CN105169094B - Indocalamus leaf total flavone extracting and purifying method - Google Patents

Indocalamus leaf total flavone extracting and purifying method Download PDF

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Publication number
CN105169094B
CN105169094B CN201510737809.7A CN201510737809A CN105169094B CN 105169094 B CN105169094 B CN 105169094B CN 201510737809 A CN201510737809 A CN 201510737809A CN 105169094 B CN105169094 B CN 105169094B
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extracting
ethanol
total flavonoids
indocalamus
indocalamus leaves
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CN105169094A (en
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乐薇
吴士筠
杨文婷
柯斌清
覃瑞
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Wuhan Technology and Business University
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Abstract

The invention belongs to the technical field of natural product separation, food additives and medicines, and particularly relates to a method for extracting and purifying total flavonoids in indocalamus leaves, which comprises the following steps: a. cleaning folium indocalami tessellati, oven drying, and pulverizing; b. pre-soaking the crushed indocalamus leaves for 20-60 min by using saturated lime water, adding an ethanol water solution, adjusting the pH value of a system to be close to neutral, heating and extracting for more than 30min, controlling the heating and extracting temperature to be 50-70 ℃, and then carrying out vacuum filtration to obtain a total flavone extracting solution; c. evaporating and concentrating the total flavone extract, adsorbing by macroporous resin, washing with water, and eluting with ethanol water solution to obtain eluate; d. evaporating and concentrating the eluent, and freeze-drying to obtain the total flavonoids. The method has the advantages of high yield of the total flavone, low concentration of ethanol solution used during extraction and low extraction temperature.

Description

Indocalamus leaf total flavone extracting and purifying method
Technical Field
The invention belongs to the technical field of natural product separation, food additives and medicines, and particularly relates to a method for extracting and purifying total flavonoids in indocalamus leaves.
Background
Modern researches show that the total flavonoids have various biological activities of resisting tumors, inflammation, bacteria and atherosclerosis, reducing blood fat, regulating immunity, eliminating free radicals and the like, and particularly have obvious inhibiting effects on occurrence, proliferation, migration, angiogenesis, multidrug resistance and the like of tumors. In addition, the total flavone is low-toxic or even non-toxic to normal cells of a human body, so that the total flavone has wide application prospects in the aspects of human health care, nutrition and disease prevention and treatment.
Indocalamus leaf is the general term of leaf of Indocalamus plant of bamboo subfamily of Gramineae, and the plant of the genus is more than 30 kinds, all produced in China, and is the package of Zongzi which is the mark food of the festival since ancient times. Modern pharmacological analysis finds that indocalamus leaf has various remarkable effects of sterilization, corrosion prevention, cancer resistance and the like, and can be widely applied to medicines, foods, daily chemical products and the like. A large amount of wild indocalamus leaf resources are available in each province in the south of Yangtze river in China, particularly, the wild indocalamus leaf resources in the mountain areas of Wuling, Hubei are extremely rich, but only a small amount of wild indocalamus leaf resources are used as food packages, so that the resource waste is large, and the processing technology content is not high. Researches prove that the total flavonoids are important active ingredients of the indocalamus leaves, so that the research on extracting and purifying the total flavonoids of the indocalamus leaves is necessary to improve the comprehensive utilization value of the indocalamus leaves.
The existing methods for extracting total flavonoids include reflux method, ultrasonic method, microwave method, etc. However, the ultrasonic method and the microwave method require special instruments, and the one-time cost investment is high; the direct reflux method needs to use ethanol water solution with higher concentration, high maintaining temperature during reflux, long reflux extraction time and higher comprehensive cost (in the research of Indocalamus leaf total flavone in the document, the research of plum water aroma and the like shows that 85 percent ethanol solution can be used for reflux extraction of Indocalamus leaf total flavone at 85 ℃ for 3 h; the research of Indocalamus leaf forest structure characteristics and bamboo leaf bioactive components in the document, the conditions for extracting the total flavone in the research of Suchufa and the like are that the ethanol concentration is 85 percent, the material-liquid ratio is 1: 20 (g: mL), the reflux temperature is 85 ℃, and the extraction time is 50 min); therefore, the development of an efficient and low-cost total flavone extraction and purification technology has better market prospect.
Disclosure of Invention
The invention aims to provide a method for extracting and purifying total flavonoids from indocalamus leaves, which is simple and convenient to operate, high in extraction rate and low in cost.
