CN105832809B - Preparation method of lespedeza total flavone - Google Patents
Preparation method of lespedeza total flavone Download PDFInfo
- Publication number
- CN105832809B CN105832809B CN201610246298.3A CN201610246298A CN105832809B CN 105832809 B CN105832809 B CN 105832809B CN 201610246298 A CN201610246298 A CN 201610246298A CN 105832809 B CN105832809 B CN 105832809B
- Authority
- CN
- China
- Prior art keywords
- lespedeza
- extraction
- eluent
- bicolor
- extract
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a preparation method of lespedeza total flavone, which comprises the following steps: an extraction step: collecting Lespedeza Seu Oriental Millettia powder, and treating with supercritical fluid CO2Extracting with organic solvent as entrainer by supercritical fluid extraction method to obtain Lespedeza bicolor extract; a purification step: purifying the lespedeza extract by adopting adsorption chromatography, eluting by using organic solvent mixed liquor as an eluent to obtain eluent, and then removing the eluent in the eluent to obtain the lespedeza total flavone. The method adopts supercritical fluid extraction to extract Lespedeza bicolor powder, can realize double functions of extraction and separation, and has high extraction efficiency; the obtained extract is separated by adopting an adsorption chromatography method, so that impurities in the extract can be effectively removed, flavonoid effective substances in the lespedeza virgata are reserved, the separation efficiency is high, and the purity of the obtained lespedeza virgata total flavonoids is high.
Description
Technical Field
The invention relates to the field of separation and purification of plants, and particularly relates to a preparation method of lespedeza total flavonoids.
Background
Chinese pasture and grassland plant resources are extremely abundant, and in recent years, in order to prevent water and soil loss, shrub half shrubs with good resistance become the first choice for water and soil conservation. At present, the plants are not utilized better, and the resource is wasted greatly. Therefore, natural compounds with remarkable activity are discovered from the plants and developed and utilized, so that the economic added value of the natural compounds is greatly improved, and the natural compounds have important ecological and social significance.
Lespedeza Hedyssaroides (Pall.) Kitag.) is a herbaceous shrub growing in the middle and dry areas, often becomes a dominant species or a companion species in meadow grassland communities in forests and forest grassland areas, and is a mountain shrub associated component in the grassland areas. The lespedeza acuminate has the characteristics of drought resistance, cold resistance, barren resistance, wide adaptability and the like, is a good water and soil retention plant, has the characteristics of early green turning, late withering and yellow, large leaf quantity, high nutritional value, good palatability and the like, is excellent pasture for improving deteriorated grasslands in arid and semi-arid regions and building artificial grasslands, is used as ecological grass and excellent pasture, and is paid great attention in recent years. In addition, the herb with root of Lespedeza virgata is recorded in the Chinese herbal medicine assembly, and the herb is bitter in taste and slightly cold, and can check diarrhea, promote urination and stop bleeding. Can be used for treating dysentery, spermatorrhea, hematemesis, and metroptosis. The inventor has carried out systematic analysis on the chemical components of lespedeza pedeza and found that the flavonoid compound is the main chemical component of lespedeza pedeza. The composition mainly contains orientin, isoorientin, vitexin, isovitexin and other carbon glycoside flavones, and the content of the compounds accounts for 90% of the total flavone content. The modern pharmacological activity research shows that the biological activity of the compound is remarkable and mainly expressed as follows: liver protecting, antioxidant, antithyroid, goiter-inducing, and antiviral effects.
Disclosure of Invention
The invention aims to provide a preparation method of lespedeza total flavone. By adopting the preparation method of the lespedeza total flavone, the extraction rate of the lespedeza total flavone is high, the purity is high, the process is simple, and the preparation method is green and environment-friendly.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a preparation method of lespedeza total flavone comprises the following steps:
an extraction step: with supercritical fluid CO2Extracting Lespedeza bicolor powder with organic solvent as entrainer by supercritical fluid extraction to obtain Lespedeza bicolor extract;
a purification step: purifying the Lespedeza kurz extract by adopting an adsorption chromatography, eluting by using an organic solvent mixed solution as an eluent to obtain an eluent, and then removing the eluent in the eluent to obtain the Lespedeza kumi total flavone.
Lespedeza is widely distributed in China, but is mainly used as pasture to prevent water and soil loss at present. The main active ingredient of the lespedeza bicolor is flavonoid compound. At present, the research method for the flavonoid compounds in lespedeza bicolor mainly comprises the following steps: extracting with organic solvent, and recovering solvent to obtain Lespedeza bicolor extract; then separating and purifying the lespedeza bicolor extract by normal phase silica gel column chromatography and polyamide chromatography. The method has the defects of low extraction efficiency, complex extraction process, large usage amount of organic solvent, no contribution to industrial production and environmental pollution.
