Food Chemistry 132 (2012) 244–251

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Food Chemistry

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Antioxidant activity of a new phenolic glycoside from Lagenaria siceraria Stand.fruits

Rahul Mohan, Rahul Birari, Aniket Karmase, Sneha Jagtap, Kamlesh Kumar Bhutani ⇑Department of Natural Products, National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar, Mohali 160 062, Punjab, India

a r t i c l e i n f o

Article history:Received 19 April 2011Received in revised form 3 October 2011Accepted 19 October 2011Available online 28 October 2011

Keywords:Lagenaria sicerariaPhenolic glycosideNMRHMBC

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.10.063

⇑ Corresponding author. Tel./fax: +91 (0)172223220E-mail address: kkbhutani@niper.ac.in (K.K. Bhuta

a b s t r a c t

The antioxidant properties of different extracts of Lagenaria siceraria (bottle gourd) fruit were evaluated.In the process, a new phenolic glycoside (E)-4-hydroxymethyl-phenyl-6-O-caffeoyl-b-D-glucopyranoside(1) was isolated and identified together with 1-(2-hydroxy-4-hydroxymethyl)-phenyl-6-O-caffeoyl-b-D-gluco-pyranoside (2), protocatechuic acid (3), gallic acid (4), caffeic acid (5) and 3,4-dimethoxy cinnamicacid (6). Their structures were elucidated by extensive NMR experiments including 1H–1H (COSY) and1H–13C (HMQC and HMBC) spectroscopy and chemical evidences. The antioxidant potential of the com-pound 1 and 2 was tested in different in vitro assay systems such as free radical scavenging assay, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, superoxide scavengingactivity, reducing power assay and linoleic acid peroxidation assay.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Redox reactions are crucial for the success of many biologicalprocesses (Liu & Nair, 2010). Although free radicals are consideredto be important for normal physiology, they cause cellular damagewhen produced in excess. Oxygen consumption inherent in cellgrowth leads to the generation of a series of reactive oxygen spe-cies (ROS). ROS include free radicals such as superoxide anion rad-icals ðO��2 Þ, hydroxyl radicals (�OH) and non-free radical speciessuch as hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Ak &Gulicn, 2008). Many antioxidant defense systems in the body suchas superoxide dismutase (in mitochondria and cytosol), catalase (inperoxisomes), glutathione peroxidase, and a-tocopherol (in mem-branes and lipoproteins), etc. limit the levels and damage causedby free radicals (Kaur & Geetha, 2006). This multiple defense sys-tem fails due to increased production of ROS or decreased levelof cellular antioxidants. ROS are capable of damaging crucial bio-molecules such as nucleic acids, lipids, proteins and carbohydratesand even causes DNA damage that can lead to mutations (Shahidi,Liyana-Pathirana, & Wall, 2006). ROS have been implicated in morethan 100 diseases including atherosclerosis, hypertension, ische-mic diseases, Alzheimer0s disease, Parkinson0s disease, cancer andinflammatory conditions (Behera, Adawadkar, & Makhija, 2003).

A number of synthetic antioxidants such as butylated hydroxy-anisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhy-droquinone (TBHQ) have been developed so far, but their use astherapeutic agents has been hampered by their toxicity (Wichi,

ll rights reserved.

8.ni).

1988). Thus, there is a need to identify newer antioxidants, whichcan scavenge several free radicals and prevent multiple diseases.Antioxidants from natural resources provide enormous scope incorrecting the imbalance. Therefore, much attention is being direc-ted to harnessing and harvesting antioxidants from natural re-sources (Gao et al., 2011; Masuda et al., 1999; Rehman, 2006;Vitalini, Braca, Passerella, & Fico, 2010).

Numerous epidemiological studies have revealed the signifi-cance of high uptake of fruits/vegetables in routine diet for preven-tion of chronic diseases. Polyphenolic compounds present in thefruits are considered to be responsible for their protective effectsagainst oxidative damage through their strong antioxidative prop-erties (Shahidi & John, 2010). Some widely consumed beverageslike tea, red wine and cocoa, well known for their high antioxidantactivities, are reported to be rich in phenolic constituents (Tabart,Kevers, Pincemail, Defraigne, & Dommesa, 2009). Additionally,such compounds display antiviral and antimicrobial activity andcan chelate iron, inhibit enzymes (matrix metalloproteinase’s),regulate gene expression, and significantly improve endothelialfunction (Lee, Kim, Lee, & Lee, 2003). Thus, exploration of edibleplants as sources of physiologically active compounds offers enor-mous opportunities for the development of novel health promotingfoods.

Lagenaria siceraria Stand. (Cucurbitaceae), popularly known asbottle gourd is a climbing perennial plant used as a common veg-etable in India (Ghule, Ghante, Saoji, & Yeole, 2009; Ghule, Ghante,Upaganlawar, & Yeole, 2006). The fruit finds its medicinal value intraditional Indian medicine and has been used as cardiotonic, aph-rodisiac, general tonic, hepatoprotective, analgesic, anti-inflamma-tory, expectorant and diuretic (Ghule, Ghante, Yeole, & Saoji, 2007).

