CN115166082A - Extraction process and component analysis method of total flavones of spinosad - Google Patents

Extraction process and component analysis method of total flavones of spinosad Download PDF

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CN115166082A
CN115166082A CN202210784343.6A CN202210784343A CN115166082A CN 115166082 A CN115166082 A CN 115166082A CN 202210784343 A CN202210784343 A CN 202210784343A CN 115166082 A CN115166082 A CN 115166082A
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郭明鑫
胡志强
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Yixing Peoples Hospital
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Abstract

The invention discloses an extraction process of total flavonoids from spinosad and a component analysis method thereof, and the extraction process comprises the following steps of adding 60-90% of ethanol into the prepared powder of the spinosad, wherein the mass-volume ratio of the powder of the spinosad to the ethanol is 1g: (10-30) ml, heating and refluxing at 60-90 ℃ for 30-120min, extracting for 2 times, collecting mixed filtrate, rotary evaporating to dryness, redissolving with 70% ethanol, and fixing volume in a 10ml volumetric flask. The optimal process for optimizing the total flavones of the spinosyns by adopting the response surface method is adopted, the extraction rate of the flavones is 0.3889% under the condition, and the experimental result is close to the prediction value of the model, so that the extraction process method is simple, accurate and feasible, and the experiment uses AB-8 type macroporous resin for purification, so that impurities with larger polarity, such as pigments, saccharides and the like, are well removed, and flavonoids are selectively adsorbed, thereby realizing the purpose of purification and enrichment.

Description

Extraction process and component analysis method of total flavones of spinosa
Technical Field
The invention relates to the technical field of extraction of total flavonoids from spinosad, in particular to an extraction process of total flavonoids from spinosad and a component analysis method thereof.
Background
The spinosa is small particles of the plant Alhagi sparsifolia belonging to the genus Alhagi of the family Leguminosae, the secretion of stem and leaf is condensed into spherical shape, the surface is yellow, the taste is sweet, and the spinosa is mainly distributed in arid desert areas such as Xinjiang, inner Mongolia and the like. The thorn sugar is a folk medicine widely used by Uygur nationality, is recorded in Uygur nationality medicinal materials, is often used independently or is compatible with other medicinal materials, and has the effects of nourishing and strengthening body, balancing body fluid, replenishing essence and tonifying yang, astringing intestines and relieving pain, relieving cough and reducing phlegm and the like.
Modern researches show that the thorn sugar is rich in flavone, polysaccharide, amino acid, a small amount of trace elements and other components, and has the effects of resisting diarrhea, resisting bacteria, protecting blood vessels, preventing and resisting cancers and the like. At present, the research on the acanthose mainly focuses on the extraction, composition and activity of polysaccharide, while the research on the extraction process of total flavonoids is less and the research method is simple. In addition, the chemical composition of the echinacea sugar is still under investigation.
Therefore, a process for extracting the total flavonoids in the acanthopanax senticosus is needed to effectively extract the total flavonoids in the acanthopanax senticosus.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides an extraction process of total flavones of spinosa and a component analysis method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the extraction process of the total flavones of the thorn sugar comprises the following steps:
s1: crushing the thorn sugar, sieving the thorn sugar by a 40-mesh sieve, degreasing the thorn sugar according to the mass ratio of the medicinal materials to the petroleum ether of 1: 3, naturally volatilizing the solvent of the degreased medicinal materials, and storing the degreased medicinal materials in a dry and cold storage place for later use;
s2: drawing and weighing rutin control in a volumetric flask, adding appropriate amount of 70% ethanol solution, ultrasonic dissolving, and fixing volume to obtain rutin control solution, scanning the rutin control solution at full wavelength, measuring absorbance at 510nm wavelength, and measuring absorbance according to NaNO 2 -AI(NO) 3 -drawing a standard curve by NaOH color development;
s3: and (3) extracting the total flavonoids of the spiny sugar, weighing the spiny sugar powder prepared in the step (S1), adding 60-90% of ethanol by volume, wherein the mass-volume ratio of the spiny sugar powder to the ethanol is 1g: (10-30) ml, heating and refluxing at 60-90 ℃ for 30-120min for extraction for 2 times, collecting mixed filtrate, performing rotary evaporation to dryness, redissolving with 70% ethanol, and fixing the volume in a 10ml volumetric flask;
s4: enriching and purifying total flavones of Saccharum spinosum by pretreating AB-8 type resin, and purifying 100g of resin is filled in a chromatography column of 2.4cm multiplied by 60cm by a wet method, the sample loading amount is 5mL, purified water with 3 times of column volume is used for washing after the adsorption is finished, finally 100mL of 70 percent ethanol is used for elution, the elution flow rate is 1.0mL min- 1 And rotationally evaporating the eluent to dryness, and determining the content of the total flavones of the spinosad after the redissolution of methanol.