Compared with the prior art, the invention has the beneficial effects that:
(1) the supercritical fluid extraction method is adopted to extract the lespedeza bicolor powder, so that the dual functions of extraction and separation can be realized, the process flow is simple, the extraction efficiency is high, no organic solvent residue is generated, the product quality is good, and no environmental pollution is caused.
(2) By supercritical fluid CO2The benefit of being an extractant is that CO2Is chemically inert and CO is present during the extraction process2Does not react with the compounds in the lespedeza and can effectively prevent the heat-sensitive substances in the lespedeza from being oxidized and dissipated in the extraction process. In addition, CO2Has low cost, high purity and high safety.
(3) Under supercritical conditions, CO2The extraction efficiency of a compound containing a large amount of polar groups and a compound having a large molecular weight is low. The organic solvent is used as an entrainer, so that the condition can be effectively changedThe solubility of the two compounds can extract the chemical components in the lespedeza as far as possible.
(4) The obtained extract is separated by adopting an adsorption chromatography method, so that impurities in the extract can be effectively removed, flavonoid effective substances in the lespedeza virgata are reserved, the separation efficiency is high, and the purity of the obtained lespedeza virgata total flavonoids is high.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment provides a preparation method of lespedeza total flavonoids, which comprises the following steps:
a pretreatment step: selecting whole Lespedeza bicolor or whole Lespedeza bicolor harvested in 7-9 months, cleaning, drying and crushing to obtain Lespedeza bicolor powder.
The inventor researches the content of the total flavonoids in the lespedeza bicolor in different periods, and finds that the content of the flavonoids in the whole lespedeza bicolor or the whole girald daphne herb harvested in the 7 month to 9 month is higher, so that the yield of the total flavonoids in the lespedeza bicolor can be improved by selecting the whole lespedeza bicolor or the whole girald daphne herb harvested in the 7 month to 9 month in the pretreatment step. In the preferred embodiment of the invention, the particle size of the lespedeza bicolor powder after being crushed is 40-60 meshes, which is beneficial to improving the extraction efficiency.
The lespedeza including lespedeza acuminata, lespedeza amurensis and lespedeza bicolor, the method provided by the invention is applicable to all lespedeza, in the embodiment, lespedeza acuminata is often selected as a research object, and the purpose is to obtain more target products and obtain more yield of total flavonoids of lespedeza.
The inventor researches the content of the total flavone in the lespedeza acuminata, the lespedeza amurensis and the lespedeza bicolor at different periods to find that the content of the total flavone in the lespedeza acuminata harvested in 7 months to 9 months is higher than that of the lespedeza acuminata and the lespedeza bicolor harvested at the same period. Therefore, in the preferred embodiment of the present invention, Lespedeza virgata is used as the subject.
An extraction step: with supercritical fluid CO2Extracting Lespedeza bicolor powder with organic solvent as entrainer by supercritical fluid extraction to obtain Lespedeza bicolor extract.
The supercritical fluid extraction method is adopted to extract the lespedeza bicolor powder, so that the dual functions of extraction and separation can be realized, the process flow is simple, the extraction efficiency is high, no organic solvent residue is generated, the product quality is good, and no environmental pollution is caused. By supercritical fluid CO2The benefit of being an extractant is that CO2Is inert to the chemical nature of CO in the extraction process2Does not react with the compound in the lespedeza, and can effectively prevent the oxidation and the escape of the heat-sensitive substance in the extraction process. In addition, CO2Has low cost, high purity and high safety. Under supercritical conditions, CO2The extraction efficiency of a compound containing a large amount of polar groups and a compound having a large molecular weight is low. The organic solvent is used as an entrainer, so that the solubility of the two compounds can be effectively changed, and the chemical components in the lespedeza virgata can be extracted to the maximum extent.
In the preferred embodiment of the present invention, the entrainer is absolute ethanol or methanol. The polarity of methanol and absolute ethanol is relatively high, and the extraction rate of the total flavonoids in the lespedeza. Compared with methanol, absolute ethanol has the advantages of small pollution and easy removal. Therefore, the entrainer is more preferably absolute ethanol.
In a preferred embodiment of the present invention, the relationship between the amount of the lespedeza virgata powder and the entrainer is as follows: adding entrainer 1-5mL per gram of Lespedeza bicolor powder. Further preferably, the volume of entrainer added per gram of lespedeza virgata powder is 3 mL. When the volume of the entrainer is 1-5mL when each gram of the lespedeza virgata powder entrainer is extracted, the extraction efficiency of the total flavonoids in lespedeza virgata is high, and especially when the volume of the entrainer is 3mL, the extraction efficiency is highest.
