CN111991452A - Method for extracting total saponins from clematis - Google Patents
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Abstract
The invention discloses a method for extracting total saponins from clematis, which comprises the following steps of S1: pretreating fresh roots and stems of clematis chinensis in Shaanxi, drying in the sun, crushing, heating and refluxing for 2 hours in 80 ℃ water bath by using petroleum ether with the material-liquid ratio of 1:5, and heating and refluxing the refluxed filter residues in a secondary water bath to obtain degreased medicine residues; s2: carrying out primary heating reflux on the degreased medicine residues for 2h by using 70% ethanol, carrying out primary ultrasonic extraction at the ultrasonic temperature of 65 ℃ for 50min, carrying out secondary heating reflux and secondary ultrasonic extraction on the filter residues, mixing primary filtrate obtained after the primary ultrasonic extraction and secondary filtrate obtained after the secondary ultrasonic extraction, centrifuging, and taking supernatant to obtain total saponin extracting solution; s3: and (4) determining the extraction rate of the total saponins in the total saponin extracting solution. The extraction method of the total saponins can improve the extraction rate of the total saponins in clematis shaanxi.
Description
Technical Field
The invention relates to the technical field of natural organic chemistry, in particular to a method for extracting total saponins from clematis.
Background
Studies show that the extract of the rhizome of the Clematis (Clematis) has the effects of resisting oxidation, reducing blood pressure and blood sugar, dredging collaterals and relieving pain, suppressing immunity, benefiting gallbladder, promoting urination, relaxing smooth muscle and the like, and is known as 'vine queen'. The clematis plants are 230 or more species in total and distributed in the northern temperate zone, and 108 species exist in China and are mainly distributed in the southwest region and the west of the central region. Qinling mountain is a suitable growing area for many medicinal plants due to its unique geographical features, abundant soil environments and unique climatic features, and the clematis plant resource is very abundant. There are studies showing that: the natural distribution of 22 and variant clematis plant resources such as clematis koilowii, clematis glaucus, clematis macroloba, clematis shaanxi and the like is found in the mountain area of Qinling mountains.
Shaanxi Clematis (Clematis shansenensis W.T.Wang) is a perennial deciduous vine of Clematis of Ranunculaceae. The branch is cylindrical and has fluffy shape, the branch is green after being dried, obvious longitudinal stripes are formed, and the base part has no scale. Feathery compound leaves with the length of 13-18 cm; 5 small leaves, oval shape, paper, 3-7cm long, 2-4cm wide, leaf tip short and tapered, leaf base heart shape or truncated shape, whole edge, sparse and short hair on the surface, dense and short hair on the back. The calyx is white in flower, the diameter is 3-5cm, the sepals are usually 4, the calyx is developed, the sepals are in a shape of inverted needles or inverted needles, the length is 1.5-2.5cm, the outer surface of the sepals is provided with white villi at the edge, the middle part of the sepals is short hair, and the inner surface of the sepals is hairless; the 3-7 forms a parasol inflorescence which is terminal or axillary; the total pedicel is 6-10cm long, and is short hair with 2 leaves of involucre; the flower stalk is 3-4cm long, is densely covered with silk hair, and has 2 linear or full-split bracts in the middle; the stamen is as long as 1cm, the filament is linear, the stamen has obvious middle pulse, no hair, the anther is linear and has no hair, and the tip is slightly provided with a short tip; most pistils, dense quilt and soft hair; the flower column has light brown feather. The flowering phase is June, and the fruit phase is unknown. Producing the final southern mountain, the Huashan mountain, the Taibaishan mountain and the like of the southern slope and the northern slope of Qinling mountains; is grown on a hillside with the elevation of about 1200 m.
