CN115177663A - Preparation method and application of Potentilla chinensis extract - Google Patents
Preparation method and application of Potentilla chinensis extract Download PDFInfo
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- CN115177663A CN115177663A CN202210965008.6A CN202210965008A CN115177663A CN 115177663 A CN115177663 A CN 115177663A CN 202210965008 A CN202210965008 A CN 202210965008A CN 115177663 A CN115177663 A CN 115177663A
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- potentilla
- eutectic solvent
- extract
- deep eutectic
- natural
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Abstract
The invention belongs to the field of natural plant component extraction and application, and particularly discloses a preparation method and application of a Potentilla nandii extract, which provides a natural deep eutectic solvent composed of natural alkaloid, sugar and natural organic acid, and extracts an active substance of Potentilla nandii by adopting a three-order ultrasonic assisted extraction method, so that the obtained Potentilla nandii extract is applied to preparation of a dipeptidyl peptidase-4 inhibitor drug/health product. The natural deep eutectic solvent adopted by the invention is completely green and nontoxic, the component proportion is designed in a targeted manner according to the physicochemical properties of the extract, the natural deep eutectic solvent has the characteristics of good stability and high extraction rate, and the natural deep eutectic solvent can provide important reference value for promoting the research on the hypoglycemic activity of the Potentilla chinensis and accelerating the reasonable utilization of the plant resources in Qinghai province.
Description
Technical Field
The invention belongs to the field of natural plant component extraction and application, and particularly discloses a preparation method and application of a Potentilla chinensis extract.
Background
Potentilla (Potentilla) is a Rosaceae (Rosaceae) plant, and has a long medicinal history. Potentilla chinensis (Potentilla bifurca var. Humileor R.) is a variation of Potentilla chinensis, and is mainly distributed in temperate zone of northern hemisphere and high mountain area. The plant is spread in short and small way, the period of flower and fruit is 5-10 months, the length of flower stem is less than 7cm, the number of leaves is usually 3-5, and the whole edge or a few top ends have two cracks. The Potentilla chinensis bunge mainly contains various active ingredients such as flavonoids, triterpenes, polysaccharides, tannins and the like, and the whole plant extract of the Potentilla chinensis bunge can be used for resisting diarrhea, tumors, viruses and hyperglycemia, and has good effects on clinically treating diseases such as hemiplegia, diabetes and the like in recent years.
Dipeptidyl peptidase-4 (DPP-4), also known as the T-cell surface antigen CD26, is a widely expressed class of transmembrane glycoproteins. Glucagon-like peptide-1 (glp-1) is an important peptide hormone, which is secreted by intestinal L cells to regulate blood glucose in vivo. The DPP-4 enzyme can decompose GLP-1 in vivo, and has a remarkable effect of promoting the increase of blood sugar level in vivo. In recent years, DPP-4 enzyme inhibitors have been used as a class of hypoglycemic drugs for treating diabetes. DPP-4 inhibitors (DPP 4 Is) can enhance GLP-1 and GIP (gastrointestinal inhibitory peptide) effects by inhibiting DPP-4 enzyme activity, thereby inhibiting glucagon production and promoting glucose metabolism and insulin secretion to realize the effect of reducing blood sugar.
In the aspect of extraction solvents, chloroform, ether and other traditional organic solvents are most widely applied, the traditional organic solvent extraction method has the defects of low efficiency, high energy consumption, high pollution, long preparation period and serious resource waste, the requirements on health and environmental protection are more and more strict when food and medicine are used for plant extraction in the application field, and the traditional organic solvents such as chloroform have higher toxicity, so that the development of low-toxicity or nontoxic green extraction solvents is more important.
Compared with the traditional solvent, the eutectic solvent is a novel green solvent, has the incomparable advantages of low cost, easy preparation, easy storage, recoverability, safety, no toxicity, biodegradability, environmental friendliness and other organic solvents, has excellent physicochemical properties, and has great potential in the aspects of extraction and separation of natural plants.
Although the toxicity of the eutectic solvent is low, research shows that the common eutectic solvent still has certain low toxicity, but for the field of medicine and food, the complete non-toxicity of the extraction solvent is a continuous pursuit direction. Meanwhile, the eutectic solvent has designability, and specific component proportion design is required to be carried out on different extraction objects according to the properties of the extract, so that the eutectic solvent is not simple and universal. At present, an ultrasonic-assisted extraction method is generally adopted for extraction of the eutectic solvent, the bonding between the eutectic solvents belongs to intermolecular hydrogen bonding, the bonding between the eutectic solvents is damaged by overhigh ultrasonic power and time in the general ultrasonic-assisted extraction method, the stability of the eutectic solvent is influenced, the ultrasonic extraction power is reduced, and the ultrasonic time is reduced, so that the extraction rate is obviously influenced.
Disclosure of Invention
Based on the method, the invention provides a natural deep eutectic solvent consisting of natural alkaloid, sugar and natural organic acid for the potentilla anserine, and the active substances of the potentilla anserine are extracted by a three-order ultrasonic assisted extraction method.
The invention provides a preparation method of a Potentilla chinensis extract, which is prepared by extracting a natural deep eutectic solvent by adopting a three-order ultrasonic assisted extraction method.
Preferably, the preparation method of the Potentilla chinensis bunge extract comprises the following steps of preparing a natural deep eutectic solvent from anhydrous betaine, sucrose and proline, wherein the molar ratio of the three components is 1-3: 1 to 2:1 to 3.
The anhydrous betaine, the sucrose and the proline are natural substances existing in nature, are all green and non-toxic, and the eutectic solvent prepared by adopting the three substances belongs to a natural green eutectic solvent and is also completely green and non-toxic.
