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Antioxidant Capacities andAcetylcholinesterase-inhibitory Activityof Hoppea fastigiataUtkarsh Ravindra Moon a , Sukanta Kumar Sen b & Adinpunya Mitra aa Agricultural and Food Engineering Department, Natural ProductBiotechnology Group , Indian Institute of Technology Kharagpur ,Kharagpur , Indiab Department of Botany, Microbiology Division , Visva-Bharati (aCentral University) , Santiniketan , IndiaPublished online: 14 Feb 2014.

To cite this article: Utkarsh Ravindra Moon , Sukanta Kumar Sen & Adinpunya Mitra (2014) AntioxidantCapacities and Acetylcholinesterase-inhibitory Activity of Hoppea fastigiata , Journal of Herbs, Spices& Medicinal Plants, 20:2, 115-123, DOI: 10.1080/10496475.2013.840711

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Journal of Herbs, Spices & Medicinal Plants, 20:115–123, 2014Copyright © Taylor & Francis Group, LLCISSN: 1049-6475 print/1540-3580 onlineDOI: 10.1080/10496475.2013.840711

Antioxidant Capacities andAcetylcholinesterase-inhibitory Activity

of Hoppea fastigiata

UTKARSH RAVINDRA MOON,1 SUKANTA KUMAR SEN,2

and ADINPUNYA MITRA1

1Agricultural and Food Engineering Department, Natural Product Biotechnology Group,Indian Institute of Technology Kharagpur, Kharagpur, India

2Department of Botany, Microbiology Division, Visva-Bharati (a Central University),Santiniketan, India

Antioxidant capacities of solvent extracts of Hoppea fasti-giata were studied by analyzing free radical scavengingactivities 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and ferric reducingantioxidant potential (FRAP). The ethanolic extract showed a com-paratively higher amount of total phenolics, free radical scavengingactivities DPPH, ABTS, and FRAP. Qualitative antioxidant activityof ethanolic extract was tested by DNA nicking assay. The ethanolicextract showed significant acetylcholinesterase (AChE)-inhibitoryactivity with the IC50 value of 1.1 mg dry weight mL−1.

KEYWORDS Hoppea fastigiata, antioxidant capacity,acetylcholinesterase-inhibitory activity

INTRODUCTION

Hoppea fastigiata (Gentianaceae) is an endemic and threatened medicinalplant of India (2). This genus has been used by the local Indian tribesfor various ailments (1). A related species, H. dichotoma, is known for its

Received April 30, 2013.Address correspondence to Adinpunya Mitra, Agricultural and Food Engineering

Department, Natural Product Biotechnology Group, Indian Institute of Technology Kharagpur,Kharagpur 721 302, India. E-mail: adin@iitkgp.ac.in; adinpunya@gmail.com

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/whsm.

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use in the Indian system of medicine for the treatment of certain men-tal disorders including epilepsy (9). Plants from Gentianaceae family havebeen analyzed for xanthones, flavonoids, flavone-C-glycosides, and phenolicacids (7). Several xanthones such as 1,7-dihydroxy-3,5-dimethoxyxanthone,1,5-dihydroxy-3,7-dimethoxyxanthone, 1,5,7-trihydroxy-3-methoxyxanthone,1,3,8-trihydroxy-7-methoxyxanthone, and 1, 3, 6-trihydroxy-8-methoxy xan-thone have been identified from extracts of H. fastigiata (6,15–17). Severalxanthones isolated from a related Gentianaceae member such as Swertialongifolia was found to be bioactive (11).

Antioxidants are the scavengers of free radicals that are formed dur-ing oxidation (5). Scavenging is done by the donation of hydrogen atomsto the free radicals. An imbalance between the antioxidants and the reac-tive oxygen species results in the oxidative stress that is responsible for thecell death leading to the formation of certain neurodegenerative diseases,such as Alzheimer’s disease (AD) (12,23). AD is the most common agingdisease related to low level of acetylcholine. AChE enzyme is responsiblefor degrading the neurotransmitter acetylcholine in the synaptic cleft and isoften related to the onset of AD (13). One approach for the treatment of thisdisease would be to retain the normal levels of acetylcholine in the brainwith the help of AChE inhibitors (24).

