HPLC chromatogram of total flavones of Lithocarpus polystachyus rehd, and it can be seen from FIG. 6 that the unknown flavones of peak 1 and peak 2 are basically consistent with the ultraviolet absorption spectra of phlorizin of peak 3 and trilobatin of peak 4, and are similar flavones of the same dihydrochalcone structural mother nucleus. FIG. 7 is a thin-layer identification chart of kaempferol and quercetin in total flavonoids of Lithocarpus polystachyus (hook. f.) Rehd, wherein spots 1, 2 and 3 are respectively 2. mu.l, 5. mu.l and 10. mu.l of sample, and spots 4 and 5 are respectively quercetin and kaempferol. FIG. 8 is a thin-layer identification chart of phlorizin in total flavonoids of Lithocarpus polystachyus Rehd.
The invention has the beneficial effects that:
1. according to the invention, through researching the preparation process of the total flavone extract in lithocarpus polystachyus rehd, the quality standard of the total flavone extract of lithocarpus polystachyus rehd is researched, the thin-layer chromatography is established to identify kaempferol, quercetin and phlorizin in the total flavone extract of lithocarpus polystachyus rehd, and the ultraviolet spectrophotometry and the ultra-high performance liquid chromatography are used for measuring the content of the total flavone, phlorizin and trilobatin of lithocarpus polystachyus rehd, so that a reference basis is provided for establishing the complete quality standard of the lithocarpus polystachyus rehd and developing and utilizing resources in the future.
2. The preparation method of the total flavone extract of lithocarpus polystachyus rehd disclosed by the invention is simple in process and low in energy consumption, and the total flavone extract of lithocarpus polystachyus rehd with higher total flavone content can be obtained by mainly extracting with methanol or ethanol in the process and separating and purifying through a macroporous adsorption resin column for one time, so that the residual quantity of harmful solvents in the raw materials is small, and the requirements of the state on food and medicines are met.
3. The method adopts an ultraviolet-visible spectrophotometry method to directly identify the total flavone of lithocarpus polystachyus rehd, is simple and accurate, can meet the requirement of industrial production, and can effectively control the quality of the total flavone extract of lithocarpus polystachyus rehd.
4. The content of phlorizin and trilobatin in the total flavone extract of lithocarpus polystachyus rehd is detected by adopting a high performance liquid chromatography and taking phlorizin and trilobatin as reference substances, and the relative percentage content of the total flavone of lithocarpus polystachyus rehd is calculated by a chromatographic area normalization method, so that the analysis and test cost can be reduced, the analysis process is simplified, and the analysis speed is increased; more effectively applied to the quality control of multiple components and indexes of the traditional Chinese medicine.
5. The method adopts the thin-layer chromatography to identify the total flavones of lithocarpus polystachyus rehd, such as kaempferol, quercetin and phlorizin, is accurate and simple, can meet the requirements of industrial production, and can effectively control the quality of the total flavones of lithocarpus polystachyus rehd.
6. Pharmacological experiments prove that the total flavone extract of lithocarpus polystachyus rehd prepared by the invention has better blood sugar reducing effect and can be applied to preparing blood sugar reducing medicines.
7. The invention establishes a preparation and quality detection method of total flavonoids of lithocarpus polystachyus rehd by researching the total flavonoids extract of lithocarpus polystachyus rehd, adopts methods such as an ultraviolet-visible spectrophotometry method, an ultra-high performance liquid chromatography method and the like to measure the content of the total flavonoids in the total flavonoids of lithocarpus polystachyus rehd, has the advantages of accuracy, stability and simple operation, is beneficial to further development and utilization of related products of lithocarpus polystachyus rehd, develops high-technology and high-added-value products for developing special medicinal materials in Guangxi, improves market competitiveness, and can generate potential and immeasurable social benefits and economic benefits.