In order to achieve the purpose, the invention adopts the following technical measures: an extraction and purification method of indocalamus leaf total flavonoids comprises the following steps: a. cleaning folium indocalami tessellati, oven drying, and pulverizing; b. pre-soaking the crushed indocalamus leaves for 20-60 min by using saturated lime water, adding an ethanol water solution, adjusting the pH value of a system to be close to neutral, heating and extracting for more than 30min, controlling the heating and extracting temperature to be 50-70 ℃, and then carrying out vacuum filtration to obtain a total flavone extracting solution; c. evaporating and concentrating the total flavone extract, adsorbing by macroporous resin, washing with water, and eluting with ethanol water solution to obtain eluate; d. evaporating and concentrating the eluent, and freeze-drying to obtain the total flavonoids. Concentrating the eluate on rotary evaporator, evaporating to remove solvent until a small amount of material liquid is left in the desolventizing bottle and can be poured out, and freeze drying to obtain folium Indocalami total flavone extract.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the drying in the step a is required to be carried out for 3-7 hours at the temperature of 40-70 ℃.
Further, the pulverization in the step a requires that the obtained indocalamus leaf particles are 12-50 meshes.
Further, the dosage of the saturated lime water in the step b is V1mL, the dosage of the indocalamus leaves is M g V1The ratio of M to M is 2 to 10: 1.
further, in the step b, the volume fraction of ethanol in the ethanol aqueous solution is 30-60%, and the dosage of the ethanol aqueous solution is V2mL, the dosage of the indocalamus leaves is M g V2The ratio of M to M is 10 to 30: 1.
further, after the ethanol water solution is added in the step b, the pH value of the system is adjusted to 6.5-7.0, and the heating extraction time is 40-60 min. When the pH value of the system is adjusted, non-oxidizing acids such as dilute acetic acid, dilute hydrochloric acid and the like can be added into the system.
Further, the evaporation concentration in step c is required to be less than 1/3 of the total flavone extract after concentration. Concentrating the total flavone extract on a rotary evaporator, and evaporating to remove the solvent, wherein the vacuum degree of the rotary evaporator is about 0.08MPa, the water bath temperature is 45-55 ℃, and the concentration is stopped until the residual feed liquid is below 1/3 of the original volume, so as to obtain the concentrated solution of the total flavone extract of indocalamus leaves.
Further, the adsorption flow rate of the macroporous resin in the step c during adsorption is 2-5 BV/h.
Furthermore, the volume fraction of ethanol in the ethanol water solution in the step c is 50-80%, the dosage of the ethanol in the step c is 3-6 BV during elution, and the elution flow rate is 2-5 BV/h.
Further, the macroporous resin in the step c is any one of HPD800, HPD600, HPD450 and AB-8.
The invention has the beneficial effects that: (1) the indocalamus leaf adopted by the invention can be discarded and newly picked fresh indocalamus leaf small leaf, damaged leaf, processed indocalamus leaf waste residue and the like; (2) the saturated limewater is adopted for presoaking, the yield of the total flavone in the extracting solution can be improved to 35 percent compared with the result without presoaking, the yield reaches 2.98 percent, and the extraction rate is high; (3) the volume percentage of ethanol added into the ethanol water solution is 30-60% during extraction, the temperature is 50-70 ℃, and compared with the ethanol used in the Indocalamus leaf total flavone extraction method reported in the literature, the ethanol concentration is low, the extraction temperature is low, the impurities such as chlorophyll are less, and the cost loss is low; (4) the macroporous resin can be regenerated and reused, and the ethanol obtained by evaporation and concentration can also be reused; (5) the extracted and purified total flavone has no harmful impurities and good bioactivity, and can be used for development of medicines, foods, etc.
Detailed Description
The features of the present invention are described below, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention.
For a better understanding of the present invention, reference is made to the following examples.
Example 1
Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. The indocalamus leaf granules are put into an extraction bottle, 5 times (mL: g, namely the multiple relation between the value of the volume of the added solution in mL and the value of the mass of the indocalamus leaf in g) of saturated lime water solution is added for pre-soaking for 0.5 hour, 20 times of ethanol water solution with the volume fraction of 50% is added, the pH of the system is adjusted to be about 6.5-7, extraction is carried out for 40min at the temperature of 60 ℃, and the total flavone extracting solution is obtained by decompression and suction filtration. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding HPD800 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with ethanol water solution with volume fraction of 70%, collecting eluate, concentrating under reduced pressure, vacuum freeze drying the concentrate to obtain total flavone with yield of 2.98%, and detecting to obtain the final product with good bioactivity.