In the preferred embodiment of the present invention, the process parameters in the supercritical fluid extraction method are as follows: the extraction pressure is 30MPa-60MPa, the extraction temperature is 40-60 ℃, and the extraction time is 60min-120 min. The technological parameters are further optimized, the extraction pressure is 40MPa-50MPa, the extraction temperature is 45 ℃ -55 ℃, and the extraction time is 80min-100 min. The inventor adopts an orthogonal test to optimize main parameters influencing the extraction efficiency of the lespedeza total flavonoids, and when the process parameters are adopted for extraction, the extraction efficiency of the lespedeza total flavonoids is the maximum, and the extraction amount of the lespedeza total flavonoids is also the maximum.
A purification step: separating the Lespedeza kurz extract by adopting an adsorption chromatography, eluting by using an organic solvent mixed solution as an eluent to obtain an eluent, and then removing the eluent in the eluent to obtain the Lespedeza kumi total flavone. The Lespedeza kummensis extract is separated by adopting an adsorption chromatography method, so that impurities in the Lespedeza kummensis extract can be effectively removed, effective flavonoid substances in Lespedeza kummensis are reserved, the separation efficiency is high, and the purity of the obtained total flavonoid in Lespedeza kummensis is high.
In a preferred embodiment of the invention, the adsorption chromatography is macroporous resin adsorption chromatography. The macroporous resin has the advantages of high stability, large adsorption capacity, good selectivity, convenient resin regeneration, mild desorption conditions, long service cycle, low cost and the like. The extract of the lespedeza bicolor is separated and purified by adopting macroporous resin adsorption chromatography, the separation efficiency is high, the purity of the lespedeza bicolor total flavonoids is high, and meanwhile, the amount of the obtained lespedeza bicolor total flavonoids is large. More preferably, D-101 type resin is selected for use in macroporous resin adsorption chromatography. The D-101 type resin has short adsorption balance time on the xanthone compound in the lespedeza, belongs to fast adsorption chromatography, and can effectively shorten the production period, reduce the production cost and improve the production efficiency.
In a preferred embodiment of the present invention, the eluent is a mixed solution of ethanol and water, wherein the volume fraction of ethanol is 60% -80%. The method adopts ethanol water as eluent, is green and environment-friendly, and has little harm to the body of an experimenter. 60% -80% ethanol water solution is used as eluent, the elution rate is high, and the reduction of the yield of the total flavonoids in the lespedeza bicolor can be effectively avoided due to dead adsorption of active substances on macroporous resin.
More preferably, the sample injection rate is 0.5mL/min to 1.5mL/min and the flow rate of the eluent is 1mL/min to 5mL/min when the Lespedeza virgata extract is purified by macroporous resin adsorption chromatography. When the flow rate of the eluent is 1mL/min-5mL/min, the high purity of the total flavonoids in the lespedeza bicolor can be ensured, the high elution rate is realized, the elution time is shortened, and the efficiency is improved. More preferably, the eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow rate is 3 mL/min. Under the condition, the elution time is short, the purity of the obtained lespedeza total flavone is high, the dead adsorption amount of the macroporous resin to the flavonoid component in lespedeza is small, and the purification effect is optimal.
Example 1
Step 1: selecting whole Lespedeza speranskia with roots collected in 7 months, cleaning, drying, and crushing to 40 meshes to obtain Lespedeza virgata powder.
Step 2: and (3) putting 5g of the lespedeza bicolor powder obtained in the step (1) into a 24mL extraction kettle, and extracting by adopting a supercritical fluid extraction method. Wherein the extractant is supercritical fluid CO2The entrainer is absolute ethyl alcohol, the extraction pressure is 30MPa, the extraction temperature is 40 ℃, the extraction time is 60min, and the volume of the entrainer added into each gram of lespedeza bicolor powder is 1 mL. And measuring the absorbance at the wavelength of 510nm of the Lespedeza bicolor extract obtained after extraction by adopting an ultraviolet spectrophotometer method, and calculating the extraction amount of flavone in the Lespedeza bicolor extract.
And 3, dissolving the Lespedeza virgata extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is a 60% ethanol water solution, the sample injection speed is 0.5mL/min, and the eluent flow speed is 1 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Example 2
Step 1: selecting 8 parts of collected whole herb of lespedeza pedeza with roots, cleaning, drying, and crushing to 50 meshes to obtain lespedeza powder.
Step 2: the lespedeza bicolor powder 5 obtained in the step 1And g, putting the mixture into a 24mL extraction kettle, and extracting by adopting a supercritical fluid extraction method. Wherein the extractant is supercritical fluid CO2The entrainer is absolute ethyl alcohol, the extraction pressure is 40MPa, the extraction temperature is 45 ℃, the extraction time is 80min, and the volume of the entrainer added into each gram of lespedeza bicolor powder is 2 mL. And measuring the absorbance at the wavelength of 510nm of the Lespedeza bicolor extract obtained after extraction by adopting an ultraviolet spectrophotometer method, and calculating the extraction amount of flavone in the Lespedeza bicolor extract.