Drawings
FIG. 1 is a flow chart of the preparation process of total flavonoids from Lithocarpus polystachyus Rehd;
FIG. 2 is a graph of the ultraviolet absorption spectrum of a control of total flavonoids and phlorizin of Lithocarpus polystachyus Rehd;
FIG. 3 is a high performance liquid chromatogram of the total flavone extract of Lithocarpus polystachyus rehd, wherein the peak components of the chromatogram are respectively 1, unknown flavone, 2, unknown flavone, 3, phlorizin, 4 and trilobatin;
FIG. 4 is a high performance liquid chromatogram of the mixed reference solution, wherein the peak components of each chromatogram are 1, phlorizin, 2, trilobatin;
FIG. 5 is a 3D spectrum of the total flavonoid extract of Lithocarpus polystachyus Rehd;
FIG. 6 is a graph showing the UV absorption spectra from peak 1 to peak 4 in the HPLC of the total flavonoids of Lithocarpus polystachyus rehd of the present invention;
FIG. 7 is a thin-layer identification chart of kaempferol and quercetin in the total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd, wherein spots 1, 2 and 3 are respectively 2. mu.l, 5. mu.l and 10. mu.l of sample, and spots 4 and 5 are respectively quercetin and kaempferol;
FIG. 8 is a thin-layer identification chart of phlorizin in the total flavone extract of lithocarpus polystachyus rehd, wherein spots 1, 2 and 3 are respectively the sample spot amounts of 2. mu.l, 5. mu.l and 10. mu.l, and spot 4 is phlorizin.
Detailed Description
In order to make the technical solutions and advantages of the present invention clearer, the following describes the technical solutions of the present invention clearly and completely in combination with the embodiments of the present invention.
Example 1
A method for preparing total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd comprises the following steps:
(1) heating and refluxing the methanol solution for extraction:
300g of lithocarpus polystachyus rehd dry leaves are crushed into coarse powder, and 10 times of methanol with the volume concentration of 70% is added according to the weight;
heating reflux extraction at 90 deg.c for 2 times of 1.5 hr each time, filtering and merging the filtrate;
③ recovering methanol from the filtrate, concentrating the filtrate until no alcohol smell exists, adding water to fix the volume to 5000ml, diluting the filtrate until the concentration of the lithocarpus polystachyus extracting solution is 0.06g crude drug/ml, and putting the extract on a column for use;
(2) separating and purifying by macroporous adsorption resin:
firstly, selecting D101 macroporous adsorption resin, filtering four layers of gauze to obtain clear liquid, filtering, and passing through a D101 type macroporous adsorption resin column;
adsorption: the concentration of the lithocarpus polystachyus rehd extract on the column is 0.05-0.06 g crude drug amount/ml, the volume of the lithocarpus polystachyus rehd extract on the column is 10 times of the volume of the filled macroporous adsorption resin, and the flow rate on the column is 2 BV/h;
flushing: the eluent is water, the dosage of the eluent is 2 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 2BV/h, and the eluent is discarded;
ethanol solution elution: the eluent is 30% ethanol, the dosage of the eluent is 15 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 1BV/h, the eluent is collected, the ethanol is recovered under reduced pressure, the concentrated solution is dried, and the total flavone extract of lithocarpus polystachyus rehd is obtained after vacuum drying at 50 ℃.
