CN115919949A - Rehmannia functional factor extract and application thereof in preparation of anti-oxidation and/or hypoglycemic drugs - Google Patents
Rehmannia functional factor extract and application thereof in preparation of anti-oxidation and/or hypoglycemic drugs Download PDFInfo
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Abstract
The invention relates to a method for extracting a rehmannia root functional factor extract, which comprises the steps of soaking rehmannia root decoction piece powder in distilled water, heating, cooling, filtering, centrifuging, and concentrating supernate to be dry to obtain a rehmannia root crude extract; soaking rehmanniae radix decoction pieces powder in distilled water, heating, cooling, filtering, centrifuging, passing the supernatant through D101 macroporous adsorbent resin column, washing with distilled water, collecting liquid, concentrating to dry to obtain rehmanniae radix water eluate, washing with ethanol, collecting ethanol extract, and concentrating to dry to obtain rehmanniae radix ethanol eluate; the rehmanniae radix functional factor extract comprises one or more of rehmanniae radix crude extract, rehmanniae radix water eluate and rehmanniae radix alcohol eluate. The invention researches the antioxidant activity and the alpha-glucosidase inhibitory activity of the rehmannia root functional factor extract by adopting an in vitro experimental method, can be used for preparing antioxidant and/or hypoglycemic drugs, and provides a theoretical basis for the development of the rehmannia root hypoglycemic functional factor and the research of hypoglycemic mechanism.
Description
Technical Field
The invention belongs to the technical field of extraction and application of Chinese herbal medicine plants, and particularly relates to a rehmannia root functional factor extract and application thereof in preparation of an antioxidant and/or hypoglycemic medicament.
Background
Rehmannia glutinosa (Latin scientific name: rehmannia glutinosa (Gaetn.) Libosch. Ex Fisch. Et Mey.) is a perennial herb of Scrophulariaceae, rehmannia, with a height of up to 30 cm, fleshy rhizome, fresh yellow, and purplish red stem under cultivation conditions. The diameter can reach 5.5 cm, the leaves are oval to oblong, the veins are sunken on the upper surface, flowers are slightly arranged into a general inflorescence at the top of the stem, the flower crown is purplish red outside, the interior is Huang Zise, the medicine chamber is rectangular and circular, the capsule is oval to oblong, and the flower and fruit period is 4-7 months.
Rehmannia root, radix rehmanniae is sweet, bitter and cold in nature, and has the functions of clearing heat, cooling blood, nourishing yin and promoting the production of body fluid. The root of underground root tuber is yellow-white, so the root of the root tuber is one of the traditional Chinese medicines, and the earliest classics is recorded in Shen nong Ben Cao Jing. The method comprises the following steps: fresh rehmannia root, dry rehmannia root and prepared rehmannia root have great difference in property and efficacy, and are classified according to the efficacy of the Chinese materia medica: fresh rehmannia root is used as a heat-clearing and blood-cooling medicine; shu Di Huang is tonifying drug.
The rehmannia root mainly contains glycosides, oligosaccharides, iridoids, phenethyl alcohol, amino acids, organic acids and the like, has the functions of resisting aging and tumors, and also has the functions of regulating the human body immunity and treating various diseases. In recent years, researchers have made extensive studies on the chemical composition of rehmannia glutinosa. The existing research results show that: many active ingredients in rehmannia can be used as medicines and can also be used for clinical treatment, such as treatment of hypertension and the like. At present, there is no report about the effects of anti-oxidation and blood sugar reduction of rehmannia.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a rehmannia root functional factor extract and application thereof in preparing an antioxidant and/or hypoglycemic medicament. The invention adopts an in vitro experimental method to research the antioxidant activity and the alpha-glucosidase inhibitory activity of the rehmannia root functional factor extract, and provides a theoretical basis for the development of the rehmannia root hypoglycemic functional factor and the research of the hypoglycemic mechanism.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting rehmanniae radix functional factor extract (DHTW) comprises soaking rehmanniae radix decoction piece powder in distilled water, heating, cooling, filtering, centrifuging, and concentrating the supernatant to dry to obtain rehmanniae radix crude extract;
soaking rehmanniae radix decoction pieces powder in distilled water, heating, cooling, filtering, centrifuging, separating the supernatant with D101 macroporous adsorbent resin column, washing with distilled water, collecting the liquid, concentrating to dry to obtain rehmanniae radix water eluate, washing with ethanol, collecting ethanol extract, and concentrating to dry to obtain rehmanniae radix ethanol eluate;
the rehmanniae radix functional factor extract comprises one or more of rehmanniae radix crude extract (DHC), rehmanniae radix water eluate (DHS) and rehmanniae radix alcohol eluate (DHY).
