IJERDInternational Journal of Environmental and Rural Development (2018) 9-2

Research article
erd

Antioxidant Activity of Crude Extract from Raw and Heattreated Moringa oleifera
NAWAPORN LAPSONGPHON*
Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi,
Phathum Thani, Thailand
Email: nawaporn_l@rmutt.ac.th

MONSICHA PINTHONG
Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi,
Phathum Thani, Thailand

PIYADA SUKDEE
Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi,
Phathum Thani, Thailand
Received 31 December 2017

Accepted 20 October 2018(*Corresponding Author)

Abstract The aim of this research was to investigate the effect of heat treatment on
antioxidant properties of crude extract from Moringa oleifera (peel, pulp, and seed). Total
phenolic content and antioxidant properties based on 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging, ferric reducing antioxidant power (FRAP), and metal chelating activity
were investigated. Results revealed that crude extract obtained from raw moringa peel
showed the highest total phenolic content and DPPH radical scavenging activity (3.65 mg
gallic acid eq./g DW and 1.35 mg Trolox eq./g DW), followed by pulp (3.44 mg gallic acideq./g DW and 1.25 mg Trolox eq./g DW) and seed (2.80 mg gallic acid eq./g DW and 0.91
mg Trolox eq./g DW), respectively. The highest FRAP were obtained from the raw moringa
peel extract (3.62 mg ferrous eq./g DW), whereas the highest metal chelating activity was
found in raw moringa pulp extract 0.42 mg EDTA eq./g DW). All samples with heat
treatment at 100 °C for 15 min showed lower total phenolic content and antioxidant
properties than crude extract from raw moringa samples (P<0.05).
Keywords Moringa oleifera, heat treatment, antioxidant activity

INTRODUCTION
In recent years, attention has been focused on antioxidants derived from natural sources, due to thefact that antioxidative activity of extracts from plants or animals, in some cases, is similar or higher
than that of commonly used synthetic antioxidants such as butylated hydroxytoluene (BHA) andbutylated hydroxylanisole (BHT), which are quite unsafe and their toxicity is a problem of concern(Hossain et al., 2008). Plant and its products are rich sources of a phytochemicals and have beenfound to possess a variety of biological activities including antioxidant potential in various
oxidative reaction systems have been discovered. The antioxidant properties include scavenging or
quenching of reactive oxygen species (ROS)/free radicals and inhibition of ROS induced oxidationof biological macromolecules such as lipids, proteins, and DNA. Other mechanisms of antioxidant
activity include transition metal chelating activity and ferric reducing power (Arabshahi-Delouee etal, 2007).
Moringa oleifera is a widely cultivated tree considered as a multi-purpose plant which can befound in tropical and subtropical climates and contains various phytochemicals such as carotenoids,vitamins, minerals, amino acids, sterols, glycosides, alkaloids, flavonoids and phenolics(Siddhuraju and Becker, 2003; Upadhyay et al., 2015). Almost all the parts of this plant such asroot, bark, gum, leaf, fruit (pods), flowers, seed and seed oil have been reported as source ofdifferent biochemical compounds with anticarcinogenic, antiinflammatory, antidiabetic, antioxidant,Ⓒ ISERD59

IJERDInternational Journal of Environmental and Rural Development (2018) 9-2

and antimicrobial effects (Chuang et al., 2007; Upadhyay et al., 2015). M. oleifera leaf was foundto have high source of protein, beta-carotene, vitamin C, iron, potassium, and other nutrients(Jongrungruangchok et al., 2010). Extracts from M. oleifera roots and flowers were found to have asignificant hepatoprotective effect (Ruckmani et al., 1998).
The effect of food processing procedure on the antioxidant activity of foods are generally theresult of different processing. Thus, the evaluation of processing factors influencing the antioxidant
activity is imperative to increase or preserve their lability (Nicoli et al., 1999). Several studies havereported the antioxidant potential of M. oleifera leaf (Jongrungruangchok et al., 2010; Sreelatha andPadma, 2009; Verma et al., 2009; Vongsak et al., 2013). Only a few studies have reported theantioxidant activity of Moringa peel, pulp, and seed and their ability after heat process. Therefore,
this research aimed to in e iga e he effec of hea ea men on an io idan p ope ie of c dee ac f om Mo inga oleife a (peel, p lp, and eed).
OBJECTIVE
The objec i e of hi
d a o in e iga e he effec of heaof c de e ac f om Mo inga oleife a (peel, p lp, and eed).

