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Glucosidase inhibitory activity and antioxidant activity of flavonoid compound and triterpenoid compound from Agrimonia Pilosa Ledeb
© Liu et al.; licensee BioMed Central Ltd. 2014
Received: 2 August 2013
Accepted: 6 January 2014
Published: 10 January 2014
In Chinese traditional medicine, Agrimonia pilosa Ledeb (APL) exhibits great effect on treatment of type 2 diabetes mellitus (T2DM), however its mechanism is still unknown. Considering that T2DM are correlated with postprandial hyperglycemia and oxidative stress, we investigated the α-glucosidase inhibitory activity and the antioxidant activity of flavonoid compound (FC) and triterpenoid compound (TC) from APL.
Entire plants of APL were extracted using 95% ethanol and 50% ethanol successively. The resulting extracts were partitioned and isolated by applying liquid chromatography using silica gel column and Sephadex LH 20 column to give FC and TC. The content of total flavonoids in FC and the content of total triterpenoids in TC were determined by using UV spectrophotometry. HPLC analysis was used to identify and quantify the monomeric compound in FC and TC. The α-glucosidase inhibitory activities were determined using the chromogenic method with p-nitrophenyl-α-D-glucopyranoside as substrate. Antioxidant activities were assessed through three kinds of radical scavenging assays (DPPH radical, ABTS radical and hydroxyl radical) & β-carotene-linoleic acid assay.
The results indicate FC is abundant of quercitrin, and hyperoside, and TC is abundant of 1β, 2β, 3β, 19α-tetrahydroxy-12-en-28-oic acid (265.2 mg/g) and corosolic acid (100.9 mg/g). The FC & the TC have strong α-glucosidase inhibitory activities with IC50 of 8.72 μg/mL and 3.67 μg/mL, respectively. We find that FC show competitive inhibition against α-glucosidase, while the TC exhibits noncompetitive inhibition. Furthermore, The FC exhibits significant radical scavenging activity with the EC50 values of 7.73 μg/mL, 3.64 μg/mL and 5.90 μg/mL on DPPH radical, hydroxyl radical and ABTS radical, respectively. The FC also shows moderate anti-lipid peroxidation activity with the IC50 values of 41.77 μg/mL on inhibiting β-carotene bleaching.
These results imply that the FC and the TC could be responsible for the good clinical effects of APL on T2MD through targeting oxidative stress and postprandial hyperglycaemia. So APL may be good sources of natural antioxidants and α-glucosidase inhibitors exhibiting remarkable potential value for the therapy of T2DM.
Type II diabetes mellitus (T2DM) as an epidemic disease, associated with increased significant social and financial burden, is a cause of very high morbidity and mortality in the world . It is well-known that the genesis and progression of T2DM is generally attributed to several factors including persistent hyperglycemia toxicity, oxidative damage and lipotoxicity, etc. . A widely held notion is that postprandial hyperglycemia (PPHG) is a primary risk factor in the development of T2DM and its complications via multi-factorial mechanisms . Meanwhile, increasing evidences suggest that oxidative stress (OS) is involved in the pathogenesis of T2DM and the development of diabetic complications through the mechanism of impairing oxidation reduction system, leading to β-cell failure and insulin resistance [3, 4].
Despite great efforts that have been made to normalize blood glucose level in clinic practice, it is still a formidable challenge. Even more difficult is the control of PPHG . Nowadays, the more attentions are paid to control PPHG based on the α-glucosidase, α-amylase, amylin analogues as targets . Among them, most therapeutic approaches for controlling PPHG are the pharmacological inhibitors with greatest effect on α-glucosidase including acorbose, miglilol, emigitate, voglibiose, etc. [6, 7]. However, the continuous use of those synthetic agents should be limited because those agents may cause side effects such as flatulence, abdominal cramp, vomiting, and diarrhea [8, 9]. Numerous studies have been carried out to screen natural agents (active natural components and crude extracts) to inhibit α-glucosidase activity without or with fewer side effects . On the other hand, overwhelming researches suggest that natural antioxidants may be used to reduce oxidative damage and decrease the occurrence of diabetic complications [11, 12]. Therefore, it is a prospective strategy that PPHG and ROS are used as dual-target to screen the natural drugs to combat the multiple disorders of T2DM.