As a special species in China, the Shaanxi clematis takes Shaanxi as a main distribution area. The plants are distributed in Hubei, Henan and Shanxi except Shaanxi, and are usually grown in mountain slopes, ravines or stone walls at the elevation of 1200 m. Shanlu located in southeast of Shaanxi is an earth-rock mountain area which spans two large watersheds of Yangtze river and yellow river and takes middle and low mountains as the leading part. The terrain is high in the northwest and low in the southeast, the average altitude is 900 meters, and three types of landforms exist: valley, low mountain and middle mountain slope trough. Due to the blocking effect of the Qinling mountains on the wind in the southeast season, the climate with warm and semi-humid areas on the north side of the Qinling mountains and the humid climate on the south side of the North subtropics are formed, and the special climate soil, the complicated and changeable landform and landform have the advantages of high forest coverage rate, no severe cold in winter, no cold in summer, clear four seasons and rain and heat in the same season, so that the Shangluo becomes one of the main habitats of a plurality of medicinal plant resources, wherein the Nelumbo ferrite belongs to a large class.
The research on the chemical components of the clematis plants shows that the rhizomes of the clematis plants contain saponins, flavonoids, alkaloids, coumarins, lignans, volatile components and a plurality of trace elements. The saponins contained in the rhizome of clematis are mainly pentacyclic triterpenoid saponins, oleanolic acid and ivy are main sapogenins of the rhizome, a few are ursolic alkane type, taraxane type and the like, and glucose, rhamnose, arabinose and ribose are main saccharides combined by the saponins. Fu et al isolated 3 new oleanane-type triterpene saponin compounds in 2013 from alcohol extract of dried rhizome of Clematis chinensis. 7 triterpene saponin compounds are separated and identified from the whole herb of clematis filamentosa (C.lasiandra) by Tian and the like, wherein 4 oleanane type triterpene saponins are novel compounds. The existing extraction processes of saponin mainly comprise a water extraction method, an alcohol extraction method, an ultrasonic extraction method, a microwave-assisted extraction method and CO2The supercritical extraction technology and the ultrahigh pressure extraction technology, but the traditional water extraction and alcohol extraction methods are simple and economic in method, have low requirements on personnel and equipment, and have low yield; and supercritical CO2Although the extraction methods such as extraction, ultrahigh pressure and the like have high yield, the methods are complex, have high requirements on personnel and equipment, and have certain difficulty in industrial production.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a method for extracting total saponins from clematis chinensis, which can improve the extraction rate of the total saponins from clematis chinensis in Shaanxi.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for extracting total saponins from clematis is characterized by comprising the following steps,
s1: pretreating fresh shanxi clematis roots to obtain degreased medicine residues;
s2: carrying out alcohol and ultrasonic extraction on the degreased dregs to obtain a total saponin extracting solution;
s3: and (4) determining the extraction rate of the total saponins in the total saponin extracting solution.
Further, the specific operation of step S1 includes,
s11: drying fresh roots of Shaanxi clematis in the sun, and crushing into medicinal powder;
s12: adding petroleum ether into medicinal material powder for degreasing, wherein the material-to-liquid ratio of the medicinal material powder to the petroleum ether is 1: 4-1: 6, and heating and refluxing in a water bath;
s13: adding petroleum ether with a material-to-liquid ratio of 1: 4-1: 6 into the filter residue obtained by the water bath heating reflux in the step S12, and carrying out secondary water bath heating reflux;
s14: and (5) filtering the filter residue obtained after heating and refluxing in the water bath for the second time in the step (S13) through a 40-mesh sieve to obtain degreased medicine residues, and drying and storing for later use.
Further, the ratio of the material to the liquid in the step S12 and the step S13 is 1: 5.
Further, the water bath heating temperature of the water bath heating reflux and the water bath heating reflux of the second time is 80 ℃, and the heating reflux time is 2 hours.
Further, the specific operation of step S2 includes,
s21: adding 70% ethanol into the defatted residue, and heating and refluxing for the first time to obtain a first extractive solution; the feed-liquid ratio of the degreased medicine residues to 70% ethanol is 1: 45-1: 55;
s22: carrying out primary ultrasonic extraction on the primary extracting solution to obtain primary ultrasonic filtrate and primary ultrasonic filter residue;
s23: adding ethanol with the concentration of 70% into the primary ultrasonic filter residue to carry out secondary heating reflux and secondary ultrasonic extraction to obtain secondary ultrasonic filtrate and secondary ultrasonic filter residue; the material-liquid ratio of the primary ultrasonic filter residue to 70% ethanol is 1: 45-1: 55;
s24: mixing the primary ultrasonic filtrate and the secondary ultrasonic filtrate, centrifuging, and taking supernatant to obtain total saponin extract.