R. Mohan et al. / Food Chemistry 132 (2012) 244–251 245

Further, antihepatotoxic activity of fruit pulp (Deshpande et al.,2008; Shirwaikar & Sreenivasan, 1996), analgesic and anti-inflam-matory activity of fruit juice have also been evaluated (Itoh,Kikuchi, Tamura, & Matsumoto, 1981; Mohale, Dewani, Saoji, &Khadse, 2009). Limited studies have been reported on the antioxi-dant activity of the fruit extract (Deshpande, Mishra, Meghre,Wadodkar, & Dorle, 2007; Erasto & Mbwambo, 2009). Phytochem-ical screening on L. siceraria fruit has revealed the presence offucosterol and compesterols (Enslin, Holzapfel, Norton, & Rehm,1967), flavonoids, cucurbitacins, saponins, polyphenolics, triterpe-noids (Chen, Chen, & Chang, 2008), C-flavone glycosides and ellag-itannins (Krauze-Baranowska & Cisowski, 1994).

As our current interest involves the evaluation of health-pro-moting phytochemicals, we investigated the total phenolic and to-tal flavonoid content of various extracts of fruits of L. siceraria.Further the antioxidant activity of these extracts and compound1 and 2 isolated from the EtOAc extract of the bottle gourd fruitswere studied in different in vitro assay systems such as free radicalscavenging assay, MTT reduction assay, superoxide scavengingactivity, reducing power assay and linoleic acid peroxidation as-say. This paper also deals with the isolation and structure elucida-tion of a new phenolic glycoside (1) together with five knowncompounds.

2. Materials and methods

2.1. Plant material

Bottle gourd (L. siceraria Stand.) fruits were obtained from a lo-cal market in Chandigarh, India. Fruits were identified in theDepartment of Natural Products (National Institute of Pharmaceu-tical Education and Research, NIPER) and a voucher specimen (Her-barium No. NIP: 151) is available in the herbarium of Departmentof Natural Products, NIPER.

2.2. Instruments

The 1H and 13C NMR spectra were recorded on Ultrashield 400NMR spectrometer (Bruker, Faellanden, Switzerland). The opticalrotations were measured on Autopol IV polarimeter (Rudolph,Flanders, NJ, USA). Mass spectra were recorded on a LCQ (Thermo-Quest Finnigan, San Jose, CA, USA). The IR spectra were performedon a FTIR system (Impact 410, Nicolet Instrument. Corp., Madison,Wisconsin, USA). Absorbances were recorded on ELISA plate reader(Labsystem, Helsinki, Finland). The extracts/compounds were driedon rotary vacuum evaporator (Buchi, Flawil, Switzerland).

2.3. Chemicals and reagents

The chemicals used in this study were of analytical reagentgrade that include: 1,1-diphenyl-2-picrylhydrazyl (DPPH�), trolox,2,20-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), di-methyl sulphoxide (DMSO), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nitro blue tetrazolium(NBT), xanthine, xanthine oxidase from bovine milk, superoxidedismutase (SOD), linoleic acid (Sigma Chemical Co., St. Louis, MO,USA); trichloroacetic acid, potassium ferricyanide, hydrogen per-oxide, ferrous sulphate, ferrous chloride (Loba chemie, Mumbai, In-dia); ammonium thiocyanate (99.99%), gallic acid, quercetindihydrate, Folin–Ciocalteau’s phenol reagent (S.D. Fine-Chem Ltd.,Mumbai, India); hydrochloric acid, sulphuric acid, aluminium tri-chloride, ferric chloride (Spectrochem, Mumbai, India); 2-thiobar-bituric acid, L-ascorbic acid (Himedia, Mumbai, India), silica gel(#60–120) (Loba chemie, Mumbai, India).

2.4. Extraction and isolation

Fruits of L. siceraria (1 kg), oven dried at 40 �C for 3 days, werepowdered using cutter mill. The powdered fruits (particle size#20) were defatted using hexane (5 L) as solvent in a Soxhletextraction unit. The defatted plant material (970 g) was air-driedand successively extracted with 5 L of each solvent to give three ex-tracts: CH2Cl2 extract (LDE; 1.45%), EtOAc extract (LEE; 2.26 %) andMeOH extract (LME; 4.88 %) respectively. The extracts were driedunder vacuum on a rotary evaporator at 40 �C to pursue furtheranalysis. LEE (20 g) was fractionated on vacuum liquid chromatog-raphy on silica gel (TLC grade 500 g). Elution was carried out suc-cessively with solvents of increasing polarity of n-hexane/EtOAc9:1, 4:1, 7:3, 3:2, 1:1 (v/v); CH2Cl2/MeOH 49:1, 19:1, 13:1, 9:1,4:1, 3:2, 1:1 (v/v). Each of 500 mL of fraction was collected. Basedon the TLC pattern [toluene/EtOAc/acetic acid: 5:4:1 (v/v/v) andEtOAc/acetic acid/formic acid/water: 100:11:11:26 (v/v/v/v)] usingNatural Product – Polyethylene glycol (NP-PEG) reagent, fractionswere pooled into seven major fractions (A to G). Fraction F (2.3 g)was subjected to liquid chromatography on silica gel (#60–120)column (30 cm � 4 cm) and eluted with gradient of CH2Cl2/MeOH49:1, 19:1, 13:1, 9:1 (v/v). CH2Cl2/MeOH (19:1) fraction was driedon rotary evaporator and further purified on Sephadex LH-20 col-umn (40 cm � 2 cm) using MeOH to yield compound 1 (25 mg)and compound 2 (15 mg). Fraction D (5.1 g) was subjected to liquidchromatography on silica gel (#60–120) column (40 cm � 4.5 cm)and eluted with gradient of n-hexane/EtOAc 9:1, 4:1, 7:3, 3:2, 1:1,2:3, 3:7, 1:4, 1:9 (v/v) to afford three major fractions D1–D3. Frac-tion D2 (1.2 g) purified by liquid chromatography on silica gel(#60–120) column (30 cm � 4 cm) using n-hexane–EtOAc gradientto yield compounds 3 (32 mg) and 4 (45 mg). Compounds 5 (30 mg)and 6 (20 mg) were obtained from purification of fraction D3 (1.4 g)using liquid chromatography on silica gel (#60–120) column(30 cm � 4 cm).

