CN113318184A - Extraction method of lilium tigrinum total saponins - Google Patents
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
A method for extracting total saponins from Lilium tigrinum L comprises: (1) pretreatment of lilium tigrinum, (2) nitrogen charging and grinding, (3) complex enzyme treatment, (4) ethanol extraction, (5) centrifugation and concentration, and (6) macroporous resin purification. The purity of the saponin extracted by the invention can reach 73-75%.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a method for extracting total saponins of lilium tigrinum.
Background
Lilium brownii is a perennial herb bulbous plant of the genus Lilium of the family Liliaceae, and is mainly distributed in temperate and cold regions of the northern hemisphere. There are about more than hundred lily varieties worldwide, and China has 39 varieties and 26 varieties, wherein 25 varieties and 19 varieties are special products in China. The lily has extremely high ornamental value and is widely applied to the aspects of flower bed cultivation, potted plant display, indoor decoration cut flower and the like. The lily bulb has edible and medicinal values, and the lily is the lilium tigrinum of the liliaceae family recorded in the pharmacopoeia of the people's republic of China (Lilium lancifofium) And lily (a)Lilium brownii F E.Brownii var．viridulum Bake) And Lilium tenuifolia (Lilium tenuifolium) (II)Lilium pumilum DC) The three dry fleshy scaly leaves have effects of nourishing yin, moistening lung, clearing heart fire and tranquilizing, and are commonly used for treating yin deficiency, dry cough and chronic coughHemoptysis, vexation, pavor, insomnia, dreaminess, absentmindedness, etc.
Lilium tigrinum is a kind of plant belonging to Lilium of Liliaceae, and has a spherical bulb with hypertrophic and fleshy bulb leaves. The lilium tigrinum contains various nutrient substances and active ingredients, and has rich nutrition and medicinal value. The saponin substance is one of the main active ingredients of the lilium tigrinum bulb. The saponin is glucoside with aglycone of triterpene or spirostane, and has antiinflammatory, antitumor, cholesterol reducing, blood glucose reducing, and immunoregulating effects.
Disclosure of Invention
The invention aims to provide an extraction method of lilium tigrinum total saponins.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for extracting total saponins from Lilium tigrinum Maxim comprises the following steps:
step 1: cleaning the lilium tigrinum scale leaves, and draining off surface water for later use;
step 2: grinding the lilium tigrinum scaly leaves obtained in the step 1 into paste with the fineness of about 80-100 meshes;
and step 3: adding cellulase and pectinase into the pasty mixture of the refined scaly leaves, and performing complex enzyme treatment for 1.5-3.0h at 35-45 deg.C and stirring speed of 15-30r/min to obtain a treated material;
and 4, step 4: mixing the enzyme treated material with ethanol at a mass ratio of 1:10-15, extracting at 65-75 deg.C for 2.5-4.0h, and collecting the extract;
and 5: centrifuging the extracted material at 3000-; then concentrating the supernatant to obtain a concentrate;
step 6: dispersing the concentrate with 5-10 times of distilled water to obtain suspension; adsorbing the suspension by AB8 macroporous resin, eluting water-soluble impurities by pure water, and eluting impurities by ethanol with volume fraction of 20% which is 3 times of the volume of AB8 macroporous resin; eluting with 80% ethanol (3 times the volume of AB8 macroporous resin), collecting eluate, mixing eluates, concentrating to obtain extract, and drying to constant weight to obtain total saponins.
The preferable technical scheme is as follows: : the enzyme activity of the cellulase is 5-20 ten thousand U, and the addition amount of the cellulase is 0.25-1.0% of the mass of the slimy refined scaly leaves; the enzyme activity of the pectinase is 5-20 ten thousand U, and the addition amount of the pectinase is 0.25-1.0% of the mass of the mud-paste refined scaly leaves.
The preferable technical scheme is as follows: : in step 4, the ethanol is ethanol with a volume fraction of 80%.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the invention adopts cellulase and pectinase to treat the scaly leaves, promotes the breaking of the cell walls of the scaly leaves and is beneficial to improving the extraction effect of saponin.
2. The purity of the saponin extracted by the invention can reach 73-75%.
Drawings
FIG. 1 is an absorption spectrum of saponin after color development.