The reasonable molar ratio among the components of the eutectic solvent is the key of the excellent physicochemical property and stability of the eutectic solvent, and meanwhile, the molar ratio is designed according to the difference of the physicochemical properties of the extraction objects, so that the eutectic solvent prepared by adopting the molar ratio has good structural stability and is not easy to generate the phenomena of structural instability such as crystallization and the like; meanwhile, when the molar ratio of the invention is adopted, the polarity of the solution is the closest to that of the active ingredients to be extracted in the potentilla bifida, so that the solubility of the active ingredients can be effectively improved.
Preferably, in the preparation method of the potentilla bifida extract, the preparation steps of the natural deep eutectic solvent comprise:
(1) Accurately weighing anhydrous betaine, sucrose and proline according to a molar ratio, mixing, adding deionized water, and stirring until the anhydrous betaine, the sucrose and the proline are completely dissolved to obtain a solution A;
(2) Placing the solution A in a water bath environment at 40-60 ℃, stirring for 6-8 h at the rotating speed of 100 r/min, and then carrying out ultrasonic oscillation on the solution A for 1-3 h by using ultrasonic with the power of 50-100W to obtain a solution B;
(3) And (3) evaporating the solution B obtained in the step (2) to remove excessive water by adopting a reduced pressure rotary evaporator to obtain a transparent viscous liquid, namely the natural deep eutectic solvent.
By adopting water bath selection stirring and ultrasonic oscillation treatment, the bonding effect between eutectic solvent members can be effectively improved, and the stability and the preparation efficiency of the eutectic solvent are improved.
Preferably, the preparation method of the potentilla anserine extract comprises the following steps:
(1) Taking the whole grass of the dwarf cinquefoil, drying, crushing and sieving the grass of the dwarf cinquefoil with the sieve hole number of 60-80 meshes to obtain the dwarf cinquefoil powder;
(2) Calculating and weighing the mass of the required Potentilla anserine powder and the volume of the natural deep eutectic solvent and water according to a certain liquid-material ratio and the volume ratio of the natural deep eutectic solvent to water;
(3) Mixing the weighed Potentilla chinensis powder with water, setting ultrasonic power and time, performing first-order ultrasonic treatment, then setting second-order ultrasonic power and time, slowly adding a natural deep eutectic solvent, performing second-order ultrasonic treatment, setting third-order ultrasonic power and time, and performing third-order ultrasonic treatment to obtain a solution C;
(4) And (4) centrifuging the solution C obtained in the step (3) at a certain rotating speed, taking supernatant, filtering the supernatant, concentrating and drying, and separating and recovering a natural deep eutectic solvent from the supernatant to obtain a finished product of the Potentilla chinensis extract.
Preferably, the preparation method of the potentilla anserine extract comprises the following steps of (2) preparing a raw material from 20 to 40:1 (ml/g), the volume ratio of the natural deep eutectic solvent to water is that of the natural deep eutectic solvent: water =1 to 9:1.
the viscosity of the eutectic solvent can obviously influence the mass transfer process of effective substances, a proper amount of water content is beneficial to reducing the viscosity of a solution system and increasing the mass transfer process of active ingredients, the water content is too low, the viscosity coefficient of the solution is large, the mass transfer speed is slow, and the water content is too high, so that the effective concentration of the solution is influenced, and the dissolution of the active ingredients is also not facilitated. Meanwhile, different component systems and different molar ratios are adopted, the self obvious viscosity coefficients of the eutectic solvent are different, the water content is matched with the component types and the molar ratios of the selected eutectic solvent, and the extraction capacity of the prepared eutectic solvent can be obviously improved.
Preferably, in the preparation method of the Potentilla chinensis extract, in the step (3), the first-order ultrasonic power is 400-500W, the first-order ultrasonic time is 20-30 min, the second-order ultrasonic power is 50-100W, the second-order ultrasonic time is 10-15 min, the third-order ultrasonic power is 150-350W, and the third-order ultrasonic time is 40-60 min
The three-order ultrasonic treatment is designed according to different system objects, the first-order ultrasonic treatment is directed to the original water solution of the material powder, and the first-order high-power ultrasonic treatment can quickly cause the damage and the rupture of plant cells through high-energy ultrasonic waves so as to be beneficial to the dissolution of effective substances in the cells; the second-order ultrasound aims at the addition and dispersion of the natural deep eutectic solvent, the second-order low-power short-time ultrasonic treatment can accelerate the dispersion effect of the natural deep eutectic solvent, the viscosity coefficient of an extraction system is rapidly reduced after the eutectic solvent is added, and the diffusion efficiency of the eutectic extraction solvent is improved; the three-order ultrasound is a natural deep eutectic solution of material powder subjected to certain damage, and the three-order ultrasound with proper power can continuously accelerate the damage of plant cells under the combined action of ultrasonic energy and the eutectic solvent, effectively promote the effective dissolution of active substances in the extraction solvent and cannot damage the bonding effect among the components of the eutectic solvent.
Preferably, the preparation method of the Potentilla chinensis extract comprises the step (4), wherein the centrifugal rotating speed is 8000-10000 r/min, the centrifugal time is 15-20 min, and a microporous membrane with the diameter of 0.5-1 mu m is used for filtering.
The high-speed centrifugation can quickly promote the separation of the extracting solution and the extracting residues in the extracting solution, and the purity of the extracting solvent can be ensured by adopting the filtration of a microporous membrane.
