CN114712416A

CN114712416A - Method for efficiently and synchronously extracting flavone, alkaloid and polyphenol from lotus leaves by water medium method

Info

Publication number: CN114712416A
Application number: CN202210103706.5A
Authority: CN
Inventors: 严守雷; 刘璐; 刘纪红; 刘延照; 李洁
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-01-24
Filing date: 2022-01-24
Publication date: 2022-07-08
Anticipated expiration: 2042-01-24
Also published as: CN114712416B

Abstract

The invention provides a method for efficiently and synchronously extracting flavone, alkaloid and phenol from lotus leaves by a water medium method. The method comprises the steps of lotus leaf powdering, lotus leaf powder enzymolysis, ultrasonic-assisted surfactant extraction, filtration, concentration and the like. The water medium method adopts glycerol and Tween 80 as medium for extraction, can efficiently extract alkaloid and quercetin in folium Nelumbinis, and glycerol has great advantages in extracting flavone and phenols. The method solves the problems that the prior multi-purpose organic solvent extraction for industrial extraction easily causes organic matter residue and is difficult to extract the quercetin, and simultaneously optimizes parameters such as enzyme preparation selection, enzymolysis pH value, ultrasonic conditions and the like. And the prepared extract has good stability. The lotus leaf extract prepared by the method is not suitable for any organic reagent in the extraction process, strictly complies with the regulation requirements, can be directly applied to beverages and other foods, and has the characteristics of greenness, safety, economy, high efficiency and the like.

Description

Method for efficiently and synchronously extracting flavone, alkaloid and polyphenol from lotus leaves by water medium method

Technical Field

The invention belongs to the field of a method for extracting effective components of plants, and particularly relates to the technical field of lotus leaf functional component extraction.

Background

Lotus leaf (Lotus leaf) is the leaf of Nelumbo nucifera (Nelumbo) of Nymphaeaceae, and has effects of removing summer-heat and promoting diuresis, and promoting hair growth and clearing yang as common Chinese medicinal material. According to the record of the compendium of materia Medica: the lotus leaf is taken orally, so that people can be thin and bad, and original qi can be generated to help spleen and stomach. In document No. 45 of the department of health of the people's republic of China (2002), 2002, 2 months, lotus leaves are listed in the list of batch 2 "food and medicine". The lotus leaves are widely distributed, the cultivation area of the lotus leaves is 900-1100 million mu in China, the lotus leaves are mainly distributed in provinces such as Hubei province and Hunan province, the area of the Hubei province reaches about 150 million mu, and the utilization rate of lotus leaf resources is less than 1%. The lotus leaves in large area are not harvested and utilized, so that the lotus leaves are allowed to decay to cause great resource waste and even environmental pollution, which is in contradiction with huge lotus leaf resources owned by China. The lotus leaves are considered to lack the research and development of related high-added-value deep-processed products, the reasonable utilization of lotus leaf resources in China is severely restricted by factors such as low market demand and the like, and the development of health-care products from plants becomes a research hotspot with the attention of consumers on health.