Example 2
A method for preparing total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd comprises the following steps:
(1) heating and refluxing the methanol solution for extraction:
firstly, 100g of lithocarpus polystachyus rehd dry leaves are crushed into coarse powder, and 15 times of methanol with the volume concentration of 80% is added according to the weight;
heating reflux extraction at 80 deg.c for 4 times of 1 hr each time, filtering and merging the filtrates;
③ recovering methanol from the filtrate, concentrating until no alcohol smell exists, adding water to a constant volume of 2000ml, diluting until the concentration of the extracting solution of lithocarpus polystachyus rehd is 0.05g crude drug/ml, and putting the extracting solution on a column for use;
(2) separating and purifying by macroporous adsorption resin:
firstly, selecting a nonpolar XAD-1600N macroporous adsorption resin column, filtering four layers of gauze to obtain a clear solution, filtering, and passing through the XAD-1600N macroporous adsorption resin column;
adsorption: the concentration of the extracting solution of lithocarpus polystachyus rehd on the upper column is 0.05g of crude drug/ml, the volume of the extracting solution of lithocarpus polystachyus rehd on the upper column is 10 times of the volume of the filled macroporous adsorption resin, and the flow rate of the extracting solution on the upper column is 2 BV/h;
flushing: the eluent is water, the dosage of the eluent is 1 time of the volume of the filled macroporous adsorption resin, the elution flow rate is 3BV/h, and the eluent is discarded;
ethanol solution elution: the eluent is 30% ethanol, the dosage of the eluent is 15 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 3BV/h, the eluent is collected, the ethanol is recovered under reduced pressure, the concentrated solution is dried, and the extract of the total flavone of the lithocarpus polystachyus rehd is obtained after freeze drying.
Example 3
A method for preparing total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd comprises the following steps:
(1) heating and refluxing the methanol solution for extraction:
50g of lithocarpus polystachyus rehd dry leaves are crushed into coarse powder, and 5 times of methanol with the volume concentration of 90% is added according to the weight;
heating reflux extraction at 80 deg.c for 3 times of 0.5 hr each time, filtering and merging the filtrate;
③ recovering methanol from the filtrate, concentrating until no alcohol smell exists, adding water to a constant volume of 1000ml, diluting until the concentration of the extracting solution of lithocarpus polystachyus rehd is 0.05g crude drug/ml, and putting the extracting solution on a column for use;
(2) separating and purifying by macroporous adsorption resin:
selecting a medium-polarity XAD-18 macroporous adsorption resin column, filtering four layers of gauze to obtain a clear solution, filtering, and passing through the XAD-18 macroporous adsorption resin column;
adsorption: the concentration of the extracting solution of lithocarpus polystachyus rehd on the upper column is 0.05g of crude drug/ml, the volume of the extracting solution of lithocarpus polystachyus rehd on the upper column is 10 times of the volume of the filled macroporous adsorption resin, and the flow rate of the extracting solution on the upper column is 4 BV/h;
flushing: the eluent is water, the dosage of the eluent is 2 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 3BV/h, and the eluent is discarded;
ethanol solution elution: the eluent is ethanol with volume concentration of 70%, the dosage of the eluent is 15 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 3BV/h, the eluent is collected, the ethanol is recovered under reduced pressure, the eluent is concentrated to be dry, and the total flavone extract of lithocarpus polystachyus rehd is obtained after vacuum drying at 60 ℃.
Example 4
A method for preparing total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd comprises the following steps:
(1) heating and refluxing the methanol solution for extraction:
firstly, drying 150g of lithocarpus polystachyus rehd leaves, crushing the lithocarpus polystachyus rehd leaves into coarse powder, and adding ethanol with the volume concentration of 70% in an amount which is 10 times the weight of the coarse powder;
heating reflux extraction at 90 deg.c for 2 times of 1 hr each time, filtering and merging the filtrates;
③ recovering ethanol from the filtrate, concentrating until no alcohol smell exists, adding water to a constant volume of 2500ml, diluting until the concentration of the extracting solution of lithocarpus polystachyus rehd is 0.06g crude drug/ml, and putting the extracting solution on a column for use;
(2) separating and purifying by macroporous adsorption resin:
selecting a medium-polarity XAD-18 macroporous adsorption resin column, filtering four layers of gauze to obtain a clear solution, filtering, and passing through the XAD-18 macroporous adsorption resin column;
adsorption: the concentration of the extracting solution of lithocarpus polystachyus rehd on the upper column is 0.06g of crude drug/ml, the volume of the extracting solution of lithocarpus polystachyus rehd on the upper column is 10 times of the volume of the filled macroporous adsorption resin, and the flow rate of the extracting solution on the upper column is 1 BV/h;
flushing: the eluent is water, the dosage of the eluent is 2 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 1BV/h, and the eluent is discarded;
ethanol solution elution: the eluent is ethanol with volume concentration of 60%, the dosage of the eluent is 15 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 1BV/h, the eluent is collected, the ethanol is recovered under reduced pressure, the ethanol is concentrated to be dry, and the lithocarpus polystachyus rehd total flavone extract is obtained after freeze drying.