Example 2:
similar to example 1, but without presoaking with saturated lime water. Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. Placing folium indocalami tessellati granule into extraction bottle, adding 20 times of 50% ethanol water solution, extracting at 60 deg.C for 40min, and vacuum filtering to obtain total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding HPD800 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 70% ethanol water solution, collecting eluate, concentrating, vacuum freeze drying the concentrate, and obtaining total flavone of 1.89%.
Example 3
Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. The indocalamus leaf granules are put into an extraction bottle and added with 2 times of saturated lime water solution for pre-soaking for 1 hour. Then adding 20 times of ethanol water with volume fraction of 50%, adjusting the pH of the system to about 6.5-7, extracting at 70 ℃ for 40min, and carrying out vacuum filtration to obtain a total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding HPD800 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with ethanol water solution with volume fraction of 70%, collecting eluate, concentrating under reduced pressure, vacuum freeze drying the concentrate to obtain total flavone with yield of 2.31%, and detecting to obtain the final product with good bioactivity.
Example 4
Similar to example 3, but without presoaking with saturated lime water. Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. Placing folium indocalami tessellati granule into extraction bottle, adding 20 times of 50% ethanol water solution, extracting at 70 deg.C for 40min, and vacuum filtering to obtain total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in desolventizing bottle is 1/3 of original volume, adding HPD800 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 70% ethanol water solution, collecting eluate, concentrating under reduced pressure, and vacuum freeze drying the concentrate to obtain total flavone yield of 1.95%.
Example 5
Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. The indocalamus leaf granules are put into an extraction bottle and added with 5 times of saturated lime water solution for pre-soaking for 0.5 h. Then adding 20 times of 30% ethanol aqueous solution by volume fraction, adjusting the pH of the system to about 6.5-7, extracting at 50 ℃ for 40min, and carrying out vacuum filtration to obtain a total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding AB-8 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 70% ethanol water solution, collecting eluate, concentrating, vacuum freeze drying the concentrate to obtain total flavone with yield of 1.78%, and detecting to obtain the final product with good bioactivity.
Example 6
Similar to example 5, but without presoaking with saturated lime water. Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. Placing folium indocalami tessellati granule into extraction bottle, adding 20 times volume of 30% ethanol water solution, extracting at 50 deg.C for 40min, and vacuum filtering to obtain total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding AB-8 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 70% ethanol water solution, collecting eluate, concentrating, vacuum freeze drying the concentrate, and obtaining total flavone of 1.09%.
Example 7:
cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. The indocalamus leaf granules are put into an extraction bottle and added with 5 times of saturated lime water solution for pre-soaking for 0.5 h. Then adding 20 times volume fraction of 60% ethanol water solution, extracting at 60 deg.C for 60min, and vacuum filtering to obtain total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding HPD450 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 70% ethanol water solution, collecting eluate, concentrating, vacuum freeze drying the concentrate to obtain total flavone with yield of 2.56%, and detecting to obtain the final product with good bioactivity.
Example 8
Similar to example 7, but without presoaking with saturated lime water. Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. Placing indocalamus leaf granules into an extraction bottle, adding 20 times of 60% ethanol aqueous solution by volume, extracting at 60 ℃ for 60min, and carrying out vacuum filtration to obtain a total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding HPD450 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 70% ethanol water solution, collecting eluate, concentrating, vacuum freeze drying the concentrate, and obtaining total flavone of 1.96%.
Example 9
Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. Placing indocalamus leaf granules into an extraction bottle, and adding 10 times of saturated lime water solution for pre-soaking for 20 min. And then adding 20 times of 60% ethanol water solution in volume fraction, and adjusting the pH of the system to about 6.5-7. Extracting at 50 deg.C for 40min, and vacuum filtering to obtain total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until the residual material liquid in the desolventizing bottle is 1/3 of the original volume, adding HPD600 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 70% ethanol water solution, collecting eluate, concentrating, vacuum freeze drying the concentrate to obtain total flavone with yield of 2.48%, and detecting to obtain the final product with good bioactivity.
