Isolation and characterization of antioxidant constituents of the fruit of Telfairia occidentalis Hook F ( Cucurbitaceae )

Purpose: To evaluate the antioxidant property of the fruit of Telfairia occidentalis and isolate the components responsible for the antioxidant activity. Methods: The fruit pericarp was macerated with methanol and the extract obtained successively partitioned with n-hexane, dichloromethane and ethyl acetate. The in vitro antioxidant activity of the extract and fractions was evaluated using 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging, reducing power, nitric oxide scavenging, total antioxidant and hydrogen peroxide scavenging assays. The n-hexane fraction, which had the highest DPPH scavenging and total antioxidant activities, was subjected to column and thin layer chromatography to isolate the components. The isolated compounds were identified by ultraviolet-visible (UV), nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy and mass spectrometry. Results: Among the fractions tested, n-hexane had the best total antioxidant activity of 99.44 % at 20 mg/ml (p < 0.05) compared to ascorbic acid at 99.71 % of 20 mg/ml. This fraction also had the highest DPPH radical scavenging activity of all the fractions (p < 0.05) at all test concentrations. For nitric oxide scavenging activity, the whole extract, and the chloroform and aqueous fractions exhibited activity ranging from 92.29 to 97.66 % compared to 98.93 % for ascorbic acid. The hydrogen peroxide scavenging activity of the extract and fractions ranged from 92.60 to 96.23 % compared with of the standard, ascorbic acid (101.68 %). The major components of the n-hexane fractions were αamyrin and β-amyrin. Conclusion: The fruit pericarp of Telfairia occidentalis possesses good DPPH radical scavenging activity. This is the first time the antioxidant activity of the fruit (pericarp) and the presence of αand βamyrins in Telfairia occidentalis have been reported.


INTRODUCTION
Telfairia occidentalis is popularly known mainly because of its nutritionally valuable leaves [1].The leaves have high nutritive value compared with other vegitables grown in the tropics.The seed is also edible.Telfairia occidentalis is popular among herbal practitioners.A review of the medicinal properties of the plant was written by Eseyin et al [2].Telfairia occidentalis possesses antioxidant property [3].
Not much work has been done on the fruit pericarp of the plant which is usually discarded as it is often considered not useful.There is therefore no available report on the antioxidant activity or isolated component of the fruit pericarp of Telfairia occidentalis.Since the leaf and seed of Telfairia occidentalis have been found to possess significant antioxidant properties, the authors decided to screen the fruit pericarp for antioxidant property and isolate the antioxidant components of the pericarp.

EXPERIMENTAL Plant collection and identification
The fruit of Telfairia occidentalis (Fluted pumpkin) was obtained from the medicinal plant farm of the Faculty of Pharmacy, University of Uyo, Akwa Ibom State, Nigeria in June 2015.The fruits were identified by Professor Margaret Bassey in the Department of Botany and Ecological Studies University of Uyo, Akwa Ibom State, Nigeria and it was assigned voucher number UUPH28 (d).A voucher specimen was kept in the faculty of Pharmacy, University of Uyo, herbarium.

Preparation and extraction of plant material
The freshly collected and identified fruits of Telfairia occidentalis were sliced opened and the pulp and the seed removed.The pericarp was chopped into small bits and air dried.Methanol (95 %, 5 litres) was poured into a container containing 500 g of the dried fruit material and macerated for 72 h at ambient temperature with intermittent shaking.The liquid extract was filtered and concentrated in vacuo.The extract was further dried in a desiccator containing selfindicating silica gel orange (Sigma-Aldrich, Germany).

Partitioning of the extract
The dried methanol extract (85 g) was dissolved in 500 mL of distilled water and partitioned successively with n-hexane, dichloromethane and ethyl acetate to obtain their respective fractions.Each of the fractions was concentrated in a rotary evaporator to dryness.