The above method for extracting rehmanniae radix functional factor comprises soaking in distilled water 10-20 times of rehmanniae radix decoction piece powder.
The above rehmanniae radix functional factor extract can be prepared by heating at 70-80 deg.C for 0.5-2 h.
The extraction method of the rehmanniae radix functional factor extract comprises filtering with 300-500 mesh gauze.
Specifically, the centrifugation can be carried out for 8-20min at 2000-4000 r/min.
Specifically, the alcohol extract can be washed by ethanol with the volume percentage concentration of 90-95% and collected.
The invention provides a rehmannia functional factor extract obtained by the method.
The invention also provides application of the rehmannia root functional factor extract in preparing anti-oxidation and/or hypoglycemic drugs.
The invention discusses the antioxidant activity and the alpha-glucosidase inhibitory activity of the rehmannia glutinosa libosch functional factor extract (DHTW) through in vitro tests, and provides a theoretical basis for the development and utilization of the rehmannia glutinosa libosch hypoglycemic functional factor. The invention adopts ultraviolet spectrophotometry (UV) to determine the ability of DHTW to eliminate DPPH, ABTS and other free radicals, and utilizes an enzyme-linked immunosorbent assay to determine the activity of DHTW to inhibit alpha-glucosidase. The results show that:
1) The DHTW has antioxidant activity, the antioxidant activity is continuously increased along with the increase of the concentration in the concentration range of 0-6 mg/mL, and the antioxidant activity tends to be stable after the concentration reaches a certain concentration;
2) Scavenging ability IC of DHTW on DPPH free radicals 50 The value size order is: alcohol eluate DHY (0.087 mg/mL) < crude extract DHC (2.987 mg/mL) < water eluate DHS (3.024 mg/mL), so the radical scavenging ability is expressed as: DHY > DHC > DHS;
3）ABTS free radical scavenging ability IC of DHTW 50 The value size order is: DHY (0.137 mg/mL) < DHC (1.463 mg/mL) < DHS (2.168 mg/mL), therefore, ABTS radical clearance appears as: DHY > DHC > DHS;
4) DHTW also has significant inhibitory effect on alpha-glucosidase. Wherein, DHY has obvious inhibiting ability, and the inhibiting activity of alpha-glucosidase is obviously better than that of DHC and DHS. And (4) conclusion: DHTW has both antioxidant and α -glucosidase inhibitory activity, with best DHY, second DHC and worst DHS.
Drawings
FIG. 1 shows the inhibitory activity of rehmannia glutinosa extract on alpha-glucosidase at different concentrations;
FIG. 2 is a graph of DPPH clearance of a positive control ascorbic acid;
FIG. 3 shows DPPH radical scavenging ability and IC of different rehmanniae radix extracts 50 A value;
FIG. 4 is ABTS free radical clearance of a positive control ascorbic acid;
FIG. 5 shows ABTS free radical scavenging ability and IC of different rehmanniae radix extracts 50 The value is obtained.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
1. Instruments and materials
1.1 Instrument for measuring the position of a moving object
Enzyme-linked immunosorbent assay (ELIASA), available from Burley, USA;
an AUW220D one ten-thousandth balance, available from shimadzu, japan;
ME204 parts per million balance, available from Mettler-Torledo instruments, inc.;
a pulverizer, available from Yongshi pharmaceutical machinery, inc., of Reian;
TU1810 ultraviolet spectrophotometry, available from Beijing Pujingyo general instruments liability Co., ltd;
TDL-5-A centrifuge, purchased from Shanghai' an pavilion scientific Instrument factory;
DGX-9143B forced air drying cabinet, from Shanghai Fuma.