Agrimonia pilosa Ledeb (APL), a Chinese traditional medicinal plant of Rosaceae family, is widely used to treat blood, tumor, gastrointestinal, gynecological, genitourinary diseases in Chinese traditional medicine . Especially, in the past several decades Chinese traditional medicine have shown the great effect of APL on T2DM in clinical practice. But it is unclear how APL acts on T2DM. Chemical composition studies reveal that the APL is abounded with polyphenols, terpenoids and coumarins etc. [14, 15]. It has been reported that some flavonoids and terpenoids from medicinal plants have the inhibitory activities of α-glucosidase . Moreover, the abundant flavonoids are mainly responsible for the antioxidant activities of many herbs . Therefore, we speculated that the APL could combat T2DM through targeting PPHG and OS.
In this study, we isolated the flavonoid compound (FC) and the triterpenoid compound (TC) from APL, and evaluated their α-glucosidase inhibition activity and antioxidative activities. Meanwhile, the inhibitory effect on α-glucosidase of the compounds with the different ratio of the FC and the TC also was tested. Furthermore, the inhibition kinetics against α-glucosidase of the FC and the TC were studied.
Butylated hydroxyl toluene (BHT), gallic acid, β-carotene, linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH·), ρ-nitrophenyl α-D-glucopyranoside(PNPG), 3,5-dinitro salicylic acid, soluble potato starch and 1-deoxyrojirimycine, α-Glucosidase (from Saccharomyces cerevisiae), HPLC grade methanol and acetonitrile were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin-Ciocalteu reagent was obtained from E. Merck Co. (Darmstadt, Germany). Standards including oleanolic acid, ursolic acid, vitexin, rutin, hyperoside, luteolin-7-O-β-D-glucopyranoside, quercitrin, quercetin, luteolin, apigenin and kaempferol were obtained from the National Institutes for Food and Drug Control (Beijing, China). 1β, 2β, 3β, 19α-tetrahydroxy-12-en-28-oic acid, tormentic acid, maslinatic acid, corosolic acid and tiliroside are isolated and identified by ourselves in lab. All other reagents were analytical grade procured from indigenous manufacturers.
Plant materials and preparation of the extract
Determination of total flavonoids content
Total flavonoids content in FC was determined by using the aluminium chloride colorimetric method. A mixture of 0.5 mL sample, 100 μL 10% aluminum chloride, 100 μL 1 M potassium acetate and 2.3 mL distilled water were incubated at room temperature for 30 min. The absorbance was measured at 415 nm. Quercetrin as a standard was used to make the calibration curve. The estimation of total flavonoids in the extracts was carried out in triplicate and the results were averaged.
Determination of total triterpenoids content
Total triterpenoid content in TC was determined by using UV spectrophotometry , with some modifications. Reference substance (0.8 mL) was moved in 10 mL volumetric flask, to evaporate ethanol in water bath at 85°C and then add 0.4 mL 5% vanillin-glacial acetic acid and 1.0 mL perchloric acid, respectively. The mixture was kept in water bath at 80°C for 20 min, cooled to room temperature in ice bath, and 5.0 mL glacial acetic acid was added. The solutions were mixed by manual shaking for 15–20 s and allowed to stand for 15 min at room temperature. Finally, the absorbance was measured at 765 nm. Ursolic as a standard was used to make the calibration curve. The estimation of total triterpenoid content in the extracts was carried out in triplicate and the results were averaged.
HPLC analysis of flavonoids in FC
HPLC analysis was performed using Agilent1260 with UV detector, and chromatographic separations were conducted on a Welch ultimate XB-C18 column (4.6 × 250 mm, 5 μm). The solvent system was a gradient of mobile phase: soln. A: 0.1% phosphate in H2O; soln. B, acetonitrile. The following gradient was used: 0–30 min, 87% A; 30–50 min, 87% A to 80% A; 50–70 min, 80% A to 60% A; 70–80 min, 60% A to 87% A; 80-90 min, 87% A. Operating conditions were as follows: flow rate, 1.0 mL/min; column temp., 30°C; injection volume, 20 μL; UV detection at 350 nm.