Further, the material-liquid ratio of the defatted residues to 70% ethanol in step S21 is 1:50, and the material-liquid ratio of the primary ultrasonic filtration residues to 70% ethanol in step S23 is also 1: 50.
Further, the temperature of the primary heating reflux and the secondary heating reflux is 65 ℃, and the reflux time is 2 hours.
Further, the ultrasonic temperature of the primary ultrasonic extraction and the secondary ultrasonic extraction is 65 ℃, and the ultrasonic extraction time is 50 min.
Further, the specific operation of step S3 is,
s31: taking 1mg of oleanolic acid standard product, and preparing 1mg/ml mother liquor by using absolute ethyl alcohol;
s32: precisely transferring 0.1 mL, 0.2 mL, 0.3 mL, 0.4mL, 0.5 mL, 0.6 mL and 0.7mL of mother liquor respectively to prepare solutions of 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL and 0.7mg/mL, adding into seven dry test tubes with plugs, and volatilizing the solvent in a water bath;
s33: respectively adding 0.4mL of 5% vanillin-glacial acetic acid solution and 1.6mL of perchloric acid into seven test tubes, and heating in a constant-temperature water bath at 70 ℃ for 20 min;
s34: cooling the test tube heated in the water bath for 3min by running water, adding 5mL of glacial acetic acid, shaking up, taking absolute ethyl alcohol as a blank control, and measuring the absorbance at 542 nm;
s35: drawing a standard curve by taking the concentration of oleanolic acid as a horizontal coordinate and the absorbance as a vertical coordinate to obtain a regression equation;
s36: preparing a total saponin extracting solution, measuring an absorbance value at the wavelength of 542nm, and obtaining the diluted saponin filtrate concentration C in the saponin extracting solution by using the regression equation in the step S35;
s37: using the formula of the extraction rate of the total saponins
Calculating the extraction rate of the total saponins in clematis schreberi in Shaanxi;
wherein, W: the extraction rate (%) of the general saponins of clematis in Shaanxi;
c: diluting the saponin filtrate to obtain a concentration;
v: volume of filtrate/mL;
n: dilution times;
m: shaanxi clematis powder mass/g.
The invention has the beneficial effects that:
1. aiming at the clematis chinensis in Shaanxi, the method for extracting the total saponins in the clematis chinensis in Shaanxi firstly pretreats the clematis chinensis in Shaanxi, degreases by adopting petroleum ether, and then can obviously improve the extraction yield of the total saponins in the clematis chinensis in Shaanxi by utilizing an alcohol and ultrasonic extraction method.
2. The method for extracting the total saponins from clematis chinensis is optimized according to specific process parameters in the processes of pretreatment and alcohol-ultrasonic extraction of clematis chinensis in Shaanxi, and the extraction process parameters are utilized to extract the total saponins from clematis chinensis in Shaanxi, so that the extraction rate of the total saponins from clematis chinensis can be improved.
3. Aiming at the current situation that the extraction method of the total saponins in clematis chinensis in Shaanxi is planted in the social and economic development of the Shanxi local, the early investment is small, the requirements on equipment and personnel are low, the yield is high, and the method has the characteristics of greenness, economy, high efficiency and the like, is easy to carry out industrial production and is very suitable for the local development level; the method has important practical significance for the sustainable development of medicinal plant resources of the phytolacca, the implementation of the strategy of 'medicine industry with market', and the implementation of the national precision poverty-relieving strategy, and has certain reference value for the industrial development of regions with underdeveloped economy similar to the phytolacca, especially the regions with the young and the old.