Example 5
A method for preparing total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd comprises the following steps:
(1) heating and refluxing the methanol solution for extraction:
280g of lithocarpus polystachyus rehd dry leaves are crushed into coarse powder, and 15 times of ethanol with the volume concentration of 95% is added according to the weight;
heating reflux extraction at 60 deg.c for 5 times of 2 hr each time, filtering and merging the filtrates;
③ recovering ethanol from the filtrate, concentrating until no alcohol smell exists, adding water to fix the volume to 5000ml, diluting until the concentration is 0.056g crude drug/ml of lithocarpus polystachyus rehd extract, and putting the lithocarpus rehd extract on a column for use;
(2) separating and purifying by macroporous adsorption resin:
firstly, selecting a nonpolar D101 macroporous adsorption resin column, filtering four layers of gauze to obtain a clear solution, filtering, and passing through the D101 macroporous adsorption resin column;
adsorption: the concentration of the extracting solution of lithocarpus polystachyus rehd on the upper column is 0.05g of crude drug/ml, the volume of the extracting solution of lithocarpus polystachyus rehd on the upper column is 20 times of the volume of the filled macroporous adsorption resin, and the flow rate of the extracting solution on the upper column is 2 BV/h;
flushing: the eluent is water, the dosage of the eluent is 3 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 3BV/h, and the eluent is discarded;
ethanol solution elution: the eluent is ethanol with volume concentration of 50%, the dosage of the eluent is 10 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 2BV/h, the eluent is collected, the ethanol is recovered under reduced pressure, the eluent is concentrated to be dry, and the total flavone extract of lithocarpus polystachyus rehd is obtained after vacuum drying at 60 ℃.
The total flavone extract of lithocarpus polystachyus rehd prepared in examples 1-5 was subjected to quality inspection, and the results are shown in table 1 below: TABLE 1 detection results of total flavone extract from Lithocarpus polystachyus (Rehd.) Rehd
The total flavone of Lithocarpus polystachyus rehd extracted by the method of the invention has pharmacological action of reducing blood sugar, and pharmacodynamic tests are carried out on the total flavone extract of Lithocarpus polystachyus rehd prepared in examples 1-5, and the results are as follows:
(1) animal model establishment and grouping
Selecting a clean-grade ICR male mouse with the body mass of 20-22 g. Adaptively feeding for 1 week, feeding with high fat feed for 4 weeks, fasting for 12h, performing intraperitoneal injection of streptozotocin at a dose of 50 mg/(kg · bw), continuously feeding with high fat feed, collecting blood in tail vein after 72h, and determining diabetic mice after fasting for 12h before blood collection and fasting blood glucose of more than or equal to 11.10 mmol/L. The high-fat feed formula (%) is as follows: 71.8 parts of base material, 18 parts of lard, 8 parts of yolk powder, 2 parts of cholesterol and 0.2 part of bile salt. The mice successfully modeled are divided into high-dose, medium-dose, low-dose and drug treatment groups and model control groups of the total flavone dosage of lithocarpus polystachyus according to the physical quality and the blood sugar value, wherein each group comprises 10 mice, and the total group comprises 6 groups of 10 healthy mice. Each group of mice is fed with rodent standard pellet feed, water is freely drunk, the daily intragastric administration dosage of the administration group is respectively 200, 150 and 100mg/(kg · bw), the daily intragastric administration dosage of the drug treatment group is 150 mg/(kg · bw) metformin hydrochloride, and the intragastric equivalent normal saline of the normal control group and the model group is continuous for 4 weeks. The environmental temperature of the mice is kept at (20 +/-2) DEG C in the research period, the day and night are 12h, and other environmental conditions are controlled to accord with GB 14923-2001. After the model is successfully made, tail vein blood is collected at the same time every week of each group of mice for determining fasting blood glucose.