1.2 Material
PNPG 4-nitrophenyl-D-glucopyranoside, available from Shanghai Anam Bright Standard technology service, inc.;
alpha-glucosidase, available from Shanghai leaf Biotech, inc.;
100808-201905 acarbose, available from the Hospital;
20160921 anhydrous sodium carbonate, purchased from Beijing chemical plant;
XK13-2010101-112 dipotassium hydrogen phosphate, available from chemical Agents works of Luoyang;
XK13-201-00115 Potassium dihydrogen phosphate, available from Tianjin Kangship chemical reagent science and technology Co., ltd;
13-201-00115 Potassium persulfate, available from Fengshan chemical reagents science and technology, inc., tianjin;
ascorbic acid, available from Hengxing Chemicals manufacturing Co., ltd, tianjin;
absolute ethanol, purchased from mikou chemical reagent of Tianjin;
2021051014 methanol, available from seijn, remote chemical agents, ltd;
1898-66-4 DPPH 1, 1-diphenyl-2-trinitrophenylhydrazine; 30931-67-0 ABTS 2,2 hydrazine-bis 3-ethylbenzothiazole-6-sulfonic acid diammonium salt.
1.3 Reagent
1.3.1 α -glucosidase solution: 0.1moL/L buffer (pH = 6.8) diluted to 0.2U/mL.
1.3.2 PNPG solution: dissolving with 0.1moL/L buffer solution to obtain 5 mmoL/L solution.
1.3.3 Sodium carbonate solution: adding deionized water, and ultrasonically dissolving to obtain 0.5 moL/L solution.
1.3.4 Phosphate buffer (pH = 6.8): 49.7 mL0.1mol/L K 2 HPO 4 And 50.3 mL of 0.1moL/L KH 2 PO 4 The mixed solution of (1).
1.3.5 DPPH solution: stock solutions of DPPH were made up to 0.1mg/mL with absolute ethanol.
1.3.6 ABTS solution: accurately weighing the ABTS powder 0.1920g, precisely weighing 50mL of methanol, and performing ultrasonic dissolution to obtain a test solution A; 0.0946 g potassium persulfate is added into 50mL distilled water in sequence to be dissolved into sample B by ultrasonic wave, then A and B are mixed for use, and the mixture is kept stand for 12 hours in dark place.
1.3.7 Sample solution: respectively weighing 0.1g of rehmannia crude extract, water eluate and alcohol eluate, adding methanol to dissolve to a constant volume of 10mL, and obtaining a sample mother liquor with a concentration of 10 mg/mL. Diluting the mother liquor to 1, 2, 4, 6, 8 and 10mg/mL to obtain a solution to be detected.
2. Preparation of rehmannia glutinosa Libosch functional factor extract (DHTW)
Weighing 200g of rehmannia decoction piece powder, soaking the powder in 15 mass times of distilled water for 5 hours, heating the powder at 75 ℃ for 1 h, filtering the powder by using 400-mesh gauze after cooling, centrifuging the powder for 10 minutes at 3000 r/min, and concentrating supernate to be dry to obtain a rehmannia crude extract (DHC), wherein the weight of the powder is about 54g, the color is tawny, and the yield is 2.7%;
weighing 200g of rehmannia decoction piece powder, soaking the powder in 15 mass times of distilled water for 5 hours, heating the powder at 75 ℃ for 1 h, filtering the powder by using 400-mesh gauze after cooling, centrifuging the powder for 10 minutes at 3000 r/min, enabling supernatant to pass through a D101 macroporous adsorption resin column, washing the column by using 1L distilled water, collecting liquid, concentrating the liquid to be dry to obtain a rehmannia water eluate (DHS), wherein the yield is 2.4%, and the color of the collected liquid is tawny; washing with 1L 95% (volume percentage) ethanol, collecting ethanol extract, and concentrating to dryness to obtain ethanol eluate (DHY) of about 40g, which is dark brown and has yield of 2%.