Drawings
FIG. 1 is a flow chart of the operation of pretreatment of fresh roots of clematis shaanxi in the present invention;
FIG. 2 is a flow chart of the ultrasonic extraction operation of the defatted residues in the present invention;
FIG. 3 is a standard curve of the relationship between oleanolic acid and absorbance in the present invention;
FIG. 4 is a graph showing the influence of ethanol concentration on the extraction rate of saponins from clematis shaanxi in the alcohol + ultrasonic extraction process in the first embodiment of the present invention;
FIG. 5 shows the effect of ultrasonic time on the extraction rate of clematis saponins in Shaanxi in the alcohol + ultrasonic extraction process in the first embodiment of the present invention;
FIG. 6 shows the effect of extraction temperature on the extraction rate of clematis saponins in Shaanxi during the alcohol + ultrasonic extraction process in the first embodiment of the present invention;
FIG. 7 is a graph showing the effect of feed liquid ratio on the extraction rate of clematis saponins in Shaanxi in the alcohol + ultrasonic extraction process in the first embodiment of the present invention;
FIG. 8 is a graph showing the effect of feed liquid ratio on the extraction rate of clematis saponins in Shaanxi during pretreatment in the third embodiment of the present invention;
FIG. 9 shows the effect of heating reflux temperature on the extraction rate of clematis saponins in Shaanxi during pretreatment in the third embodiment of the present invention;
FIG. 10 is a graph showing the effect of heating reflux time on the extraction rate of clematis saponins in Shaanxi during the pretreatment process in the third embodiment of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following further describes the technical solution of the present invention with reference to the drawings and the embodiments.
The first embodiment is as follows:
as shown in attached drawings 1 and 2, taking the clematis in Shanxi province as an example, the extraction of the total saponins comprises the following steps,
s1: pretreating fresh shanxi clematis roots to obtain degreased medicine residues;
specifically, S11: drying fresh roots of Shaanxi clematis in the sun, and crushing into medicinal powder;
s12: taking 5g of dried medicinal material powder, adding petroleum ether into the medicinal material powder, wherein the material-to-liquid ratio of the medicinal material powder to the petroleum ether is 1:5, and heating and refluxing in a water bath; the water bath heating temperature of the water bath heating reflux is 80 ℃, and the heating reflux time is 2 hours;
s13: carrying out secondary water bath heating reflux on the filter residue obtained in the step S12 through heating reflux; the water bath heating temperature of the secondary water bath heating reflux is 80 ℃, and the heating reflux time is 2 hours;
s14: and (5) filtering the filter residue obtained after heating and refluxing in the water bath for the second time in the step (S13) through a 40-mesh sieve to obtain degreased medicine residues, and placing the degreased medicine residues in a glass bottle for drying and storage for later use.
S2: carrying out alcohol and ultrasonic extraction on the degreased dregs to obtain a total saponin extracting solution;
specifically, S21: taking 2g of degreasing dregs, adding 70% ethanol into the degreasing dregs, and heating and refluxing for the first time to obtain a first extracting solution;
the temperature of the primary heating reflux is 65 ℃, and the reflux time is 2 hours;
the feed-liquid ratio of the degreased medicine dregs to 70% ethanol is 1: 50;
s22: carrying out primary ultrasonic extraction on the primary extracting solution to obtain primary ultrasonic filtrate and primary ultrasonic filter residue;
the ultrasonic temperature of the primary ultrasonic extraction is 65 ℃, and the ultrasonic extraction time is 50 min;
s23: adding ethanol with the concentration of 70% into the primary ultrasonic filter residue to carry out secondary heating reflux and secondary ultrasonic extraction to obtain secondary ultrasonic filtrate and secondary ultrasonic filter residue;
the temperature of the secondary heating reflux is 65 ℃, and the reflux time is 2 hours;
the ultrasonic temperature of the secondary ultrasonic extraction is 65 ℃, and the ultrasonic extraction time is 50 min;
s24: mixing the primary ultrasonic filtrate and the secondary ultrasonic filtrate, centrifuging, and taking supernatant to obtain total saponin extract.
S3: and (4) determining the extraction rate of the total saponins in the total saponin extracting solution.