(2) Index measurement
Blood sugar and blood fat: fasting plasma glucose was measured with a glucometer.
(3) As a result: the influence of the total flavone extract of lithocarpus polystachyus on the fasting blood glucose of diabetic mice is shown in table 1, after high-fat diet and streptozotocin injection modeling, the fasting blood glucose of the mice in a normal group is obviously increased (P is less than 0.01) compared with that of the mice in other groups; after the gavage for 1 week, compared with the model group, the blood sugar of each dose group and the drug group of the total flavone of the lithocarpus polystachyus rehd is obviously reduced (P is less than 0.05); after 4 weeks of gavage, the total flavonoids of Lithocarpus polystachyus Rehd were high, the fasting blood glucose of the medium dose group and the drug group were continuously decreased and approached to the normal group, compared to the model group. The test result proves that the total flavone extract of lithocarpus polystachyus rehd has the function of reducing blood sugar. The results are shown in table 2 below:
in conclusion, the preparation method of the total flavone extract of lithocarpus polystachyus rehd is reasonable and stable, has short production period and is suitable for industrial production. The method adopts the thin-layer chromatography identification, the ultraviolet-visible spectrophotometry, the high-performance liquid chromatography external standard method and the area normalization method to measure the content of the total flavone of lithocarpus polystachyus rehd, has the advantages of accuracy, stability, simple operation and low analysis and test cost, can meet the requirements of industrial production, and can effectively control the quality of the total flavone extract of lithocarpus polystachyus rehd. The total flavone extract of lithocarpus polystachyus rehd has good blood sugar reducing effect and good development prospect.
Claims (9)
1. A preparation method of total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd is characterized by comprising the following steps: the method comprises the following steps:
(1) heating and refluxing a methanol or ethanol solution:
firstly, drying leaves of lithocarpus polystachyus rehd, crushing, and adding 5-20 times of methanol or ethanol with the volume concentration of 50-90% by weight;
heating, refluxing and extracting for 2-4 times at the temperature of 60-90 ℃, each time for 0.5-2.5 hours, filtering, and combining the filtrates;
③ recovering methanol or ethanol from the filtrate, concentrating the filtrate until no alcohol smell exists, adding water to dilute the filtrate until the concentration of the extract of lithocarpus polystachyus rehd is 0.05-0.06 g crude drug amount/ml, and putting the extract on a column for use;
(2) separating and purifying by macroporous adsorption resin:
selecting medium polarity or non-polarity macroporous adsorption resin;
adsorption: the concentration of the lithocarpus polystachyus rehd extract on the upper column is 0.05-0.06 g crude drug amount/ml, the volume of the lithocarpus polystachyus rehd extract on the upper column is 8-16 times of the volume of the filled macroporous adsorption resin, and the flow rate on the upper column is 1-4 BV/h;
flushing: the eluent is water, the dosage of the eluent is 1-3 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 1-3 BV/h, and the eluent is discarded;
ethanol solution elution: and the eluent is ethanol with the volume concentration of 30-90%, the dosage of the eluent is 10-20 times of the volume of the filled macroporous adsorption resin, the elution flow rate is 1-3 BV/h, the eluent is collected, the ethanol is recovered under reduced pressure, the concentrated solution is dried, and then the vacuum drying or freeze drying is carried out, so that the lithocarpus polystachyus rehd total flavone extract is obtained.
2. The method for preparing the total flavonoid extract of lithocarpus polystachyus rehd as claimed in claim 1, wherein: the macroporous adsorption resin in the step (2) is moderate-polarity XAD-18 or nonpolar XAD-1600N, D101 macroporous adsorption resin.