Preferably, the Potentilla anserine extract contains
1) Quercetin-3-O-beta-D-glucuronic acid pyranoside (Quercetin-3-O-beta-D-glucopyranoside) is abbreviated as QC3G,
2) Myricetin (Myricetin) is abbreviated as MC,
3) Quercetin-3-O-beta-D-xylopyranoside (Quercetin-3-O-beta-D-xylose) is abbreviated as QC3X,
4) Quercetin-3-O- (6 '-O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (Quercetin-3-O- (6' -O-trans-p-coumaroyl) -beta-D-glucoside) is abbreviated as QC3GP, beta-D-glucoside,
5) Quercetin (Quercetin) is abbreviated as QC,
6) (-) -epicatechin ((-) -epicatechin) abbreviated EC, and EC,
7) 1-O-coumaroyl-beta-D-glucose (1-O-coumaroyl-beta-D-glucose) is abbreviated CG.
The invention also provides application of the potentilla bifida extract in preparation of a medicament/health-care product of the dipeptidyl peptidase-4 inhibitor.
Preferably, the Potentilla anserine extract is an enzyme competitive inhibitor.
The activity of the Potentilla chinensis Franch extract is further researched by establishing a DPP-4 enzyme reaction system, and the result shows that MC, QC3X, QC3G and QC3GP show obvious inhibitory activity to DPP-4 enzyme. On the basis, 5 active compounds are subjected to an inhibitor type determination experiment, and the result shows that MC, QC3X, QC3G and QC3GP belong to competitive inhibitors.
Compared with the prior art, the invention has the following beneficial effects:
(1) The natural deep eutectic solvent prepared from natural alkaloid, cane sugar and natural organic acid is completely green and nontoxic, the proportion of components is designed according to the physicochemical properties of the extract of the Potentilla chinensis, and the preparation method has the characteristics of strong pertinence, good stability and high extraction rate.
(2) According to different extraction system objects, a three-order ultrasonic auxiliary extraction method is designed, and the extraction rate of the deep eutectic solvent is obviously improved.
(3) The invention discloses application of a Potentilla chinensis extract in preparation of a medicament/health-care product of a dipeptidyl peptidase-4 inhibitor for the first time.
(4) The Potentilla anserine extract has a remarkable inhibiting effect on DPP-4 enzyme. By combining the characteristics of wide distribution and less research of the potentilla anserine in Qinghai-Tibet plateau regions, the method can provide important data reference value for promoting the research of the hypoglycemic activity of the potentilla anserine and accelerating the reasonable utilization of plant resources in Qinghai province.
Drawings
FIG. 1 is a DPP-4 enzyme labeling standard curve;
FIG. 2 is a DPP-4 enzyme inhibition rate curve of 7 extracts and positive drugs of Potentilla chinensis Franch;
FIG. 3 is a diagram of reciprocal double plots of myricetin;
FIG. 4is a graph of reciprocal double-reciprocal Quercetin-3-O- β -D-xylose;
FIG. 5 is a graph of the reciprocal double plots of quercetin;
FIG. 6 is a graph of reciprocal double plots of quercetin-3-O- β -D-glucuronide;
FIG. 7 is a graph of reciprocal double plots of quercetin-3-O- (6' -O-trans-p-coumaroyl) - β -D-glucoside.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The reagents or instruments used in the examples of the present invention are not indicated by manufacturers, and are conventional reagents that are commercially available.
The following are abbreviated descriptions of compounds:
1) Quercetin-3-O-beta-D-glucuronic acid pyranoside (Quercetin-3-O-beta-D-glucopyranoside) is abbreviated as QC3G,
2) Myricetin (Myricetin) is abbreviated as MC,
3) Quercetin-3-O-beta-D-xylopyranoside (Quercetin-3-O-beta-D-xylose) is abbreviated as QC3X,
4) Quercetin-3-O- (6 '-O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (Quercetin-3-O- (6' -O-trans-p-coumaroyl) -beta-D-glucoside) abbreviated as QC3GP, beta-D-glucoside and D-glucoside,
5) Quercetin (Quercetin) is abbreviated as QC,
6) (-) -epicatechin ((-) -epicatechin) abbreviated EC, and EC,
7) 1-O-coumaroyl- β -D-glucose (1-O-coumaroyl- β -D-glucose) is abbreviated CG;
example 1:
according to the weight ratio of anhydrous betaine: sucrose: the molar ratio of proline is 1:1: accurately weighing the mass of the anhydrous betaine, the sucrose and the proline, mixing the three components, adding the ionized water, stirring until the three components are completely dissolved, placing the obtained solution in a water bath environment at 40 ℃, stirring at a rotating speed of 100 r/min for 8 hours, ultrasonically oscillating the solution for 3 hours by using ultrasonic waves with a power of 60W, evaporating redundant water of the obtained solution by using a reduced-pressure rotary evaporator to obtain a transparent viscous liquid, namely the natural deep eutectic solvent.
Taking the whole herb of Potentilla chinensis, drying, pulverizing, and sieving with 60 mesh sieve to obtain Potentilla chinensis powder; according to the following steps of 20:1 (ml/g), and the volume ratio of the natural deep eutectic solvent to water is 4:1, calculating and weighing the mass of the required Potentilla chinensis powder and the volume of a natural deep eutectic solvent and water; mixing weighed Potentilla chinensis powder with water, setting the ultrasonic power to 400W and the ultrasonic time to 30min, performing first-order ultrasonic treatment, then setting the second-order ultrasonic power to 50W and the ultrasonic time to 15min, slowly adding a natural deep eutectic solvent, performing second-order ultrasonic treatment, then setting the third-order ultrasonic power to 150W and the ultrasonic time to 60min, performing third-order ultrasonic treatment, centrifuging the solution after ultrasonic treatment at the rotating speed of 8000 rpm for 20min, taking supernatant, filtering the supernatant with a 0.5 mu m microporous membrane, concentrating, drying, separating and recovering the natural deep eutectic solvent from the supernatant, and thus obtaining the Potentilla chinensis extract finished product.