Specifically, S31: taking 1mg of oleanolic acid standard product, and preparing 1mg/ml mother liquor by using absolute ethyl alcohol;
s32: precisely transferring 0.1 mL, 0.2 mL, 0.3 mL, 0.4mL, 0.5 mL, 0.6 mL and 0.7mL of mother liquor respectively to prepare solutions of 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL and 0.7mg/mL, adding into seven dry test tubes with plugs, and volatilizing the solvent in a water bath;
s33: respectively adding 0.4mL of 5% vanillin-glacial acetic acid solution and 1.6mL of perchloric acid into seven test tubes, and heating in a constant-temperature water bath at 70 ℃ for 20 min;
s34: cooling the test tube heated in the water bath for 3min by running water, adding 5mL of glacial acetic acid, shaking up, taking absolute ethyl alcohol as a blank control, and measuring the absorbance at 542 nm;
s35: drawing a standard curve by using the concentration of oleanolic acid as abscissa and absorbance as ordinate, and obtaining a regression equation of y 0.1986x +0.0183, R20.9890, where y is the absorbance and x is the concentration of oleanolic acid, in units of: mg/mL.
S36: preparing a total saponin extract solution, putting 1mL of the saponin extract into a 10mL volumetric flask (diluted by 10 times), taking 1mL out of the 10mL volumetric flask, adding 0.4mL of 5% vanillin-glacial acetic acid solution and 1.6mL of perchloric acid, and heating in a constant-temperature water bath at 70 ℃ for 20 min; cooling the test tube heated in water bath for 3min with flowing water, adding glacial acetic acid 5mL, shaking, measuring absorbance at wavelength of 542nm, and calculating x by using the regression equation in step S35 to obtain the concentration C of diluted saponin filtrate in the saponin extract solution;
s37: using the formula of the extraction rate of the total saponins
Calculating the extraction rate of the total saponins in clematis schreberi in Shaanxi;
wherein, W: the extraction rate (%) of the general saponins of clematis in Shaanxi;
c: diluting the saponin filtrate to obtain a concentration;
v: the volume/mL of the filtrate, in this example, the body fluid of the total saponin extract extracted in step S2;
n: dilution factor, 10;
m: the mass of the powder of the clematis in Shaanxi is 5g, and the mass of the powder of the dried medicinal materials in the embodiment is 5 g.
Further, in this embodiment, optimization experiments are also performed on different process parameters in the alcohol + ultrasonic extraction process, specifically:
1.1 controlling the ratio of the material to the liquid of the first heating reflux to the second heating reflux to be 1: 10, the ultrasonic extraction temperature is 70 ℃, the ultrasonic extraction time is 40min, the influence of ethanol concentration of 55%, 65%, 75%, 85% and 95% on the extraction rate of saponin in clematis shaanxi is respectively researched, and the results are shown in table 1 and attached figure 4.
TABLE 1 Effect of different ethanol concentrations on Total Saponin extraction
As can be seen from table 1 and fig. 4, the extraction rate of saponins in clematis shaxi increases with the increase of ethanol concentration between 55% and 75% by mass of ethanol, and the extraction rate of saponins is the highest when the mass of ethanol is 75%, probably because the polarity of saponins in clematis shaxi is relatively close to the polarity of ethanol with 75% by mass; then, as the concentration of the ethanol is gradually increased, the extraction rate of the saponin is reduced, and some impurities in the clematis shaanxi are possibly dissolved in the ethanol, so that the extraction rate of the saponin is reduced. Therefore, in the experiment, the ethanol with the mass fractions of 70%, 75% and 80% is selected for orthogonal experimental analysis.
1.2, controlling the mass fraction of ethanol to be 95%, the extraction temperature to be 70 ℃, and the ratio of material to liquid to be 1: 10, the influence of the ultrasonic time of 20min, 30 min, 40min, 50min and 60min on the extraction rate of saponins in clematis shaanxi is respectively studied, and the results are shown in table 2 and attached figure 5.
TABLE 2 Effect of different ultrasonic extraction times on the extraction yield of Total saponins
As can be seen from table 2 and fig. 5, the extraction rate of the clematis shakei saponin is obviously increased within the range of 20-50min, and the extraction rate is the highest at 50min and reaches 1.560%, but when the extraction time is more than 40min, the increase of the extraction rate of the saponin is relatively gentle, because the saponin in the clematis shakei is basically proposed, the ultrasonic time is prolonged, but no effect is generated on the extraction rate of the experiment, and more time is required for the experiment. Due to the operability of the experiment, ultrasonic time of 45min, 50min and 55min is selected for orthogonal experimental analysis.