And 3, dissolving the Lespedeza virgata extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow speed is 2 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Example 3
Step 1: selecting 8 parts of collected whole herb of lespedeza pedeza with roots, cleaning, drying, and crushing to 50 meshes to obtain lespedeza powder.
Step 2: and (3) putting 5g of the lespedeza bicolor powder obtained in the step (1) into a 24mL extraction kettle, and extracting by adopting a supercritical fluid extraction method. Wherein the extractant is supercritical fluid CO2The entrainer is absolute ethyl alcohol, the extraction pressure is 45MPa, the extraction temperature is 50 ℃, the extraction time is 90min, and the volume of the entrainer added into each gram of lespedeza bicolor powder is 3 mL. And measuring the absorbance at the wavelength of 510nm of the Lespedeza bicolor extract obtained after extraction by adopting an ultraviolet spectrophotometer method, and calculating the extraction amount of flavone in the Lespedeza bicolor extract.
And 3, dissolving the Lespedeza virgata extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow speed is 3 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Example 4
Step 1: selecting 8 parts of collected whole herb of lespedeza pedeza with roots, cleaning, drying, and crushing to 50 meshes to obtain lespedeza powder.
Step 2: and (3) putting 5g of the lespedeza bicolor powder obtained in the step (1) into a 24mL extraction kettle, and extracting by adopting a supercritical fluid extraction method. Wherein the extractant is supercritical fluid CO2The entrainer is absolute ethyl alcohol, the extraction pressure is 50MPa, the extraction temperature is 55 ℃, the extraction time is 100min, and the volume of the entrainer added into each gram of lespedeza bicolor powder is 4 mL. And measuring the absorbance at the wavelength of 510nm of the Lespedeza bicolor extract obtained after extraction by adopting an ultraviolet spectrophotometer method, and calculating the extraction amount of flavone in the Lespedeza bicolor extract.
And 3, dissolving the Lespedeza virgata extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow speed is 4 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Example 5
Step 1: selecting 9 parts of collected whole herb of lespedeza pedeza with roots, cleaning, drying, and crushing to 60 meshes to obtain lespedeza powder.
Step 2: and (3) putting 5g of the lespedeza bicolor powder obtained in the step (1) into a 24mL extraction kettle, and extracting by adopting a supercritical fluid extraction method. Wherein the extractant is supercritical fluid CO2The entrainer is absolute ethyl alcohol, the extraction pressure is 60MPa, the extraction temperature is 60 ℃, the extraction time is 120min, and the volume of the entrainer added into each gram of lespedeza bicolor powder is 5 mL. And measuring the absorbance at the wavelength of 510nm of the Lespedeza bicolor extract obtained after extraction by adopting an ultraviolet spectrophotometer method, and calculating the extraction amount of flavone in the Lespedeza bicolor extract.
And 3, dissolving the Lespedeza virgata extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is an 80% ethanol water solution, the sample injection speed is 1.5mL/min, and the eluent flow speed is 5 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Example 6
Step 1: selecting 8 parts of collected whole herb of the brachypodium dauricum with roots, cleaning, drying, and crushing to 50 meshes to obtain the lespedeza virgata powder.
Step 2: and (3) putting 5g of the lespedeza bicolor powder obtained in the step (1) into a 24mL extraction kettle, and extracting by adopting a supercritical fluid extraction method. Wherein the extractant is supercritical fluid CO2The entrainer is methanol, the extraction pressure is 45MPa, the extraction temperature is 50 ℃, the extraction time is 90min, and the volume of the entrainer added into each gram of lespedeza virgata powder is 3 mL. And measuring the absorbance at the wavelength of 510nm of the Lespedeza bicolor extract obtained after extraction by adopting an ultraviolet spectrophotometer method, and calculating the extraction amount of flavone in the Lespedeza bicolor extract.
And 3, dissolving the Lespedeza virgata extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow speed is 3 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Example 7
Step 1: selecting 8 parts of collected bicolor lespedeza bicolor whole grass with roots, cleaning, drying and crushing to 50 meshes to obtain lespedeza bicolor powder.
Step 2: and (3) putting 5g of the lespedeza bicolor powder obtained in the step (1) into a 24mL extraction kettle, and extracting by adopting a supercritical fluid extraction method. Wherein the extractant is supercritical fluid CO2The entrainer is methanol, the extraction pressure is 45MPa, the extraction temperature is 50 ℃, the extraction time is 90min, and the volume of the entrainer added into each gram of lespedeza virgata powder is 3 mL. And measuring the absorbance at the wavelength of 510nm of the Lespedeza bicolor extract obtained after extraction by adopting an ultraviolet spectrophotometer method, and calculating the extraction amount of flavone in the Lespedeza bicolor extract.