Preferably, rutin is weighed in the standard curve drawing process, 20.00mg of rutin reference substance is precisely weighed firstly, a proper amount of 70% ethanol solution is added into a 50mL volumetric flask, the mixture is completely dissolved by ultrasonic treatment, and the volume is determined to be a scale, so that the ion-doped rutin with the concentration of 0.40 mg/mL is obtained 1 The rutin comparison solution is precisely measured, 0.20 mL, 0.48 mL, 0.96 mL, 1.44 mL, 1.92 mL, 2.4 mL and 2.88mL of the rutin comparison solution are respectively placed in a 10mL volumetric flask for constant volume to scale, and the obtained concentration is 8.00, 19.20, 38.40, 57.60, 76.80, 96.00, 115.20 mu g-mL- 1 Rutin control solutions with a series of concentrations are obtained by scanning appropriate amount of rutin control solution with full wavelength to obtain maximum absorption at 510nm, so that absorbance is measured at 510nm wavelength according to NaNO 2 -AI(NO) 3 -drawing a standard curve by NaOH color development, and performing linear regression on absorbance A by using mass concentration C to obtain: a =11.193C-0.0085.
Preferably, the standard curve is plotted using chromatographic conditions, i.e. column: waters CORTECS UPLC-C18; mobile phase: a:0.1% aqueous formic acid solution, B: acetonitrile; gradient elution procedure: 0-20.0 min,5% -20% of (B); 20.0 to 40.0min, and 20 to 55 percent of B;40.0 to 45.0min, and 55 to 80 percent of B; 45.0-48.0 min,80% -95% of (B); 48.0 to 50.0min,95 percent of B; flow rate: 0.3mL min- 1 Column temperature: 25 ℃, sample introduction: 3 μ L.
Preferably, the standard curve plots the mass spectrometry conditions employed, i.e. mass spectrometry conditions in which the ion source: ESI source, collecting positive and negative ion mode, and correcting mass number with leucine-enkephalin; taper hole voltage: 15V; capillary voltage: 2800V; desolvation temperature: 300 ℃; source temperature: 100 ℃; fragmentation energy: 15V and the scanning range is 100-2000 Da.
The invention has the beneficial effects that:
1. in the invention, the optimal process for optimizing the total flavonoids in the spiny sugar is optimized by adopting a response surface method, the extraction rate of the flavonoids is 0.3889% under the condition, and the experimental result is close to the prediction value of a model, so that the extraction process method is simple, accurate and feasible, and the experiment uses AB-8 type macroporous resin for purification, so that impurities with high polarity, such as pigments, saccharides and the like, are well removed, and flavonoids are selectively adsorbed, thereby realizing the purpose of purification and enrichment.
2. In the invention, the UPLC-Q-TOF-MS technology is adopted for analyzing the main components of the spinosyn extract for the first time, a camel thorn plant compound database is established, the analysis and identification are carried out through the network database and the masslynx4.1 workstation, 40 chemical components are identified from the spinosyn, wherein 22 chemical components are reported for the first time, and the query and analysis processing of follow-up workers can be greatly facilitated through the data statistics and analysis of the network database.