3. The method for preparing the total flavonoid extract of lithocarpus polystachyus rehd as claimed in claim 1, wherein: the prepared total flavone content of the total flavone extract of lithocarpus polystachyus rehd is more than 50 percent of the total weight of the extract in terms of phlorizin, the main components of the total flavone extract are phlorizin and trilobatin with a dihydrochalcone structure mother nucleus and other two unknown compounds with a flavone mother nucleus similar to the phlorizin in structure, the four compounds account for more than 50 percent of the total flavone component of lithocarpus polystachyus rehd, and the phlorizin and the trilobatin account for more than 30 percent of the total flavone component.
4. The method for detecting the quality of the total flavonoid extract of lithocarpus polystachyus rehd as claimed in claim 1, wherein the method comprises the following steps: the method for measuring the content of total flavonoids in lithocarpus polystachyus rehd adopts an ultraviolet spectrophotometry and comprises the following detection steps:
(1) preparation of control solutions: precisely weighing appropriate amount of phlorizin reference substance, and adding methanol to obtain solution containing 0.7mg per 1 ml;
(2) preparation of a standard curve: precisely sucking 1ml, 2ml, 3ml, 4ml, 5ml, 6ml and 7ml of phlorizin reference substance solution, respectively placing the phlorizin reference substance solution in a 100ml measuring flask, adding methanol to dilute the solution to a scale, shaking the solution uniformly, measuring the absorbance at 284 +/-2 nm by using an ultraviolet-visible spectrophotometry with the methanol as a blank, drawing a standard curve by using the concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and obtaining a regression equation of Y =0.0376X +0.0252 and r = 0.9998;
(3) the determination method comprises the following steps: precisely weighing 25mg of total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd, placing in a 25ml measuring flask, adding appropriate amount of methanol to dissolve and dilute to scale, shaking, precisely weighing 1ml, placing in a 50ml measuring flask, adding appropriate amount of methanol to dissolve and dilute to scale, shaking, measuring absorbance at 284 + -2 nm with methanol as blank, wherein the total flavone content is not less than 50.0% calculated as phlorizin.
5. The method for detecting the quality of the total flavonoid extract of lithocarpus polystachyus rehd as claimed in claim 1, wherein the method comprises the following steps: the content determination of total flavonoids of Lithocarpus polystachyus (Rehd.) Rehd adopts high performance liquid chromatography, and the detection steps are as follows:
(1) chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking 30: 70-90: 10 methanol A-0.1% formic acid aqueous solution B as a mobile phase, and carrying out gradient elution, wherein the elution gradient is as follows: 0-2 min, 25% A → 35% A; 2-20 min, 35% A → 90% A; the flow rate is 0.2-1 ml/min, and the detection wavelength is 284 nm; the number of theoretical plates is not less than 3000 calculated by phlorizin and trilobatin;
(2) preparation of a test solution: precisely weighing 16 mg of total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd, placing in a 100ml volumetric flask, adding 50% methanol solution, performing ultrasonic treatment for 10min to dissolve, treating at 320W and 80KHz frequency, adding 50% methanol solution to scale, shaking, filtering, and collecting filtrate;
(3) preparation of control solutions: accurately weighing appropriate amount of phlorizin and trilobatin reference substances, and adding methanol to obtain mixed reference substance solution containing phlorizin 35 μ g and trilobatin 16 μ g per 1 ml;
(4) the determination method comprises the following steps: respectively sucking mixed reference solution and sample solution 10 μ l each, injecting into liquid chromatograph, measuring, and calculating total amount of phlorizin and trilobatin not less than 30.0% by external standard method with phlorizin and trilobatin as reference.