Taking a certain amount of Potentilla chinensis Franch extract, preparing into 60% ethanol solution with ethanol, measuring the light absorption value of the solution at 510nm with an ultraviolet spectrophotometer, comparing with rutin standard curve, calculating the total flavone content of Potentilla chinensis Franch extract, and calculating to obtain the total flavone extraction rate of Potentilla chinensis Franch extract of 47.5mg/g.
Example 2:
according to the weight ratio of anhydrous betaine: sucrose: the molar ratio of proline is 2:1: accurately weighing the mass of the anhydrous betaine, the sucrose and the proline, mixing the three components, adding the ionized water, stirring until the three components are completely dissolved, placing the obtained solution in a water bath environment at 50 ℃, stirring at a rotating speed of 100 r/min for 7h, ultrasonically oscillating the solution for 2h by using ultrasound with a power of 80W, evaporating redundant water of the obtained solution by using a reduced-pressure rotary evaporator to obtain a transparent viscous liquid, namely the natural deep eutectic solvent.
Taking the whole herb of Potentilla chinensis, drying, pulverizing, and sieving with 70 mesh sieve to obtain Potentilla chinensis powder; according to the weight ratio of 30:1 (ml/g), and the volume ratio of the natural deep eutectic solvent to water is 2.3:1, calculating and weighing the mass of the required Potentilla chinensis powder and the volume of a natural deep eutectic solvent and water; mixing weighed Potentilla chinensis powder with water, setting the ultrasonic power to be 450W and the ultrasonic time to be 25min, performing first-order ultrasonic treatment, then setting the second-order ultrasonic power to be 75W and the ultrasonic time to be 12min, slowly adding a natural deep eutectic solvent, performing second-order ultrasonic treatment, then setting the third-order ultrasonic power to be 250W and the ultrasonic time to be 40min, performing third-order ultrasonic treatment, centrifuging the solution after ultrasonic treatment at a rotating speed of 9000 r/min for 18min, taking supernatant, filtering the supernatant with a 0.8 mu m microporous membrane, concentrating and drying, and separating and recovering the natural deep eutectic solvent from the supernatant to obtain the Potentilla chinensis extract finished product.
Taking a certain amount of Potentilla chinensis extract, preparing 60% ethanol solution with ethanol, measuring the light absorption value of the solution at 510nm by using an ultraviolet spectrophotometer, comparing with a rutin standard curve, calculating the total flavone content of the Potentilla chinensis extract, and calculating to obtain the total flavone extraction rate of the Potentilla chinensis extract of 49.3mg/g.
Example 3:
according to the weight ratio of anhydrous betaine: sucrose: the molar ratio of proline is 3:1:2, accurately weighing the mass of the anhydrous betaine, the sucrose and the proline, mixing the three components, adding the ionized water, stirring until the components are completely dissolved, placing the obtained solution in a water bath environment at 60 ℃, stirring for 6 hours at a rotating speed of 100 r/min, ultrasonically oscillating the solution for 1 hour by using 100W power ultrasound, evaporating redundant water of the obtained solution by using a reduced pressure rotary evaporator to obtain a transparent viscous liquid, namely the natural deep eutectic solvent.
Taking the whole herb of Potentilla chinensis, drying, pulverizing, and sieving with 80 mesh sieve to obtain Potentilla chinensis powder; according to the following steps of 40:1 (ml/g), and the volume ratio of the natural deep eutectic solvent to water is 1.5:1, calculating and weighing the mass of the required Potentilla chinensis powder and the volume of a natural deep eutectic solvent and water; mixing weighed Potentilla chinensis bunge powder with water, setting the ultrasonic power to be 500W and the ultrasonic time to be 20min, carrying out first-order ultrasonic treatment, then setting the second-order ultrasonic power to be 100W and the ultrasonic time to be 10min, slowly adding a natural deep eutectic solvent, carrying out second-order ultrasonic treatment, setting the third-order ultrasonic power to be 350W and the ultrasonic time to be 40min, carrying out third-order ultrasonic treatment, carrying out centrifugal treatment on the solution subjected to ultrasonic treatment at the rotating speed of 10000 r/min for 15min, taking supernatant, filtering the supernatant by using a microporous membrane of 1 mu m, concentrating and drying, and separating and recovering the natural deep eutectic solvent from the supernatant to obtain the Potentilla chinensis bunge extract finished product.
Taking a certain amount of Potentilla chinensis extract, preparing 60% ethanol solution with ethanol, measuring the light absorption value of the solution at 510nm by using an ultraviolet spectrophotometer, comparing with a rutin standard curve, calculating the total flavone content of the Potentilla chinensis extract, and calculating to obtain the total flavone extraction rate of the Potentilla chinensis extract, wherein the total flavone extraction rate of the Potentilla chinensis extract is 48.6mg/g.
Comparative example 1:
taking the whole grass of the Potentilla chinensis Franch, drying, crushing and sieving to obtain Potentilla chinensis Franch powder with the sieve hole number of 70 meshes; according to the weight ratio of 30:1 (ml/g), and the volume ratio of absolute ethyl alcohol to water is 2.3:1, calculating and weighing the mass of the required Potentilla chinensis powder and the volume of absolute ethyl alcohol and water; mixing weighed Potentilla chinensis bunge powder with water, setting the ultrasonic power to be 450W and the ultrasonic time to be 25min, carrying out first-order ultrasonic treatment, then setting the second-order ultrasonic power to be 75W and the ultrasonic time to be 12min, slowly adding a natural deep eutectic solvent, carrying out second-order ultrasonic treatment, setting the third-order ultrasonic power to be 250W and the ultrasonic time to be 40min, carrying out third-order ultrasonic treatment, carrying out centrifugal treatment on the solution subjected to ultrasonic treatment at the rotating speed of 9000 r/min for 18min, taking supernatant, filtering the supernatant by using a 0.8 mu m microporous membrane, concentrating and drying, and separating ethanol from the supernatant to obtain the Potentilla chinensis bunge extract finished product.
