CN103751269A - Application of herba rabdosiae rubescentis extract to alpha-glucosidase inhibitor - Google Patents
Application of herba rabdosiae rubescentis extract to alpha-glucosidase inhibitor Download PDFInfo
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- ethyl acetate
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- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 title claims abstract description 18
- 239000003888 alpha glucosidase inhibitor Substances 0.000 title claims abstract description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 77
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000004615 ingredient Substances 0.000 claims abstract description 11
- 241001646826 Isodon rubescens Species 0.000 claims description 42
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 239000002024 ethyl acetate extract Substances 0.000 claims description 23
- 239000003112 inhibitor Substances 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 6
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Abstract
The invention belongs to the technical field of application of Chinese herb extracts, and particularly relates to application of a herba rabdosiae rubescentis extract to an alpha-glucosidase inhibitor. The invention finds that an ethyl acetate part and a methyl alcohol part both have a certain inhibition to alpha-glucosidase, wherein the ethyl acetate part is strongest in inhibition activity, and the methyl alcohol part is stronger in inhibition activity; an effective ingredient oridonin has a strong function of inhibiting the alpha-glucosidase, which belongs to non-competitive inhibition.
Description
technical field
The invention belongs to Chinese medicine extract applied technical field, be specifically related to a kind of Rabdosia rubescens extract and exist
αapplication in-glucosidase inhibitor.
background technology
Rabdosia rubescens is the Labiatae Rabdosia plant fork of cracking rice
rabdosia rubescens(Hemsl.) dry aerial parts of Hara, originate in the ground such as Hubei (cry not only Herba microtoenae insuavis flower), Sichuan (cry not only Pyrola rotundifolia ssp.chinensis), Guizhou (cry not only Isodonpubescens (Hemsl.) C. Y. Wuet Hsuan, snowflake grass and Herba microtoenae insuavis), Shanxi, Henan (but also make Rabdosia rubescens, June make and mountain weak), Hebei and Hunan, be born in hillside, bushes, the area without shade such as forest land, gravel ground and roadside, height above sea level 100-2800m.According to < < Chinese Pharmacopoeia > > (2010 editions), record, its decoction pieces nature and flavor are bitter, sweet, be slightly cold, the effect with heat-clearing and toxic substances removing, promoting blood circulation and stopping pain, can be used for treating laryngopharynx swelling and pain, mass in the abdomen mass in the abdomen, snake bite and insect sting.Chemical research shows, contains the chemical compositions such as terpenoid (diterpene and triterpene), alkaloid, steroidal, flavone, volatile oil, organic acid in Rabdosia rubescens.Pharmacological research shows that it has the pharmacological actions such as antitumor, antibacterial, antiinflammatory, enhancing immunity, antioxidation, mutation, blood pressure lowering, especially antitumor, function well, and research report is many, toxicity is not obvious, be described as " paclitaxel second ", its main anticancer active constituent is rubescensine A and rubescensine B.Have no at present document to Rabdosia rubescens extract and compound
αthe inhibiting report of-glucosidase.
Rubescensine A (Oridonin) is the main effective ingredient of Diterpenes in Rabdosia rubescens, the leading indicator of the evaluation of Chang Zuowei Rabdosia rubescens quality, another name: rubescensin, isodonin; Molecular formula: C
20h
28o
6; Molecular weight: 364.43; CAS accession number: 28957-04-2.This product is a kind of Ent-kauran alkane type diterpene-kind compound, and taste is extremely bitter, is slightly soluble in water, dissolves in methanol, ethyl acetate, acetone and other organic solvent; Rubescensine A has multiple pharmacologically active, as antitumor, anti-inflammation, parasite killing heat-clearing and toxic substances removing, immunostimulant, antioxidation, blood pressure lowering and stomach invigorating are invigorated blood circulation etc.Clinical experiment shows, rubescensine A has obvious inhibition or lethal effect to kinds of tumor cells, has stronger antibacterial and anti-inflammation functions, toxic and side effects is less, but due to its poorly water-soluble, is orally difficult to reach effective blood drug level, reduce clinical efficacy, limited its clinical practice.For finding the drug candidate with clinical value, Chinese scholars has been carried out structural modification to rubescensine A.Do not find rubescensine A pair
αthe inhibiting report of-glucosidase.
α-glucosidase inhibitor is a class new oral hypoglycemic drug of researching and developing in the seventies later stage, and its mechanism of action is: pass through competitive inhibition
αthe activity of-glucosidase, retardance disaccharidase is hydrolyzed into monosaccharide, delays sugared absorption, makes blood glucose steadily and maintains lentamente certain level.
α-glucosidase inhibitor can effectively be controlled the rising of post-prandial glycemia, and the generation of prevent diabetes reduces diabetic complication, reduces mortality rate.