Drawings
FIG. 1 is a total ion flow diagram of a polysaccharide extract in a positive ion mode according to the extraction process of polysaccharide total flavonoids and the component analysis method thereof provided by the invention;
FIG. 2 is a total ion flow diagram of the extract of the spinosa in negative ion mode according to the extraction process of the total flavonoids in the spinosa and the component analysis method thereof provided by the invention;
FIG. 3 is a response surface diagram of the interaction among the influencing factors of the extraction process of the total flavones of spinosa and the component analysis method thereof.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1, referring to fig. 1 to 3, the process for extracting total flavones from spinosad comprises the following steps:
s1: pulverizing the acanthose sugar, sieving with a 40-mesh sieve, degreasing according to the mass ratio of the medicinal material to the petroleum ether of 1: 3, naturally volatilizing the solvent from the degreased medicinal material, and storing in a dry and cold storage place for later use;
wherein the reagents used include: absolute ethyl alcohol, sodium hydroxide, aluminum nitrate, drechsler drinking water, sodium nitrite, rutin standard, acetonitrile, methanol and formic acid;
s2: drawing and weighing rutin control in a volumetric flask, adding appropriate amount of 70% ethanol solution, ultrasonic dissolving, and fixing volume to obtain rutin control solution, scanning the rutin control solution at full wavelength, measuring absorbance at 510nm wavelength, and measuring absorbance according to NaNO 2 -AI(NO) 3 -drawing a standard curve by NaOH color development;
s3: and (3) extracting the total flavonoids of the spiny sugar, weighing the spiny sugar powder prepared in the step (S1), adding 60-90% of ethanol by volume, wherein the mass-volume ratio of the spiny sugar powder to the ethanol is 1g: (10-30) ml, heating and refluxing at 60-90 ℃ for 30-120min for extraction for 2 times, collecting mixed filtrate, performing rotary evaporation to dryness, redissolving with 70% ethanol, and fixing the volume in a 10ml volumetric flask;
s4: enriching and purifying the total flavonoids of the spinosyns, preprocessing AB-8 type resin, filling 100g of the resin into a chromatographic column with the length of 2.4cm multiplied by 60cm by a wet method, wherein the sample loading amount is 5mL, washing by purified water with the volume of 3 times of the column after the adsorption is finished, and finally eluting by 100mL of 70% ethanol with the elution flow rate of 1.0 mL-min- 1 And the eluent is dried by rotation, and the content of the total flavones of the spinosa is measured after the redissolution of the methanol.
The adopted instruments comprise an UPLC-ESI-Q/TOF-MS liquid phase mass spectrometer, a CORTECS UPLC-C18 column, a Masslynx4.1 data software processing system, a DU-800 ultraviolet visible spectrophotometer, a rotary evaporator, an HH-4 digital display constant temperature water bath kettle, a BP210D ten thousandth analysis balance and a YB-1500 high-speed pulverizer.
The rutin is weighed in the standard curve drawing process, the rutin reference substance of 20.00mg is precisely weighed firstly, a proper amount of 70 percent ethanol solution is added into a 50mL volumetric flask, the mixture is completely dissolved by ultrasonic treatment, and the volume is fixed to scale, so that the ion-doped rutin with the concentration of 0.40 mg/mL is obtained 1 The rutin control solution is precisely measured, 0.20 mL, 0.48 mL, 0.96 mL, 1.44 mL, 1.92 mL, 2.4 mL and 2.88mL of the rutin control solution are respectively placed in a 10mL volumetric flask for constant volume to scale, and the obtained concentration is 8.00, 19.20, 38.40, 57.60, 76.80, 96.00, 115.20 mu g.mL- 1 Rutin control solutions with series concentrations are prepared by collecting appropriate amount of rutinScanning the solution at full wavelength to obtain maximum absorption at 510nm, measuring absorbance at 510nm according to NaNO 2 -AI(NO) 3 -drawing a standard curve by NaOH color development, and performing linear regression on absorbance A by using mass concentration C to obtain: a =11.193C-0.0085, which shows that rutin is 8.00-115.20 mu g-mL- 1 The absorbance in the range is in a good linear relationship with concentration.