And 3, dissolving the Lespedeza virgata extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow speed is 3 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Comparative example 1
Step 1: selecting 8 parts of collected lespedeza complete grass with roots, cleaning, drying, and crushing to 50 meshes to obtain lespedeza powder.
Step 2: and (3) extracting 5g of the lespedeza bicolor powder obtained in the step (1) by adopting a water decoction method for three times, wherein each time lasts for 2 hours, so as to obtain three parts of extracting solution. Mixing the three extractive solutions, and concentrating under reduced pressure to obtain Lespedeza bicolor extract. Measuring absorbance at wavelength of 510nm by ultraviolet spectrophotometer, and calculating flavone extraction amount in Lespedeza Seu Oriental extract.
And 3, dissolving the lespedeza bicolor extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow speed is 3 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Comparative example 2
Step 1: selecting 8 parts of collected lespedeza complete grass with roots, cleaning, drying, and crushing to 50 meshes to obtain lespedeza powder.
Step 2: and (3) extracting 5g of the lespedeza bicolor powder obtained in the step (1) by adopting an ethanol reflux method for three times, wherein each time lasts for 2 hours, so as to obtain three parts of extracting solution. Mixing the three extractive solutions, and concentrating under reduced pressure to obtain Lespedeza bicolor extract. Measuring absorbance at wavelength of 510nm by ultraviolet spectrophotometer, and calculating flavone extraction amount in Lespedeza Seu Oriental extract.
And 3, dissolving the lespedeza bicolor extract obtained in the step 2, and purifying by adopting D-101 type macroporous resin adsorption chromatography, wherein an eluent is 70% ethanol water solution, the sample injection speed is 1mL/min, and the eluent flow speed is 3 mL/min. Collecting the eluent, and then removing the ethanol water solution in the eluent to obtain the lespedeza total flavone.
Experimental example 1
The total flavonoids in lespedeza bicolor were purified by the methods described in examples 1 to 5 (experimental group) and comparative examples 1 and 2 (control group), respectively. Measuring absorbance at 510nm by ultraviolet spectrophotometer, and calculating total flavone extraction amount and refining degree of Lespedeza Seu Cylindrica extract.
The method for measuring the extraction amount of the total flavonoids in the lespedeza bicolor extract comprises the following steps: and (3) measuring the absorbance of rutin at 510nm by taking rutin as a standard substance, and making a standard curve to obtain a regression equation. And (3) taking the Lespedeza bicolor extract, measuring the absorbance of the Lespedeza bicolor extract at 510nm, and substituting the measured absorbance into the regression equation to calculate the corresponding flavone concentration. And multiplying the volume of the solution of the lespedeza bicolor extract to obtain the mass of the total flavone in the product, which is marked as A. The total mass of lespedeza bicolor powder used in the extraction was marked as B. The total flavone extraction amount in the lespedeza virgata extract liquid in the product is calculated according to the following formula, wherein the total flavone extraction amount is A/B.
The purity of the lespedeza total flavone obtained in the step 3/the purity of the lespedeza extract is multiplied by 100 percent
In the above formula, the method for determining the purity of the lespedeza flavone comprises the following steps: and (3) measuring the absorbance of rutin at 510nm by taking rutin as a standard substance, and making a standard curve to obtain a regression equation. And (3) taking the extract of the lespedeza bicolor, measuring the absorbance of the extract at 510nm, and substituting the absorbance into the regression equation to calculate the corresponding flavone concentration. And multiplying the volume of the extracting solution to obtain the mass of the total flavone in the product, which is marked as A. And then drying the extracting solution by using an electrothermal constant-temperature drying oven, and measuring the dried quality of the product, and recording as B. The purity of the total flavone in the product is calculated according to the following formula:
purity (%) ═ a/B × 100%.
TABLE 1 comparison of preparation methods of total flavonoids of lespedeza in experimental group and control group
As can be seen from table 1:
1. compared with the traditional extraction process, such as the decoction method in the comparison example 1 and the ethanol reflux method in the comparison example 2, the supercritical fluid extraction method adopted by the invention can obviously improve the extraction amount of the total flavone in the lespedeza bicolor extract (the experimental group is 1.02mg/g-1.45mg/g, and the comparison group is 3.16mg/g-3.69 mg/g). In addition, the purity of the total flavone in the lespedeza bicolor extract is also obviously improved (the experimental group is 2.51% -3.47%, and the control group is 10.54% -11.79%).