2.5. Acid hydrolysis and sugar analysis

Compound was dissolved in 0.1 M H2SO4 (5 mL) and refluxedfor 2 h. After cooling the reaction mixture was extracted withEtOAc. The aqueous layer was neutralised with NaHCO3, concen-trated to dryness, and extracted with pyridine.

2.6. Characterisation

2.6.1. (E)-4-hydroxymethyl phenyl-6-O-caffeoyl-b-D-glucopyranoside(1)

Pale yellow powder (25 mg); ½a�20D �77.78 (c 0.25, MeOH); ESI-

MS: m/z 471 [M+Na]+, HR-ESI-MS (C22H24O10) m/z 471.1262[M+Na]+, (calculated for C22H24O10Na, 471.1267); IR mKBr

max cm�1

3399, 2926, 1725, 1378, 1275, 1071, 826, 750; 1H NMR (CD3OD,400 MHz): d ppm 7.5 (1H, d, J = 15.9 Hz, H-70), 7.2 (2H, d,J = 8.4 Hz, H-300, H-500), 7.05 (1H, d, J = 2.1 Hz, H-20), 7.03 (2H, dJ = 8.4 Hz, H-200, H-600), 6.95 (1H, dd, J = 8.2, 2.1 Hz, H-60), 6.78(1H, d, J = 8.2 Hz, H-50), 6.2 (1H, d, J = 15.9 Hz, H-80), 4.5 (1H, dd,J = 11.8, 2.0 Hz, H-6a), 4.4 (2H, s, H-700), 4.3 (1H, dd, J = 11.8,7.2 Hz, H-6b), 3.7 (1H, m, H-5), 3.47–3.49 (2H, overlapping, H-2,H-3), 3.40 (1H, m, H-4); 13C NMR (CD3OD, 100 MHz): d ppm168.9 (C-90), 158.2 (C-100), 149.6 (C-40), 147.1 (C-70), 146.8 (C-30),136.6 (C-400), 129.4 (C-300, C-500), 127.7 (C-10), 123.0 (C-60), 117.7(C-200, C-600), 116.5 (C-50), 115.2 (C-80), 114.9 (C-20), 102.2 (C-1),77.9 (C-3), 75.5 (C-5), 74.8 (C-2), 71.9 (C-4), 64.6 (C-6), 64.7 (C-700).

1H NMR ([(CD3)2 CO], 400 MHz): d ppm 7.56 (1H, d, J = 15.9 Hz,H-70), 7.28 (2H, d, J = 8.6 Hz, H-300, H-500), 7.17 (1H, d, J = 2 Hz, H-20),7.08 (1H, dd, J = 8.6, 1.7 Hz, H-60), 7.05 (2H, d, J = 8.6 Hz, H-20, H-600),6.89 (1H, d, J = 8.2 Hz, H-50), 6.33 (1H, d, J = 15.9 Hz, H-80); 4.9 (1H,d, J = 7.4 Hz, H-1), 4.63 (1H, dd, J = 11.8, 2.1 Hz, H-6a), 4.5 (2H, s,

246 R. Mohan et al. / Food Chemistry 132 (2012) 244–251

H-700), 4.27 (1H, dd, J = 11.8, 7.3 Hz, H-6b), 3.81 (1H, m, H-5),3.46–3.52 (3H, m, H-2, H-3, H-4).

2.6.2. 1-(2-hydroxy-4-hydroxymethyl) phenyl-6-O-caffeoyl-b-D-glucopyranoside (2)

Yellow powder (14.8 mg), ESI-MS: m/z 463 [M�1]+, IR mKBrmax cm�1

3399, 2928, 2241, 1733, 1601, 1451, 1275, 1129, 1067, 826, 750;1H NMR (CD3OD, 400 MHz): d ppm 7.5 (1H, d, J = 15.8 Hz, H-70),7.10 (1H, d, J = 8.2 Hz, H-600), 7.05 (1H, d, J = 1.8 Hz, H-20), 6.9 (1H,dd, J = 8.2, 1.8 Hz, H-60), 6.88 (1H, d, J = 1.9 Hz, H-300), 6.8 (1H, d,J = 8.2 Hz, H-50), 6.67 (1H, dd, J = 8.2, 1.9 Hz, H-500), 6.3 (1H, d,J = 15.8 Hz, H-80), 4.7 (1H, d, J = 7.12 Hz, H-1), 4.5 (1H, dd, J = 2.0,12.0 Hz, H-6a), 4.4 (2H, s, H-700), 4.3 (1H, dd, J = 7.0, 12.0 Hz, H-6b), 3.7 (1H, m, H-5), 3.5 (1H, m, H-2), 3.49 (1H, m, H-3), 3.42(1H, m, H-4); 13C NMR (CD3OD, 100 MHz): d ppm 168.9 (C-90),149.7 (C-30), 148.2 (C-200), 147.2 (C-70), 146.8 (C-40), 145.8 (C-100),138.4 (C-400), 127.7 (C-10), 123.1 (C-60), 119.6 (C-500), 118.5 (C-600),116.5 (C-50), 116.0 (C-300), 115.2 (C-80), 114.8 (C-20), 104.1 (C-1),77.5 (C-3), 75.7 (C-5), 74.8 (C-2), 71.8 (C-4), 64.8 (C-700), 64.6 (C-6).