α-glucosidase inhibitor not only has definite curative effect to diabetes, and obesity, chronic viral hepatitis B, acquired immune deficiency syndrome (AIDS) are also had to certain therapeutical effect.
At present, clinical for
αthis type of medicine of-glucosidase inhibitor has acarbose, voglibose and miglitol, in natural product
α-glucosidase inhibitor is the focus of Recent study.Research discovery,
αthe major structural types of-glucosidase inhibitor is flavonoid, alkaloids and saponins, also has in addition tea polyphenols.Flavone compound is mainly by polyhydroxy structure performance inhibitory action, and the saccharification meeting of hydroxyl group weakens compound pair
αthe inhibitory action of-glucosidase.Alkaloids is also that polyhydroxylated alkaloid is inhibited.The extract of some Chinese herbal medicine also has good inhibition
αthe effect of-glucosidase, as green tea extract and Radix Et Rhizoma Rhei, Fructus Corni, Radix Paeoniae Rubra, Galla Chinensis water boiling and precipitation with ethanol extract, and the water extract of the full powder of Guangxi Sanguis Draxonis and substep extract, Fructus Schisandrae Chinensis and Rhizoma Polygoni Cuspidati etc.Yet existing this type of medicine cost is higher, and manufacturer seldom, is attended by intestinal side effect simultaneously.
summary of the invention
The object of the present invention is to provide a kind of Rabdosia rubescens extract to exist
αapplication in-glucosidase inhibitor.
The present invention is by the following technical solutions:
Rabdosia rubescens extract exists
αapplication in-glucosidase inhibitor.
Described Rabdosia rubescens extract is ethyl acetate extract and/or methanol position.
The effective ingredient of described Rabdosia rubescens extract is rubescensine A.
Described ethyl acetate extract, methanol position refer to Rabdosia rubescens total ethanol extractum silica gel mixed sample, use successively ethyl acetate and methanol-eluted fractions, reclaim solvent, and gained eluent ethyl acetate thing is ethyl acetate extract, and gained methanol-eluted fractions thing is methanol position.
The preparation method of described Rabdosia rubescens total ethanol extractum is: Rabdosia rubescens leaf dries in the shade, pulverizes, and with ethanol (65～80V%) room temperature lixiviate 2-3 time, each 2-4 d, merges lixiviating solution, and decompression and solvent recovery, obtains Rabdosia rubescens total ethanol extractum.
Ethyl acetate extract or methanol position obtain rubescensine A by the means separation such as silica gel column chromatography, Sephadex LH-20 gel filtration chromatography, high performance liquid preparative chromatography, recrystallization repeatedly respectively.
The present invention finds that ethyl acetate extract, methanol position, the rubescensine A of Rabdosia rubescens all have certain
α-glucosidase inhibitor is active, the IC at ethyl acetate extract, methanol position
50be respectively 475.85 and 990.08 μ g/mL, and right
αthe suppression ratio of-glucosidase is all dose dependent, and, along with the increase of mass concentration, suppression ratio constantly increases; The extract rubescensine A of Rabdosia rubescens also has well
α-glucosidase inhibitor effect (IC
50be 21.48 μ g/mL), and belong to noncompetitive inhibition, within the scope of finite concentration, right
αthe suppression ratio of-glucosidase is all dose dependent, after this increases control of the concentration rate again and changes little.
accompanying drawing explanation
Fig. 1 is ethyl acetate extract, methanol position, acarbose
α-Glucosidase inhibitor rate is with the variation diagram of drug level;
Fig. 2 is rubescensine A
α-Glucosidase inhibitor rate is with the variation diagram of drug level;
Fig. 3 is rubescensine A
α-the Lineweave-Burk curve of glucosidase activity.
the specific embodiment
Rabdosia rubescens leaf dries in the shade, pulverizes, with 65V% ethanol room temperature lixiviate 2 times, each 4d, merges lixiviating solution, decompression and solvent recovery, obtain Rabdosia rubescens total ethanol extractum, Rabdosia rubescens total ethanol extractum silica gel mixed sample, uses ethyl acetate and methanol-eluted fractions successively, reclaims solvent, gained eluent ethyl acetate thing is ethyl acetate extract, and gained methanol-eluted fractions thing is methanol position;
Ethyl acetate extract or methanol position obtain rubescensine A by the means separation such as silica gel column chromatography, Sephadex LH-20 gel filtration chromatography, high performance liquid preparative chromatography, recrystallization repeatedly respectively.
rubescensine A: white crystals, mp 245-247 ℃.IR (KBr, cm
-1): 3433,3382,3304,1711,1647,1095,1080,1068.