The main reason is that the lespedeza bicolor is rich in a large amount of protein, and the protein in the lespedeza bicolor is leached out by adopting a water decoction method, so that the subsequent purification difficulty is increased; moreover, the flavonoids in lespedeza bicolor are mostly carbon glycoside flavones, and the carbon glycoside flavones are not easy to dissolve in water, so the extraction rate is low. If the ethanol reflux method is adopted for extraction, a large amount of ethanol solution is consumed, and chlorophyll in lespedeza bicolor is easily leached during extraction, so that the purity of total flavonoids is influenced. Therefore, the supercritical fluid extraction method can integrate extraction and separation, effectively improve the purity and extraction amount of the total flavonoids in the lespedeza, shorten the experimental period and facilitate the realization of industrial production.
2. By adopting the preparation method of the lespedeza total flavone provided by the invention, the extract liquid of the tip leaf total flavone can be effectively purified. The purity of the extract of lespedeza bicolor is improved by about 300 times after the extract is purified by the step 3 (the purity of the extract is 10.54 to 11.75 percent before purification, and the purity of the total flavone is 30.45 to 35.59 percent after purification). The purification degree in comparative examples 1 and 2 was higher than that in the experimental group, mainly due to the low purity of the extract in the method, and even after purification, the total flavone purity was only half of that in the experimental group, i.e., the purity was 18.05% -20.18%.
3. As can be seen from examples 6 and 7, the total flavonoids in the lespedeza kwangsiensis and lespedeza bicolor prepared by the method have the extraction amounts of 3.53mg/g and 3.49mg/g respectively, and the refining degrees of the lespedeza bicolor total flavonoids are 289.88% and 295.56% respectively. The method can be used for well preparing the total flavonoids in the two plants of the lespedeza kwangsiensis and the lespedeza bicolor, and the obtained lespedeza total flavonoids have high purity and high yield.
Experimental example 2
The experimental example adopts a supercritical carbon dioxide fluid extraction method to extract the total flavonoids in the lespedeza bicolor, and the method is similar to the method in the example 3, except that the value of the process parameter in the supercritical fluid extraction method is changed. Orthogonal experiments are carried out on 4 parameters of extraction temperature, extraction pressure, extraction time and entrainer concentration, and the optimum scheme of carbon dioxide supercritical fluid extraction is determined by measuring the content of flavone in the extract by adopting a spectrophotometry.
The experimental method comprises the following steps: accurately weighing 5g of crushed Lespedeza bicolor powder, putting into a 24mL extraction kettle, extracting the dry Lespedeza bicolor powder by using CO2 in a supercritical state under specified conditions by using absolute ethyl alcohol as an entrainer in a CO2 supercritical extraction system, collecting an extract, measuring a light absorption value, and calculating the extraction amount of flavone. Selecting L9(34) Orthogonal table is used for designing test scheme, the factor level is shown in table 2, and the influence of different levels of each factor on the extraction amount of flavone is determined by adopting orthogonal test. Thereby obtaining the best condition for extracting the total flavone in the lespedeza bicolor.
TABLE 2 factor and horizon
TABLE 3 supercritical fluid extraction orthogonality results table
TABLE 4 ANOVA TABLE
| Source of variation | Sum of squares of deviation | Degree of freedom | Mean square | F value |
| A pressure of extraction | 5.55 | 2 | 2.78 | 7.62 |
| B temperature of extraction | 0.28 | 2 | 0.138 | 0.379 |
| C extraction time | 0.034 | 2 | 0.017 | 0.047 |
| D amount of entrained substance | 0.65 | 2 | 0.3262 | 0.896 |
| Error of the measurement | 0.72 | 2 | 0.364 |
According to the analysis of the orthogonal test results shown in tables 3 and 4, the influence degree of each factor on the extraction of the total flavone is sequentially the extraction pressure, the extraction temperature, the dosage of the entrainer, the extraction time and the optimal process condition A of the extraction of the total flavone3B3C3D3Namely, the extraction pressure is 45Mpa, the extraction temperature is 50 ℃, the extraction time is 90min, and the dosage of the entrainer is 3 mL/g. The influence of the extraction pressure on the extraction amount reaches a significant level according to the analysis of variance table.
And (3) process verification test: the test was repeated five times under the optimum conditions obtained in the above manner, and the results of the verification test are shown in Table 5. Therefore, the process method is stable, and the extraction amount of the total flavone in the lespedeza bicolor extract obtained by the process method is large.