ea men on an io idan p ope ie

METHODOLOGY
The Moringa oleifera pods were purchased from a commercial market in Phathumtani province,
Thailand and then separated into Moringa oleifera peel, pulp, and seed samples and cut into small
pieces (Fig. 1). Each sample was divided into raw and heat-treated samples (in a boiling water bathfor 15 min.). All samples were incubated at 60 ºC for 24 hours. The dried samples were powderedand passed through sieve no. 18 and analyzed for moisture content (AOAC, 2000). Each sample
was extracted with 50% ethanol at a ratio of 1:20 (w/v). The mixture was then shaken at room
temperature for 24 hours with a shaking speed of 150 rpm. Supernatant was collected and filteredthrough Whatman No.1 filter paper. Then, this crude extract was subjected to total phenolic content
and antioxidant activity assays.

(A)

(B)

(C)

Fig. 1 Moringa peel (A), pulp (B), and seed (C) samples used in this study
Total phenolic compound was determined by Folin-Ciocalteu method (Kähkönen et al., 1999).
B iefl , 500 L of each ample e e mixed well with 2.5 mL of 0.2 M Folin Ciocalteu reagent,followed by the addition of 2 mL of 7.5% (w/v) sodium carbonate. The mixture was allowed atroom temperature for 60 min and absorbance was measured at 7650 nm. The total phenolic content
was calculated from the calibration curve, and the results were expressed as mg gallic acidequivalents/g dry weight (mg gallic acid eq./g DW).
2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity assay (DPPH assay) was performed
according to Burits and Bucar (2000) with some modifications. Sample (1 mL) were mixed with 1
mL of 0.1 mM DPPH solution. The reaction tubes were wrapped in aluminum foil and incubated atⒸ ISERD

60

IJERDInternational Journal of Environmental and Rural Development (2018) 9-2

room temperature for 10 min in dark. The absorbance was monitored at 517 nm. DPPH assay wasexpressed as mg Trolox equivalents/g dry weight (mg Trolox eq./g DW).
Ferric reducing antioxidant power assay (FRAP assay) was performed according to Benzieand S ain (1996) i h ome modifica ion . Sample (100 L) e e mi ed i h 1900 L of FRAP
reagent. The absorbance of the reaction mixture was monitored at 593 nm after incubating at 37 ºC
for 10 min. FRAP value of each sample was expressed as mg ferrous equivalents/g dry weight (mgferrous eq./g DW).
Metal chelating activity assay was performed according to Decker and Welch (1990) withome modifica ion . Sample (100 L) e e mi ed i h 1,400 L of di illed a e and 100 L of 2mM FeCl2.4H2O. The reaction mixture was incubated at room temperature for 3 min. Then, theeac ion mie a added i h 400 L of 5 mM ferrozine and incubated at room temperature for10 min. The absorbance was monitored at 562 nm. Metal chelating activity of each sample wasexpressed as mg EDTA equivalents/g dry weight (mg EDTA eq./g DW).
All the extraction processes and the analyses on each sample were done in duplicate. Theexperiments followed a completely randomized design (CRD) comprising six crude extract
samples. Differences among mean values were established using Duncan Multiple Range Test(DMRT) at P<0.05.
RESULTS AND DISCUSSION
Moisture content of raw moringa peel, pulp, and seed powder were 9.13, 9.90, and 9.97,respectively. Whereas, moisture content of heat-treated moringa peel, pulp, and seed powder were
8.65, 8.88, and 8.92, respectively. Total phenolic content and DPPH radical scavenging activity ofraw and heat-treated moringa peel, pulp, and seed extracts were shown in Table 1. The results
showed that the highest total phenolic content was found in moringa peel (3.65 mg gallic acid eq./gDW), followed by pulp (3.44 mg gallic acid eq./g DW) and seed (2.85 mg gallic acid eq./g DW),
respectively. After heat treatment, moringa peel, pulp, and seed samples showed lower total
phenolic content than their respective raw samples. Siddhuraju and Becker (2003) reported that themain phenolic compounds in drumstick leaves and its extracts are flavonoid groups such as
quercetin and kaempferol. They also contained 3-caffeoylquinic and 5-caffeoylquinic acid. The
whole pods are reported to contain nitriles, isothiocyanate and thicarbamates (Faizi et al., 1995)
and has antioxidant activity, which is due to the presence of carotenoid compounds (Kumar et al.,2007).
Table 1 Total phenolic content and DPPH radical scavenging activity of raw andheat-treated Moringa oleifera peel, pulp, and seed extracts
SampleRaw moringa peel
Raw moringa pulp
Raw moringa seed
Heat-treated moringa peelHeat-treated moringa pulp
Heat-treated moringa seedTotal phenolic content(mg gallic acid eq./g DW)
3.65a ±0.03
3.44b ±0.25
2.80c ±0.02
1.38d ±0.01
1.19de±0.02
0.91e ±0.01

DPPH radical scavenging activity(mg Trolox eq./g DW)
1.35a ±0.02
1.25b ±0.03
0.91c ±0.04
0.78d ±0.03
0.41e ±0.04
0.23f ±0.01

Means of the same column with different superscripts indicate significant difference (P<0.05).