The compendium of materia medica records that lotus leaves are taken and are thin and bad, so that the efficacy of the lotus leaves in reducing fat and losing weight is accepted by people from old times. A great deal of modern scientific research shows that the lotus leaves have the efficacy of reducing blood fat. Vannantingting (Vannantingting 2013) establishes a high-fat rat model, and the influence of the lotus leaf extract on the weight and serum lipid of a rat is observed, so that the lotus leaf alkaloid has an obvious lipid-lowering effect. The lipid-lowering effect of lotus leaves is researched by schwaning and the like (schwaning and the like 2003), alkaloid in the lotus leaves is extracted, a high-fat model is established by selecting a white mouse, the lotus leaf extract concentrated solution is perfused, and the lotus leaf extract has no significant difference compared with a positive control group (myotron hydrochloride tablets), and the result proves that the lotus leaf extract has the lipid-lowering effect. The Guangxin et al (Guangxin et al 2003) make the lotus leaf extract into capsule, and carry on human body test, observe its lipid-lowering effect, the result shows, the lotus leaf extract has obvious lipid-lowering effects on human body. The main substances with physiological activity in folium Nelumbinis include flavonoids and alkaloids. The influence of lotus leaf water extract on serum cholesterol and triglyceride is observed by using acute hyperlipemia mice as an integral model, and the results show that the lotus leaf water extract has obvious lipid regulation effect, and the active ingredient of the lotus leaf water extract is mainly lotus leaf flavone and then lotus leaf alkaloid. Due to the special molecular structure of polyphenol, polyphenol has various physiological activities of bacteriostasis, antianaphylaxis, hemostasis, antitumor, anti-aging and the like. Lee and other researches indicate that the polyphenol substances in the lotus leaves have the activity of inhibiting Vascular Endothelial Growth Factor (VEGF) from inducing angiogenesis, and meanwhile, the polyphenol substances in the lotus leaves have stronger total reducing power, DPPH-free radical scavenging activity and nitrite scavenging activity.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for efficiently and synchronously extracting flavone, alkaloid and polyphenol from lotus leaves by a water medium method.

The invention adopts the specific scheme that:

s1 pulverizing dried folium Nelumbinis, and sieving with 60 mesh sieve;

s2 soaking the pulverized lotus leaf powder in 50 ℃ water for 30min, adjusting the pH value with citric acid, adding an enzyme preparation, and performing enzymolysis for 2h at 50 ℃;

s3 adding water solution of surfactant after enzymolysis, and carrying out ultrasonic extraction at proper temperature for a proper time;

s4 ultrasonic extracting, filtering, and concentrating the extract by using a rotary evaporator;

no organic solvent is used in the extraction process.

The enzyme preparation is cellulase and/or pectinase. Preferably one or more of summers liquid cellulase SPE0-17, summers solid cellulase, summers pectinase, Nuoweixin KTN02309 complex enzyme and Nuoweixin KON10061 pectinase.

The dosage of the enzyme preparation is 0.5-2 wt% of the dosage of lotus leaves, and cellulase and pectinase with dosage ratio of 1:1 are preferably used.

The pH value in step S2 is 4.0-6.0, preferably 4.0, 4.5, 5.0, 5.35.

The surfactant in step S3 is one or more of glycerol, tween 20, tween 60, tween 80, span 80, polyglycerol laurate and polyglycerol caprylic decanoate, and preferably tween 80 and/or glycerol.

In the step S3, the ultrasonic time is 30-60min, and the ultrasonic temperature is 50-80 ℃.

The lotus leaf dosage in the preparation method is 5-20g/250 mL.

Compared with the prior art, the invention has the beneficial effects that:

1. compared with the traditional method for extracting by using organic solvent (multi-purpose ethanol), the method has no organic residue and no risk of flammability and explosiveness, and the chlorophyll in the lotus leaves is dissolved out and has darker color when the organic solvent is used for extracting, and the pigment is separated out after concentration and accompanied with the precipitation of other substances, thereby causing certain influence on the sense;

2. in the method, Tween 80 and glycerol used in a water medium method supplement each other, the extraction of Tween 80 in the method can efficiently extract alkaloid and quercetin, both have good fat-reducing effect, the extraction of glycerol has the advantage which is difficult to achieve by a common method in the extraction of phenolic substances and flavone, and simultaneously, the problem of low regulation limit caused by single extraction of Tween 80 (for example, the Tween 80 limit is 0.05 percent in plant beverage) is solved;

3. compared with the traditional method for extracting the flavone, the alkaloid and the phenolic substance by using the organic solvent in the industrialized extraction, the method has different advantages in the extraction of the flavone, the alkaloid and the phenolic substance, and is particularly reflected in the content of the quercetin, the alkaloid and the phenolic substance, probably because the organic substance is evaporated during the extraction and concentration of the organic substance, the alkaloid and some flavonoid compounds are not easy to dissolve in water, and the prepared extract has good stability, does not precipitate and does not separate out. In the method, the addition of cellulase, pectinase and the like can open the barrier of the cell wall, glycerol and tween 80 have solubilization, and partial alkaloid flavone phenolic substances can be dissolved to a great extent by means of auxiliary ultrasound and the like.