3. Method and results
3.1 Alpha-glucosidase activity inhibition assay
The method for testing the inhibition of alpha-glucosidase activity by the rehmannia root functional factor extract can be referred to the existing literature (XUY, NIUX, LIUN, et al. Characterisation, antioxidant and hypo-macromolecular activities of graded polysaccharides from black-range (Ribes nigrum L.) from J. Food Chemistry, 2018, 243: 26-35), and the like, and can be specifically referred to as follows:
the experiment was performed in a 96-well culture plate, while a blank group, a blank control group, a sample blank group, and a sample group were set. Acarbose (DZT) was used as a positive control. Firstly, 60 uL phosphate buffer is added to a sample group (100 uL phosphate buffer is added to a blank group, 80 uL phosphate buffer is added to both a blank group and a sample blank group), then 1, 2, 4, 6, 8, 10mg/mL of sample solution 20 uL is added to the sample group and the blank group, 20 uL alpha-glucosidase solution is added to the sample group and the blank group, the sample group and the blank group are placed in a blast drying box at 37 ℃ for about 10 min, then 20 uL PNPG solution is added to each group uniformly, reactants are placed in the blast drying box (37 ℃) again for 30 min, 80 uL sodium carbonate solution is added to stop reaction, and then absorbance is measured at nm by a microplate reader (specifically, related reagents can be added in sequence to carry out reaction according to different groups of the reaction mode in Table 1). Inhibition was calculated for 3 replicates per sample. The results are shown in Table 2 and FIG. 1.
TABLE 1 inhibition test reaction System for alpha-glucosidase Activity
The calculation method comprises the following steps: enzyme activity inhibition ratio (%) = [1- (A) 1 -A 2 ）/（A 3 -A 0 ）］×100
In the formula: a. The 1 Absorbance for the sample set; a. The 2 Absorbance for sample blank set; a. The 3 Blank absorbance; a. The 0 Absorbance of blank control.
3.2 Research on anti-oxidation activity of rehmannia root functional factor extract
3.2.1 determination of the ability to scavenge DPPH free radicals
DHS, DHC, DHY and the positive control ascorbic acid were each performed as follows.
(1) Pressing 6 test tubes of 10mL into A0 1 -A0 6 Numbering, adding exactly 2.0 mL of DPPH solution and 2.0 mL absolute ethyl alcohol, mixing well, and storing in dark for 30 min.
(2) Taking 6 test tubes of 10mL and pressing A1 1 - A1 6 The mixture was numbered, 2.0 mL of sample solution and 2.0 mL of DPPH solution were added at 1, 2, 4, 6, 8, and 10mg/mL, respectively, shaken, and left to react at room temperature (25 ℃) in the dark for 30 min.
(3) 6 test tubes of 10mL are numbered A2 1 、A2 2 、A2 3 、A2 4 、A2 5 、A2 6 Sequentially adding 2.0The sample solution with the concentration of 1 mL,2, 4, 6, 8 and 10mg/mL and the absolute ethyl alcohol of 2.0 mL are fully mixed, and the mixture is placed at room temperature and protected from light for reaction for 30 min.
(4) After the reaction is finished, absolute ethyl alcohol is used as a blank reagent, the absorbance is measured at 517 nm, the absorbance value of each group is recorded, and the parallel experiment is repeated for three times. Respectively calculating the clearance rates corresponding to different concentrations, and drawing a curve of the clearance rates to the concentrations.
DPPH clearance =1- ((A1-A2)/A0). Times.100%
A0:2 mL absolute ethyl alcohol +2 mL of DPPH solution;
a1:2 mL sample solution +2 mL DPPH solution absorbance;
a2: absorbance of 2 mL sample solution +2 mL absolute ethanol.
3.2.2 Determination of the ability to scavenge ABTS free radicals
(1) 6 test tubes of 10mL were according to A0 1 -A0 6 Numbering, adding 3.0 mL ABTS solution and 0.5mL methanol respectively, mixing, and standing in dark for 30 min;
(2) 6 test tubes of 10mL were used according to A 1 -A 6 Numbering, respectively adding 0.5mL, 1, 2, 4, 6, 8, 10mg/mL sample solution and 3.0 mL ABTS solution, shaking, and reacting for 30 min at room temperature (25 ℃) in a dark place;
(3) After the reaction, methanol is used as a blank reagent, the absorbance is measured at 734 nm, the absorbance value of each group is recorded,
three replicates were performed. A linear regression equation of concentration versus clearance is plotted.
ABTS radical clearance = (A0-A)/A0X 100 (%)
A0: represents the light absorption value of 0.5mL of methanol +3mL of BTS detection solution;
a: represents the absorbance of 0.5mL of sample +3mL of assay solution.