MATERIALS AND METHODS

Plant Material Collection and Identification

H. fastigiata plants were collected from the Reserve Forest of theGovernment of West Bengal at Santiniketan, (adjacent to Visva-Bharaticampus), India. The plant was identified on the basis of reproductive mor-phology (19) by the Department of Botany, Visva-Bharati, and a voucherspecimen collection no. VBH-129-89 was deposited in the herbarium.

Extract Preparation

The plant material was washed and dried thoroughly and kept for dryingin an oven at 40◦C. The dried plants were powdered in a grinder, and100 mg was extracted with 2 mL of solvents (water, ethanol, ethylacetate, andpetroleum ether) in an ultrasonic bath for 45 min, centrifuged at 10,000 rpmfor 10 min (MiniSpinplus), and the supernatant used for the study. All datawere expressed as mean ± SD.

Determination of Total Phenolic Content

Estimation of total phenolic content was done as described previously (3).Crude extract of plant (100 µL) was mixed with 10% Folin-Ciocalteu reagent

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(200 µL) and 800 µL of Na2CO3 (700 mM) and vortexed vigorously. Thetubes were incubated for 2 h at room temperature. Increase in absorbancewas then measured at 765 nm. Total phenolic content value was obtainedby comparing the absorbance change of solution containing the extract at765 nm with that of calibration plot of gallic acid and was expressed as mggallic acid equivalent (GAE).g−1 fresh mass.

2,2-diphenyl-1-picrylhydrazyl Free Radical Scavenging Assay

A 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was determined as previouslydescribed (5). The free radical scavenging activity of the extracts was testedusing DPPH free radical. Crude plant extracts (0.1 mL) were mixed withthe 0.9 mL ethanolic solution of DPPH (0.1 M). The mixture was vortexedand kept in dark for 30 min at room temperature. The decrease in O.D wasmonitored at 517 nm. The donation of protons by antioxidants to free radicalscaused the decolorization of DPPH solution. This decrement in color wastaken as a measure of scavenging activity. Concentrations (0.06–0.4 mg.mL−1)of four extracts were tested for the determination of IC50 value.

ABTS Free Radical Scavenging Assay

ABTS assay was performed as previously described with slight modifications(20). ABTS was dissolved in water to 7 mM concentration. The ABTS freeradical (ABTS–) was prepared by adding 2.45 mM potassium persulfate toABTS solution and incubating in dark for 12 to 16 h. For working solution,absorbance of ABTS solution was adjusted by diluting it with ethanol to0.7 (± 0.02) at 734 nm. The reaction mixture consisted of working solution(995 µL) and crude extract (5 µL). The mixture was then vortexed and incu-bated for 6 min at room temperature and decrease in absorbance monitoredat 734 nm. ABTS free radical imparts blue color, which is formed by theaction of ABTS salt and potassium persulfate. The donation of protons toABTS free radicals causes decolorization, which is used as the measure ofscavenging potential.

Ferric-Reducing Antioxidant Power Assay

The reducing power of the extract was determined by ferric-reducingantioxidant power (FRAP) (4) with slight modifications. Stock solutionsincluded TPTZ (2,4,6-tripyridyl-s-triazine) solution (10 mM), which was pre-pared in HCl (40 mM), FeCl3.6H2O (20 mM), and acetate buffer (300 mM,pH 3.6). The fresh working solution was prepared by mixing 25 mL acetatebuffer, 5 mL TPTZ, and 5 mL FeCl3.6H2O. The extracts (3 µL) were allowed to

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react with FRAP working solution (997 µL). The mixture was then incubatedfor 6 min at 37◦C and absorbance recorded at 593 nm.

DNA Nicking Assay For Non-Site Specific–OH Scavenging Activity

The nicking assay was performed as previously described with slight mod-ifications (14). The reaction mixture consists of 13 µL of KH2PO4 buffer(50 mM, pH 7.4); 4 µL of EDTA-Na2 (30 mM); 4 µL of H2O2 (30 mM); 4 µLof FeSO4 (16 mM), 1 µL of pUC18 plasmid; and 4 µL of crude extract. Thereaction mixture was incubated at 37◦C for 1 h. Then 6 x Fermentas loadingdye was added, and the reaction mixture was analyzed by agarose horizontalslab gel electrophoresis at 100 V.