1.3, controlling the mass fraction of ethanol to be 95 percent, and the feed-liquid ratio to be 1: 10. the ultrasonic treatment time is 40min, and the influence of the ultrasonic treatment time on the extraction rate of saponins in clematis shaanxi under the conditions of extraction temperature of 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃ is respectively researched, and the results are shown in table 3 and fig. 6.
TABLE 3 Effect of different ultrasonic extraction temperatures on Total Saponin extraction yield
As can be seen from table 3 and fig. 6, the extraction rate of total saponins in clematis shakei is gradually increased with the increase of extraction temperature between 50 ℃ and 70 ℃, and the dissolution and diffusion rates of total saponins in clematis shakei are further increased and the extraction rate is increased due to the increase of temperature and the increase of molecular motion, which leads to the increase of cell rupture speed. The extraction temperature is 70 ℃, and the extraction rate of the total saponins in the clematis in Shaanxi is the highest. The extraction rate of the saponin is reduced along with the increase of the temperature, probably because the heat-sensitive components in the clematis shaanxi are damaged due to the higher temperature, the ultrasonic temperature is 65 ℃, 70 ℃ and 75 ℃ for orthogonal test analysis.
1.4 controlling the ethanol mass fraction to be 95%, controlling the ultrasonic time to be 40min, setting the ultrasonic temperature to be 70 ℃, and respectively studying the conditions that the material-liquid ratio is 1: 10. 1: 30. 1: 50. 1: 70. 1: the effect on the extraction rate of saponins from clematis shaanxi under 90 conditions is shown in table 4 and fig. 7.
TABLE 4 influence of different feed liquid ratios on total saponin extraction yield
As can be seen from table 4 and fig. 7, the selection of the material-liquid ratio and the amount of the solvent directly affect the extraction rate of the saponin compounds, which is an economic indicator that cannot be underestimated in the industrial production process, and the extraction effect of different material-liquid ratios based on the minimum amount of ethanol just submerging the raw material shows that the extraction rate of the saponin increases with the increase of the amount of the solvent, when the material-liquid ratio exceeds 1: after 50, the extraction rate is increased by a small amount and even has a tendency of decreasing, so that the ratio of the extracting solution is 1: the orthogonal assay was performed for standard 50.
1.5 based on the test results of 3.1-3.4 single factors, determining three factors influencing the extraction process of the clematis shashanxi total saponins by ethanol mass fraction/%, ultrasonic time/min and extraction temperature/DEG C as investigation objects, taking the saponin yield as an investigation index, and carrying out L9(33) Orthogonal experiments, factors and levels are shown in table 5.
TABLE 5 Single factor and level
9 parts of the same defatted residue samples were prepared, orthogonal experimental design was performed according to table 5, and visual analysis was performed using saponin yield as an investigation index, with the results shown in table 6.
Table 6 orthogonal test design visual analysis table
Note: k is the average of the sum of some-level results of the factors; r is the worst outcome at some level of each factor.
The significance analysis results of the Shaanxi clematis total saponin extraction orthogonal test are shown in Table 7.
TABLE 7 significance analysis results of orthogonal test
Dependent variable: the yield of saponin is%
The orthogonal test data analysis shows that the factors influencing the extraction effect of the clematis chinensis general saponin in Shaanxi are ultrasonic time, ethanol concentration and extraction temperature, and the optimal extraction conditions are as follows: a. the1B2C1The ethanol concentration is 70%, the ultrasonic time is 50min, the extraction temperature is 65 ℃, and the material-liquid ratio is 1: 50. The verification test shows that the yield of the obtained total saponins of clematis shaanxi is 2.42%.
Example two:
the difference between the operation steps of the method for extracting the total saponins from clematis chinensis in the embodiment and the operation steps of the method for extracting the total saponins from clematis chinensis in the embodiment is that the clematis chinensis in Shaanxi is not pretreated, alcohol and ultrasonic extraction is directly performed, and the comparison between the finally obtained average value of the extraction rate of the total saponins and the average value of the extraction rate of the total saponins in the embodiment is shown in Table 8.