6. The method for detecting the quality of the total flavonoid extract of lithocarpus polystachyus rehd as claimed in claim 1, wherein the method comprises the following steps: the method for measuring the percentage content of the total flavonoids in lithocarpus polystachyus rehd adopts a high performance liquid chromatography area normalization method, and comprises the following detection steps:
(1) determination of each chromatographic peak in the liquid chromatogram of the test solution: injecting 10 μ l of the mixed reference solution and test solution into a liquid chromatograph, measuring chromatogram and 3D spectrogram, and comparing relative retention time with 3D spectrogram to determine that 3 and 4 chromatographic peaks in the test solution are phlorizin and trilobatin respectively; comparing with 3D spectrogram, wherein the spectral characteristics and maximum absorption values of No. 1 and No. 2 chromatographic peaks in the sample solution are substantially consistent with those of No. 3 and No. 4 chromatographic peaks, and are flavonoids; 1. the No. 2 chromatographic peak is basically consistent with the ultraviolet absorption spectrogram of phlorizin, and the No. 1 and No. 2 chromatographic peaks are determined to be flavonoid substances;
(2) the determination method comprises the following steps: recording the chromatogram; measuring the area of the No. 1-4 peak and the total chromatographic peak area except the solvent peak on the chromatogram according to a chromatographic area normalization method, and calculating the percentage of the sum of the No. 1-4 peak areas in the total peak area; the relative percentage content of total flavone of Lithocarpus polystachyus (Rehd.) Rehd is not less than 50.0% based on the sum of peak areas of No. 1-4 chromatographic peaks.
7. The method for detecting the quality of total flavonoids of lithocarpus polystachyus rehd as claimed in claim 1, which detects kaempferol and quercetin, wherein the method comprises the following steps: the thin-layer chromatography identification of kaempferol and quercetin in the total flavone extract of lithocarpus polystachyus rehd comprises the following steps:
(1) preparation of a test solution: adding methanol into the total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd to obtain a sample solution containing about 3mg of total flavone per 1 ml;
(2) preparation of control solutions: adding methanol into kaempferol reference substance to obtain kaempferol reference substance solution containing kaempferol 0.05mg per 1 ml; adding methanol into quercetin control to obtain quercetin control solution containing quercetin 0.06mg per 1 ml;
(3) the determination method comprises the following steps: taking the above test solution and control solution, dropping 5 μ l each on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate-formic acid (10: 9: 0.5) as developing agent, spraying with 3% aluminum trichloride ethanol solution, and observing under ultraviolet light 365nm to obtain the same fluorescent spot on the corresponding position of the sample chromatogram and the control chromatogram.
8. The method for detecting the quality of the total flavonoid extract of lithocarpus polystachyus rehd as claimed in claim 1, which detects phlorizin, characterized in that: the thin-layer chromatography identification of phlorizin in the total flavone extract of lithocarpus polystachyus rehd comprises the following steps:
(1) preparation of a test solution: adding methanol into the total flavone extract of Lithocarpus polystachyus (Rehd.) Rehd to obtain a sample solution containing about 3mg of total flavone per 1 ml;
(2) preparation of control solutions: adding methanol into phlorizin reference substance to obtain phlorizin reference substance solution containing 0.6mg of phlorizin per 1 ml;
(3) the determination method comprises the following steps: taking the above test solution and control solution, dropping 5 μ l each on the same silica gel G thin layer plate, developing with ethyl acetate-formic acid-water (7: 0.5: 0.5) as developing agent, spraying with 3% aluminum trichloride ethanol solution, and observing under ultraviolet light 365nm to obtain the same fluorescent spot at the corresponding position of the sample chromatogram and the control chromatogram.
9. The use of the total flavonoid extract of lithocarpus polystachyus rehd as claimed in claim 1 in the preparation of a hypoglycemic medicament.
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| CN113041282A (en) * | 2021-04-01 | 2021-06-29 | 广西壮族自治区中医药研究院 | Application of lithocarpus polystachyus rehd extract in preparation of medicine for improving glucagon-like peptide-1 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112225768A (en) * | 2020-11-18 | 2021-01-15 | 成都农业科技中心 | Method for extracting trilobatin and phlorizin from lithocarpus litseifolius |
| CN113041282A (en) * | 2021-04-01 | 2021-06-29 | 广西壮族自治区中医药研究院 | Application of lithocarpus polystachyus rehd extract in preparation of medicine for improving glucagon-like peptide-1 |
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