The standard curve plots the chromatographic conditions employed, i.e. the column: waters CORTECS UPLC-C18; mobile phase: a:0.1% aqueous formic acid solution, B: acetonitrile; gradient elution procedure: 0-20.0 min,5% -20% of (B); 20.0 to 40.0min, and 20 to 55 percent of B;40.0 to 45.0min, and 55 to 80 percent of B;45.0 to 48.0min,80 to 95 percent; 48.0 to 50.0min,95 percent of B; flow rate: 0.3mL min- 1 Column temperature: 25 ℃, sample introduction: 3 μ L.
The mass spectrum condition adopted by standard curve drawing, namely the mass spectrum condition, the ion source: ESI source, collecting positive and negative ion mode, and correcting mass number with leucine-enkephalin; taper hole voltage: 15V; capillary voltage: 2800V; desolventizing temperature: 300 ℃; source temperature: 100 ℃; fragmentation energy: 15V and the scanning range is 100-2000 Da.
And (3) carrying out UPLC-Q-TOF-MS methodology investigation, and investigating the precision of the instrument, the repeatability of the method and the stability of the sample respectively, wherein the RSD of the retention time of the main chromatographic peak under each investigation project is less than 0.3%, and the RSD of the relative peak area is less than 3%, which shows that the precision of the instrument is good, the sample is stable and the repeatability of the method is good.
Embodiment 2, this embodiment is a further supplementary optimization to embodiment 1, specifically:
the extraction process of total flavones from Saccharum sinensis Roxb also needs single factor test method, which comprises performing single factor test by respectively adopting ethanol concentration of 60%, 70%, 80%, 95%, liquid-material ratio of 1: 10, 1: 15, 1: 20, 1: 30, extraction time of 30, 60, 90, 120min, extraction temperature of 60, 70, 80, 90 deg.C 4 factors, and taking average value of 3 times of test as determination result.
The analysis result shows that: the percentage content of the total flavone is higher when the concentration of the ethanol is 70% than that of other concentrations, the percentage content of the total flavone tends to increase firstly and then decrease with the increase of the extraction temperature and the feed-liquid ratio, the percentage content of the total flavone reaches the maximum value when the extraction temperature is 80 ℃ and the feed-liquid ratio is 1: 20, meanwhile, the percentage content of the total flavone increases in different degrees with the continuous increase of the extraction time, and the percentage content of the total flavone does not increase obviously when the extraction time exceeds 90 min.
Embodiment 3, this embodiment is a further optimization process of embodiment 2, specifically:
on the basis of single-factor experimental screening, response surface test factor Design is required, and the steps are that ethanol concentration A, material-liquid ratio B, extraction time C and extraction temperature D are selected as influence factors, the percentage content of total flavone is used as an evaluation index, design-Expert 8.0.6 software is applied, a Box-Behnken response surface Design model is selected, 4-factor 3 horizontal center combined test conditions are designed, see Table 1, and the percentage content of total flavone is measured under different conditions according to the test conditions designed by the model.
Figure BDA0003731343430000071
TABLE 1 response surface analysis factors and level design
The percentage content of total flavonoids extracted was determined under different conditions according to the experimental conditions designed by the model, and the experimental arrangement and results are shown in table 2.
Figure BDA0003731343430000072
Figure BDA0003731343430000081
TABLE 2 response surface design and results
Analyzing the response surface model variance and the response surface graph, referring to the data in table 3, it can be known that P =0.0003 of the model is less than 0.01, the difference has statistical significance, and P =2.29> -0.05 of the mismatching term of the model indicates that the mismatching term has no significant difference, which indicates that the model is successfully established, and as can be seen from the analysis result of variance, B2, D2 and a, C, A2, C2 in the equation have statistical significance on the extraction influence of the total flavonoids (P <0.05 and P < 0.01), while the influence of the extraction temperature on the extraction of the total flavonoids is not significant.