TABLE 8 comparison of the mean extraction yields of total saponins in example two and example one
As can be seen from table 8, after pretreatment of clematis shaanxi, alcohol + ultrasonic extraction is performed, and the yield of total saponin extraction is 0.76% higher than that of total saponin extraction directly performed by alcohol + ultrasonic extraction, because the lipid in the medicinal material can be removed by using petroleum ether for pretreatment, impurities can be effectively removed, and the yield of saponin can be improved.
Example three:
the operation steps of the clematis total saponin extraction method in the embodiment are the same as those in the first embodiment, except that the material-liquid ratio, the reflux temperature and the reflux time of water bath heating reflux in the pretreatment process of fresh Shaanxi clematis roots in the step S1 are adopted; the method specifically comprises the following steps:
respectively weighing 5.00g of clematis chinensis dry powder, performing degreasing and degreasing pretreatment, performing alcohol + ultrasonic extraction according to the specific operation in the first embodiment after degreasing treatment, setting the ethanol concentration to be 70%, and the material-liquid ratio to be 1: 10, extracting at 70 ℃ for 40min by ultrasonic extraction, performing vacuum filtration after extraction, measuring the content of saponin in filtrate, and calculating the yield according to a formula.
3.1 controlling the heating reflux time to be 2h and the heating reflux temperature to be 70 ℃ in the pretreatment, and respectively researching the reaction conditions of the raw materials and the liquid in the ratio of 1: 3. 1: 4. 1: 5. 1: the effect on the extraction rate of saponins in clematis shaanxi under the condition of 6 is shown in table 9 and attached figure 8.
TABLE 9 Effect of different feed liquid ratios on total saponin extraction in pretreatment
When the ratio of the materials to the liquids is 1, it can be seen by combining table 9 and fig. 8: and 5, the extraction rate reaches an extreme value of 1.80, and then the feed-liquid ratio is increased, so that the increase amplitude of the extraction rate is not large, and even the extraction rate tends to be reduced, and the feed-liquid ratio is proper to be 1: 5.
3.2 controlling the ratio of the heating reflux to the liquid in the pretreatment to be 1:4, the reflux time to be 2h, respectively researching the influence of the heating reflux temperature to the extraction rate of the saponin in the clematis shaanxi at 60 ℃, 70 ℃, 80 ℃ and 90 ℃, and the results are shown in Table 10 and attached figure 9.
TABLE 10 Effect of different reflux temperatures on Total Saponin extraction yield during pretreatment
As can be seen by combining the table 10 and the attached figure 9, the extraction rate of the total saponins in the clematis shaanxi is the highest when the temperature is 80 ℃, and then the extraction rate of the saponins is reduced along with the increase of the temperature, so that the temperature is properly selected to be 80 ℃.
3.3 the feed-liquid ratio of heating reflux in the pretreatment is 1:4, the reflux temperature is 80 ℃, the influence on the extraction rate of saponin in clematis shaanxi when the heating reflux time is 1h, 2h, 3h and 4h is respectively researched, and the results are shown in Table 11 and attached drawing 10.
TABLE 11 Effect of different reflux temperatures on Total Saponin extraction yield during pretreatment
As can be seen from Table 11 and FIG. 10, when the heating reflux time reaches 2h, the extraction rate reaches an extreme value, and then the extraction time is prolonged, the extraction rate of total saponins is relatively gentle and even has a tendency of decreasing, and 2h is most suitable according to the economic principle.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (9)
1. A method for extracting total saponins from clematis is characterized by comprising the following steps,
s1: pretreating fresh shanxi clematis roots to obtain degreased medicine residues;
s2: carrying out alcohol and ultrasonic extraction on the degreased dregs to obtain a total saponin extracting solution;
s3: and (4) determining the extraction rate of the total saponins in the total saponin extracting solution.