Drawings

FIG. 1 effect of different enzyme preparations on extraction yield.

FIG. 2 is a HPLC chromatogram of the Xiaheng and Novoxin enzyme preparation extract. A. Extracting after liquid cellulase enzymolysis; B. extracting solution after enzymolysis of the Novoxil compound enzyme; 1. nuciferine; 2. quercetin.

FIG. 3 shows the effect of different pH values on the extraction rate.

Figure 4 effect of ultrasound time on extraction rate.

Figure 5 effect of ultrasound temperature on extraction rate.

FIG. 6 shows the color of the extract of lotus leaves with different concentrations in 1% Tween 80.

FIG. 7 the effect of different concentrations of lotus leaves on the extraction yield. A. The content of the total flavone in the lotus leaf extracting solution with different concentrations; B. the content of total phenols in the lotus leaf extracting solution with different concentrations; C. the content of total alkaloids in lotus leaf extract with different concentrations; D. the content of nuciferine in the lotus leaf extract with different concentrations; E. the content of the quercetin in lotus leaf extract with different concentrations.

FIG. 8 HPLC chromatograms of Tween 80 and glycerol extracts. A.1% concentration Tween 80 material-liquid ratio is 5g lotus leaf/250 mL extracting solution; b, the ratio of 10% glycerol to liquid is 20g lotus leaves/250 mL extracting solution; 1. nuciferine; 2. quercetin.

FIG. 9 color difference between organic extraction and aqueous media extraction:

a. extracting solutions with different extraction schemes; b. extracting solutions of different extraction schemes are not centrifuged after being concentrated; c. concentrating the extracting solution according to different extraction schemes and then centrifuging; d. after the extracting solution of different extraction schemes is concentrated, the extracting solution is centrifugally diluted by 5 times by water;

1. extracting with 5g folium Nelumbinis 1% Tween; 2. extracting with 5g folium Nelumbinis 75% ethanol; 3. extracting with 20g lotus leaf 10% glycerol; 4. extracting with 20g folium Nelumbinis 75% ethanol; 5. extracting with 5g folium Nelumbinis 75% ethanol, and adjusting pH to 4.

FIG. 10 the effect of organic extraction and aqueous medium method on extraction yield.

FIG. 11 HPLC chromatograms from organic extraction and aqueous medium extraction.

Extracting with 5g folium Nelumbinis 1% Tween; B. extracting with 5g folium Nelumbinis 75% ethanol; C. extracting with 20g lotus leaf 10% glycerol; D. extracting with 20g folium Nelumbinis 75% ethanol; E. extracting with 5g of 75% ethanol from lotus leaves, wherein the pH is 4; 1. nuciferine 2 quercetin.

Detailed Description

The present invention will be more readily understood by reference to the following examples, which are given without limiting the scope of the invention.

< method for detecting bioactive substance >

The following bioactive substance detection methods were used to test and evaluate the test samples, and the evaluation results are shown in the figure.

1. Determination of Total Flavonoids content

5% sodium nitrite solution, 10% aluminum nitrate solution and 5% sodium hydroxide solution are prepared as colorimetric solution.

Precisely absorbing 30ul of each concentrated extract sample in turn, respectively placing in 10mL measuring flasks, adding water to 4mL, adding 0.4mL of 5% sodium nitrite solution, shaking, and standing for 6 min; then adding 0.4mL of 10% aluminum nitrate solution, shaking uniformly and standing for 6 min; adding 4mL of 5% sodium hydroxide solution, adding water to the scale, shaking uniformly, and standing for 15min with the corresponding reagent as blank;

the absorbance was measured at a wavelength of 510nm and the content was calculated against a standard curve, 3 times for each sample, with absorbance values between 0.2 and 0.8.