Acetylcholinesterase Inhibitory Activity

Acetylcholinesterase (AChE) inhibitory property of different solvent extractswas studied as described previously with slight modifications (8). Thereaction mixture consisted of 500 µL of 3 mM DTNB (5, 5-dithiobis [2-nitrobenzoic acid]), 46 µL of 15 mM ATCI (acetylthiocholine iodide), 356 µLof 50 mM Tris-HCl buffer (pH 8.0), sample extract (concentration required),100 µL 0.28 U/mL AChE. The reaction mixture was monitored at 405 nmfor 5 min. For IC50 value calculations, the absorbance was recorded afterincubating reaction mixtures for 10 min. A standard AChE inhibitor eserinesalicylate was used as positive control.

RESULTS

Total Phenolic Content

Results showed that with increase in polarity, TPC increased, except inaqueous extract (Table 1). High concentration of TPC was obtained from

TABLE 1 Antioxidant Capacities and TPC Contents of Hoppea fastigiata

Sample/Extract DPPHa ABTSb FRAPc TPCd

Aqueous 1.21 ± 0.2 21 ± 0.03 10.83 ± 0.4 27.36 ± 1.1Ethanol 0.27 ± 0.03 45.9 ± 3.23 40.8 ± 1 134.4 ± 3.1Ethyl acetate 1.22 ± 0.14 38.4 ± 1.2 21.075 ± 0.63 15 ± 0.1Petroleum ether 4.53 ± 0.6 18.3 ± 1.3 6.75 ± 0.1 0.72 ± 0.1Ascorbic acid 0.00207 ± 0.0009 – – –

aDPPH IC50 value (mg DW mL−1).bABTS value in terms of AEAC (mM/g DW ascorbic acid equivalent).cFRAP value (mM Fe2+ equivalent/g DW).dTotal phenolic content (mg GAE/g DW).Note: All data are expressed as mean ± SD (n = 3).

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ethanolic extract (134.4 ± 3.1 mg GAE.g−1 DW). After ethanolic extract,aqueous extract showed more TPC (27.36 ± 1.1 mg GAE.g−1 DW), but itwas very less as compared to TPC of ethanolic extract (see Table 1).

Antioxidant Capacities

DPPH FREE RADICAL SCAVENGING ASSAY

The radical scavenging potential of the extracts was dose-dependent(Figure 1). It was observed that ethanolic extracts showed higher potentialfor scavenging DPPH free radical with the lowest IC50 value of 0.27 ± 0.03 mgdry weight m−1 compared to aqueous, ethylacetate, and petroleum ether sol-vent extracts, which showed comparatively less scavenging ability with theIC50 value of 1.21 ± 0.2, 1.22 ± 0.14, and 4.53 ± 0.6 mg dry weight mL−1,respectively (Table 1).

ABTS FREE RADICAL SCAVENGING ASSAY

Ascorbic acid equivalent antioxidant capacity (AEAC) value for the ethanolicextract was found to be 45.9 ± 3.23 mM ascorbic acid equivalent/g dryweight, which is higher as compared to other solvent extracts that showed21 ± 0.03 (aqueous), 38.4 ± 1.20 (ethylacetate), and 18.3 ± 1.30 (petroleumether) mM ascorbic acid equivalent.g−1 dry weight (Table 1).

FRAP ASSAY

Reducing power of the extracts was represented in terms of FRAP value,which also indicated that ethanolic extracts has highest potency for reducing

FIGURE 1 DPPH radical scavenging activity of Hoppea fastigiata in solvent extracts.

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Form II

Form III

Form I

Lane 1 Lane 2 Lane 3

FIGURE 2 Non-site-specific •OH scavenging activity of Hoppea fastigiata (ethanolic extract)as measured by DNA nicking assay. Lane 1, Control Plasmid DNA (pUC 18); Lane 2, Fenton’sreagent + Plasmid DNA; Lane 3, Fenton’s reagent + Plasmid DNA + ethanolic crude extract(2.5mg.µL−1). (Form II- Linear DNA; Form III- double stranded nicked and linear DNA; FormI- supercoiled form).

ferric ions. The FRAP value for ethanolic extracts was found to 40.8 ±1 mM Fe2– equivalent/g dry weight, which was highest as compared toother solvent extracts that showed 10.83 ± 0.4 (aqueous), 21.075 ± 0.63(ethylacetate), and 6.75 ± 0.1 (petroleum ether) mM Fe2– equivalent.g−1 dryweight (Table 1).