HPLC analysis of triterpenoids in TC
HPLC analysis was performed using Agilent 1260 with UV detector, and chromatographic separations were conducted on a Welch ultimate XB-C18 column (4.6 × 250 mm, 5 μm). The solvent system was a gradient of mobile phase: soln. A: 0.1% formic acid in H2O; soln. B: acetonitrile and methnol (2:1, v/v). The following gradient was used: 0–10 min, 25% A; 10–15 min, 25% A to 30% A; 15–30 min, 30%A. Operating conditions were as follows: flowrate, 1.0 mL/min; column temp., 30°C; injection volume, 20 μL; UV detection at 210 nm.
Inhibition assay for α-glucosidase activity
All the tests were run in triplicate. The IC50 values (concentration required to inhibit 50% enzyme activity) were calculated by applying logarithmic regression analysis from the mean inhibitory values.
Kinetics of inhibition against α-glucosidase
Inhibition mode of the APL against α-glucosidase activities was measured with the increasing concentration of p-nitrophenyl-α-D-glucopyranoside as a substrate in the absence or presence of FC or TC at different concentrations. Inhibition type was determined by Lineweaver-Burk plot analysis of the data, which was calculated from the result according to Michaelis-Menten kinetics.
1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity
Where AS was the absorbance of the sample and A0 was the absorbance of the blank (without sample).
Hydroxyl radical scavenging activity
Where AS, absorbance of the sample; Ac, absorbance of control solution containing 1,10-phenanthroline, FeSO4 and H2O2 without sample; A0, absorbance of blank solution containing 1,10-phenanthroline and FeSO4 without H2O2 and sample.
ABTS radical scavenging assay
Where Ac was the absorbance of the blank (without sample) and As was the absorbance of the sample.
Determination of the antilipid peroxidation activity with the β-carotene bleaching assay
All tests were performed in triplicate. Results were expressed as the means ± SD (n = 3). The IC50 values were calculated from linear regression analysis. A paired-samples T test was used for the difference analysis between groups by using SPSS 19.0 software. Difference with a value of p < 0.01 were considered statistically significant.
Results and discussion
Despite a great of efforts have been made in treatment of T2DM in clinic, poor effect accounted for a single target is still a major challenge. More and more researches suggested that the multi-target therapy combining with the control of postprandial hyperglycaemia and oxidative stress may become a promising therapy strategy because oxidative stress is mainly induced by postprandial hyperglycaemia . There is increasing interest in screening of bioactive compounds from herbal plant with the ability to delay or prevent glucose absorption, and to reduce oxidative damage. APL, as Chinese traditional medicine, has been used to treat T2DM clinically for several decades, but its pharmaco-mechanism is not so clear. To address the mechanism at least to some degree, both α-glucosidase inhibiting activity and antioxidant activity of the FC and the TC from APL were studied. Many traditionally medicinal plants and natural products have been tested for their inhibition of α-glucosidase activities and anti-diabetic potential , but few researches about APL have been reported.