Therefore, the order of the influence of all factors on the total flavone extraction content can be obtained as follows: drawing a three-dimensional graph of a response surface according to a regression equation, wherein the highest point exists in the selected ranges of the ethanol concentration, the liquid-material ratio and the extraction time with reference to the attached figure 3; the influence of the temperature is small, the interaction of the ethanol volume fraction, the liquid-material ratio, the extraction time and the liquid-material ratio and the extraction time is obvious, and the interaction of the extraction temperature, the ethanol volume fraction, the liquid-material ratio and the extraction time is not obvious.
Figure BDA0003731343430000091
TABLE 3 results of analysis of variance of response surface model
And (3) obtaining the optimal extraction process conditions by optimizing the response surface method and considering the actual operability: the volume fraction of ethanol is 67%, the liquid-material ratio is 25 times, the extraction time is 75min, 3 parallel experiments are carried out under the condition, the average percentage content of the obtained extracted total flavonoids is 0.3889%, and the method is similar to a predicted value and shows that the equation is well fitted.
Example 4, further optimization treatment of example 1, specifically qualitative analysis of chemical components of the echinitol extract:
the experiment carries out primary full scanning (m/z 100-2000 Da) of positive ions and negative ions on the enriched and purified acanthose extract, summarizes chemical component information of the acanthose, including molecular formula, substance structure, relative molecular mass, mass spectrum data, cracking rule and the like, establishes a database containing 102 related compounds by consulting domestic and foreign documents and applying online database search, searches each chromatographic peak by utilizing a masslynx4.1 workstation to obtain the molecular formula with the error range less than or equal to 5ppm, identifies 40 chemical components from the acanthose, including 7 flavones and flavonoid glycosides, 20 flavonol and flavonol glycosides, 4 isoflavones, 2 phenylpropanoids and glycosides, 2 flavanones, 2 steroids, 2 phenols and 1 other component, wherein the component comprises pinoresinol, quercetin-3-O-alpha-L-arabinoside, isorhamnetin-3-O-beta-D-arabinorutinoside, quercetin-3-O-rutinoside, isoswertiamarin, hesperidin, quercetin-3-O-rhamnose rutinoside, stigmasterol, isorhamnetin-3-O-beta-D-arabinoside, isorhamnetin-3-O-robioside, hyperoside, isorhamnetin-3-O-robioside, syringin-3-O-beta-D-glucoside, quercetin-3-O-bisrhamnoside, proanthocyanidin B-1, kaempferide, syringaresinol-4-O-beta-D-glucoside, and so as to obtain the final product, 22 ingredients such as formononetin, phytolaccin, bizarin, 3', 7-dihydroxy-4', 8-dimethyl isoflavone, apigenin and the like are reported in the acanthose for the first time.
The flavonoid compounds in the thorn sugar are mainly characterized in that flavonoid aglycone and one or more sugars form a glycoside structure, and characteristic flavonoid mother nucleus fragment quercetin can be seen in a secondary mass spectrum: m/z303.0937[ M + H ] +, isorhamnetin: m/z 317.0658[ m ] +H ] +, kaempferol: m/z 287.0452[ m ] +H ] + and the like.
In addition, neutral lost glycosyl moieties can be seen, mainly glucose, rhamnose, galactose, rutinose and the like, such as syringin-3-O-beta-D-glucoside, at the anion mode molecular ion peak m/z 507.1444[ M-H ] -, and the lost glycosyl moieties form fragment ion m/z 344.0191[ M-Glu ] -; identifying molecular ion peak m/z 611.1313, M + H ] + as rutin, and m/z 303.0456 as a characteristic fragment formed by eliminating rhamnose and glucose; the molecular ion peak is M/z595.1350[ M + H ] +, and the secondary generation of M/z 287.0452 fragment is probably formed after losing M/z 308 rutinoside, and is identified as kaempferol-3-O-rutinoside.
In summary, the response surface method fits the functional relationship between the dependent variable and the response value through a multiple quadratic regression equation to obtain the optimal process conditions which are simpler than those of the orthogonal test method and more comprehensive than those of the uniform design method, and the optimal process conditions for optimizing the total flavonoids in the spinosad by applying the response surface method in the test are as follows: the concentration of ethanol is 67%, the liquid-material ratio is 1: 25, the extraction time is 75min, the extraction temperature is 75 ℃, the extraction rate of flavone is 0.3889% under the condition, and the experimental result is close to the prediction value of a model, which shows that the extraction process method is simple, convenient, accurate and feasible.