2. Determination of the Total phenol content

And (3) determining the total phenol content of the lotus leaf extract by adopting a Folin-Ciocalteu colorimetric method.

Taking 1mL of lotus leaf extract, diluting to a 10mL centrifuge tube, precisely measuring 0.03mL of extract diluent in a 10mL volumetric flask, adding water to dilute to 6mL, shaking up, adding 0.5mL of forrin reagent, fully shaking up, and standing for 3 min; then 20 percent of anhydrous Na is added₂CO₃Shaking up 1.5mL of the solution, then fixing the volume of water to the scale and shaking up; reacting in water bath at 35 deg.C for 50min, cooling, and taking blank liquid as reference;

the absorbance values were measured at 760nm and the content was calculated against a standard curve, 3 times for each sample, with absorbance values between 0.2 and 0.8.

3. Determination of total alkaloid content

Potassium hydrogen phthalate buffer: drying potassium hydrogen phthalate at 110 ℃ for 2h, weighing 5.10g of potassium hydrogen phthalate, adding a proper amount of distilled water for dissolving, and then fixing the volume to 500mL, wherein the pH value of the solution is 4.0;

bromocresol green solution: weighing 0.10g of bromocresol green, adding 7.15mL of 0.02mol/L NaOH solution for dissolving, and fixing the volume to 250mL by using distilled water;

taking 10 mul of the lotus leaf extract concentrated solution, diluting to 5mL with chloroform (chloroform), pouring into a 10mL test tube, adding 1mL of potassium hydrogen phthalate buffer solution and 1mL of bromocresol green solution, oscillating for 30s, and standing for 30 min. Taking 4mL of the lower chloroform layer, measuring absorbance value at 414nm, measuring each sample for 3 times, and calculating total alkaloid content according to the standard curve.

4. High Performance Liquid Chromatography (HPLC) for detecting content of nuciferine and quercetin

Standards and reagents used in the experiments: nuciferine standard (Shanghai-source leaf biotechnology limited not less than 98%), quercetin standard (Shanghai-source leaf biotechnology limited not less than 98%), formic acid, and acetonitrile are all chromatographically pure.

An Agilent C18 column; the mobile phase was water (a, containing 0.4% formic acid) -acetonitrile (B); the volume flow is 1.0 mL/min; the detection wavelength is 270 nm; column temperature: room temperature; and (3) an elution mode: gradient elution

Sample preparation: the preparation method of the test solution comprises precisely weighing 2mL each of folium Nelumbinis samples, placing in conical flask with plug, precisely adding 28mL methanol, plugging plug tightly, ultrasonically treating for 30min, standing, cooling, collecting supernatant, filtering with 0.22 μm organic microporous membrane, and collecting filtrate, wherein each sample is measured for 3 times, and the sample concentration is calculated according to nuciferine and quercetin standard curve.

Example 1

In the plant extraction, cellulase and pectinase are used for treatment to break the cell wall barrier and release alkaloids and flavonoids in lotus leaves.

Taking 250mL of the extract as an example, 6.25g of lotus leaves are added with 200mL of water and soaked for 30min at 50 ℃, and the pH value is adjusted to 4.5 by using a citric acid solution; the enzyme preparation in each scheme is activated by using a proper amount of water and then added, enzymolysis is carried out for 2 hours, and then 2.5g of Tween 80 (1%) is added to be constant volume to 250 mL; performing ultrasonic treatment at 60 deg.C and 480w for 40min, vacuum filtering, and concentrating the filtrate to 20-40g with rotary evaporator; detecting the content of total flavone, total phenol and total alkaloid in the enzyme preparation extract of different schemes, and quantitatively analyzing the content of nuciferine and quercetin by high performance liquid chromatography.