4. Results and analysis
4.1 Effect of different Mass concentrations of DHTW on alpha-glucosidase inhibitory Activity
As is clear from Table 2, the results of the α -glucosidase inhibitory activity at the same concentration (10 mg/mL) using acarbose (DZT) as a positive control show that DHY, and DZT,The inhibition rates of DHS and DHC respectively reach 92.59 percent (IC) 50 ：3.896 mg/mL）、76.76%（IC 50 ：3.450 mg/mL）、54.54%（IC 50 :8.352 mg/mL). The inhibition activity of the rehmannia glutinosa functional factor extract DHTW on alpha-glucosidase is as follows from small to big: DHC < DHS < DHY. As can be seen from table 2 and fig. 1: the alpha-glucosidase inhibitory activity of DHY is close to that of DZT, so DHY has better alpha-glucosidase inhibitory action.
Table 2 different mass concentrations of DHTW on α -glucosidase inhibitory activity (n = 5)
The result shows that the inhibition activity of the rehmannia glutinosa libosch functional factor extract DHTW on alpha-glucosidase is positively correlated with the concentration, namely the inhibition activity of alpha-glucosidase is enhanced along with the increase of the concentration, wherein the concentration of DHY is linearly increased at 1-10 mg/mL, and the concentration tends to be stable when the concentration is increased to 10 mg/mL. Within the range of 1-10 mg/mL, the inhibition rate of acarbose is increased along with the increase of concentration, and the alpha-glucosidase inhibition activity of DHY is obviously higher than that of DHC and DHS.
4.2 DPPH Positive control antioxidant Activity
The DPPH radical scavenging rate of the positive control was determined as described above, and the DPPH radical scavenging effect increased with increasing VC concentration in the range of 0.001-0.030 mg/mL using ascorbic acid (VC) as the positive control, as shown in FIG. 2.
4.3 Assay for antioxidant Activity of DHS, DHC and DHY (DPPH method)
According to FIG. 3 it is shown that: the DPPH antioxidant activity of DHY is far higher than that of DHS and DHC; the antioxidant activity of DHS is higher than that of DHC when the concentration is in the range of 0.1 mg/mL-3 mg/mL, and the antioxidant activity of DHC is higher than that of DHS when the concentration is in the range of 3 mg/mL-6 mg/mL. IC according to FIG. 3 50 Value, overall, IC for free radical scavenging ability by DHTW DPPH method 50 The value size order is: VC (0.005 mg/mL)<DHY（0.087 mg/mL）<DHC（2.987 mg/mL）<DHS (3.024 mg/mL), IC of DHY 50 The value is close to VC, so the antioxidant activity DHY is best, DHC is second, and DHS is worst.
4.4 ABTS positive control antioxidant Activity
The clearance of the positive control to ABTS free radicals was determined as described above, and when VC was used as the positive control and the clearance was in the range of 0.001-0.030 mg/mL, the clearance of ABTS free radicals increased with the increase of VC concentration, as shown in FIG. 4.
4.5 Assay for antioxidant Activity of DHS, DHC and DHC (ABTS method)
As shown in FIGS. 4 and 5, when the antioxidant activity of the rehmannia glutinosa libosch functional factor extract is measured by ABTS method, the antioxidant activity of DHY is slightly lower than that of VC; IC of DHTW 50 VC (0.016 mg/mL)<DHY（0.137 mg/mL）<DHC（1.463 mg/mL）<DHS (2.168 mg/mL), oxidation activity was shown as: DHY> DHC > DHS。
4.6 Statistical method
Half maximal Inhibitory Concentration (IC) was calculated using IBM SPSS statics 26 statistical software 50 ) All experimental data are mean values of 3 replicates.
5. Final phrase
The rehmannia glutinosa Libosch functional factor extract DHTW has antioxidant activity and alpha-glucosidase inhibitory activity, and can be used for preparing antioxidant and/or hypoglycemic drugs, wherein DHY is the best, DHC is the second best, and DHS is the worst. Experiments show that the DHTW has good utilization and development values. The UPLC-MS is used for carrying out qualitative and quantitative analysis on the functional factors, the mechanism of the hypoglycemic effect is deeply researched, the foundation is laid for establishing an alpha-glucoside inhibition kinetic model and a Hep G2 cell insulin resistance model in the next step, and a new idea is provided for developing rehmannia and novel medicaments for treating diabetes.