2.7. Determination of total phenolic in the plant extracts

Total phenolics were determined using the reported method(Garzon, Riedl, & Schwartz, 2009). A 20 lL sample aliquot of ex-tract or gallic acid/tannic acid standard (50–500 lg/mL) was mixedwith distilled water (1.58 mL) followed by 2 N Folin–Ciocalteau’sreagent (100 lL). After vortexing and incubating at room tempera-ture for 8 min, aqueous sodium carbonate solution (20%, 300 lL)was added. Samples were vortexed and kept at room temperaturefor 2 h. Absorbance of the blue-colour solution was recorded at725 nm. The experiment was performed in triplicate. The totalphenolic content was expressed as gallic acid equivalent (GAE)and tannic acid equivalent (TAE).

2.8. Determination of total flavonoid content in the plant extracts

Total flavonoid content was determined by the aluminium chlo-ride colorimetric method using quercetin dihydrate as a standard(Ismail, Chan, Mariod, & Ismail, 2010; Quettier-Deleu et al.,2000). Briefly, the extracts and compounds were individually dis-solved in DMSO. Then, the sample solution (100 lL) was mixedwith an aqueous solution of anhydrous aluminium chloride (2%,100 lL). After 10 min of incubation at ambient temperature, theabsorbance of the supernate was measured at 435 nm. The exper-iment was performed in triplicate. The total flavonoid content wasexpressed as quercetin dihydrate equivalents.

2.9. Antioxidant activity

2.9.1. DPPH radical scavenging assayFree radical scavenging activity of the test compounds/extracts

was determined by the DPPH assay described by Gautam, Srivast-ava, Jachak, and Saklani (2010) and Shahidi et al. (2006). Briefly200 lL reaction mixture contained methanolic solution of DPPH�

(190 lL) and 10 lL of different concentrations of test com-pounds/extracts in MeOH. The reaction mixture was incubatedfor 1 h at room temperature and the absorbance was measuredat 517 nm. The change in absorbance with respect to the control(containing DPPH� only without sample, expressed as 100% freeradicals) is calculated as percentage scavenging and the IC50 valuewas calculated. Trolox and ascorbic acid were used as positive con-trols. The assay was performed in triplicate.

2.9.2. ABTS radical scavenging assayABTS radical scavenging activity of the test compounds was

determined using an ABTS�+ decolorisation assay (Lee, Kim, Jang,

Jung, & Yun, 2007). To the ABTS liquid substrate system 2.45 mMpotassium persulphate was added in a stoichiometric ratio of1:0.5 (v/v). The mixture was allowed to stand in the dark at roomtemperature for 8 h. In the ABTS�+ solution (190 lL) different con-centrations of test compounds/extracts (10 lL) were added. Thereaction mixture was incubated for 6 min at room temperatureand the absorbance was measured at 734 nm. The change in absor-bance with respect to the control (containing ABTS�+ solution onlywithout sample, expressed as 100% free radicals) was calculated aspercentage scavenging and the IC50 value was calculated. Theknown antioxidants Trolox and ascorbic acid were used as positivecontrols.

2.9.3. NBT assay for superoxide inhibitionSuperoxide radicals were generated by the xanthine/xanthine

oxidase system (Liu, Chen, Shang, Jiao, & Huang, 2009). Briefly,200 lL reaction mixture contained sodium pyrophosphate buffer(80 mM, pH 7.5), xanthine (120 lM), xanthine oxidase (0.1 U/mL;from bovine milk), NBT (100 mM) and test compounds/extracts.After 10 min of incubation at 25 �C the absorbance was measuredat 570 nm. The known antioxidant ascorbic acid was also studiedin the assay. SOD was used as a positive control. The experimentwas performed in triplicate.

2.9.4. MTT antioxidant assayMTT antioxidant assay was performed as per the reported pro-

cedure (Liu & Nair, 2010). Briefly a stock solution of test extracts/compounds (1 mg/mL) in DMSO and an aqueous solution of MTT(1 mg/mL) were prepared. In a capped glass vial (5 mL) mixtureof aqueous solution of MTT (190 lL) and different concentrationsof test compound/extract (10 lL) was vortexed. To it DMSO(200 lL) was added and was vortexed again. The reaction mixturewas incubated at 37 �C for 6 h. After the incubation 200 lL of thereaction mixture were taken in 96-well cell culture plate, and theabsorbance was read at 570 nm. Each sample was assayed in trip-licate and the average absorbance was noted. In control the testsolution was replaced with 10 lL of DMSO. Quercetin dihydratewas used as a positive standard.