TABLE 5 verification test results Table
| Sample number | 1 | 2 | 3 | 4 | 5 | Mean value of | RSD％ |
| Amount of extraction | 3.689 | 3.702 | 3.695 | 3.689 | 3.692 | 3.6934 | 1.465 |
Experimental example 3
A similar procedure as described in example 3 was used in this experimental example, except that the D-101 type macroporous resin was replaced with another 8 types of macroporous resin, i.e., HP-20, XAD7HP, AB-8, XAD16, LS-300, LS-302, LS-303, and LS-300B types of macroporous resin. The adsorption and desorption effects of the 9-type macroporous resin on lespedeza virgata flavone are inspected by adopting a static adsorption method, and experiments show that:
TABLE 6 nine kinds of macroporous resin adsorption and desorption energy charts
As can be seen from Table 6, the macroporous resins of AD7HP, D-101, XAD16 and LS-303 types have better flavone adsorption capacity, wherein the adsorption rate of the macroporous resins of XAD16 type is up to 77.8%, but the desorption capacity of the macroporous resins of XAD7HP and XAD16 type is not strong. HP-20, D-101, LS-300, LS-303 and LS-300B type macroporous resins have good desorption capacity, but LS-300 and LS-300B type macroporous resins have weak adsorption capacity.
Comprehensively analyzing the adsorption and desorption capacity of the 9 kinds of resins on the lespedeza virgata flavone, and preliminarily selecting three kinds of resins of XAD16, D-101 and LS-303 type macroporous resins to perform adsorption kinetics tests according to the quantity of obtained flavone. The results are shown in Table 7:
TABLE 7 dynamic adsorption of resins
From Table 7, the macroporous resin XAD16 type had a large initial adsorption amount, 6 hours were required for reaching equilibrium, and the saturated adsorption amount was large. The adsorption capacity of the D-101 type macroporous resin is rapidly increased along with the time extension in 0-1 hour, and the adsorption equilibrium time is about 3 hours. LS-303 and D-101 type macroporous resins have similar adsorption equilibrium time of flavonoids compounds, about 3 hours, but the saturated adsorption capacity of LS-303 is not as good as that of D-101 type macroporous resins. Because the D-101 type macroporous resin belongs to the fast adsorption resin, the time can be saved, and the production period can be shortened, so from the aspect of industrial production cost, the D-101 type macroporous resin is selected as the special resin in the lespedeza virgata flavone refining process, the production cost can be reduced, and the production efficiency can be improved.
Experimental example 4
This experimental example used the procedure described in example 3,
the process method for purifying the picrorhiza kurroa total flavone by using the D-101 type macroporous adsorption resin is verified.
Taking 25mL (15.32mg/mL) of Lespedeza virgata extract, passing through a treated adsorption column (diameter-height ratio is 1:10) filled with 10g D-101 type macroporous adsorption resin at the flow rate of 1mL/min, performing dynamic adsorption, washing with distilled water, eluting with 110mL of ethanol solution with percentage content of 70% at the flow rate of 3mL/min, collecting eluent, determining the content of total flavonoids in the eluent according to a content determination method, evaporating the ethanol eluent to dryness, and determining the weight of dry paste. The results of 3 parallel experiments were obtained by calculating the elution rate and purity and examining the reproducibility according to the relative standard deviation, the degree of refinement (purity of dried extract after elution/purity of sample. times.100%), and are shown in Table 8.
TABLE 8 process verification result table for purifying lespedeza flavone by using D-101 type macroporous adsorbent resin
The result shows that the effect of purifying the total flavonoids in the lespedeza bicolor by using the D-101 macroporous adsorption resin is good, the refining degree is higher, and the reproducibility is good.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (3)
1. A preparation method of the lespedeza total flavone is characterized by comprising the following steps:
an extraction step: with supercritical fluid CO2Extracting Lespedeza bicolor powder with organic solvent as entrainer by supercritical fluid extraction to obtain Lespedeza bicolor extract;
a purification step: purifying the lespedeza bicolor extract by adopting an adsorption chromatography, eluting by using an organic solvent mixed solution as an eluent to obtain an eluent, and then removing the eluent in the eluent to obtain lespedeza total flavone, wherein the adsorption chromatography adopts D-101 type macroporous resin; the lespedeza bicolor is lespedeza acuminate;
in the extraction step, the entrainer is absolute ethyl alcohol or methanol;
the dosage relationship of the lespedeza bicolor powder and the entrainer is as follows: extracting and adding 1-5mL of entrainer into each gram of lespedeza bicolor powder;
the supercritical fluid extraction method comprises the following process parameters: the extraction pressure is 30MPa-60MPa, the extraction temperature is 40-60 ℃, and the extraction time is 60min-120 min;
in the purification step, the eluent is a mixed solution of ethanol and water, wherein the volume fraction of the ethanol is 60-80%;
in the purification step, the sample injection speed is 0.5-1.5 mL/min when the Lespedeza bicolor extract is purified by adopting the adsorption chromatography, and the flow speed of the eluent is 1-5 mL/min.
2. The method for preparing lespedeza bicolor total flavonoids according to claim 1, characterized by further comprising a pretreatment step before the extraction step, wherein the pretreatment step is to select whole lespedeza bicolor or whole lespedeza bicolor harvested in 7-9 months, wash, dry and pulverize to obtain lespedeza bicolor powder.