Claims (8)
1. A method for extracting rehmannia root functional factor extract is characterized in that rehmannia root decoction piece powder is soaked in distilled water, heated, cooled, filtered and centrifuged, and supernatant is concentrated to be dry to obtain a rehmannia root crude extract;
soaking rehmanniae radix decoction pieces powder in distilled water, heating, cooling, filtering, centrifuging, separating the supernatant with D101 macroporous adsorbent resin column, washing with distilled water, collecting the liquid, concentrating to dry to obtain rehmanniae radix water eluate, washing with ethanol, collecting ethanol extract, and concentrating to dry to obtain rehmanniae radix ethanol eluate;
the rehmanniae radix functional factor extract comprises one or more of rehmanniae radix crude extract, rehmanniae radix water eluate and rehmanniae radix alcohol eluate.
2. The method for extracting the rehmannia glutinosa libosch functional factor extract as claimed in claim 1, wherein the amount of distilled water used for soaking is 10-20 times of the rehmannia glutinosa libosch decoction piece powder by mass.
3. The method for extracting the rehmannia glutinosa Libosch functional factor extract as claimed in claim 1, wherein the extract is heated at 70-80 ℃ for 0.5-2 h.
4. The method for extracting the rehmannia glutinosa libosch functional factor extract as claimed in claim 1, wherein the filtering is performed using 300-500 mesh gauze.
5. The method for extracting rehmanniae radix functional factor extract as claimed in claim 1, wherein the centrifugation is carried out at 2000-4000 r/min for 8-20min.
6. The method for extracting rehmanniae radix functional factor extract as claimed in claim 1, wherein the washing is carried out with ethanol having a concentration of 90-95 vol.%.
7. The rehmannia glutinosa Libosch functional factor extract obtained by the method of any one of claims 1 to 6.
8. The use of the rehmannia glutinosa libosch functional factor extract according to claim 7 for the preparation of an antioxidant and/or hypoglycemic agent.
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|---|---|---|---|---|
| CN101385807A (en) * | 2007-09-14 | 2009-03-18 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Use of rehmannia root whole crude medicine in preventing and treating hyperlipemia and hyperglycaemia |
| CN101704864A (en) * | 2009-10-20 | 2010-05-12 | 南京荣世医药科技有限公司 | Method for preparing catalpol |
| US20100197618A1 (en) * | 2007-07-18 | 2010-08-05 | Ling Zhang | Extract of rehmannia glutinasa libosch for reducing blood sugar, reducing blood fat, treating leukemia, and preparation method uses thereof |
| CN102127131A (en) * | 2010-11-03 | 2011-07-20 | 张守力 | Catalpol extracting method |
| CN102863489A (en) * | 2011-07-04 | 2013-01-09 | 苏州玉森新药开发有限公司 | Method for extracting catalpol from rehmannia stem |
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2022
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| US20100197618A1 (en) * | 2007-07-18 | 2010-08-05 | Ling Zhang | Extract of rehmannia glutinasa libosch for reducing blood sugar, reducing blood fat, treating leukemia, and preparation method uses thereof |
| CN101385807A (en) * | 2007-09-14 | 2009-03-18 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Use of rehmannia root whole crude medicine in preventing and treating hyperlipemia and hyperglycaemia |
| CN101704864A (en) * | 2009-10-20 | 2010-05-12 | 南京荣世医药科技有限公司 | Method for preparing catalpol |
| CN102127131A (en) * | 2010-11-03 | 2011-07-20 | 张守力 | Catalpol extracting method |
| CN102863489A (en) * | 2011-07-04 | 2013-01-09 | 苏州玉森新药开发有限公司 | Method for extracting catalpol from rehmannia stem |
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| Title |
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| ZHU HUIFENG等: "Antidiabetic and antioxidant effects of catalpol extracted from Rehmannia glutinosa (Di Huang) on rat diabetes induced by streptozotocin and high-fat, high-sugar feed", CHINESE MEDICINE, vol. 11, no. 1, pages 25 * |
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