FIG. 2 is a standard curve of saponin.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-2. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are provided for a better understanding of the present invention, and are not intended to limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1: extraction method of lilium tigrinum total saponins
The total saponins of lilium tigrinum are extracted according to the technical method of the invention, and the purity of the saponins is detected by adopting the following method.
(1) Selection of measurement wavelength
Preparing a dioscin standard solution: accurately weighing 2.5mg dioscin standard substance, dissolving with methanol, and diluting to 25ml volume to obtain 0.1mg/ml dioscin standard solution.
Preparing a sample solution to be detected: 3.5mg of a sample to be detected (namely the lilium tigrinum total saponin prepared by the method of the invention) is accurately weighed, dissolved by 80 percent ethanol, and the volume is determined to be 25ml, thus obtaining a sample solution to be detected of 0.14 mg/ml.
Preparation of 5% vanillin glacial acetic acid: weighing vanillin 0.5g, dissolving with glacial acetic acid, and diluting to 10 ml.
Determination of optimal measurement wavelength: accurately transferring 0.2ml of dioscin standard solution and sample solution to be detected into a test tube with a plug by using a pipettor, volatilizing the solvent, respectively adding 0.2ml of 5% vanillin glacial acetic acid and 0.8ml of perchloric acid into the test tube, carrying out water bath at 60 ℃ for 20 minutes, cooling in the ice water bath, adding 5ml of glacial acetic acid, uniformly mixing, standing for 10 minutes, and scanning the absorption spectra of the dioscin standard solution after color development and the sample solution to be detected after color development by using a T6 new century ultraviolet-visible spectrophotometer at the wavelength of 400-650 nm, wherein the scanning result is shown in figure 1. After the dioscin reference substance solution and the lily saponin solution are developed, the corresponding absorption peak is positioned at 470 nm. Therefore, 470nm is selected as the wavelength of the incident light.
(2) Preparation of saponin Standard Curve
Accurately transferring 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1.0ml of dioscin standard solution into a test tube with a plug by using a pipette, volatilizing the solvent, respectively adding 0.2ml of 5% vanillin glacial acetic acid and 0.8ml perchloric acid into the test tube, carrying out water bath at 60 ℃ for 20 minutes, cooling in the ice water bath, adding 5ml glacial acetic acid, uniformly mixing, standing for 10 minutes, using a reagent blank as a control, and measuring the light absorption value at 470nm by using a T6 new century ultraviolet visible spectrophotometer. And (4) taking the concentration of the dioscin as an X axis and the light absorption value as a Y axis to prepare a standard curve. The dioscin is used as a standard substance to establish a standard curve, and the equation of the standard curve is as follows: y =19.842x +0.006, linear correlation coefficient is R = 0.9999. The linear relationship was good in the range of 0.02mg to 0.08 mg. The results are shown in FIG. 2.
The extraction method of the lilium tigrinum total saponins is characterized by comprising the following technical steps.
(1) Pretreatment of lilium tigrinum
The lilium tigrinum raw material is mature fresh lilium tigrinum scaly leaves without insect pest, mildew and mechanical damage.
And (3) fully cleaning the lilium tigrinum scaly leaves with clear water meeting the food sanitation requirement to remove silt on the surface, and draining the surface water for later use.
(2) Grinding by charging nitrogen
And (3) putting the scaly leaves washed and drained in the step (1) into a closed grinding device, pumping air in the grinding device, filling nitrogen, and fully grinding until the fineness of the material is below 100 meshes to obtain the pasty refined scaly leaves for later use.
(3) Complex enzyme treatment
Adding cellulase and pectinase into the refined scaly leaves in the step (2), putting the mixed material into stirring type enzyme treatment equipment with controllable temperature, controlling the temperature at 40 ℃ and the stirring speed at 20r/min, and treating with complex enzyme for 2.0h to obtain an enzyme treated material for later use. The cellulase has the enzyme activity of 10 ten thousand U, and the addition amount of the cellulase is 0.5 percent of that of the refined scaly leaves. The enzyme activity of the pectinase is 10 ten thousand U, and the addition amount of the pectinase is 0.4 percent of that of the refined scaly leaves.
(4) Ethanol extraction
Mixing the enzyme treated material in the step (3) and ethanol solution according to the mass ratio of 1:12, putting the mixture into a thermal extraction device with a distillate reflux device, thermally extracting for 3.1h at the temperature of 70 ℃, and collecting the extracted material for later use. Preferably, the ethanol solution used is an 80% ethanol solution by volume fraction.