2.9.5. Reducing power assayThe reducing power assay was done by reported protocol

(Amarowicz, Pegg, Rahimi-Moghaddam, Barld, & Weilc, 2004;Yen & Chen, 1995). Briefly each extract/compound (0.125–1 mg)was dissolved in 1.0 mL of methanol. To this extract solution, phos-phate buffer (0.2 M, 2.5 mL, pH 6.6) was added followed by addi-tion of a solution of potassium ferricyanide (1%, 2.5 mL). Thereaction mixture was incubated in water bath at 50 �C for20 min. After incubation trichloroacetic acid (10% w/v, 2.5 mL)was added to the reaction mixture and was centrifuged at7500 rpm (5471 g) for 10 min. To the aliquot of the upper layer(2.5 mL) distilled water (2.5 mL) and a solution of ferric chloride(0.1%, 0.5 mL) was added. Finally the absorbance of the reactionmixture was measured at 700 nm. Increased absorbance of thereaction mixture indicates greater reducing power. The experimentwas performed in triplicate. Quercetin dihydrate was used as a po-sitive standard.

2.9.6. Total antioxidant test2.9.6.1. Ferric thiocyanate (FTC) test. FTC test was performedaccording to the reported method (Kikuzaki & Nakatani, 1993;Kumar, Ganesan, & Subba Rao, 2008; Liu & Yao, 2007). Briefly,1 mg/mL solution of each extract/compound was prepared. Then,the test solutions (4 mL) were respectively mixed with linoleic acid(4.1 mL), 0.1 M sodium phosphate buffer pH 7.0 (8 mL) and dis-tilled water (3.9 mL). These mixtures were then kept in screw-cap glass vials at 40 �C in the dark. In order to determine the FTC

R. Mohan et al. / Food Chemistry 132 (2012) 244–251 247

values, 0.1 mL of these mixtures was, respectively, added into of75% ethanol (9.7 mL) and of 30% ammonium thiocyanate(0.1 mL). After 3 min of incubation 0.02 M ferrous chloride solution(0.1 mL) in 3.5% HCl was added to the reaction mixture and theabsorbance of the samples was measured at 500 nm. This proce-dure was repeated every 48 h until the control sample reachedits maximum absorbance value. Ascorbic acid was used as a posi-tive standard in this test. The control and standard were subjectedto the same procedure.

2.9.6.2. 2-Thiobarbituric acid (TBA) test. The TBA test was preformedaccording to Mackeen et al. (2000) immediately after the controlsample from FTC test reached its maximum absorbance value.Briefly, 1.0 mL of 20% aqueous trichloroacetic acid and 2.0 mL of0.67% aqueous 2-thiobarbituric acid were added to 2 mL of samplesolutions acquired from FTC test. The mixtures were then placed ina boiling water bath for 10 min. After cooling under running tapwater, the mixtures were centrifuged at 8000 rpm (6225 g) for30 min. Finally, the absorbance of supernate was measured at532 nm. Ascorbic acid was used as a positive standard.

2.10. Statistical analysis

All experiments were performed in triplicates and the data isrepresented as mean ± standard error of mean (SEM).

3. Result and discussion

3.1. Isolation and characterisation

LEE (20 g) was subjected to vacuum liquid chromatography onsilica gel. Fractionation and purification on silica gel (#60–120) fol-lowed by purification on Sephadex LH-20 afforded a new phenolicglycoside (E)-4-hydroxymethyl-phenyl-6-O-caffeoyl-b-D-gluco-pyranoside (1) along with the known 1-(2-hydroxy-4-hydroxy-methyl)-phenyl-6-O-caffeoyl-b-D-gluco-pyranoside (2) and proto-catechuic acid (3), gallic acid (4), caffeic acid (5) and 3,4dimethoxycinnamic acid (6) (Fig. 1). Compound 1 was obtained asa pale yellow powder with an optical rotation ½a�20

D �77.78 (c 0.25,MeOH) and gave positive test with NP-PEG reagent on TLC whichindicated that it might be phenolic type of compound. The meltingpoint was determined to be 178–179 �C. The IR spectrum suggestedthe presence of hydroxyl group (3399 cm�1) and conjugated COOR(1725 cm�1).

ESI-MS of compound 1 revealed a quasi-molecular ion at m/z471 [M+Na]+, corresponding to a molecular formula C22H24O10. Thismolecular formula was determined by HR-ESI-MS (C22H24O10)

HO

HO 4

3'

HO

HO

OH

O

OH

HO

HO

O

H

HO

H

HO

H

HOH

H

O

O

OHO

HO

HO

1'

6'5'

4'

3'

2' 7'

8'

9'

12

3

4

5

6

1'' 2''

3''

4''5''

6''

7''1

HO

HO

OH

O

3 4

Fig. 1. Compounds (1–6) from

[M+Na]+ at m/z 471.1262, (calculated for C22H24O10Na, 471.1267)and confirmed by 1H and 13C NMR experiments. The structure ofcompound 1 was elucidated by detailed analysis of 1H and 13CNMR chemical shifts and by COSY, HMQC, and HMBC experiments.