The antiradical activity was measured as ability to reduce DPPH radical by crude extract fromraw and heat-treated M. oleifera (peel, pulp, and seed). As shown in Table 1, raw moringa peel
showed the highest DPPH radical scavenging activity (1.35 mg Trolox eq./g DW), followed bypulp (1.25 mg Trolox eq./g DW) and seed (0.91 mg Trolox eq./g DW), respectively. The lower
DPPH radical scavenging activity was also found in heat-treated samples. These results are inagreement with Arabshahi-Delouee et al. (2007). Incubating moringa leaves extract at 100 ºC for
15 min resulted in a significant decrease in inhibition of lipid peroxidation by 17% usingⒸ ISERD61IJERDInternational Journal of Environmental and Rural Development (2018) 9-2

thiobarbituric acid (TBA) assay. Heat processing may have resulted in degradation of antioxidants
present in moringa leaves extract, thereby decreasing the activity.
FRAP of raw and heat-treated moringa peel, pulp, and seed extracts were shown in Table 2.
FRAP of raw moringa peel (3.62 mg ferrous eq./g DW), seed (3.32 mg ferrous eq./g DW), and pulp
extracts (3.28 mg ferrous eq./g DW) was comparable (P>0.05) and showed higher FRAP than allheat-treated samples (P<0.05). This method is based on the ability of a compound to donate one
electron to Fe3+ to reduce it to Fe2+, whereas DPPH radical scavenging assay is involved hydrogen
atom transfer.
Table 2 Ferric reducing antioxidant power and metal chelating activity of raw andheat-treated Moringa oleifera peel, pulp, and seed extracts
SampleRaw moringa peel
Raw moringa pulp
Raw moringa seed
Heat-treated moringa peelHeat-treated moringa pulp
Heat-treated moringa seed

Ferric reducing antioxidantpower (mg ferrous eq./g DW)
3.62a ±0.083.28ab±0.25
3.32ab±0.09
2.13c ±0.28
2.87b ±0.30
2.05c ±0.01

Metal chelating activity(mg EDTA eq./g DW)
0.41a ±0.04
0.42a ±0.06
0.19b ±0.01
0.15b ±0.01
0.13b ±0.01
0.01c ±0.00

Means of the same column with different superscripts indicate significant difference (P<0.05).

Metal chelating activity of raw moringa pulp (0.42 mg EDTA eq./g DW) showed the highest
content and was comparable with the raw moringa peel (0.41 mg EDTA eq./g DW) (P 0.05).
Heating proceed resulted in a significant decrease in metal chelating activity of all samples(P<0.05). Transition metals act as catalysts that promote the generation of the first few radicals,
which initiate the oxidative chain reaction. Thus, the chelating of transition metal ions by these
extracts would reduce available transition metals, rendering the inhibition of the radical-mediatedoxidative chain reactions (Zaid et al., 2012).
The decrease in FRAP and metal chelating activity of all samples might be due to the loss ofnaturally occurring antioxidants present in the extract or formation of novel compounds havingprooxidant activity upon heat processing at 100 ºC for 15 min. These results demonstrated thatantioxidant activities including DPPH radical scavenging activity, FRAP, and metal chelating
activity of moringa peel, pulp, and seed samples depends on heat treatment.
CONCLUSION
Crude extract obtained from raw moringa peel showed the highest total phenolic content and DPPH
radical scavenging activity followed by pulp and seed, respectively. The highest FRAP wereobtained from the raw moringa peel extract, whereas the highest metal chelating activity was foundin raw moringa pulp extract. Moringa peel, pulp and seed samples with heat treatment at 100 °C for
15 min showed lower total phenolic content and antioxidant properties than crude extract from raw
moringa samples.
ACKNOWLEDGEMENT
This work was supported by Faculty of Agricultural Technology, Rajamangala University ofTechnology Thanyaburi (RMUTT). The author would like to thank all the staff members of thedivision of food science and technology for all supports and assistance.