(5) Centrifuging and concentrating
And (4) centrifuging. Centrifuging the material extracted in the step (4) at the rotating speed of 4000r/min for 12min, and collecting the supernatant for later use.
And (4) concentrating in vacuum. And putting the supernatant into vacuum concentration equipment, and boiling and concentrating under the condition of vacuum gauge pressure of-0.095 MPa until the material is pasty for later use.
(6) Purification by macroporous resins
Dispersing the pasty material obtained in the step (5) by using distilled water with the mass being 8 times that of the pasty material to obtain suspension; adsorbing the suspension by AB8 macroporous resin, eluting water-soluble impurities by using a large amount of pure water, and eluting impurities with larger polarity by using 3 times of column volume and 20% ethanol; eluting with 3 times of column volume of 80% ethanol, collecting and mixing 80% ethanol eluates, concentrating under reduced pressure to obtain extract, and drying to constant weight to obtain total saponins of Lilium tigrinum L.
The vacuum concentration is carried out under the operating conditions of vacuum gauge pressure of-0.08 MPa and temperature of 60 ℃.
(3) Determination of content and purity of total saponins of lily
Weighing 3.5mg of lilium tigrinum total saponin extracted by the method of the invention, dissolving to a constant volume of 10ml, putting 0.4ml in a test tube, volatilizing the solvent, respectively adding 0.2ml of 5% vanillin glacial acetic acid and 0.8ml of perchloric acid into the test tube, carrying out water bath at 60 ℃ for 20 minutes, cooling in the ice water bath, adding 5ml glacial acetic acid, mixing uniformly, standing for 10 minutes, and measuring the light absorption value to be 0.348 at 470nm by using a T6 new century ultraviolet visible spectrophotometer.
Bringing the absorbance to y =19.842x +0.006, the total saponin content was 2.6 mg.
The purity of lily total saponin = (calculating total saponin content/weighing total saponin content) × 100%. And calculating the purity of the total saponins of lily, wherein the purity of the total saponins is 74.3%.
Example 2: extraction method of lilium tigrinum total saponins
A method for extracting total saponins from Lilium tigrinum Maxim comprises the following steps:
step 1: cleaning the lilium tigrinum scale leaves, and draining off surface water for later use;
step 2: putting the lilium tigrinum scaly leaves obtained in the step 1 into a closed grinding device, pumping out air in the grinding device, filling nitrogen, and grinding until the fineness of the materials is below 100 meshes to obtain mud-paste refined scaly leaves;
and step 3: adding cellulase and pectinase into the pasty thinned scaly leaves, and carrying out complex enzyme treatment on the mixed material for 1.5h at the temperature of 35 ℃ and the stirring speed of 15r/min to obtain an enzyme treated material;
and 4, step 4: mixing the enzyme treated material with ethanol at a mass ratio of 1:10, extracting at 65 deg.C for 2.5h, and collecting the extract;
and 5: centrifuging the extract at 3000r/min for 10min, and collecting supernatant; then concentrating the supernatant to obtain a concentrate;
step 6: dispersing the concentrate with 5 times of distilled water to obtain suspension; adsorbing the suspension by AB8 macroporous resin, eluting water-soluble impurities by pure water, and eluting impurities by ethanol with volume fraction of 20% which is 3 times of the volume of AB8 macroporous resin; eluting with 80% ethanol (3 times the volume of AB8 macroporous resin), collecting eluate, mixing eluates, concentrating to obtain extract, and drying to constant weight to obtain total saponins.
The preferred embodiment is: the enzyme activity of the cellulase is 5 ten thousand U, and the addition amount of the cellulase is 1.0 percent of the mass of the slimy refined scaly leaves; the enzyme activity of the pectinase is 5 ten thousand U, and the addition amount of the pectinase is 1.0 percent of the mass of the mud-like refined scaly leaves.
The preferred embodiment is: in step 4, the ethanol is ethanol with a volume fraction of 80%.