The contents of flavonoids in flavonoid compound (FC)
24.2 ± 1.2
10.8 ± 0.4
9.8 ± 0.5
8.4 ± 0.8
73.6 ± 2.6
13.9 ± 0.8
31.2 ± 0.9
14.7 ± 1.1
11.3 ± 0.4
2.7 ± 0.5
The contents of triterpenoids in triterpenoid compounds (TC)
6.7 ± 0.4
28.2 ± 0.4
53.7 ± 0.5
100.9 ± 7.5
22.2 ± 1.7
1β, 2β, 3β, 19α-tetrahydroxy-12-en-28-oic acid
265.2 ± 2.0
α-glucosidase inhibition activity
Previously some plant extracts have been reported to inhibit α-glucosidase activity, such as the ethanol exract of Andrographis paniculata (IC50 = 17.20 mg/mL) , the ethanol extracts of Mangifera indica bark (IC50 = 314 μg/mL) , and the butanol extract of Acosmium panamense (IC50 = 109 μg/mL) . Compared with these reported natural extracts, the FC and the TC from APL showed excellent α-glucosidase inhibitory activities. In addition, some natural products could exhibit different inhibition modes against α-glucosidase, such as the methanol fraction of Bitter melon in an uncompetitive manner with an IC50 value of 2.60 mg/mL . Our results also show that the FC and the TC from APL inhibit α-glucosidase in different modes, i.e. the FC in competitive mode and the TC in noncompetitive inhibition mode. In addition, it was reported that corosolic acid from Lagerstroemia apeciosa leaves exhibit a nocompetitive mode with an IC50 value of 3.53 μg/mL . And corosolic acid with the content of 100.9 mg/g also is the second abundant triterpenoid in the TC (Table 2). Furthermore, the inhibition activities against α-glucosidase of the complexes with different mass ratio of FC and TC were assayed based on their different inhibitory modes. The result reveals that the combination with mass ratio of FC and TC as 4:1 has superiority inhibition activity against α-glucosidase to the other complexes with mass ratio of FC and TC as 1:4 and 1:1 at the concentration range of 4.17- 33.33 μg/mL. But when the concentration is greater than 33.33 μg/mL, the complexes (FC: TC = 1:4, g/g) has better α-glucosidase inhibition activity than others. Based on the results mentioned above, we could propose that, at the low concentration, the competitive inhibition is dominant, but the noncompetitive inhibition has a little advantage at the high concentration. So we think that the APL has a potential treating effect on T2MD, which is attributed partly to the α-glucosidase inhibition activities of the FC and the TC for the control of the postprandial hyperglycaemia.
In vitro antioxidant activity
In order to obtain the credible conclusion, four assays including DPPH scavenging assay, hydroxyl radical scavenging assay, ABTS radical scavenging assay and β-carotene-linoleic acid assay were employed to evaluate antioxidant activities of the FC and the TC.
The EC 50 values and IC 50 values of flavonoid compound (FC) and triterpenoid compounds (TC) from agrimonia pilosa ledeb
Radical scavenging (EC50, μg/mL)
β-carotene bleaching (IC50, μg/mL)
7.7 ± 0.4
3.6 ± 0.2
5.9 ± 0.4*
41.8 ± 1.0*
35.2 ± 0.3*
98.5 ± 1.2*
7.7 ± 0.2
3.1 ± 0.1
1.8 ± 0.2
2.5 ± 0.1
Among the quantified 10 flavonoids in FC, 6 flavonoids including quercetin, luteolin, rutin, hyperoside, luteolin-7-O-β-D-glucopyranoside and quercitrin have excellent scavenging activities on radical due to the o-catechol group (3′, 4′–OH) in B ring . These 6 flavonoids making up 65.4% of the quantified 10 flavonoids could be responsible for the FC’s significant scavenging activities on DPPH radical, hydroxyl radical and ABTS radical. Radicals induced by postprandial hyperglycaemia or other reasons [4, 31], such as superoxide anion radical and hydroxyl radical, have the very high reactivity which enables them to react with a wide range of molecules, such as protein, lipids, and nucleotides leading to occurrence and development of a variety of diseases including T2DM [3, 32]. The remarkable scavenging activities of the FC on radical imply that the FC could be responsible for the good clinical effects of APL on T2MD targeting oxidative stress.
In conclusion, our findings suggest that the FC and the TC could be responsible for the good clinical effects of APL on T2MD through targeting oxidative stress and postprandial hyperglycaemia. So APL may be good sources of natural antioxidants and α-glucosidase inhibitors exhibiting remarkable potential application in the therapy of T2DM. Further investigations should be taken to illustrate the pharmaco-mechanism deeply and to isolate the active components.
This work was supported by the Fundamental Research Funds for the Central Universities (Project No. CQDXWL-2012-130), Program for Innovation Team Building at Institutions of Higher Education in Chongqing (KJTD201325), the Natural Science Foundation Project of CQ CSTC (No. 2011BB5109), and Visiting Scholar Foundation of Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education (CQKLBST-2012-007).
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