Selecting summertime liquid cellulase SPE0-17, summertime solid cellulase, summertime pectinase, Nuoweixin KTN02309 complex enzyme and Nuoweixin KON10061 pectinase, and dividing the materials into 5 groups for experiment; the experimental protocol is shown in table 1.

TABLE 1

As can be seen from FIG. 1, the enzyme preparation had a large effect on the extraction of the active substance. Comparing the scheme I, the scheme II and the scheme III, the scheme III has great advantages in the content of total flavonoids and quercetin; as can be seen from fig. 2, the High Performance Liquid Chromatography (HPLC) chart also clearly shows that the use of scheme three (norwexin KTN02309 complex enzyme) has significant advantages in the extraction of quercetin compared with scheme one (summertime liquid cellulase SPE 0-17); compared with the third scheme and the fifth scheme, the addition of the Novoverin KON10061 pectinase obviously improves the content of total alkaloids in the extracting solution, and other bioactive substances are improved to different degrees.

Example 2

Taking 250mL of the extract as an example, 6.25g of lotus leaves are added with 200mL of water and soaked for 30min at 50 ℃, and the pH value is adjusted to 4.0-5.35 by citric acid solution; adding 0.05g of Novoxin KTN02309 complex enzyme and 0.05g of Novoxin KON10061 pectinase after activating, uniformly stirring, performing enzymolysis for 2 hours, adding 2.5g of Tween 80 (1%) and fixing the volume to 250 mL; performing ultrasonic treatment at 60 deg.C and 480w for 40min, vacuum filtering, and concentrating the filtrate to 20-40g with rotary evaporator;

detecting the content of total flavone, total phenol and total alkaloid in the extracting solution with different pH values of enzymolysis, and quantitatively analyzing the content of nuciferine and quercetin by high performance liquid chromatography.

As can be seen from FIG. 3, with the decrease of pH, the total phenol, total flavone and total alkaloid content fluctuates in a certain range, the overall growth is not obvious, the quercetin content increases when the pH is not adjusted to 5.35 to 5.0, the fluctuation of pH4.0 to 5.0 is small, and the nuciferine content gradually increases with the decrease of pH.

Example 3

Taking 250mL of the extract as an example, 6.25g of lotus leaves are added with 200mL of water and soaked for 30min at 50 ℃, and the pH value is adjusted to 4.5 by using a citric acid solution; adding 0.05g of Xiasang liquid cellulase SPE0-17 and 0.05g of Xiasang pectinase after activation, uniformly stirring, carrying out enzymolysis for 2h, adding the surfactant, wherein the concentration of glycerol is 10%, the rest concentration is 1%, and fixing the volume to 250 mL; performing ultrasonic treatment at 60 deg.C and 480w for 40min, vacuum filtering, and concentrating the filtrate to 20-40g with rotary evaporator.

The method comprises the steps of selecting glycerol, Tween 20, Tween 60, Tween 80, span 80, polyglycerol laurate and polyglycerol caprylic caprate with a solubilizing effect as extraction solvents, detecting the content of total flavonoids, total phenols and total alkaloids in different surfactant extracting solutions, and quantitatively analyzing the content of nuciferine and quercetin by high performance liquid chromatography.

TABLE 2 Effect of different surfactants on extraction yield

As can be seen from table 2, in tween 20, tween 60 and tween 80, tween 80 has different degrees of advantages in the extraction of various substances, and in the specification of GB 2760 plant beverage, the limited amount of tween series is 0.5g/kg product, which means that in a bottle of 300mL plant beverage, the lotus leaf extract required by tween limitation is added, the nuciferine content in the final product is less than 1 mg/bottle, and the requirement that the lipid-lowering efficacy is not less than 2mg nuciferine per day in the prior art cannot be met, while glycerol (glycerol) has no limited requirement when used as a food additive/processing aid in GB 2760; comparing the extraction of 1

% Tween

80 and 10% glycerol, Tween 80 has a great advantage in the content of total alkaloids and quercetin, glycerol is extracted in the content of total phenols, and the content difference of nuciferine in the two is not great, so that in the actual extraction process, when the glycerol is used as an extraction solvent, the color of an extracting solution is clear and transparent, the extracting solution is wine red after concentration, and has no impurities, and the sensory evaluation is good.

Example 4

Taking 250mL of the extract as an example, 6.25g of lotus leaves are added with 200mL of water and soaked for 30min at 50 ℃, and the pH value is adjusted to 4.5 by using a citric acid solution; 0.05g of Xiasang liquid cellulase SPE0-17 and 0.05g of Xiasang pectinase are added after being activated, the mixture is stirred evenly and is subjected to enzymolysis for 2 hours, and 2.5g of Tween 80 (1%) is added to the mixture until the volume is 250 mL; performing ultrasonic treatment at 50-80 deg.C and 480w for 30-60min, vacuum filtering, and concentrating the filtrate to 20-40g with rotary evaporator.

Selecting different ultrasonic conditions of ultrasonic time 30min, ultrasonic temperature 50 deg.C, ultrasonic temperature 60 deg.C, ultrasonic temperature 70 deg.C, and ultrasonic temperature 80 deg.C, detecting total flavone, total phenol and total alkaloid content in the extractive solution under different ultrasonic conditions, and quantitatively analyzing the content of nuciferine and quercetin by high performance liquid chromatography.

As can be seen from FIG. 4, at an ultrasonic temperature of 60 ℃ and within a time range of 30min-60min, at 40min, the contents of total flavonoids and nuciferine reach maximum values, and the content of quercetin fluctuates in a small amplitude, so that 40min is selected as the optimal ultrasonic time in the invention; as can be seen from FIG. 5, the total flavone and quercetin contents reach the maximum values at the ultrasonic temperature of 60 ℃ within the ultrasonic temperature range of 50-80 ℃ within 40min, the nuciferine content fluctuates in a small range, and the total phenol content has little difference, so that 60 ℃ is selected as the optimal ultrasonic temperature in the invention.

Example 5

Taking 250mL of the extract as an example, 5g of lotus leaf, 7.5g of lotus leaf, 10g of lotus leaf, 12.5g of lotus leaf, 15g of lotus leaf and 20g of lotus leaf are respectively added, soaked in 200mL of water at 50 ℃ for 30min, and the pH value is adjusted to 4.5 by using a citric acid solution; adding 0.05g of Novoxin KTN02309 complex enzyme and 0.05g of Novoxin KON10061 pectinase after activating, uniformly stirring, performing enzymolysis for 2 hours, adding 1.3.3 of the preferable surfactant, and keeping the volume to 250 mL; performing ultrasonic treatment at 60 ℃ and 480w for 40min, performing suction filtration, concentrating the filtrate to 20-40g by using a rotary evaporator, and comparing the difference of a 1% Tween 80 extracting solution before and after centrifugation because a small amount of white precipitate is formed after the Tween extracting solution is placed for a period of time; detecting the content of total flavone, total phenol and total alkaloid in the extracting solutions with different material-liquid ratios, and quantitatively analyzing the content of nuciferine and quercetin by high performance liquid chromatography.

As can be seen from FIG. 6, in the 1% Tween 80 system, from left to right, the yellow-green color of the extract became lighter and the solution became turbid from clear with the increase of the concentration of lotus leaves.

As can be seen from fig. 7, in the 1% tween 80 solution, with the increase of the concentration of lotus leaves, the total flavonoids, total phenols and total alkaloids do not increase regularly, and show a state of first decreasing and then increasing, the total does not increase, but the content of quercetin shows a sharp falling trend; comparing the content of each active substance before and after centrifugation, wherein the content of partial substances after centrifugation is slightly reduced and not large, and in order to fully ensure the stability of the extracting solution, 5g of lotus leaves/250 ml of the extracting solution is selected as the optimal concentration in a 1% Tween system;

as can be seen from FIG. 7, in 10% glycerol solution, with the increase of folium Nelumbinis concentration, total flavone, total phenol, and total alkaloid all show a gradual increase trend, the total quercetin content fluctuates within a small range, and the maximum value is reached when the content of nuciferine fluctuates slightly and then in 20g folium Nelumbinis/250 mL of extract; for comprehensive consideration, 20g of lotus leaves/250 ml of extracting solution is selected in a 10% glycerol system.

Compared with the extraction of 1

% Tween

80 and 10% glycerol, 1% Tween 80 has great advantages in the content of total alkaloids and quercetin, and the glycerol system is helpful for the extraction of total phenols and total flavonoids; when the concentration of the lotus leaves is 20g of lotus leaves per 250mL of extracting solution, 10% of glycerin is used as an extracting solvent for extraction, the content of total flavonoids can reach about 800 mg/part at most, and the content of total phenols can reach about 600 mg/part. FIG. 8 shows that when 1% Tween 80, 5g lotus leaf/250 ml extract and 10% glycerol, 20g lotus leaf/250 ml extract were used, the content of nuciferine and quercetin was quantitatively analyzed by HPLC, and it is evident that the former is more advantageous for the extraction of quercetin.

Example 6

Pulverizing dried folium Nelumbinis, and sieving with 60 mesh sieve; soaking pulverized folium Nelumbinis powder in 50 deg.C water for 30min, adjusting pH with citric acid, and performing enzymolysis on folium Nelumbinis at 50 deg.C for 2 hr at pH of 4.0 with 0.05g of Novoxin KTN02309 complex enzyme and 0.05g of Novoxin KON10061 pectinase; then adding tween 80 with the concentration of 1% and glycerol with the concentration of 10% respectively, performing ultrasonic treatment at 60 ℃ and 480w for 40 minutes, filtering, and concentrating the extracting solution to an appropriate volume through a rotary evaporator. Wherein 1% Tween 80 folium Nelumbinis concentration is 5g folium Nelumbinis/250 mL extractive solution, and 10% glycerol folium Nelumbinis concentration is 20g folium Nelumbinis/250 mL extractive solution.

Control group 1: selecting lotus leaves with the same concentration as that of the lotus leaves in the example 6, namely 5g of lotus leaves/250 mL of extracting solution and 20g of lotus leaves/250 mL of extracting solution; the pH is not adjusted;

control group 2: selecting an extraction method with the pH value of 4 of 5g of lotus leaves/250 mL of extracting solution; the remaining parameters were the same as in control 1.

Detecting the content of total flavone, total phenol and total alkaloid in the extracting solutions with different material-liquid ratios, and quantitatively analyzing the content of nuciferine and quercetin by high performance liquid chromatography.

As can be seen from FIG. 9a, the extract was clear before concentration, the extraction color of glycerol was light, the extraction of Tween was yellow-green, the extraction of 75% ethanol was very dark when the concentration of lotus leaf was 20g/250mL, because the dissolution of chlorophyll was dark green; as can be seen from fig. 9b, after the extract was concentrated, ethanol-extracted nos. 2, 4 and 5 all had many precipitates, which were analyzed as the precipitates of chlorophyll and some water-insoluble flavones, alkaloids and other substances, with a small amount of white precipitate at the bottom of 1, and glycerin-extracted No. 3 having a wine-red color without precipitates; FIG. 9c is the color of the concentrate of FIG. 9b after centrifugation; FIG. 9d shows the color of FIG. 9c after being reconstituted by 5 times by dilution with water, which shows that the clarity of 5 extracts after reconstitution is good;

as can be seen from fig. 10, compared with organic extraction, the tween extraction in the method of the present invention has significant advantages in the extraction of quercetin and total alkaloids, while the glycerol extraction in the method of the present invention has incomparable advantages in the content of total flavonoids and total phenols, when 75% ethanol is extracted, the content of each substance whose concentration of lotus leaf is increased to 20g/250mL is significantly increased compared to 5g/250mL, when the pH value is initially adjusted to 4, the content of total phenols and nuciferine is significantly increased, fig. 11 is a graph in which the difference in content of nuciferine and quercetin is analyzed by using high performance liquid chromatography, which shows significantly the advantages when fig. 11A tween is used to extract quercetin, whereas fig. 11B, fig. 11D and fig. 11E are not effective in extracting nuciferine by using 75% ethanol, and when fig. 11E is adjusted to 4.0, the content of nuciferine is significantly increased.

Example 7

Pulverizing dried folium Nelumbinis, and sieving with 60 mesh sieve; adding 400mL of water into 10g of lotus leaves, soaking at 50 ℃ for 30min, adjusting the pH value to 4.0 by using a citric acid solution, and carrying out enzymolysis on the lotus leaves at the pH of 4.0 and the temperature of 50 ℃ for 2h by using 0.1g of the activated Novoxin KTN02309 complex enzyme and 0.1g of the Novoxin KON10061 pectinase; then adding 2.5g of Tween 80 (1%) and 25g of glycerol to a constant volume of 500mL, carrying out ultrasonic treatment at 60 ℃ and 480w for 40min, filtering, and concentrating the extracting solution to an appropriate volume through a rotary evaporator. The test calculation obtains the yield of the total flavone, the total phenol, the total alkaloid and the quercetin.

Example 8

Pulverizing dried folium Nelumbinis, and sieving with 60 mesh sieve; adding 200mL water into 40g folium Nelumbinis, soaking at 50 deg.C for 30min, adjusting pH to 4.0 with citric acid solution, and performing enzymolysis on folium Nelumbinis at 50 deg.C for 2 hr at pH4.0 using activated 0.1g Nuoweixin KTN02309 complex enzyme and 0.1g Nuoweixin KON10061 pectinase; then adding 2.5g of Tween 80 (1%) and 25g of glycerol to a constant volume of 500mL, carrying out ultrasonic treatment at 60 ℃ and 480w for 40min, filtering, and concentrating the extracting solution to an appropriate volume through a rotary evaporator. The test calculation is carried out to obtain the yield of the total flavone, the total phenol, the total alkaloid and the quercetin.

TABLE 3

The extraction amount is mg/g	Flavone	Phenol and its salts	Alkaloids	Nuciferine	Quercetin
						Example 7	105.33	50.74	26.47	3.36	6.54
Example 8	112.64	56.73	22.39	3.54	5.12

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. A method for efficiently and synchronously extracting flavone, alkaloid and polyphenol from lotus leaves by a water medium method is characterized by comprising the following steps:

s1 pulverizing dried folium Nelumbinis, and sieving with 60 mesh sieve;

no organic solvent is used in the extraction process.

2. The method according to claim 1, characterized in that the enzyme preparation is a cellulase and/or pectinase.

3. The method of claim 2, wherein said enzyme preparation is one or more of xiasheng liquid cellulase SPE0-17, xiasheng solid cellulase, xiasheng pectinase, norvisin KTN02309 complex enzyme, and norvisin KON10061 pectinase.

4. The method of claim 3, wherein said enzyme is a combination of Novoxin KTN02309 complex enzyme, Novoxin KON10061 pectinase.

5. The method according to claim 1, characterized in that the enzyme preparation is used in an amount of 0.5-2 wt% of the amount of lotus leaves, preferably cellulase and pectinase in an amount ratio of 1: 1.

6. The method according to claim 1, characterized in that the pH in step S2 is 4.0-6.0, preferably 4.0, 4.5, 5.0, 5.35.

7. The method according to claim 1, wherein the surfactant in step S3 is one or more of glycerol, tween 20, tween 60, tween 80, span 80, polyglycerol laurate and polyglycerol caprylate caprate, preferably tween 80 and/or glycerol.

8. The method according to claim 1, wherein the sonication time in step S3 is 30-60min, and the sonication temperature is 50-80 ℃.

9. The method of claim 1, wherein the amount of lotus leaf used in the preparation method is 5-20g/250 mL.