Example 10
Similar to example 9, but without presoaking with saturated lime water. Cleaning folium indocalami tessellati raw material, drying at 40 deg.C for 5h, adding into a traditional Chinese medicine pulverizer, pulverizing uniformly, and sieving with a 20-mesh sieve to obtain folium indocalami tessellati granules. Placing Indocalamus leaf granules into an extraction bottle, adding 20 times of 60% ethanol water solution, extracting at 50 deg.C for 40min, and vacuum filtering to obtain total flavone extract. Concentrating the total flavone extractive solution under reduced pressure on a rotary evaporator (vacuum degree of 0.095Mpa, temperature of 50 deg.C) until no substance is separated out from the rest material liquid in the desolventizing bottle, adding HPD600 resin column for adsorption, loading at flow rate of 2BV/h, eluting with 5BV column volume water after adsorption saturation, eluting with 50% volume fraction ethanol water solution, collecting eluate, concentrating under reduced pressure, and vacuum freeze drying the concentrate to obtain total flavone yield of 1.80%.
The embodiment shows that the method for extracting and purifying the total flavonoids in indocalamus leaves provided by the invention has the advantages that the yield of the total flavonoids is obviously improved after the total flavonoids are pre-soaked by saturated lime water, the volume fraction of ethanol in the used ethanol aqueous solution is lower (30-60%), and the extraction temperature is also lower (50-70 ℃).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (8)

1. A method for extracting and purifying total flavonoids in indocalamus leaves is characterized by comprising the following steps:
a. cleaning folium indocalami tessellati, oven drying, and pulverizing;
b. pre-soaking the crushed indocalamus leaves for 20-60 min by using saturated lime water, adding an ethanol water solution, adjusting the pH value of a system to be close to neutral, heating and extracting for more than 30min, controlling the heating and extracting temperature to be 50-70 ℃, and then carrying out vacuum filtration to obtain a total flavone extracting solution;
c. evaporating and concentrating the total flavone extract, adsorbing by macroporous resin, washing with water, and eluting with ethanol water solution to obtain eluate;
d. evaporating and concentrating the eluent, and freeze-drying to obtain total flavonoids;
in the step b, the volume fraction of ethanol in the ethanol aqueous solution is 30-60%, and the dosage of the ethanol aqueous solution is V2mL, the dosage of the indocalamus leaves is M g V2The ratio of M to M is 10 to 30: 1;
and (c) adding an ethanol water solution in the step (b), adjusting the pH value of the system to 6.5-7.0, and heating and extracting for 40-60 min.
2. The method for extracting and purifying total flavonoids in indocalamus leaves as claimed in claim 1, wherein the drying in step a is carried out at 40-70 ℃ for 3-7 h.
3. The method for extracting and purifying total flavonoids from indocalamus leaves as claimed in claim 1, wherein the pulverization in step a is carried out in order to obtain indocalamus leaves with particle size of 12-50 mesh.
4. The method for extracting and purifying total flavonoids in indocalamus leaves as claimed in claim 1, wherein the amount of saturated limewater in step b is V1mL, the dosage of the indocalamus leaves is M g V1The ratio of M to M is 2 to 10: 1.
5. the method for extracting and purifying total flavonoids in indocalamus leaves as claimed in claim 1, wherein the evaporation concentration in step c is required to be less than 1/3 of the total flavonoids extract after concentration.
6. The method for extracting and purifying total flavonoids in indocalamus leaves as claimed in claim 1, wherein the adsorption flow rate of the macroporous resin in the step c is 2-5 BV/h.
7. The method for extracting and purifying total flavonoids in indocalamus leaves as claimed in claim 1, wherein the volume fraction of ethanol in the ethanol aqueous solution in step c is 50-80%, the amount of ethanol used in elution is 3-6 BV, and the elution flow rate is 2-5 BV/h.
8. The method for extracting and purifying total flavonoids from Indocalamus leaves as claimed in any one of claims 1 to 7, wherein the macroporous resin used in step c is any one of HPD800, HPD600, HPD450 and AB-8.
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CN105920415A (en) * 2016-06-20 2016-09-07 沈丹虹 Response surface methodology chequer-shaped indocalamus leaf general flavones ultrasonic extraction method
CN105998792B (en) * 2016-06-20 2019-07-02 嘉兴职业技术学院 A method of extracting general flavone from indocalamus leaf
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