Taking a certain amount of Potentilla chinensis extract, preparing 60% ethanol solution with ethanol, measuring the light absorption value of the solution at 510nm by using an ultraviolet spectrophotometer, comparing with a rutin standard curve, calculating the total flavone content of the Potentilla chinensis extract, and calculating to obtain the total flavone extraction rate of the Potentilla chinensis extract, wherein the total flavone extraction rate of the Potentilla chinensis extract is 32.4mg/g.
Comparative example 2:
according to the weight ratio of choline chloride: the molar ratio of ethylene glycol is 1:2, accurately weighing the mass of choline chloride and ethylene glycol, mixing the two components, adding ionized water, stirring until the two components are completely dissolved, placing the obtained solution in a water bath environment at 50 ℃, stirring for 7 hours at a rotating speed of 100 revolutions per minute, carrying out ultrasonic oscillation on the solution for 2 hours by using ultrasonic waves with 80W power, evaporating redundant water of the obtained solution by using a reduced-pressure rotary evaporator to obtain a transparent viscous liquid, namely the deep eutectic solvent.
Taking the whole herb of Potentilla chinensis, drying, pulverizing, and sieving with 70 mesh sieve to obtain Potentilla chinensis powder; according to the proportion of 30:1 (ml/g), the volume ratio of the deep eutectic solvent to water is 2.3:1, calculating and weighing the mass of the required Potentilla chinensis powder and the volume of a deep eutectic solvent and water; mixing weighed Potentilla chinensis powder with water, setting the ultrasonic power to be 450W and the ultrasonic time to be 25min, performing first-order ultrasonic treatment, then setting the second-order ultrasonic power to be 75W and the ultrasonic time to be 12min, slowly adding a deep eutectic solvent, performing second-order ultrasonic treatment, then setting the third-order ultrasonic power to be 250W and the ultrasonic time to be 40min, performing third-order ultrasonic treatment, centrifuging the solution after ultrasonic treatment at a rotating speed of 9000 r/min for 18min, taking supernatant, filtering the supernatant with a 0.8 mu m microporous membrane, concentrating and drying, and separating and recovering the deep eutectic solvent from the supernatant to obtain the Potentilla chinensis extract finished product.
Taking a certain amount of Potentilla chinensis extract, preparing 60% ethanol solution with ethanol, measuring the light absorption value of the solution at 510nm by using an ultraviolet spectrophotometer, comparing with a rutin standard curve, calculating the total flavone content of the Potentilla chinensis extract, and calculating to obtain the total flavone extraction rate of the Potentilla chinensis extract of 42.1mg/g.
Comparative example 3:
according to the weight ratio of anhydrous betaine: sucrose: the molar ratio of proline is 2:1: accurately weighing the mass of the anhydrous betaine, the sucrose and the proline, mixing the three components, adding the ionized water, stirring until the three components are completely dissolved, placing the obtained solution in a water bath environment at 50 ℃, stirring at a rotating speed of 100 r/min for 7h, ultrasonically oscillating the solution for 2h by using ultrasound with a power of 80W, evaporating redundant water of the obtained solution by using a reduced-pressure rotary evaporator to obtain a transparent viscous liquid, namely the natural deep eutectic solvent.
Taking the whole herb of Potentilla chinensis, drying, pulverizing, and sieving with 70 mesh sieve to obtain Potentilla chinensis powder; according to the weight ratio of 30:1 (ml/g), and the volume ratio of the natural deep eutectic solvent to water is 2.3:1, calculating and weighing the mass of the required Potentilla chinensis powder and the volume of a natural deep eutectic solvent and water; diluting a natural deep eutectic solvent into an aqueous solution by using water, mixing weighed potentilla nandii powder with the natural deep eutectic solvent aqueous solution, setting the ultrasonic power to be 250W and the ultrasonic time to be 60min, carrying out ultrasonic treatment, centrifuging the solution after the ultrasonic treatment for 18min at the rotating speed of 9000 r/min, taking supernatant, filtering the supernatant by using a microporous membrane of 0.8 mu m, concentrating and drying, and separating and recovering the natural deep eutectic solvent from the supernatant to obtain the finished product of the potentilla nandii extract.
Taking a certain amount of Potentilla chinensis extract, preparing 60% ethanol solution with ethanol, measuring the light absorption value of the solution at 510nm by using an ultraviolet spectrophotometer, comparing with a rutin standard curve, calculating the total flavone content of the Potentilla chinensis extract, and calculating to obtain the total flavone extraction rate of the Potentilla chinensis extract of 42.9mg/g.