In the experiment, the AB-8 type macroporous resin is used for purification, so that impurities with high polarity, such as pigment, saccharides and the like, are well removed, and flavonoids are selectively adsorbed, so that the aim of purification and enrichment is fulfilled.
The experiment adopts UPLC-Q-TOF-MS technology to analyze the main components of the spinosyn extract for the first time, a camel thorn plant compound database is established, the analysis and identification are carried out through a network database and a masslynx4.1 workstation, 40 chemical components are identified from the spinosyn, wherein 22 chemical components are reported for the first time, and the identification of the compound structure is found by utilizing NMR technology, so that the flavonoid compounds in the camel thorn plants are mainly connected with monosaccharide or polysaccharide through beta-glycosidic bonds, such as isorhamnetin-3-O-beta-D-arabinoside and 3-methylquercetin-3-O-alpha-L-rhamnose-beta-D-galactopyranose-7-O-beta-D-glucoside.
Therefore, the research provides scientific basis for further research of the spinosyn drug effect substances and establishment of quality standards.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (1)

1. The extraction process of the total flavones of the thorn sugar is characterized by comprising the following steps:
s1: crushing the thorn sugar, sieving the thorn sugar by a 40-mesh sieve, degreasing the thorn sugar according to the mass ratio of the medicinal materials to the petroleum ether of 1: 3, naturally volatilizing the solvent of the degreased medicinal materials, and storing the degreased medicinal materials in a dry and cold storage place for later use;
s2: drawing standard curve, weighing rutin control, adding 70% ethanol solution, ultrasonic dissolving, and determiningCollecting rutin control solution, scanning with full wavelength, measuring absorbance at 510nm wavelength according to NaNO 2 -AI(NO) 3 -drawing a standard curve by a NaOH color development method;
s3: and (3) extracting the total flavonoids of the spiny sugar, weighing the spiny sugar powder prepared in the step (S1), adding 60-90% of ethanol by volume, wherein the mass-volume ratio of the spiny sugar powder to the ethanol is 1g: (10-30) ml, heating and refluxing at 60-90 ℃ for 30-120min for extraction for 2 times, collecting mixed filtrate, performing rotary evaporation to dryness, redissolving with 70% ethanol, and fixing the volume in a 10ml volumetric flask;
s4: enriching and purifying the total flavonoids of the spinosyns, preprocessing AB-8 type resin, filling 100g of the resin into a chromatographic column with the length of 2.4cm multiplied by 60cm by a wet method, wherein the sample loading amount is 5mL, washing by purified water with the volume of 3 times of the column after the adsorption is finished, and finally eluting by 100mL of 70% ethanol with the elution flow rate of 1.0 mL-min- 1 And rotationally evaporating the eluent to dryness, and determining the content of the total flavones of the spinosad after the redissolution of methanol.
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苏小川;黄昀;赵永昕;阿吉艾克拜尔・艾萨;: "骆驼刺中黄酮类化合物的提取工艺研究", 时珍国医国药, no. 06, pages 1333 - 1334 *
赛那瓦尔・芒思尔;韩克飞;艾买提江・阿依甫别克;邹国安;阿吉艾克拜尔・艾萨;: "骆驼刺药材质量标准研究", 医药导报, no. 08, pages 1074 - 1076 *
郭明鑫 等: "刺糖总黄酮的提取工艺优化及其成分的快速分析", 中药新药与临床药理, vol. 31, no. 09, pages 1110 - 1116 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115645397A (en) * 2022-10-25 2023-01-31 新疆师范大学 Application of russianolive leaf active composition in preparation of anti-melanin synthesis medicine
CN115645397B (en) * 2022-10-25 2024-02-09 新疆师范大学 Application of oleaster leaf active composition in preparation of anti-melanin synthesis medicine

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Application publication date: 20221011