The 1H NMR spectrum of compound 1 in CD3OD showed twotrans-olefinic protons signal at dH 7.5 (1H, d, J = 15.9 Hz, H-70)and 6.2 (1H, d, J = 15.9 Hz, H-80), aromatic protons as signals atdH 7.0 (1H, d, J = 2.1 Hz, H-20), 6.7 (1H, d, J = 8.2 Hz, H-50), 6.9 (1H,dd, J = 8.2, 2.1 Hz, H-60) together with carbon resonance at dc

127.7 (C-10), 114.9 (C-20), 149.6 (C-40), 146.8 (C-30), 116.5 (C-50),123.0 (C-60), 147.1 (C-70), 115.2 (C-80), 168.9 (C-90) suggested thepresence of caffeoyl moiety. Further, the occurrence of aromaticprotons as an A2B2 type pattern signals at dH 7.2 (d, J = 8.4 Hz, H-300 and H-500) and 7.0 (d, J = 8.4 Hz, H-20 0and H-600) along with thecarbon resonance at dC 158.2 (C-100), 117.7 (C-200, C-600), 129.4 (C-300, C-500), 136.6 (C-400) indicated a 1,4-disubstituted phenyl moiety.Proton singlet at dH 4.4 (2H, s, H-700) and corresponding carbon at dC

64.7 indicates the attached methylene group to the phenyl ring. E-geometry of C-70/C-80 double bond was proved by the J (H-70/H-80)value (15.9 Hz).

The 1H NMR display the proton signals at dH 3.47–3.49 (overlap-ping, H-2 and H-3), 3.40 (m, H-4), 3.7 (m, H-5) for four protons, twodouble doublets at dH 4.5 (dd, J = 11.8, 1.96 Hz, H-6a) and 4.3 (dd,J = 11.8, 7.2 Hz, H-6b) along with the carbon resonances at dC

102.2 (C-1), 74.8 (C-2), 77.9 (C-3), 71.9 (C-4), 75.5 (C-5) and 64.6(C-6) revealed a glucose moiety. NMR spectra in CD3OD did not re-veal the anomeric proton due to presence of moisture in same re-gion dH (4.8–5), so its 1H NMR in (CD3)2CO was taken. It clearlyindicated the anomeric proton at dH 4.9 (1H, d, J = 7.4 Hz, H-1).The coupling constant of anomeric proton (J = 7.4 Hz) confirmedthe presence of b-glucose (Ishii & Yanagisawa, 1998).

Further, to confirm the presence of glucose, acid hydrolysis wascarried out. The sugar obtained was identified as glucose whenanalysed on silica gel TLC (EtOAc/MeOH/H2O/acetic acid:13:3:3:4, v/v/v/v) using anisaldehyde–sulphuric acid reagent withthe authenticated standard of D-glucose (Kanchanapoom, Kasai, &Yamasaki, 2001). The ESI-MS spectrum of compound 1 also showedpeak at m/z 161, a characteristic fragment ion peak due to the glu-cose moiety.

The attachment of glucose at C-10 0 of the phenyl moiety wasconfirmed by the HMBC. Correlation was (Fig. 2) observed betweendH [(CD3)2CO] 4.9 (H-1; Glc) and dC 158.2 (C-100) proving the C-100

glycosylation. HMBC correlations between dH 4.3 (1H, dd, J = 11.8,7.2 Hz, H-6b) with dC 168.9 (C-90) inferred the attachment of C-6of glucose to the caffeoyl moiety. The absence of any long rangecorrelations of proton from H-1 at d 4.9 to C-700 at dC 64.79 andthe occurrence of long-range correlations of hydroxymethyl at dH

O

H

HO

H

HO

H

HOH

H

O

O

O

HO

1'

6'5'

'

2' 7'

8'

9'

12

3

4

5

6

1'' 2''

3''

4''5''

6''

7''

OH

OH

O

O

O

OH

O

2

5 6

Lagenaria siceraria fruits.

HO

O

O

H

HO

H

HO

H

HOHH

O

O

H

H

H

OH

H

H

HOH

H

H

H

H

H

O

HOH

H

HO

H

HOH

O

O

Hb HaH

OH

O

HO

HO

(A) (B)

Fig. 2. (A) HMQC and (B) HMBC correlations of compound 1.

Table 1The extractive value, total phenolic (mg GAE/g extract DW; mg TAE/g extract DW)and flavonoid contents (mg quercetin/g extract DW) of various extracts of bottlegourd.

Extracts Extractivevalue (%)

Total phenolic content Total flavonoid contentmg quercetin/g extractDWamg GAE/g

extractDWa

mg TAE/gextractDWa

LDE 0.5 68.5 ± 4.4 23.6 ± 2.1 11.7 ± 2.5LEE 0.7 402.5 ± 1.8 233.4 ± 3.0 43.4 ± 2.8LME 10.1 42.7 ± 2.5 9.5 ± 1.2 0.49 ± 0.2

GAE – gallic acid equivalent; TAE – tannic acid equivalent; DW – dry weight; LDE –CH2Cl2 extract, LEE – EtOAc extract; LME – MeOH extract.

a Values are expressed in mean ± SEM.

248 R. Mohan et al. / Food Chemistry 132 (2012) 244–251

4.4 (2H, s, H-700) with dC 136.6 (C-400) and 129.4 (C-300, C-500) con-firmed the position of hydroxymethyl group at C-400.

Thus, the structure of compound 1 was deduced as (E)-4-hydroxymethyl phenyl-6-O-caffeoyl-b-D-glucopyranoside.