Example 4
Preparation of the Compounds
The following compounds were prepared by referring to the isolation method of Potentilla sessiliflorae described in the applicant's previously published patent (CN 110256512A, an alpha-glucosidase inhibitor extracted from Potentilla sessiliflorae) or the research paper of the subject group (research on chemical composition of Potentilla sessiliflorae. Chapter chemical composition research on chemical composition of Potentilla sessiliflorae. Chapter. Paper):
1) Quercetin-3-O-beta-D-glucuronic acid pyranoside (Quercetin-3-O-beta-D-glucopyranoside) is abbreviated as QC3G,
2) Myricetin (Myricitin) is abbreviated as MC, myricetin,
3) Quercetin-3-O-beta-D-xylopyranoside (Quercetin-3-O-beta-D-xylose) is abbreviated as QC3X,
4) Quercetin-3-O- (6 '-O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (Quercetin-3-O- (6' -O-trans-p-coumaroyl) -beta-D-glucoside) is abbreviated as QC3GP, beta-D-glucoside,
5) Quercetin (Quercetin) is abbreviated as QC,
6) (-) -epicatechin ((-) -epicatechin) abbreviated EC, and EC,
7) 1-O-coumaroyl-beta-D-glucose (1-O-coumaroyl-beta-D-glucose) is abbreviated CG.
Example 5:
determination of DPP-4 enzyme inhibitory Activity of Potentilla anserine extract
Reagents and materials:
instruments and equipment:
1. preparation of related Agents
1.1 preparation of Tris-HCl buffer:
the buffer solution of the reaction system is selected from Tris-HCl mixed solution with the pH value of 7.8 and the concentration of 100mM, and the specific operation steps are as follows:
(1) Weighing 6.05g of Tris (hydroxymethyl) aminomethane (Tris) by using an electronic balance;
(2) Putting the weighed Tris into a beaker, adding ultrapure water into the beaker, stirring the mixture by using a glass rod until the ultrapure water is completely dissolved, and then adding hydrochloric acid to adjust the pH value to about 7.8;
(3) Pouring the dissolved liquid into a 500mL volumetric flask, fixing the volume, and shaking up to obtain 100mM Tris-HCl buffer solution with the pH value of 7.8.
1.2 preparation of related solution of an enzyme testing system:
preparing a DPP-4 enzyme solution: DPP-4 (human-derived, 1U/visual) was dissolved in 1mL of a prepared buffer (pH: 7.8C: 100mM) to prepare 1U. ML -1 The enzyme solution of (a) was stored in EP tubes in an ultra-low temperature refrigerator at-80 ℃ for future use. When in use, 1 U.mL is added according to the need -1 The enzyme solution is diluted to the desired concentration.
Preparing an enzyme substrate preservation solution: 5mg of glycylproline p-nitroaniline powder was weighed and dissolved completely in 760. Mu.L of the buffer solution to obtain an enzyme reaction substrate having a concentration of 20 mM. The substrate solution should be protected from light during the preparation process and storage.
Preparation of enzyme substrate reaction solution: the enzyme substrate preservation solution with the concentration of 20mM is added with buffer solutions with different amounts to be diluted into four concentration gradients of 1mM, 2mM, 3 mM and 4mM according to requirements.
Preparation of positive reaction solution: 1.2mg of sitagliptin was weighed and added with 237. Mu.L of DMSO to dissolve sufficiently, thereby obtaining a positive control solution with a concentration of 10 mM. Stored in EP tubes at ambient temperature. When in use, the DMSO solution is added to dilute to 1, 0.5, 0.25, 0.125, 0.0625, 0.0313 and 0.0156mM according to needs.
Preparing a sample solution: weighing a certain amount of QC, MC, EC, CG, QC3X, QC3G and QC3GP, dissolving in DMSO to prepare 2mM extract solution, and storing in an EP tube at normal temperature. When in use, the DMSO solution is added according to the needs to be diluted to 1, 0.5, 0.25, 0.125, 0.0625, 0.0313 and 0.0156mM.
2. Establishment of enzyme Activity reaction System
The reaction was carried out in a non-detachable 96 well plate and enzyme inhibition was measured using a fully automatic enzyme scalePreparing the activity. The reaction system is selected to have a concentration of 0.02 U.mL -1 Is established on the basis of the DPP-4 enzyme and substrate at a concentration of 2 mM. Because the added reagent such as medicine has influence on the absorbance, in order to eliminate the interference of other objective factors such as color, a blank control group and a sample control group are additionally arranged besides the sample group and the enzyme control group. The specific grouping is as follows:
blank control group: 140. Mu.L of buffer, 10. Mu.L of dimethyl sulfoxide, 50. Mu.L of substrate (2 mM);
enzyme control group: buffer 90. Mu.L, dimethyl sulfoxide 10. Mu.L, DPP-4 enzyme (0.02 U.mL) -1 ) 50 μ L, substrate (2 mM) 50 μ L;
sample control group: 140. Mu.L of buffer, 10. Mu.L of sample at different concentrations, 50. Mu.L of substrate (2 mM);
sample group: buffer 90. Mu.L, samples of different concentrations 10. Mu.L, DPP-4 enzyme (0.02 U.mL) -1 ) 50 μ L, substrate (2 mM) 50 μ L.
After the constant temperature reaction in a 37 ℃ water bath kettle for 20min, the established DPP-4 enzyme reaction system is placed in a fluorescence multi-mode enzyme labeling instrument, and OD values of all groups are measured at 405 nm. Each sample was replicated in triplicate at the same concentration and averaged.
3. Drawing of Standard Curve
According to the method in the establishment of the enzyme activity reaction system, samples are not added, and the concentration gradients of the DPP-4 enzyme are 0,0.01,0.02,0.04,0.06,0.08 and 0.1U.mL respectively -1 The reaction is carried out. After the reaction is finished, determining the OD value of each group at 405nm in a fluorescent multi-mode enzyme-labeling instrument. In calculation, the average OD value of each group was subtracted by the blank group (DPP-4 enzyme concentration 0U. ML) -1 ) The average OD value serves as a correction value. And (4) drawing by taking the enzyme concentration as an abscissa and the corresponding OD value as an ordinate, and drawing a DPP-4 enzyme standard curve. The action curve of DPP-4 enzyme is shown in figure 1, the equation of the curve is Y =9.3932X +0.0103 2 =0.999.DPP-4 enzyme is in 0.1 U.mL -1 In the range, the OD value gradually increases with the increase of the enzyme concentration, and the OD valueThe values show a good linear relationship with enzyme concentration.