Evaluation of antioxidant properties
The DPPH antioxidant capacity of the extracts, vitamin C and E were evaluated by the method of Enujiugha [4].A dose of 0.2 mL of the extracts was added to 3.8 mL ethanol solution of DPPH radical until a final concentration of 0.1 mM was obtained.The mixture was agitated vigorously for 1 min and left to stand at room temperature for 30 min.The absorbance of each sample (As) was measured on a UV spectrophotometer at 517 nm against ethanol blank.Negative control (A) was taken after adding DPPH solution to 0.2 mL of the extracts.DPPH scavenging activity (D) of the sample was calculated as in Eq 1.
where Ac and As are the absorbance of control and test compound, respectively.
The reducing power assay was estimated by the method of Athukorala et al [5].1.0 mL extract was mixed with 2.5 mL of phosphate buffer (20 mM, pH 6.6) and 2.5 mL of potassium ferricyanide (30 mM) and incubated at 50 o C for 20 minutes.Thereafter, 2.5 mL of trichloroacetic acid (600 mM) was added to the reaction mixture, centrifuged for 10 minutes at 3000 rpm.The upper layer of the solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl 3 (6 mM) and absorbance was measured at 700 nm.Nitric oxide (NO) scavenging activity was estimated by the method of Green et al [6].Sodium nitroprusside (10 mM, 3 mL) in phosphate buffer was added to 2.0 mL of extract and reference compound in different concentrations (20 -100 g/ml).
The resulting solutions were then incubated at 25 o C for 60 minutes.A similar procedure was repeated with methanol as blank, which served as control.To 2.0 mL of the incubated sample, 5.0 mL of Griess reagent (1 % sulphanilamide, 0.1 % naphthyethylene diamine dihydrochloride in 2 % H 3 PO 3 ) was added and absorbance of the chromophore formed was measured at 540 nm.The total antioxidant activity was evaluated using the method of Lingnert [7].Each extract (0.1-20 mg/ml) in water or ethanol (100 µl) was mixed with 2.0 mL of 10 mM linoleic acid emulsion in 0.2 M sodium phosphate buffer (pH 6.6) in a test tube and kept in the dark at 37 o C to accelerate oxidation.After incubation for 15 h, 0.1 mL from each tube was mixed with 7.0 mL of 80 % methanol in deionized water and the absorbance of the mixture was measured at 234 nm against a blank in a spectrophotometer.Antioxidant activity (D) was calculated as as in Eq 2. D (%) = {(Ac -As)/Ac} ………….(1) where Ac and As are the absorbance of control and test compound, respectively.
Hydrogen peroxide scavenging activity was evaluated according to the method of Ruch et al [8].A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (50 mM, pH 7.4).The concentration of hydrogen peroxide was determined by absorption at 230 nm using a spectrophotometer.Extract (20 -60 g/mL) dissolved in distilled water was added to hydrogen peroxide and absorbance at 230 nm was determined after 10 min against a blank without hydrogen peroxide.Hydrogen peroxide scavenging H (%) was calculated as in Eq 3.
where Ac and As are the absorbance of control and test compound, respectively.

Isolation and purification of compounds in nhexane fraction
The dried n-hexane fraction (9.13 g) which had the highest DPPH and total antioxidant activities was subjected to open column chromatography (Rotaflo glass column (Quick-fit, England) measuring 3 x 80 cm; silica gel-G mesh 60 -120 (Burgoyne, India).The flow rate of the eluent was kept constant at about 1 mL/min and the fractions were collected in 20 mL test tube.The sample was eluted using solvent systems that consisted of varying percent of two of the following solvents at a time in the specified order Pentane, n -hexane, CHCl 3 , EtOAC and, MeOH to obtain 291 eluates.The eluates were monitored by TLC (silica gel; solvent system: pentane 10: n -hexane 9: ethyl acetate 1) and visualized using Ultraviolet lamp (366 nm) (Allen, London) and appropriate detecting reagents such as 1 % anisaldehyde in 10 % H 2 SO 4 , 5 % FeCl 3 and conc.H 2 SO 4 .
Eluates with same R f values were pooled together to obtain fractions A, B, C, D, E, F and G. Fraction D (3500 mg) was further purified using open column chromatography (silica gel-G; 60 -120 mesh).The fraction was separated using n-hexane and CHCl 3 (starting with 100 % n-hexane, varying ratio of the two solvents and 100 % CHCl 3 ) to obtain 40 eluates which were pooled together based on similarity in Rf values to obtain different fractions.Fraction D2 (eluate 12 -36; 3300 mg) gave a spot tailing on TLC (silica gel; solvent system: pentane 10: nhexane 5: ethyl acetate 1) under UV: 366 nm.Fraction D2 was purified further using Sephadex LH-20 (Pharmacia Fine Chemicals, Sweden) as the stationary phase while ethanol (100 %) was used to elute the fraction to obtain BEN-4 (pure white compound, 1951 mg) which gave a single spot on TLC.

Ultraviolet spectroscopy
The UV spectra of isolated compounds were obtained using a UV spectrophotometer (Unico UV-2100 Spectrophotometer, Shanghai Instruments Co, Ltd., China).

Nuclear magnetic resonance
The isolates where weighed (10 mg) and dissolved in deuterioted chloroform (CDCl 3 ).The solution was introduced into sample tubes, and was inserted into the NMR spectrometer (Bruker Avance AV 400, 400.033MHz) equipped with a 5mm DUL 13C-1 z-gradient probe head.The machine was operated by means of a computer to obtain the different NMR spectra.The proton and Carbon NMR (including 2-dimensional Carbon NMR: COSY and NOESY) spectra were acquired.