Compound 2 was isolated as a yellow powder, ESI-MS: m/z 463[M�1]+ and gave positive test with NP-PEG reagent which sug-gested that it might be phenolic type of compound. The IR spec-trum suggested the presence of hydroxyl group (3399 cm�1),conjugated COOR (1733 cm�1) and phenyl group (1601 cm�1).The 1H and 13C NMR spectra of compound 2 were similar to thoseof compound 1 and showed the presence of caffeoyl, b-glucopyran-osyl moieties (anomeric signals: dH 4.7, J = 7.1 Hz and dC 104.1) anda substituted benzyl alcohol moiety. Proton signals at dH 6.88 (d,1H, J = 1.9 Hz, H-300), 7.1 (1H, d, J = 8.2 Hz, H-600) and 6.67 (1H, dd,J = 8.2, 1.9 Hz, H-500) indicated the presence of 1,2,4-trisubstitutedphenyl moiety. Further dH 4.4 (2H, s, H-700) and corresponding dC

(64.8) indicates methylene group attached to the phenyl ring.The glucose moiety was further confirmed by the acid hydrolysisexperiment. The glucosyl linkage was established by HMBC exper-iments. HMBC correlations were observed between dH 4.5 (dd, 1H,J = 2.0 Hz and 12 Hz, H-6a) and dC 168.9 (C-90); dH 4.7 (d, J = 7.1 Hz,H-1) and dC 145.8 (C-10 0); dH 4.4 (s, H-70 0) and dC 116.0 (C-30 0), 119.6(C-50 0) and 138.4 (C-40 0).

The 1H and 13C NMR data and 2D NMR analysis confirmed thestructure of 2 as 1-(2-hydroxy-4-hydroxymethyl) phenyl-6-O-caf-feoyl-b-D-glucopyranoside (Fig. 1). This compound was previouslyreported in Crinum asiaticum L. (Sun, Zhang, Shen, Zhang, & Li,2008).

Compounds 3 and 4 were confirmed to be protocatechuic acidand gallic acid, respectively, from their spectral data while caffeicacid (5) and 3,4-dimethoxycinnamic acid (6) were confirmed com-paring the spectral data with that of literature (Kelley, Harruff, &Carmack, 1976). All these compounds (1–6) were identified forthe first time as bottle gourd constituents.

3.2. Determination of total phenol and total flavonoids

The total phenolic content of different extracts from fruits ofbottle gourd was assayed by the Folin–Ciocalteau’s method usinggallic acid and tannic acid as standards as described in Section 2.The Folin–Ciocalteau’s assay is a frequently used screening methodfor measurement of antioxidant capacity of food products and die-tary supplements. The data presented in Table 1 indicates high to-tal phenol content of 402.5 ± 1.8 mg GAE/g of dried sample in theLEE while the lowest total phenol content of 42.7 ± 2.5 mg GAE/gof dried sample was obtained in LME. The phenolic content ofLEE was found to be higher than most of the common fruits, suchas cranberries, raspberries, strawberries and apple (Kahkonenet al., 1999). As the antioxidant activity of the dietary food is di-rectly correlated with its total phenolic content the LEE is expectedto have strong antioxidant activity.

The total flavonoid content of different extracts from bottlegourd fruits was assayed by aluminium chloride colorimetric assayas described in Section 2. The total flavonoid content was ex-pressed as mg of quercetin equivalents/g of dried samples. Thedata presented in Table 1 indicates that the highest flavonoid con-tent of 43.4 ± 2.8 mg quercetin/g of dried samples was observed inthe LEE and the lowest content was observed in the LME i.e.0.49 ± 0.2 mg quercetin/g of dried samples. The order of total phen-olics and flavonoid content was found to be LEE > LDE > LME.

3.3. Antioxidant activity

The antioxidant activity of the extracts, compounds 1 and 2were assayed using the different assay systems; (1) scavengingactivity of free radicals based on chemical trapping using DPPHand ABTS assay methods, (2) inhibitory activity on superoxide an-ion generation by the xanthine/xanthine oxidase system, (3) MTTantioxidant assay, (4) reducing power assay and (5) total antioxi-dant assay.

3.3.1. Radical scavenging activityThe free radical chain reaction is widely accepted as a common

mechanism of lipid peroxidation. Radical scavengers may directlyreact with and quench peroxide radicals to terminate the peroxida-tion chain reactions. Assays based upon the use of DPPH� andABTS�+ are among the most popular spectrophotometric methodsfor determination of the antioxidant capacity of foods, beveragesand vegetable extracts. Both chromogens and radical compoundscan directly react with antioxidants (Ak & Gulicn, 2008).

In the DPPH assay, the stable DPPH� in alcoholic solution re-duces to the yellow-coloured diphenyl-picrylhydrazine in presenceof antioxidant due to the formation of the non-radical form DPPH-H in the reaction. DPPH� is a stable free radical and accepts an elec-tron or hydrogen radical to become a stable diamagnetic molecule.The results are shown in Table 2. Compound 1 was found to be

Table 2Radical scavenging activity and the superoxide radical scavenging activity of thedifferent extracts of bottle gourd and compound 1 and 2.

Extracts/compounds Radical scavenging activity IC50

(lg/mL)Superoxide radicalscavenging activityIC50 (lg/mL)

DPPH�a ABTS�+a

LDE 48.5 ± 2.3 73.9 ± 1.1 >100LEE 5.2 ± 0.5 9.5 ± 0.8 35.7 ± 1.2LME >100 >100 >100Compound 1 2.1 ± 0.3 8.3 ± 1.3 7.8 ± 0.7Compound 2 5.8 ± 1.1 10.3 ± 0.8 33.4 ± 1.1Ascorbic acid 3.8 ± 0.4 4.7 ± 1.1 >50Trolox 3.4 ± 0.9 4.1 ± 1.3 –

DPPH – 1,1-diphenyl-2-picrylhydrazyl radical; ABTS – 2,20-azino-bis (3-ethyl-benzthiazoline-6-sulphonic acid) radical; LDE – CH2Cl2 extract, LEE – EtOAc extract;LME – MeOH extract.

a Values are expressed in mean ± SEM.