Example 3: extraction method of lilium tigrinum total saponins
A method for extracting total saponins from Lilium tigrinum Maxim comprises the following steps:
step 1: cleaning the lilium tigrinum scale leaves, and draining off surface water for later use;
step 2: putting the lilium tigrinum scaly leaves obtained in the step 1 into a closed grinding device, pumping out air in the grinding device, filling nitrogen, and grinding until the fineness of the materials is below 100 meshes to obtain mud-paste refined scaly leaves;
and step 3: adding cellulase and pectinase into the pasty thinned scaly leaves, and carrying out complex enzyme treatment on the mixed material for 3.0h at the temperature of 45 ℃ and the stirring speed of 30r/min to obtain an enzyme treated material;
and 4, step 4: mixing the enzyme treated material with ethanol at a mass ratio of 1:15, extracting at 75 deg.C for 4.0h, and collecting the extract;
and 5: centrifuging the extract at 5000r/min for 15min, and collecting supernatant; then concentrating the supernatant to obtain a concentrate;
step 6: dispersing the concentrate with distilled water 10 times of the concentrate to obtain suspension; adsorbing the suspension by AB8 macroporous resin, eluting water-soluble impurities by pure water, and eluting impurities by ethanol with volume fraction of 20% which is 3 times of the volume of AB8 macroporous resin; eluting with 80% ethanol (3 times the volume of AB8 macroporous resin), collecting eluate, mixing eluates, concentrating to obtain extract, and drying to constant weight to obtain total saponins.
The preferred embodiment is: the enzyme activity of the cellulase is 20 ten thousand U, and the addition amount of the cellulase is 0.25 percent of the mass of the slimy refined scaly leaves; the enzyme activity of the pectinase is 20 ten thousand U, and the addition amount of the pectinase is 0.25 percent of the mass of the slimy refined scaly leaves.
The preferred embodiment is: in step 4, the ethanol is ethanol with a volume fraction of 80%.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (3)
1. A method for extracting total saponins of lilium tigrinum has the following characteristics: comprises the following steps:
step 1: cleaning the lilium tigrinum scale leaves, and draining off surface water for later use;
step 2: grinding the lilium tigrinum scaly leaves obtained in the step 1 into paste with the fineness of about 80-100 meshes;
and step 3: adding cellulase and pectinase into the pasty mixture of the refined scaly leaves, and performing complex enzyme treatment for 1.5-3.0h at 35-45 deg.C and stirring speed of 15-30r/min to obtain a treated material;
and 4, step 4: mixing the enzyme treated material with ethanol at a mass ratio of 1:10-15, extracting at 65-75 deg.C for 2.5-4.0h, and collecting the extract;
and 5: centrifuging the extracted material at 3000-; then concentrating the supernatant to obtain a concentrate;
step 6: dispersing the concentrate with 5-10 times of distilled water to obtain suspension; adsorbing the suspension by AB8 macroporous resin, eluting water-soluble impurities by pure water, and eluting impurities by ethanol with volume fraction of 20% which is 3 times of the volume of AB8 macroporous resin; eluting with 80% ethanol (3 times the volume of AB8 macroporous resin), collecting eluate, mixing eluates, concentrating to obtain extract, and drying to constant weight to obtain total saponins.
2. The extraction method of lilium tigrinum total saponins according to claim 1, characterized in that: the enzyme activity of the cellulase is 5-20 ten thousand U, and the addition amount of the cellulase is 0.25-1.0% of the mass of the slimy refined scaly leaves; the enzyme activity of the pectinase is 5-20 ten thousand U, and the addition amount of the pectinase is 0.25-1.0% of the mass of the mud-paste refined scaly leaves.
3. The extraction method of lilium tigrinum total saponins according to claim 1, characterized in that: in step 4, the ethanol is ethanol with a volume fraction of 80%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107412546A (en) * | 2017-07-14 | 2017-12-01 | 百色学院 | A kind of method of Lilium lancifo1ium Thunb extraction saponin(e |
| CN110143996A (en) * | 2019-05-23 | 2019-08-20 | 佛山市欧若拉生物科技有限公司 | One kind of multiple Lilium brownie preparation methods |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107412546A (en) * | 2017-07-14 | 2017-12-01 | 百色学院 | A kind of method of Lilium lancifo1ium Thunb extraction saponin(e |
| CN110143996A (en) * | 2019-05-23 | 2019-08-20 | 佛山市欧若拉生物科技有限公司 | One kind of multiple Lilium brownie preparation methods |
Non-Patent Citations (1)
| Title |
|---|
| 张璐 等: ""超声酶法提取百合总皂苷的研究"", 《饮料工业》 * |
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