IC50 values of the extracts were determined from SPSS20.0, and the enzyme reaction system correlation curves were prepared using Origin Pro9.1 and Excel.
The fluorescence substrate method is a method which is most widely applied to DPP-4 enzyme experiments at present, and is favored by researchers due to the advantages of strong sensitivity and low time cost. In the application, a DPP-4 enzyme reaction system is measured by adopting a fluorogenic substrate method, namely, the DPP-4 catalyzes Gly-Pro-PNA to hydrolyze under the condition of pH 7.8, and the generated product has an absorption peak at the wavelength of 405 nm. If the Potentilla chinensis bunge extract has an inhibiting effect on DPP-4 enzyme, the catalytic rate of the Potentilla chinensis bunge extract is reduced, and the absorbance of a reaction system is reduced due to the reduction of the generation amount of p-nitroaniline. The activity of the DPP-4 enzyme is reflected by the OD value measured at 405nm by a fluorescent multi-mode microplate reader.
According to the determination, the DPP-4 enzyme inhibition activity of the MC, QC3X, QC3G and QC3GP groups is gradually enhanced along with the increase of the concentration of the extract in the concentration ranges of 0.031, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mM. Wherein, under the condition of maximum concentration, MC expresses the inhibition activity stronger than that of the positive drug group, and QC, QC3X, QC3G, QC3GP expresses the inhibition activity weaker than that of the positive drug group under the condition of maximum concentration. In addition, as the concentration of the extract increases, the EC and CG groups have weaker DPP-4 enzyme inhibition activity, as shown in figure 2.
The IC50 refers to the concentration of the drug when the enzyme inhibition activity reaches 50%, and the lower the IC50 value is, the stronger the DPP-4 enzyme inhibition activity of the drug is. Although different extracts of Potentilla nandii showed different inhibitory activities on DPP-4 enzyme, the IC50 values (0.52, 1.21, 65.90, 1.32, 41.20, 0.74, 0.38mM respectively) were higher for 7 extracts than for the sigma delta group (0.05 mM). The IC50 values of the EC group and the CG group are 65.90mM and 41.20mM respectively, and the DPP-4 enzyme inhibitory activity is lowest (Table 1).
TABLE 1 inhibitory Activity of extracts and sitagliptin on DPP-4 enzyme
Example 6:
determination of enzyme inhibitor type of Potentilla chinensis extract
The enzyme substrate stock solution (20 mM) prepared in example 4 was mixed in the following ratio of 1: 5. 1:6.7, 1: 10. 1:20 to give a gradient of 4, 3, 2, 1mM of enzyme substrate reaction solution, and reacting with 1, 0.5, 0.25mM of monomeric compound according to the method for establishing the enzyme activity reaction system in example 4 (90. Mu.L of buffer solution + 10. Mu.L of sample + 50. Mu.L of DPP-4 enzyme + 50. Mu.L of substrate). Adding enzyme, incubating in warm water for 5min, adding substrate, reading OD value in an enzyme-labeling instrument at 405nm, recording as OD1, placing in warm water, incubating for 20min, measuring OD value, recording as OD2, and performing three groups of parallel experiments to obtain average value. The reciprocal of the reaction rate is plotted on the ordinate and the reciprocal of the substrate concentration is plotted on the abscissa, to plot the reaction tendency of the compound. The reciprocal of the rate in the system is represented by the formula
When the enzyme reaction time was determined, Δ T was 20min. Performing analysis according to the kinetic characteristics of related enzymes, and determining the inhibitor type of the extract.
IC50 values of the compounds were determined from SPSS20.0, and enzyme reaction system correlation curves were prepared using Origin Pro9.1 and Excel.
Vmax and Km can be obtained from the 1/V and 1/S double reciprocal curves. The determination result of the activity of the enzyme inhibitor shows that MC, QC3X, QC3GP and QC3G in the Potentilla chinensis bunge extract show higher inhibition activity on DPP-4 enzyme, and extracts EC and CG have weaker inhibition activity on DPP-4 enzyme. Based on the above results, the types of MC, QC3X, QC3GP, and QC3G inhibitors were determined for the present study. As shown in FIGS. 3 to 7 and Table 2, since Vm of these 5 extracts was unchanged and Km increased with increasing concentration, MC, QC3X, QC3GP and QC3G were competitive inhibitors.
TABLE 2 enzyme inhibition type screening of the 5 extracts
And (4) conclusion: the DPP-4 enzyme is a serine protease distributed on the cell surface, and the over-activation thereof is an important factor in the development of diabetes. The DPP-4 inhibitor can achieve the effect of reducing blood sugar in vivo by inhibiting the activity of DPP-4 enzyme, increasing the sensitivity of insulin, promoting the secretion of insulin at meals and reducing the blood sugar level after meals. The above results show that the screening of DPP-4 inhibitors from plants and activity research are the trend of future development, and the research on DPP-4 inhibitors has become a main direction for treating DM diseases. In the network pharmacology research, DPP-4 is an important potential target of the Potentilla anserine extract.
The results of the examples show that CG compounds and EC compounds have poor inhibitory activity on DPP-4 enzyme, and MC, QC3X, QC3G and QC3GP compounds show obvious inhibitory activity on DPP-4 enzyme. On the basis, 5 active extracts are subjected to an inhibitor type determination experiment, and the result shows that the extracts MC, QC3X, QC3G and QC3GP all belong to competitive inhibitors.