Fourier transform infra-red spectroscopy (FTIR)
The isolates where weighed (10 mg) and dissolved in dichloromethane.The solution was introduced into sample tubes, and was inserted into the infra-red spectrometer (JASCO 302-A) to obtain the IR spectra.

Mass spectrometry (MS)
The isolates were weighed (10 mg) and dissolved in dichloromethane.The solution was introduced into sample tubes, and was inserted into the mass spectrometer (ionization voltage 70 eV; JEOL JMS-600H).
The machine was operated by means of a computer to obtain the MS spectra.

Statistical analysis
The data obtained are expressed as mean ± SEM and were subjected to one-way analysis of variance (ANOVA) using GraphPad Prism 5.01 (USA).Values of p < 0.05 were considered significant.

RESULTS
The yield of methanol extract, n -hexane, chloroform, ethyl acetate and aqueous fractions are 15.7, 11.87, 5.49, 26.60 and 51.63 %, There were significant variations in the antioxidant activity of the different fractions based on the method used.Some fractions showed very high antioxidant activity in one or more methods but less in other models.Table 1 shows the results for DPPH scavenging property of extract and fractions of the pericarp of Telfairia occidentalis.Table 2 and Table 3 show the results for reducing power and nitric oxide scavenging activities for extract and fractions, respectively, while those for hydrogen peroxide scavenging and total antioxidant activities of extract and fractions are depicted in Table 4 and Table 5, respectively.Table 6 displays the results for antioxidant activity of the isolated compounds based on DPPH-radical scavenging.
The n -hexane fraction which had the highest total antioxidant and DPPH-radical scavenging activities was purified further on column chromatography to obtain two compounds coded BEN-4 and BEN-7.From the proton NMR spectra, COSY, NOESY, MS and IR spectra, BEN-4 was identified as alpha-amyrin (1) and BEN-7 as beta-amyrin (2).

DISCUSSION
N -hexane had the highest DPPH inhibitory activity (Figure 1).The activity was comparable (p < 0.05) to that of the reference compound (Vitamin C).This correlates with the report of Nkereuwem et al [9] on the DPPH inhibiting activity of fractions of the leaf extract of Telfairia occidentalis.None of the fractions had significant reducing power activity (Table 2).For Nitric Oxide (NO) radical scavenging model, the whole extract had the least activity of 95.15 % at 20 mg/mL while the chloroform and aqueous fraction had similar activity of 92.29 and 97.66 % at 20 mg/mL compared to ascorbic acid of 98.93 % at 20 mg/mL (Figure 3).Hydrogen peroxide (H 2 O 2 ), although not very reactive, but it may be toxic to cell due to increase in hydroxyl radical concentration in the cells.Thus, removal of H 2 O 2 as well as superoxide anion leads to survival of the cell life and its components.The scavenging ability of the extract (95.46 %), chloroform (96.23%), ethyl acetate (92.60 %) and aqueous (94.77 %) on hydrogen peroxide is comparable with that of the standard ascorbic acid (101.68 %) at 20 mg/mL (Figure 4).n -hexane had the best total antioxidant activity of 99.44 % at 20 mg/mL compared to the ascorbic acid at 99.71 % of 20 mg/mL.Amyrins are abundant naturally occurring two isomeric pentacyclic triterpenoids which are isolated from the leaves and barks of plants.Plants from which the amyrins have been reported since 2008 to possess α-amyrin, βamyrin and α, β-amyrin mixture in minor amounts include Boswellia carterii, Populus euramericana.The amyrins have been isolated from the nhexane extract of Melastoma malabathricum L, Pocirus trifoliate, Antiaris africana, Amelanchier alnifolia, Nelumbo nucifera Ficus cordata, Ficus cordata and Byrsonima crassa.
Alpha-and β-amyrins showed in vitro and in vivo antihyperglycemic, hypolipidemic [12] and antioxidant [13] activities.Biological activities of the amyrins and the pharmacological potential of their modified products has been reviewed [14].
Supplementary data: Details on the antioxidant assays and results, FTIR, NMR and MS data are available from the corresponding author on request.

CONCLUSION
The findings of the present study indicate that the pericarp of Telfairia occidentalis is a potential source of natural antioxidants and a rich source of alpha-and beta-amyrins.

Table 1 :
DPPH-radical scavenging activity of extract and fractions of the fruit of T. occidentalis

Table 2 :
Reducing power activity of extract and fractions of the fruit of T. occidentalis

Table 3 :
Nitric oxide scavenging assay of extract and fractions of the fruit of T. occidentalis

Table 4 :
Hydrogen peroxide scavenging activity of the extract and fractions of the fruit of T. occidentalis

Table 5 :
Total antioxidant assay of the extract and fractions of the fruit of T. occidentalis Absorbance values are expressed as mean ± standard deviation.% scavenging activity is expressed in bracket.

Table 6 :
DPPH-radical scavenging assay of the isolated compounds