1h-NMR (Acetone-d
6) δ: 6.17,5.57 (each 1H, s, H-17), 5. 04 (1H, s, H-14), 4.38,4.13 (each 1H, d,
j=10.0 Hz, H-20), 3.81 (1H, d, J=7.0 Hz, H6), 3.62 (1H, dd, J=11.6 Hz, H-1), 1.19,1.15 (each 3H, s, C4-Me2).
13C-NMR?(Acetone-d6)?δ:?210?(C-15),?153.8?(C-16),?119.6?(C-17),?98?(C-7),?74.9?(C-6),?74.1?(C-14),?73.8?(C-1),?64.2?(C-20),?63.0?(C-8),?61.2?(C-5),?55.2?(C-9),?44.5?(C-13),?42.3?(C-10),?40.0?(C-3),?34.6?(C-4),?33.6?(C-18),?31.6?(C-12),?30.7?(C-2),?22.6?(C-19),?20.8?(C-11)。
Embodiment 2
As different from Example 1: concentration of alcohol is 75V%, lixiviate 3 times, each 2d.
Embodiment 3
As different from Example 1: concentration of alcohol is 80V%, lixiviate 3 times, each 3d.
effect test
test one, Rabdosia rubescensethyl acetate extract, methanol position, rubescensine A
external
αthe test of-glucosidase activity
1.1 test methods: Microdilution plate method
1.1.1 principle: α-glucoside enzymatic hydrolysis 4-Nitrobenzol-
α-D-pyranglucoside (PNPG), produces nitrophenol (PNP, yellow substance have absorption maximum at 405 nm),
α-glycosidase inhibitor can suppress
αthereby-glucosidase and Binding Capacity reduce the burst size of PNP, with the changes of contents of PNP in reaction system in certain hour, calculate the enzyme inhibition activity of extract.
instrument:multiskan MK3 microplate reader (Thermo Electron); LRH-150 constant incubator (Shanghai Yi Heng Science and Technology Ltd.); DELTA 320 type pH meters (Mettler-Toledo); Electronic balance (Mettler-Toledo); Rotary Evaporators (Heidolph).
reagent: α-glucosidase (Sigma company, EC 3.2.1.20), 4-nitrobenzophenone-
α-
d-pyranglucoside (PNPG, Sigma company, lot number: 026K1516), kaliumphosphate buffer (pH 6.8), acarbose (Acarbose, Zhongmei Huadong Pharmaceutical Co., Ltd. Hangzhou, lot number: 110704), other reagent are analytical pure.
detection method and date processing
The configuration of sample solution:
Ethyl acetate extract, methanol position, acarbose: the sample that takes certain mass adds the DMSO of certain volume, being made into concentration is 30 mg/mL solution; Rubescensine A: take the rubescensine A of certain mass, add the DMSO of certain volume, being made into concentration is 2 mg/mL solution.
Blank group: 8 μ LDMSO+152 μ L kaliumphosphate buffers, under 405 nm wavelength, survey OD value; Negative control group: 8 μ LDMSO+20 μ L
αμ L kaliumphosphate buffer+20 ,-glucosidase+112 μ LPNPG surveys OD value under 405 nm wavelength;
Sample matched group: 8 μ L sample solution+152 μ L kaliumphosphate buffers, under 405 nm wavelength, survey OD value; Sample sets: 8 μ L sample solution+20 μ L
αμ L kaliumphosphate buffer+20 ,-glucosidase+112 μ LPNPG surveys OD value under 405 nm wavelength.(detailed process: 112 μ L kaliumphosphate buffers (pH 6.8), add 20 μ L 0.2 U/mL
α-glucosidase, 8 μ L sample solutions, 37 ℃ of constant temperature 15 min, add 20 μ L 2.5 mmol/L PNPG, 37 ℃ of isothermal reaction 15 min.The terminator Na that adds again 80 μ L 0.2 mol/L
2cO
3solution is surveyed OD value under 405 nm wavelength.）
Do negative control group under same system, blank group, sample sets and sample matched group simultaneously, by method below, calculate suppression ratio, and obtain corresponding IC with Origin6.0 software
50value.
Suppression ratio
Utilize respectively said method to measure the IC of Rabdosia rubescens effective site in embodiment 1 (ethyl acetate extract and methanol position) and effective ingredient rubescensine A and positive control acarbose
50.
test two, Rabdosia rubescens effective ingredient external
αthe test of-Glucosidase inhibitor type
Foundation
test onemensuration, get respectively 2 suitable variable concentrations of rubescensine A (40 μ g/mL, 50 μ g/mL), PNPG gets 5 variable concentrations (0.625,1.25,2.5,5,10 mmol/L), respectively assaying reaction speed.Press Lineweave-Burk graphing method, take 1/[S] be abscissa, 1/V is vertical coordinate, with Graphpad Prism 5 Software on Drawings, goes out inhibitory action kinetic curve.