3. The method for preparing the lespedeza total flavone as claimed in claim 1, wherein the supercritical fluid extraction method comprises the following process parameters: the extraction pressure is 40MPa-50MPa, the extraction temperature is 45-55 ℃, and the extraction time is 80-100 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610246298.3A CN105832809B (en) | 2016-04-19 | 2016-04-19 | Preparation method of lespedeza total flavone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610246298.3A CN105832809B (en) | 2016-04-19 | 2016-04-19 | Preparation method of lespedeza total flavone |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105832809A CN105832809A (en) | 2016-08-10 |
| CN105832809B true CN105832809B (en) | 2020-06-09 |
Family
ID=56588723
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610246298.3A Active CN105832809B (en) | 2016-04-19 | 2016-04-19 | Preparation method of lespedeza total flavone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105832809B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361786B (en) * | 2008-08-25 | 2010-10-27 | 中国农业科学院草原研究所 | Total-flavone extraction and purification and its monomer separation method from Lespedaza bedysaroides |
| CN102406694B (en) * | 2011-12-02 | 2013-11-27 | 安徽省药物研究所 | Anti-tumor effective part of traditional Chinese medicine lespedeza as well as preparation method and application thereof |
-
2016
- 2016-04-19 CN CN201610246298.3A patent/CN105832809B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 超临界CO2 萃取技术在中药有效成分提取中的应用新进展;周雪晴等;《海南大学学报自然科学版》;20070331;第25卷(第1期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105832809A (en) | 2016-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105998109A (en) | Mango seed polyphenol extract and preparation method thereof | |
| CN101167775B (en) | Method for extracting chrysanthemum total flavone | |
| CN101333203A (en) | Method for extracting dihydroquercetin form larch | |
| CN103520233A (en) | Preparation method and application of apocynum venetum leaf total polyphenol | |
| Dong et al. | The evaluation of five bioactive compounds content and in vitro antioxidant of Caryophylli Flos extracts obtained by natural deep eutectic solvents | |
| CN104569195B (en) | A kind of method of flavonoids effective constituent content in toothpaste detected containing baikal skullcap root | |
| CN103183616A (en) | Method for preparing chlorogenic acid from leaves of lonicera hypoglauca miq | |
| CN106138130B (en) | Mango seed flavone extract and preparation method thereof | |
| CN105832809B (en) | Preparation method of lespedeza total flavone | |
| CN104140909B (en) | A kind of rose flower wine and preparation method thereof | |
| CN106617060A (en) | Fructus phyllanthi polyphenols enriching and purifying method | |
| CN101845236B (en) | Method for extracting melanin from black waxy corn | |
| CN107163059B (en) | A kind of preparation method of mango core ellagic acid | |
| CN106187975B (en) | A kind of preparation for improving rice bran aldehydes matter bioactivity and purification process | |
| CN104606297B (en) | Method for extracting active ingredients in cistanche tubulosa | |
| CN107468746A (en) | A kind of method that middle extraction polyphenol and flavones are spent from Zhu Ying | |
| CN113512021A (en) | Method for simultaneously extracting procyanidine and polysaccharide from rosa davurica pall seeds | |
| CN102670634A (en) | C-glycosylflavones composition, preparation method and application thereof | |
| CN108623642B (en) | Deep eutectic solvent water mixture for synchronously extracting salidroside and tyrosol from rhodiola rosea, and preparation method and extraction method thereof | |
| CN106266168A (en) | A kind of preparation method of Fructus Schisandrae Sphenantherae lignanoid extract | |
| CN115032287A (en) | Quantitative analysis method of tetrahydrocannabinol and cannabidiol | |
| CN101780130A (en) | Industrial extraction method of bo-chrysanthemum | |
| Li et al. | Enrichment and isolation of syringin and oleuropein from Syringa reticulata subsp. amurensis branch bark ionic liquid extract via macroporous resin adsorption and desorption | |
| CN108159112A (en) | A kind of preparation method of mongolian oak leaf extractive of general flavone | |
| CN113813305B (en) | Method for integrated extraction of active ingredients in schisandra chinensis fruits |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wu Hongxin Inventor after: A Lamusi Inventor after: Chang Chun Inventor after: Dai Yating Inventor after: Zhang Xiaoqing Inventor after: Zhao Jinmei Inventor after: Wu Lanbateer Inventor after: Sun Juanjuan Inventor after: Liu Honglin Inventor before: A Lamusi Inventor before: Wu Hongxin Inventor before: Chang Chun Inventor before: Dai Yating Inventor before: Zhang Xiaoqing Inventor before: Zhao Jinmei Inventor before: Wu Lanbateer Inventor before: Sun Juanjuan Inventor before: Liu Honglin |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |