CN114712416B - Method for efficiently and synchronously extracting flavone, alkaloid and polyphenol in lotus leaves by using water-borne method - Google Patents
Method for efficiently and synchronously extracting flavone, alkaloid and polyphenol in lotus leaves by using water-borne method Download PDFInfo
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Abstract
The invention provides a method for efficiently and synchronously extracting flavone, alkaloid and phenol in lotus leaves by an aqueous medium method. The method comprises the steps of powdering lotus leaves, carrying out enzymolysis on the lotus leaf powder, extracting with an ultrasonic-assisted surfactant, filtering, concentrating and the like. The aqueous medium method is to extract with glycerin and tween 80 as medium, which can extract alkaloid and quercetin from lotus leaf with high efficiency, while glycerin has great advantage in flavone and phenols extraction. The method solves the problems that the existing industrial extraction multipurpose organic solvent extraction is easy to cause organic matter residues and difficult to extract quercetin, and 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 meets the requirement of regulations, can be directly applied to beverages and other foods, and has the characteristics of green, safety, economy, high efficiency and the like.
Description
Technical Field
The invention belongs to the field of extraction methods of plant active ingredients, and particularly relates to the technical field of extraction of lotus leaf functional ingredients.
Background
Lotus leaf (Lotus leaf) is leaf of Lotus (Nelumbo) belonging to Nymphaeaceae (Nymphaeaceae), and has effects of removing summer heat, promoting diuresis, and improving hair and clearing yang as common Chinese medicinal materials. According to the description of Ben Cao gang mu: the lotus leaf is taken with the effects of reducing the weight, promoting the generation of primordial qi and helping the spleen and stomach. In Wei Jian (2002) document No. 45 of the ministry of health of the people's republic of China, month 2 of 2002, lotus leaves are listed in the list of "both food and medicine" of lot 2. The lotus leaves are widely distributed, the cultivation area of the lotus leaves is 900-1100 mu nationwide, the lotus leaves are mainly distributed in Hubei provinces, hunan provinces and the like, the Hubei provinces reach about 150 mu, and the utilization rate of lotus leaf resources is less than 1%. The large area of lotus leaves are not harvested and utilized, and the decay of the lotus leaves causes great resource waste and even environmental pollution, which is in contradiction with huge lotus leaf resources owned by China. The research and development of the relevant high-added-value deep-processing products of lotus leaves are considered, the reasonable utilization of lotus leaf resources in China is severely restricted by factors such as low market demand, and along with the attention of consumers to health, the development of health-care products from plants becomes a research hotspot.
The materia medica outline records that lotus leaves are taken with poor appetite, so that the efficacy of lotus leaves in reducing blood fat and losing weight is recognized from ancient times. Modern scientific researches have shown that lotus leaves have the effect of reducing blood fat. Fan Tingting (Fan Tingting 2013) establishes a high-fat rat model, observes the influence of lotus leaf extract on rat weight and serum lipid, and shows that the lotus leaf alkaloid has obvious lipid-lowering effect. Xu Laying et al (Xu Laying et al 2003) studied the lipid lowering effect of lotus leaf, extracted alkaloids from lotus leaf, selected mice to build a high-lipid model, and the concentrated solution of the gastric lavage lotus leaf extract was compared with the positive control group (myo-agarol hydrochloride tablet) without significant difference, and the results prove that the lotus leaf extract has the lipid lowering effect. Guan Zhangshun (Guan Zhangshun and 2003) is prepared by making lotus leaf extract into capsule, and performing human body test to observe the lipid-lowering effect, and the result shows that the lotus leaf extract has obvious lipid-lowering effect on human body. The main substances with physiological activity in the lotus leaf comprise flavonoid compounds and alkaloids. Du Lijun and the like observe the influence of lotus leaf aqueous extract on serum cholesterol and triglyceride by taking an acute hyperlipidemic mouse as an integral model, and the result shows that the lotus leaf aqueous extract has obvious lipid regulating effect, and the active ingredients of the lotus leaf aqueous extract are mainly lotus leaf flavone and secondarily lotus leaf alkaloid. The polyphenol has various physiological activities such as bacteriostasis, antiallergic, hemostasis, anti-tumor, anti-aging and the like due to the special molecular structure of the polyphenol. Lee et al research indicates that the lotus leaf polyphenol substance has the activity of inhibiting Vascular Endothelial Growth Factor (VEGF) from inducing vascular regeneration, and simultaneously the polyphenol substance in the lotus leaf has stronger total reducing power, DPPH-free radical activity removal and nitrite removal activity.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for efficiently and synchronously extracting flavone, alkaloid and polyphenol in lotus leaves by using an aqueous medium method.
The invention adopts the following specific scheme:
s1, crushing dried lotus leaves, and sieving with a 60-mesh sieve for standby;
s2, soaking the crushed lotus leaf powder in water at 50 ℃ for 30min, then adjusting the pH value by using citric acid, adding an enzyme preparation, and carrying out enzymolysis for 2h at 50 ℃;
s3, adding an aqueous solution of a surfactant after enzymolysis, and performing ultrasonic extraction within a proper temperature time;
s4, filtering after ultrasonic extraction, and concentrating the extracting solution by using a rotary evaporator;
the extraction process does not use organic solvent.
The enzyme preparation is cellulase and/or pectase. Preferably one or more of summer liquid cellulase SPE0-17, xia Cheng solid cellulase, xia Cheng pectase, noveXin KTN02309 complex enzyme and NoveXin KON10061 pectase.
The dosage of the enzyme preparation is 0.5-2wt% of that of lotus leaf, and preferably, cellulase and pectase with the dosage ratio of 1:1 are used.
The pH in step S2 is 4.0-6.0, preferably 4.0, 4.5, 5.0, 5.35.
The surfactant in the step S3 is one or more of glycerol, tween 20, tween 60, tween 80, span 80, polyglycerol laurate and polyglycerol caprylate, preferably Tween 80 and/or glycerol.
In the step S3, the ultrasonic time is 30-60min, and the ultrasonic temperature is 50-80 ℃.
The dosage of lotus leaves in the preparation method is 5-20g/250mL.
Compared with the prior art, the invention has the beneficial effects that:
1. compared with the traditional industrialized extraction using an organic solvent (multi-purpose ethanol extraction), the method has no risk of residual organic matters, combustibility and explosiveness, and chlorophyll in lotus leaves is dissolved out, the color is darker, pigment is separated out after concentration and other substances are precipitated, so that a certain influence is caused on sense organs;
2. tween 80 and glycerol used by the water-borne method complement each other, and the application of the Tween 80 in the method can efficiently extract the alkaloid and the quercetin, both have good fat reducing effect, and the glycerol extraction has the advantage that the common method is difficult to achieve in the extraction of phenolic substances and flavones, and simultaneously solves the problem that the regulation limit is low in the single Tween 80 extraction (for example, in plant beverage, the limit of the Tween 80 is 0.05 percent);
3. compared with the traditional industrialized extraction using organic solvents, the method has different advantages when extracting flavone, alkaloid and phenols, and is particularly reflected in the content of quercetin, alkaloid and phenols, probably because the organic matters are evaporated when extracting and concentrating the organic matters, the alkaloid and some flavonoid compounds are not easy to dissolve in water, and the prepared extract has good stability, no precipitation and no precipitation. In the method, the addition of cellulase pectase and the like can unwrap the barrier of the cell wall, glycerol and tween 80 have solubilization, and the modes of ultrasound assistance and the like can greatly dissolve part of alkaloid flavone phenolic substances.
Drawings
FIG. 1 influence of different enzyme preparations on extraction yield.
Figure 2 Xia Chengji HPLC chromatogram of novelixir enzyme preparation extract. A. Extracting after enzymolysis of liquid cellulase; B. extracting solution after the enzymolysis of the Norwestin complex enzyme; 1. nuciferine; 2. quercetin.
FIG. 3 influence of different enzymatic pH values on extraction yield.
FIG. 4 influence of ultrasound time on extraction yield.
FIG. 5 influence of ultrasound temperature on extraction yield.
FIG. 6 color of extracts of lotus leaves at different concentrations in 1% Tween 80.
Figure 7 effect of different concentrations of lotus leaf on extraction yield. A. The content of total flavonoids in lotus leaf extract with different concentrations; B. the total phenol content of lotus leaf extract with different concentrations; C. the content of total alkaloids in lotus leaf extract with different concentrations; D. the nuciferine content of the lotus leaf extract with different concentrations; E. the content of quercetin in lotus leaf extract with different concentrations.
FIG. 8 HPLC chromatogram of Tween 80 and glycerol extract. The ratio of the tween 80 with the concentration of 1 percent to the liquid is 5g of lotus leaf/250 mL of extracting solution; the ratio of 10% glycerol to liquid is 20g lotus leaf/250 mL extracting solution; 1. nuciferine; 2. quercetin.
Fig. 9 organic extraction and aqueous extraction color difference:
a. extracting solutions with different extraction schemes; b. concentrating the extractive solutions of different extraction schemes, and not centrifuging; c. concentrating the extractive solutions with different extraction schemes, and centrifuging; d. concentrating the extractive solutions with different extraction schemes, centrifuging, and diluting with water for 5 times;
1. extracting with 5g of 1% Tween of lotus leaf; 2. extracting with 5g of 75% ethanol of lotus leaf; 3. extracting with 20g 10% glycerol of folium Nelumbinis; 4. extracting with 20g of 75% ethanol of lotus leaf; 5. 5g of lotus leaf 75% ethanol is used for extraction, and the pH is 4.
FIG. 10 is a graph showing the effect of organic extraction and aqueous media on extraction yield.
FIG. 11 HPLC chromatograms of organic and aqueous extraction.
Extracting with 5g of 1% Tween of lotus leaf; B. extracting with 5g of 75% ethanol of lotus leaf; C. extracting with 20g 10% glycerol of folium Nelumbinis; D. extracting with 20g of 75% ethanol of lotus leaf; E. extracting with 5g of 75% ethanol of lotus leaf with pH of 4;1. nuciferine 2. Quercetin.
Detailed Description
The 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 experimental samples are tested and evaluated by the following bioactive substance detection method, and the evaluation results are shown in the attached drawing.
1. Determination of total flavone content
A 5% sodium nitrite solution, a 10% aluminum nitrate solution, and a 5% sodium hydroxide solution were prepared as a colorimetric solution.
Sequentially and precisely sucking 30ul of concentrated extract samples respectively, placing in 10mL measuring flask, adding water to 4mL, adding 0.4mL of 5% sodium nitrite solution, shaking, and standing for 6min; then adding 0.4mL of 10% aluminum nitrate solution, shaking uniformly and standing for 6min; adding 4mL of 5% sodium hydroxide solution, adding water to the scale, shaking uniformly, and standing for 15min, and taking the corresponding reagent as a blank;
absorbance was measured at 510nm wavelength, the content was calculated against a standard curve, 3 times per sample, and absorbance value was between 0.2 and 0.8.
2. Determination of total phenol content
Folin-Ciocalteu colorimetric method is used for determining total phenol content of folium Nelumbinis extract.
Diluting 1mL of lotus leaf extract to a 10mL centrifuge tube, precisely measuring 0.03mL of extract diluent in a 10mL volumetric flask, adding water to dilute to 6mL, shaking uniformly, adding 0.5mL of Fu Lin Fen reagent, shaking uniformly, and standing for 3min; then 20% anhydrous Na is added 2 CO 3 Shaking 1.5mL of the solution uniformly, and then fixing the volume of water to the scale and shaking uniformly; carrying out water bath reaction at 35 ℃ for 50min, cooling, and taking blank liquid as a reference;
the absorbance was measured at 760nm, the content was calculated against a standard curve, 3 times per sample, and the absorbance was between 0.2 and 0.8.
3. Determination of total alkaloid content
Potassium hydrogen phthalate buffer: drying potassium hydrogen phthalate at 110 ℃ for 2 hours, weighing 5.10g of potassium hydrogen phthalate, adding a proper amount of distilled water for dissolution, and then fixing the volume to 500mL, wherein the pH value of the solution is 4.0;
bromocresol green solution: 0.10g of bromocresol green is weighed, 7.15mL of 0.02mol/L NaOH solution is added for dissolution, and distilled water is used for constant volume to 250mL;
taking 10 mu l of lotus leaf extraction concentrated solution, fixing the volume to 5mL by using chloroform, pouring the solution 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 30min. The lower chloroform layer was taken at 4mL and absorbance was measured at 414nm, 3 times per sample, and total alkaloid content was calculated from the standard curve.
4. High Performance Liquid Chromatography (HPLC) method for detecting nuciferine and quercetin content
Standard and reagent used in the experiment: nuciferine standard (more than or equal to 98% Shanghai Yuan Ye Biotechnology Co., ltd.), quercetin standard (more than or equal to 98% Shanghai Yuan Ye Biotechnology Co., ltd.), formic acid and acetonitrile are all chromatographic purities.
An Agilent C18 column; the mobile phase is water (A, containing 0.4% formic acid) -acetonitrile (B); volume flow rate is 1.0mL/min; the detection wavelength is 270nm; column temperature: room temperature; elution mode: gradient elution
Sample preparation: the preparation of the test sample solution comprises precisely weighing 2mL of lotus leaf samples, placing in a conical bottle with a plug, precisely adding 28mL of methanol, plugging the plug, performing ultrasonic treatment for 30min, standing still for cooling, collecting supernatant, filtering with 0.22 μm organic microporous membrane, collecting filtrate, measuring each sample for 3 times, and calculating the sample concentration according to nuciferine and quercetin standard curve.
Example 1
In the plant extraction, the cell wall barrier can be loosened by using cellulase and pectase and the like to release alkaloids and flavonoid substances in lotus leaves.
Taking 250mL of extracting solution as an example, adding 200mL of water into 6.25g of lotus leaves, soaking for 30min at 50 ℃, and regulating the pH value to 4.5 by using citric acid solution; the enzyme preparation of each scheme is added into the activated water after the activation of a proper amount of water, and is subjected to enzymolysis for 2 hours, and then 2.5g of Tween 80 (1%) is added into the activated water to reach 250mL; ultrasonic filtering at 60deg.C and 480w for 40min, and concentrating the filtrate to 20-40g by rotary evaporator; detecting the contents of total flavone, total phenol and total alkaloid in the enzyme preparation extract of different schemes, and quantitatively analyzing the contents of nuciferine and quercetin by high performance liquid chromatography.
Selecting Xia Cheng liquid cellulase SPE0-17, xia Cheng solid cellulase, xia Cheng pectase, norwestin KTN02309 complex enzyme and Norwestin KON10061 pectase, and dividing into 5 groups for experiments; the experimental protocol is shown in table 1.
TABLE 1
As can be seen from fig. 1, the enzyme preparation has a large influence on the extraction of the active substance. Compared with the first scheme, the second scheme and the third scheme, the third scheme has great advantages in the content of total flavone and quercetin; as can be seen from fig. 2, the High Performance Liquid Chromatography (HPLC) chart also shows that the use of scheme three (novelin KTN02309 complex enzyme) has significant advantages over the use of scheme one (Xia Cheng liquid cellulase SPE 0-17) in the extraction of quercetin; compared with the third and fifth schemes, the addition of the Norwestin KON10061 pectase 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 extracting solution as an example, adding 200mL of water into 6.25g of lotus leaves, soaking for 30min at 50 ℃, and regulating the pH value to 4.0-5.35 by using citric acid solution; activating 0.05g of Norwegian KTN02309 complex enzyme and 0.05g of Norwegian KON10061 pectase, adding the complex enzyme, stirring uniformly, carrying out enzymolysis for 2 hours, and adding 2.5g of Tween 80 (1%) to a constant volume of 250mL; ultrasonic filtering at 60deg.C and 480w for 40min, and concentrating the filtrate to 20-40g by rotary evaporator;
detecting total flavone, total phenol and total alkaloid content in the extracting solution with different pH values, and quantitatively analyzing nuciferine and quercetin content by high performance liquid chromatography.
As shown in FIG. 3, as the pH value is reduced, the total phenol, total flavone and total alkaloid content fluctuates within a certain range, the overall growth is not obvious, the quercetin content increases when the pH value is not adjusted to 5.35 to 5.0, the fluctuation of pH value is 4.0 to 5.0 is small, and the nuciferine content gradually increases along with the reduction of the pH value.
Example 3
Taking 250mL of extracting solution as an example, adding 200mL of water into 6.25g of lotus leaves, soaking for 30min at 50 ℃, and regulating the pH value to 4.5 by using citric acid solution; activating 0.05g Xia Cheng liquid cellulase SPE0-17 and 0.05g Xia Cheng pectase, adding into the activated liquid cellulase SPE0-17 and the activated liquid pectase, stirring uniformly, performing enzymolysis for 2 hours, adding the surfactant, wherein the concentration of glycerol is 10%, and the rest concentration is 1%, and fixing the volume to 250mL; ultrasonic treatment is carried out at 60 ℃ and 480w for 40min, suction filtration is carried out, and the filtrate is concentrated to 20-40g by using a rotary evaporator.
And (3) selecting glycerol, tween 20, tween 60, tween 80, span 80, polyglycerol laurate and polyglycerol caprylic caprate with solubilization as extraction solvents, detecting the total flavone, total phenol and total alkaloid content in the extracting solutions of different surfactants, and quantitatively analyzing the nuciferine and quercetin content by high performance liquid chromatography.
TABLE 2 Effect of different surfactants on extraction yield
As can be seen from table 2, tween 20, tween 60 and tween 80 have advantages of different degrees in the extraction of various substances, and in the specification of GB 2760 plant beverage, the limit of tween series is 0.5g/kg of product, which means that in one 300mL of plant beverage, lotus leaf extract required by tween limit is added, the nuciferine content in the final product is less than 1 mg/bottle, the requirement that the lipid-lowering efficacy is not less than 2mg nuciferine per day in the prior art cannot be met, and the glycerol (glycerol) has no limit requirement when being used as a food additive/processing aid in GB 2760; compared with the extraction of 1% Tween 80 and 10% glycerin, the Tween 80 has the advantages of higher total alkaloids and quercetin content, the glycerin extraction has higher total phenol content, the nuciferine content of the Tween 80 and the nuciferine is not very different, and the actual extraction process shows that when glycerin is used as an extraction solvent, the color of the extraction solution is clear and transparent, and the concentrated solution is reddish with no impurities and has good sensory evaluation.
Example 4
Taking 250mL of extracting solution as an example, adding 200mL of water into 6.25g of lotus leaves, soaking for 30min at 50 ℃, and regulating the pH value to 4.5 by using citric acid solution; activating 0.05g Xia Cheng liquid cellulase SPE0-17 and 0.05g Xia Cheng pectase, adding into the activated liquid cellulase SPE0-17 and the activated liquid pectase, uniformly stirring, performing enzymolysis for 2 hours, and fixing the volume of 2.5g Tween 80 (1%) to 250mL; after ultrasonic treatment for 30-60min at 50-80 ℃ and 480w, suction filtration is carried out, and the filtrate is concentrated to 20-40g by using a rotary evaporator.
Selecting ultrasonic time of 30min,40min,50min and 60min, detecting total flavone, total phenol and total alkaloid content in the extracting solution under different ultrasonic conditions at the ultrasonic temperature of 50 ℃,60 ℃,70 ℃ and 80 ℃, and quantitatively analyzing nuciferine and quercetin content by high performance liquid chromatography.
As can be seen from FIG. 4, at the ultrasonic temperature of 60 ℃ for 30min-60min, the total flavone and nuciferine contents reach the maximum value at 40min, and the quercetin content fluctuates slightly, so that 40min is selected as the optimal ultrasonic time in the invention; as shown in FIG. 5, the ultrasonic time is 40min, the ultrasonic temperature is 50-80 ℃, the total flavone and quercetin contents reach the maximum value when the ultrasonic temperature is 60 ℃, the nuciferine content fluctuates slightly, and the total phenol content is not greatly different, so that the invention selects 60 ℃ as the optimal ultrasonic temperature.
Example 5
Taking 250mL of extracting solution as an example, adding 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 respectively, soaking in 200mL of water at 50 ℃ for 30min, and regulating the pH value to 4.5 by using citric acid solution; activating 0.05g of Norwestin KTN02309 complex enzyme and 0.05g of Norwestin KON10061 pectase, adding the activated Norwestin KTN02309 complex enzyme and the activated Norwestin KON10061 pectase, stirring the mixture uniformly, carrying out enzymolysis for 2 hours, and adding the surfactant which is preferable in the embodiment 3 to the volume of 250mL; after ultrasonic treatment at 60 ℃ and 480w for 40min, suction filtration is carried out, the filtrate is concentrated to 20-40g by using a rotary evaporator, and the difference of 1% Tween 80 extract before and after centrifugation is compared due to a small amount of white precipitate after the Tween extract is placed for a period of time; detecting total flavone, total phenol and total alkaloid content in the extracting solutions with different feed liquid ratios, and quantitatively analyzing the nuciferine and quercetin content by high performance liquid chromatography.
From fig. 6, in the 1% tween 80 system, the yellow-green color of the extract becomes lighter as the concentration of the lotus leaves increases, and the solution becomes clear and turbid.
As can be seen from fig. 7, with increasing lotus leaf concentration in the 1% tween 80 solution, total flavonoids, total phenols and total alkaloids do not increase regularly, and show a situation of descending and then rising, and the total quercetin content does not increase, but shows a steep decline trend; comparing the contents of active substances before and after centrifugation, wherein the content of partial substances after centrifugation is slightly reduced, and the amplitude is not large, so as to fully ensure the stability of the extracting solution, and selecting 5g of lotus leaf/250 ml extracting solution as the optimal concentration in a 1% Tween system;
as shown in fig. 7, with the increase of the lotus leaf concentration in the 10% glycerol solution, the total flavonoids, total phenols and total alkaloids all show a gradual increase trend, the total quercetin content fluctuates in a small range, and the nuciferine content reaches the maximum value when 20g of lotus leaf/250 mL of the extracting solution after the small fluctuation; in the 10% glycerol system, 20g of lotus leaf/250 ml of extract is selected for comprehensive consideration.
Compared with extraction of 1% tween 80 and 10% glycerol, 1% tween 80 has great advantages in total alkaloids and quercetin content, and the glycerol system is conducive to extraction of total phenols and total flavonoids; when the lotus leaf concentration is 20g lotus leaf/250 mL extracting solution, 10% glycerol is used as an extracting solvent for extraction, the total flavone content can reach about 800 mg/part, and the total phenol content can reach about 600 mg/part. FIG. 8 shows that the high performance liquid chromatography quantitatively analyzes nuciferine and quercetin content using 1% Tween 80 lotus leaf concentration of 5g lotus leaf/250 ml extract and 10% glycerol lotus leaf concentration of 20g lotus leaf/250 ml extract, and it is evident that the former is more advantageous for quercetin extraction.
Example 6
Pulverizing dried folium Nelumbinis, and sieving with 60 mesh sieve; soaking crushed lotus leaf powder in 50 ℃ water for 30min, regulating the pH value by using citric acid, and carrying out enzymolysis on lotus leaves for 2h at 50 ℃ at the pH value of 4.0 by using 0.05g of Norwestin KTN02309 complex enzyme and 0.05g of Norwestin KON10061 pectase; then adding Tween 80 with concentration of 1% and glycerin with concentration of 10% respectively, filtering after ultrasonic treatment at 60deg.C and 480w for 40min, and concentrating the extractive solution to a proper volume by rotary evaporator. Wherein, the concentration of Tween 80 lotus leaf with 1% concentration is 5g lotus leaf/250 mL extract, and the concentration of glycerin lotus leaf with 10% concentration is 20g lotus leaf/250 mL extract.
Control group 1: the same lotus leaf concentration as in example 6 was selected, namely 5g lotus leaf/250 mL extract and 20g lotus leaf/250 mL extract; the pH is not adjusted;
control group 2: selecting an extraction method of 5g of lotus leaves per 250mL of extracting solution with the pH value of 4; the remaining parameters were the same as control 1.
Detecting total flavone, total phenol and total alkaloid content in the extracting solutions with different feed liquid ratios, and quantitatively analyzing the nuciferine and quercetin content by high performance liquid chromatography.
As can be seen from FIG. 9a, the extraction solution is clear before concentration, the glycerol extraction color is lighter, tween extraction is yellowish green, 75% ethanol extraction is darker due to the dissolution of chlorophyll, and the color is quite dark when the lotus leaf concentration is 20g/250 mL; as can be seen from fig. 9b, after the extract is concentrated, the ethanol extracted No. 2, no. 4 and No. 5 have a plurality of precipitates, and the precipitates are analyzed to be chlorophyll, some water-insoluble flavone, alkaloid and other substances, the bottom of the 1 is a small amount of white precipitate, and the glycerol extracted No. 3 is a reddish wine without precipitate; FIG. 9c is the color of the concentrate of FIG. 9b after centrifugation; FIG. 9d shows the color of FIG. 9c after 5-fold reconstitution with water, showing good clarity of the 5 extracts after reconstitution;
as can be seen from fig. 10, compared with the 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 total flavone and total phenol contents, and when the concentration of lotus leaves is increased to 20g/250mL compared with the content of 5g/250mL, the content of each substance is significantly increased when the concentration of lotus leaves is 75% ethanol extraction, and when the pH value is adjusted to 4 initially, the content of total phenols and nuciferine is significantly increased, fig. 11 shows the difference between the nuciferine and quercetin content by using high performance liquid chromatography, and the advantages when fig. 11A extracts quercetin by using tween in the method of the present invention are obviously seen, while fig. 11B, fig. 11D and fig. 11E do not extract them effectively by using 75% ethanol extraction, and when the pH is adjusted to 4.0 in fig. 11E.
Example 7
Pulverizing dried folium Nelumbinis, and sieving with 60 mesh sieve; adding 400mL of water into 10g of lotus leaves, soaking for 30min at 50 ℃, regulating the pH value to 4.0 by using citric acid solution, and carrying out enzymolysis on the lotus leaves for 2h at 50 ℃ at the pH value of 4.0 by using activated 0.1g of novelin KTN02309 complex enzyme and 0.1g of novelin KON10061 pectase; then adding 2.5g of Tween 80 (1%), 25g of glycerin to 500mL, carrying out ultrasonic treatment at 60 ℃ and 480w for 40min, filtering, and concentrating the extract to a proper volume by a rotary evaporator. And testing and calculating 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; soaking 40g of lotus leaves in 200mL of water at 50 ℃ for 30min, regulating the pH value to 4.0 by using citric acid solution, and carrying out enzymolysis on the lotus leaves for 2h at 50 ℃ at the pH value of 4.0 by using activated 0.1g of novelin KTN02309 complex enzyme and 0.1g of novelin KON10061 pectase; then adding 2.5g of Tween 80 (1%), 25g of glycerin to 500mL, carrying out ultrasonic treatment at 60 ℃ and 480w for 40min, filtering, and concentrating the extract to a proper volume by a rotary evaporator. And testing and calculating the yield of the total flavone, the total phenol, the total alkaloid and the quercetin.
TABLE 3 Table 3
| Extraction amount mg/g | Flavone | Phenol | Alkaloid | 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 are 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 the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various 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 (2)
1. A method for efficiently and synchronously extracting flavone, alkaloid and polyphenol in lotus leaves by a water-borne method is characterized by comprising the following steps of:
s1, crushing dried lotus leaves, and sieving with a 60-mesh sieve for standby;
s2, soaking the crushed lotus leaf powder in water at 50 ℃ for 30min, then adjusting the pH value by using citric acid, adding an enzyme preparation, and carrying out enzymolysis for 2h at 50 ℃;
s3, adding a surfactant to the mixture to fix the volume to 250mL after enzymolysis, and performing ultrasonic extraction at the ultrasonic temperature of 50-80 ℃ for 30-60 min;
s4, filtering after ultrasonic extraction, and concentrating the extracting solution by using a rotary evaporator;
the dosage of lotus leaves in the extraction method is 5-20g;
the pH value in the step S2 is 4.0-6.0;
the surfactant in the step S3 is Tween 80 and glycerol; the dosage of the enzyme preparation is 0.5-2wt% of the dosage of the lotus leaf;
the enzymes are cellulase and pectase with the dosage ratio of 1:1.
2. The method of claim 1, wherein the enzyme is a combination of novelin KTN02309 complex and novelin KON10061 pectinase.
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