DNA nicking assay was also performed to check the abilities of ethanolicextract to protect the plasmid DNA against the Fenton’s reagents –OH radicaldamage. The .OH radical cleaved pUC18 plasmid DNA in the non-site-specific manner leading to the formation of linear DNA and degradationof supercoiled DNA. Presence of peroxidizing agents such as Fenton reagentin the reaction mixture (Lane 2) caused the breakage of supercoiled formof plasmid DNA into double stranded nicked form (Form III) or linearform (Form II). The cleavage of plasmid DNA is prevented by the com-pounds scavenging free radicals. Presence of ethanolic extract of H. fastigiata(0.5 mg µL−1) in the reaction mixture (Lane 3) scavenged the –OH rad-ical, preventing the breakage of plasmid DNA (see Figure 2). Thus, thesupercoiled form (Form I) of plasmid DNA is retained in Lane 3.

ACETYLCHOLINESTERASE INHIBITORY ACTIVITY

Extracts of H. fastigiata were also screened for the acetylcholinesteraseinhibitory activities. The ethanolic crude extract inhibited AChE enzymeeffectively with the IC50 of 1.1 mg dry weight. mL−1 than other solvents,which showed IC50 value of 9.8, 3.3, 2.8 mg.dry weight mL−1 for aqueous,ethylacetate, and petroleum ether, respectively (see Table 2).

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TABLE 2 Acetylcholinesterase Inhibitory Activities of Hoppea fastigiata Solvent Extracts

Sample/Extract Concentrationa % Inhibition IC50 valueb

Aqueous 1 5.92 ± 0.2 9.8Ethanol 1 48.9 ± 3.1 1.1Ethylacetate 1 22.2 ± 1.9 3.3Petroleum ether 1 14.1 ± 0.8 2.8Eserine salicylatec in (H2O) 1 100 0.0013

aConcentration (mg dry weight.mL−1).bIC50 value (mg dry weight.mL−1).cPositive control.Note: Data are expressed as mean ± SD (n = 3).

DISCUSSION

Plant extracts with higher levels of total phenolics and flavonoids wereknown to exhibit greater free radical scavenging activities (10). It wasobserved that among all the solvents, ethanol was the most effective sol-vent for the whole plant extraction of H. fastigiata in terms of antioxidantcapacities, TPC, and AChE inhibition. Because of the presence of hydroxylgroup in the aromatic ring of the compounds present in the extract, thesecompounds mediate redox reactions and thus are capable of scavenging freeradicals (18,21). The ability to dissolve a special group of antioxidant com-pounds altered upon changes in solvent polarity, which in turn affect theantioxidant capacity estimation (25). Thus, the extracts obtained using morepolar solvent, as ethanol, showed higher antioxidant capacities compared toless polar solvents, as ethyl acetate and petroleum ether.

Xanthones isolated from several genera of Gentianaceae were reportedto have potent inhibitory properties against acetylcholinesterase andmonoamine oxidase enzymes (22). Presence of xanthones from wholeplant extracts of H. fastigiata was reported earlier (6,15–17). The chemi-cal structure of the xanthone compounds reported from H. fastigiata arebroadly similar to those found in several species of Gentiana showing thoseinhibitory activities. This suggested that AChE inhibitory activity of H. fasti-giata extract could be due to the presence of xanthone compounds inthe extract. Ethanolic H. fastigiata extract showed very less IC50 value andindicated a strong AChE inhibitory potential. This observation indicated apositive relationship between the TPC and AChE inhibition of the ethanolicextract. The AChE inhibitory activities of ethylacetate and petroleum etherwere almost the same, but the TPC of ethylacetate extract was more or less20 times higher than the petroleum ether extract.

A xanthone having a rare substitution pattern was isolated frompetroleum ether extract of the whole plant of H. fastigiata and was sub-sequently identified on the basis of spectral and chemical properties, as1,7-dihydroxy-3,5-dimethoxyxanthone (15). This particular xanthone present

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in the petroleum ether extract may contribute to the inhibition of AChE activ-ity in vitro. Thus, this study indicated that H. fastigiata nature-grown plantshave the potential to scavenge free radicals and to inhibit AChE enzymeresponsible for Alzheimer’s disease.

FUNDING

Utkarsh Ravindra Moon thanks University Grants Commission, India for theaward of an individual junior research fellowship [F. No. 2-16/98 (SA-I), dated04-21-2011].

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