R. Mohan et al. / Food Chemistry 132 (2012) 244–251 249

potent free radical scavenger with an IC50 value of 2.1 ± 0.3 lg/mL(4.6 lM). Ascorbic acid and Trolox were used as standards for theassay. The DPPH� scavenging activity was found to be in order ofCompound 1 > Trolox > ascorbic acid > LEE > Compound 2 > LDE >LME.

Similarly, compound 1 exhibited effective radical cation scav-enging activity in ABTS assay with an IC50 value of 8.3 ± 1.3 lg/mL(18.5 lM). LEE strongly scavenged the ABTS radicals with an IC50

value of 9.5 ± 0.8 lg/mL. The results are reported in Table 1. TheDPPH� and ABTS radical scavenging activity of bottle gourdextracts showed similar trend with the result of total phenolic con-tent and total flavonoid content, indicating that radical scavengingactivity of bottle gourd extracts is related to the amount of pheno-lic compounds present in the extracts.

Fig. 3. (A) Dose response curve for MTT assay; (B) dose response curve for reducingpower assay.

3.3.2. Superoxide radicals scavenging activity by enzymatic systemThe reduction of NBT by superoxide anion generated by xan-

thine oxidase was effectively inhibited by compound 1 in a dose-dependent manner. It exhibited potent activity with an IC50 valueof 7.8 ± 0.7 lg/mL (16.5 lM). The LEE extract also inhibited thereduction with an IC50 of 35.7 ± 1.2 lg/mL stronger than ascorbicacid. Ascorbic acid inhibited 40% of NBT reduction at a final con-centration of 50 lg/mL. The results are shown in Table 2. SOD isa body defense mechanism against the superoxide anion and wasused as a positive control. 1 U of the SOD inhibited the superoxideanion produced in the assay by 56.71% in 200 lL of reactionmixture.

3.3.3. MTT assayThe MTT assay is an established colorimetric assay for measur-

ing the activity of the mitochondrial enzymes present in healthycells by monitoring the absorbance of purple formazan formed asthe enzymatic reduction product of MTT. A recently publishedantioxidant assay using MTT was followed for quantitative deter-mination of antioxidant activity of the extracts and isolated com-pounds (Liu & Nair, 2010). The results are shown as the doseresponse curve in Fig. 3A. Quercetin dihydrate was used as a posi-tive control. Compound 1 showed comparable activity as com-pared to the positive standard.

3.3.4. Reducing power assayIn the reducing power assay, the presence of reductants (antiox-

idants) in the samples results in the reduction of the Fe3+/ferricya-nide complex to its ferrous form. Fig. 3B shows the extent ofreduction, in terms of absorbance values at 700 nm, for the extractsand compounds ranging in concentration from 0.125 to 1 mg/mL.The reducing power of LEE, compound 1 and 2 was comparableto that of quercetin dihydrate, the positive control.

Fig. 4. (A) Hydroperoxides inhibitory activity of test extracts/compounds throughFTC test; (B) 2-thiobarbituric acid reactive substances inhibitory activity of testextracts/compounds measured by TBA test.

250 R. Mohan et al. / Food Chemistry 132 (2012) 244–251

3.3.5. Total antioxidant activityThe active LEE, compounds 1 and 2 were studied for the deter-

mination of FTC value. The extract as well as test compounds sig-nificantly retarded the formation of hydroperoxides throughoutthe incubation period when compared with the control. The resultsare shown in Fig. 4A. The absorbance value of control reached to itsmaximum value on day 10 of incubation.

After the control reached its maximum absorbance value in FTCtest, samples were studied for TBA test. TBA test determines the 2-thiobarbituric acid reactive substances content at a later stage oflipid oxidation, involving the quantification of the secondary prod-ucts formed from lipid oxidation. Low absorbance value indicateshigher 2-thiobarbituric acid reactive substances inhibitory activity.Fig. 4B shows that the strength of 2-thiobarbituric acid reactivesubstances inhibitory activity of LEE, compounds 1 and 2 was com-parable to that of the standard ascorbic acid. A similar trend wasobserved for TBA and FTC test compounds.

4. Conclusion

In present study, the ethyl acetate extract of bottle gourd wasfound to have potent antioxidant activity in different in vitro as-says including reducing power assay, radical scavenging (DPPHand ABTS) assay, superoxide scavenging assay, MTT reduction as-say and lipid peroxidation inhibition assay. Other extracts of bot-tle gourd were not as effective antioxidants as that of ethylacetate extract. Further, for the first time we reported isolationand structure elucidation of compound 1, which also exhibitedpotent antioxidant activity. The antioxidant activity of ethyl ace-tate extract thus, can be attributed to compound 1 and othersuch phenolic constituents present in it. Observed antioxidantactivity of the ethyl acetate extract and compound 1 was similaror stronger than the known antioxidants. Thus, the bottle gourdcan be considered as a source of natural antioxidants for foodand nutraceutical products.

Acknowledgement

Authors acknowledge the efforts of Mr. Shahid Khan for obtain-ing the plant material.

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