2. The method for extracting total saponins in clematis according to claim 1, wherein the specific operation of step S1 includes,
s11: drying fresh roots of Shaanxi clematis in the sun, and crushing into medicinal powder;
s12: adding petroleum ether into medicinal material powder for degreasing, wherein the material-to-liquid ratio of the medicinal material powder to the petroleum ether is 1: 4-1: 6, and heating and refluxing in a water bath;
s13: adding petroleum ether with a material-to-liquid ratio of 1: 4-1: 6 into the filter residue obtained by the water bath heating reflux in the step S12, and carrying out secondary water bath heating reflux;
s14: and (5) filtering the filter residue obtained after heating and refluxing in the water bath for the second time in the step (S13) through a 40-mesh sieve to obtain degreased medicine residues, and drying and storing for later use.
3. The method for extracting total saponins in clematis according to claim 2, wherein the ratio of the material to the liquid in step S12 to that in step S13 is 1: 5.
4. The method for extracting the total saponins in clematis according to claim 2, wherein the water bath heating temperature of the water bath heating reflux and the water bath heating reflux of the second time is 80 ℃, and the heating reflux time is 2 hours.
5. The method for extracting total saponins in clematis according to claim 1, wherein the step S2 comprises the specific steps of,
s21: adding 70% ethanol into the defatted residue, and heating and refluxing for the first time to obtain a first extractive solution; the feed-liquid ratio of the degreased medicine residues to 70% ethanol is 1: 45-1: 55;
s22: carrying out primary ultrasonic extraction on the primary extracting solution to obtain primary ultrasonic filtrate and primary ultrasonic filter residue;
s23: adding ethanol with the concentration of 70% into the primary ultrasonic filter residue to carry out secondary heating reflux and secondary ultrasonic extraction to obtain secondary ultrasonic filtrate and secondary ultrasonic filter residue; the material-liquid ratio of the primary ultrasonic filter residue to 70% ethanol is 1: 45-1: 55;
s24: mixing the primary ultrasonic filtrate and the secondary ultrasonic filtrate, centrifuging, and taking supernatant to obtain total saponin extract.
6. The method for extracting total saponins in clematis according to claim 5, wherein the material-to-liquid ratio of the defatted residues to 70% ethanol in step S21 is 1:50, and the material-to-liquid ratio of the primary ultrasonic residues to 70% ethanol in step S23 is also 1: 50.
7. The method for extracting total saponins in clematis according to claim 5, wherein the temperature of the first heating reflux and the second heating reflux is 65 ℃ and the reflux time is 2 h.
8. The method for extracting total saponins in clematis according to claim 5, wherein the ultrasonic temperature of the primary ultrasonic extraction and the ultrasonic temperature of the secondary ultrasonic extraction are 65 ℃ and the ultrasonic extraction time is 50 min.
9. The method for extracting total saponins in clematis according to claim 1, wherein the specific operation of step S3 is,
s31: taking 1mg of oleanolic acid standard product, and preparing 1mg/ml mother liquor by using absolute ethyl alcohol;
s32: precisely transferring 0.1 mL, 0.2 mL, 0.3 mL, 0.4mL, 0.5 mL, 0.6 mL and 0.7mL of mother liquor respectively to prepare solutions of 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL and 0.7mg/mL, adding into seven dry test tubes with plugs, and volatilizing the solvent in a water bath;
s33: respectively adding 0.4mL of 5% vanillin-glacial acetic acid solution and 1.6mL of perchloric acid into seven test tubes, and heating in a constant-temperature water bath at 70 ℃ for 20 min;
s34: cooling the test tube heated in the water bath for 3min by running water, adding 5mL of glacial acetic acid, shaking up, taking absolute ethyl alcohol as a blank control, and measuring the absorbance at 542 nm;
s35: drawing a standard curve by taking the concentration of oleanolic acid as a horizontal coordinate and the absorbance as a vertical coordinate to obtain a regression equation;
s36: preparing a total saponin extracting solution, measuring an absorbance value at the wavelength of 542nm, and obtaining the diluted saponin filtrate concentration C in the saponin extracting solution by using the regression equation in the step S35;
s37: using the formula of the extraction rate of the total saponins
Calculating the extraction rate of the total saponins in clematis schreberi in Shaanxi;
wherein, W: the extraction rate (%) of the general saponins of clematis in Shaanxi;
c: diluting the saponin filtrate to obtain a concentration;
v: volume of filtrate/mL;
n: dilution times;
m: shaanxi clematis powder mass/g.
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