experimental result
1.2.1 Rabdosia rubescens effective site and effective ingredient is external
α-glucosidase activity result
The different samples of table 1
α-glucosidase inhibitor is active
| Sample | Primary dcreening operation concentration (μ g/mL) | Primary dcreening operation suppression ratio (I%) | IC 50Value (μ g/mL) |
| | 1500 | 101.87 | 475.85 |
| | 1500 | 76.34 | 990.08 |
| | 100 | 95.09 | 21.48 |
| Acarbose | 1500 | 52.74 | 1358.34 |
As can be seen from Table 1, Rabdosia rubescens ethyl acetate extract, methanol position pair
α-active (the IC of Glucosidase inhibitor
50be respectively 475.85 and 990.08 μ g/mL) be all better than positive control drug acarbose (IC
50be 1358.34 μ g/mL), this shows to extract two active sites (ethyl acetate extract, methanol position) from Rabdosia rubescens and all has certain
α-glucosidase inhibitor effect.Wherein, the IC of ethyl acetate extract
50being worth minimumly, showing that its inhibition is best, is secondly methanol position.From ethyl acetate or methanol effective site, isolated rubescensine A also has well
α-glucosidase inhibitor effect (IC
50be 21.48 μ g/mL), activity is better than ethyl acetate extract, methanol position.
Fig. 1 shows, Rabdosia rubescens ethyl acetate extract of the present invention, methanol position pair
αthe suppression ratio of-glucosidase is all dose dependent, and, along with the increase of mass concentration, suppression ratio constantly increases.Fig. 2 demonstration, within the scope of finite concentration, effective ingredient rubescensine A pair
αthe suppression ratio of-glucosidase is all dose dependent, and when concentration reaches 25 μ g/mL, its suppression ratio 83.86%, after this increases control of the concentration rate again and change not quite, and when concentration is increased to 100 mg/mL, its suppression ratio has just reached maximum 95.09%.
rabdosia rubescens effective ingredient external
α-Glucosidase inhibitor types results
Fig. 3 is rubescensine A pair
α-the Lineweave-Burk curve of glucosidase activity.As seen from the figure: rubescensine A pair
α-glucosidase inhibitor effect belongs to noncompetitive and suppresses, response speed V
maxalong with the increase of inhibitor concentration, diminish, Michaelis constant K
mremain unchanged.Press noncompetitive inhibition kinetics equation: 1/V '
max=1/V
max(1+[I]/K
i), can obtain its K
ivalue is: 5.56 μ g/mL.Illustrate it both can and enzyme, also can with enzyme-substrate complex combination, thereby reduce enzymatic activity, reach and reduce blood glucose effect.
Generally speaking, above-mentioned external Inhibiting enzyme activity test confirmation, two groups of Rabdosia rubescens effective sites that the present invention provides (ethyl acetate extract, methanol position) are right
α-glucosidase all has certain inhibitory action, and wherein ethyl acetate extract inhibition activity is the strongest, is secondly methanol position; Effective ingredient rubescensine A pair
α-glucosidase also has strong inhibitory action, and belongs to noncompetitive inhibition.
Claims (6)
1. Rabdosia rubescens extract exists
αapplication in-glucosidase inhibitor.
2. Rabdosia rubescens extract as claimed in claim 1 exists
αapplication in-glucosidase inhibitor, is characterized in that: described Rabdosia rubescens extract is ethyl acetate extract and/or methanol position.
3. the application of the effective ingredient of Rabdosia rubescens extract as claimed in claim 1 in alpha-glucosidase inhibitor, is characterized in that: the effective ingredient of described Rabdosia rubescens extract is rubescensine A.
4. Rabdosia rubescens extract as claimed in claim 2 exists
αapplication in-glucosidase inhibitor, it is characterized in that: described ethyl acetate extract, methanol position refer to Rabdosia rubescens total ethanol extractum silica gel mixed sample, use successively ethyl acetate and methanol-eluted fractions, reclaim solvent, gained eluent ethyl acetate thing is ethyl acetate extract, and gained methanol-eluted fractions thing is methanol position.
5. Rabdosia rubescens extract as claimed in claim 4 exists
αapplication in-glucosidase inhibitor, is characterized in that: the preparation method of described Rabdosia rubescens total ethanol extractum is: Rabdosia rubescens leaf dries in the shade, pulverizes, with ethanol room temperature lixiviate 2-3 time, each 2-4 d, merge lixiviating solution, decompression and solvent recovery, obtains Rabdosia rubescens total ethanol extractum.
6. Rabdosia rubescens extract as claimed in claim 5 exists
αapplication in-glucosidase inhibitor, is characterized in that: concentration of alcohol is 65～80V%.
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Cited By (2)
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| CN103751269B (en) | 2016-08-17 |
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