Ⓒ ISERD

62

IJERDInternational Journal of Environmental and Rural Development (2018) 9-2

REFERENCES
AOAC. 2000. Official methods of analysis. 15th ed. Wilson Boulevard Arlington, Verginia, USA.
Arabshahi-Delouee, S., Devi, D.V. and Urooj, A. 2007. Evaluation of antioxidant activity of some plant
extracts and their heat, pH and storage stability. Food Chem., 100, 1100-1105.
Benzie, I.F.F. and Strain, J.J. 1999. Ferric reducing/antioxidant power assay: Direct measure of total
antioxidant activity of biological fluids and modified version for simultaneous measurement of total
antioxidant power and ascorbic acid concentration. Methods Enzymol., 299, 15-27.
Burits, M. and Bucar, F. 2000. Antioxidant activity of Nigella sativa essential oil. Phytother Res., 14, 323328.
Chuang, P.H., Lee, C.W., Chou, J.Y., Murugan, M., Shieh, B.J. and Chen, H.M. 2007. Anti-fungal activity ofcrude extracts and essential oil of Moringa oleifera Lam. Bioresour. Technol., 98, 232-236.
Decker, E.A. and Welch, B. 1990. Role of ferritin as a lipid oxidation catalyst in muscle food. J. Agri. Food
Chem., 38, 674-677.
Faizi, S., Siddiqui, B.S., Saleem, R., Siddiqui, S., Aftab, K. and Gilani, A.H. 1995. Fully acetylated
carbamate and hypotensive thiocarbamate glycosides from Moringa oleifera. Phytochemistry, 38, 957963.
Hossain, M.B., Brunton, N.P., Barry-Ryan, C., Martin-Diana, A.B. and Wilkinson, M. 2008. Antioxidant
activity of spices extracts and phenolics in comparison to synthetic antioxidants. Rasayan J. Chem., 4,751-756.
Jongrungruangchok, S., Bunrathep, S. and Songsak, T. 2010. Nutrients and minerals content of elevendifferent samples of Moringa oleifera cultivated in Thailand. J. Health Res., 24, 123-127.
Kähkönen, M.P., Hopia, A.I., Vuorela, J., Rauha, J.P., Pihlaja, K., Kujala, T.S. and Heinonen, M. 1999.
Antioxidant activity of plant extracts containing phenolic compounds. J. Agri. Food Chem., 47 (1), 39543962.
Kumar, S., Kumar, D., Singh, N. and Vasisht, B.D. 2007. In-vitro free radicals scavenging and antioxidant
activity of Moringa oleifera pods. J. Herb. Med. Toxicol., 1, 117-122.
Nicoli, M.C., Anese, M. and Parpinel, M. 1999. Influence of processing on the antioxidant properties of fruits
and vegetables. Trends Food Sci. Technol., 10, 94-100.
Ruckmani, K., Kavimani, S., Anandan, R. and Jaykar, B. 1998. Effect of Moringa oleifera Lam onparacetamol-induced hepatoxicity. Indian J. Pharm. Sci., 60, 33-35.
Siddhuraju, P. and Becker, K. 2003. Antioxidant properties of various solvent extracts of total phenolicconstituents from three different agro-climatic origins of drumstick tree (Moringa oleifera Lam.). J. Agr.
Food Chem., 51 (8), 2144-2155.
Sreelatha, S. and Padma, P.R. 2009. Antioxidant activity and total phenolic content of Moringa oleifera
leaves in two stages of maturity. Plant Foods Hum. Nutr., 64, 303-311.
Upadhyay, P., Yadav, M.K., Mishra, S., Sharma, P. and Purohit, S. 2015. Moringa oleifera: A review of themedical evidence for its nutritional and pharmacological properties. Int. J. Res. Pharm. Sci., 5 (2), 12-16.
Verma, A.R., Vijayakumar, M., Mathela, C.S. and Rao, C.V. 2009. In vitro and in vivo antioxidant propertiesof different fractions of Moringa oleifera leaves. Food Chem. Toxicol., 47, 2196-2201.
Vongsak, B., Sithisarn, P., Mangmool, S., Thongpraditchote, S., Wongkrajang, Y. and Gritsanapan, W. 2013.
Maximizing total phenolics, total flavonoids contents and antioxidant activity of Moringa oleifera leaf
extract by the appropriate extraction method, Ind. Crops Prod., 44, 566-571.
Zaid, A.A., Farooqui, M. and Janrao, D.M. 2012. Study of stability constant of biological active molecules(drug) using potentiometric titration technique. J. Chem. Bio. Phy. Sci., 2, 67-81.

Ⓒ ISERD63