The results show that the potentilla anserine extract has a remarkable inhibitory effect on DPP-4 enzyme. By combining the characteristics of wide geographical distribution and less research of the Potentilla chinensis Franch on the Qinghai-Tibet plateau, the application can promote the research of the hypoglycemic activity of the Potentilla chinensis Franch and has great potential in preparing medicaments/health care products of dipeptidyl peptidase-4 inhibitors.
The foregoing is only a preferred embodiment of the present invention. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the Potentilla chinensis extract is characterized in that the Potentilla chinensis extract is prepared by extracting a natural deep eutectic solvent by a three-order ultrasonic assisted extraction method.
2. The preparation method of Potentilla anserine extract according to claim 1, wherein the natural deep eutectic solvent comprises a ternary natural solvent of anhydrous betaine, sucrose and proline, and the molar ratio of the components of the ternary natural solvent is 1-3: 1 to 2:1 to 3.
3. The method for preparing Potentilla anserine extract according to claim 2, wherein the natural deep eutectic solvent is prepared by:
(1) Accurately weighing anhydrous betaine, sucrose and proline according to a molar ratio, mixing, adding deionized water, and stirring until the anhydrous betaine, the sucrose and the proline are completely dissolved to obtain a solution A;
(2) Placing the solution A in a water bath environment at 40-60 ℃, stirring for 6-8 h at the rotating speed of 100 r/min, and then carrying out ultrasonic oscillation on the solution A for 1-3 h by using ultrasonic with the power of 50-100W to obtain a solution B;
(3) And (3) evaporating the solution B obtained in the step (2) to remove excessive water by adopting a reduced pressure rotary evaporator to obtain a transparent viscous liquid, namely the natural deep eutectic solvent.
4. The method of preparing Potentilla anserina extract according to claim 1, wherein the method comprises the steps of:
(1) Taking the whole herb of Potentilla chinensis, drying, crushing and sieving to obtain Potentilla chinensis powder with the number of sieve holes of 60-80 meshes;
(2) Calculating and weighing the mass of the required Potentilla anserine powder and the volume of the natural deep eutectic solvent and water according to a certain liquid-material ratio and the volume ratio of the natural deep eutectic solvent to water;
(3) Mixing the weighed Potentilla chinensis powder with water, setting ultrasonic power and time, performing first-order ultrasonic treatment, then setting second-order ultrasonic power and time, slowly adding a natural deep eutectic solvent, performing second-order ultrasonic treatment, setting third-order ultrasonic power and time, and performing third-order ultrasonic treatment to obtain a solution C;
(4) And (4) centrifuging the solution C obtained in the step (3) at a certain rotating speed, taking supernatant, filtering the supernatant, concentrating and drying, and separating and recovering a natural deep eutectic solvent from the supernatant to obtain a finished product of the Potentilla chinensis extract.
5. The method for preparing Potentilla anserine extract according to claim 4, wherein in the step (2), the liquid-to-material ratio is 20-40: 1 (ml/g), the volume ratio of the natural deep eutectic solvent to water is that of the natural deep eutectic solvent: water =1 to 9:1.
6. the preparation method of Potentilla anserine extract according to claim 4, wherein in the step (3), the first-order ultrasonic power is 400-500W, the first-order ultrasonic time is 20-30 min, the second-order ultrasonic power is 50-100W, the second-order ultrasonic time is 10-15 min, the third-order ultrasonic power is 150-350W, and the third-order ultrasonic time is 40-60 min.
7. The method for preparing Potentilla anserine extract according to claim 4, wherein in the step (4), the centrifugal rotation speed is 8000-10000 r/min, the centrifugal time is 15-20 min, and a microporous membrane of 0.5-1 μm is used for filtration.
8. The method of claim 4, wherein the Potentilla nanseri extract of step (4) comprises quercetin-3-O- β -D-glucuronic acid pyranoside, myricetin, quercetin-3-O- β -D-xylopyranoside, quercetin-3-O- (6' -O-trans-p-hydroxycinnamoyl) - β -D-glucoside, quercetin, (-) -epicatechin, 1-O-coumaroyl- β -D-glucose.
9. Use of the extract of Potentilla anserina according to any one of claims 1-8 for the preparation of a medicament/nutraceutical for dipeptidyl peptidase-4 inhibitors.
10. Use according to claim 9, wherein the Potentilla nandii extract is an enzyme competitive inhibitor.
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|---|---|---|---|---|
| WO2018092042A2 (en) * | 2016-11-15 | 2018-05-24 | Landlab S.R.L. | Solvent and method for extracting active compound from plants and use of such a solvent |
| CN110256512A (en) * | 2019-07-09 | 2019-09-20 | 青海师范大学 | A kind of alpha-glucosidase restrainer extracted from short raw Potentilla bifurca |
| CN113549497A (en) * | 2021-07-19 | 2021-10-26 | 上海应用技术大学 | Method for extracting perilla leaf essential oil by using ultrasonic-assisted natural deep eutectic solution and application of perilla leaf essential oil |
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2022
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018092042A2 (en) * | 2016-11-15 | 2018-05-24 | Landlab S.R.L. | Solvent and method for extracting active compound from plants and use of such a solvent |
| CN110256512A (en) * | 2019-07-09 | 2019-09-20 | 青海师范大学 | A kind of alpha-glucosidase restrainer extracted from short raw Potentilla bifurca |
| CN113549497A (en) * | 2021-07-19 | 2021-10-26 | 上海应用技术大学 | Method for extracting perilla leaf essential oil by using ultrasonic-assisted natural deep eutectic solution and application of perilla leaf essential oil |
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