Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts.

Theeffects of four extracting solvents [absolute ethanol, absolute methanol, aqueous ethanol (ethanol: water, 80:20 v/v) and aqueous methanol (methanol: water, 80:20 v/v)] and two extraction techniques (shaking and reflux) on the antioxidant activity of extracts of barks of Azadirachta indica, Acacia nilotica, Eugenia jambolana, Terminalia arjuna, leaves and roots of Moringa oleifera, fruit of Ficus religiosa,and leaves of Aloe barbadensis were investigated. The tested plant materials contained appreciable amounts of total phenolic contents (0.31-16.5 g GAE /100g DW), total flavonoid (2.63-8.66 g CE/100g DW); reducing power at 10 mg/mL extract concentration (1.36-2.91), DPPH(.) scavenging capacity (37.2-86.6%), and percent inhibition of linoleic acid (66.0-90.6%). Generally higher extract yields, phenolic contents and plant material antioxidant activity were obtained using aqueous organic solvents, as compared to the respective absolute organic solvents. Although higher extract yields were obtained by the refluxing extraction technique, in general higher amounts of total phenolic contents and better antioxidant activity were found in the extracts prepared using a shaker.

Introduction

Plant-derived antioxidants, especially, the phenolics have gained considerable importance due to their potential health benefits. Epidemiological studies have shown that consumption of plant foods containing antioxidants is beneficial to health because it down-regulates many degenerative processes and can effectively lower the incidence of cancer and cardio-vascular diseases [1].

Recovery of antioxidant compounds from plant materials is typically accomplished through different extraction techniques taking into account their chemistry and uneven distribution in the plant matrix. For example, soluble phenolics are present in higher concentrations in the outer tissues (epidermal and sub-epidermal layers) of fruits and grains than in the inner tissues (mesocarp and pulp) [2]. Solvent extraction is most frequently used technique for isolation of plant antioxidant compounds. However, the extract yields and resulting antioxidant activities of the plant materials are strongly dependent on the nature of extracting solvent, due to the presence of different antioxidant compounds of varied chemical characteristics and polarities that may or may not be soluble in a particular solvent. Polar solvents are frequently employed for the recovery of polyphenols from a plant matrix. The most suitable of these solvents are (hot or cold) aqueous mixtures containing ethanol, methanol, acetone, and ethyl acetate [3]. Methanol and ethanol have been extensively used to extract antioxidant compounds from various plants and plant-based foods (fruits, vegetables etc.) such as plum, strawberry, pomegranate, broccoli, rosemary, sage, sumac, rice bran, wheat grain and bran, mango seed kernel, citrus peel, and many other fruit peels. Other studies have also demonstrated the efficacy of ethyl acetate to extract phenolic compounds from onion and citrus peel [3,4,5,6]. Bonoli et al. [7] reported that maximum phenolic compounds were obtained from barley flour with mixtures of ethanol and acetone. Similarly, aqueous methanol was found to be more effective in recovering highest amounts of phenolic compounds from rice bran [8], and Moringa oleifera leaves [9]. Anwar et al. [10] extracted antioxidant compounds from various plant materials including rice bran, wheat bran, oat groats and hull, coffee beans, citrus peel and guava leaves using aqueous 80% methanol (methanol: water, 80:20 v/v).

The medicinal plants selected for the present investigation, which included Moringa oleifera, Azadirachta indica, Terminalia arjuna, Acacia nilotica, Eugenia jambolana, Aloe barbadensis etc. have long been used in the folk medicine due to their potential health promoting and pharmacological attributes, which are mainly ascribed to the presence of antioxidant constituents such as phenolic acids and flavonoids [9,11,12,13]. It is important to establish appropriate means to evaluate and quantify effective antioxidant principles of medicinally or economically viable plant materials. The present study therefore was conducted with the main objective of investigating the most effective solvent/technique for extracting potent antioxidant compounds, especially phenolics from different parts of selected medicinal plants native to Pakistan.

Results and Discussion

Effects of extracting solvent/technique on the extracts yields from different medicinal plant materials

Amounts (g/100g of dried plant material) of the antioxidant extract determined for different medicinal plant materials, using four different solvents (absolute methanol and aqueous methanol (methanol: water, 80:20 v/v); absolute ethanol and aqueous ethanol, (ethanol: water, 80:20 v/v) and two extracting techniques: shaker and reflux are shown in Table 1.

Table 1

Effects of extracting solvent/technique on the extract yield (g/100 g of DW) of medicinal plant materials.

Medicinal plant organs	Extraction by shaker
	Absolute methanol	Aqueous (80%) methanol	Absolute ethanol	Aqueous (80%) ethanol
Moringa oleifera leaves	9.61 ± 0.39dc	17.9 ± 0.18ca	8.94 ± 0.27dc	12.6 ± 0.51db
Moringa oleifera root	3.24 ± 0.14eb	6.65 ± 0.19ea	2.23 ± 0.12eb	3.63 ± 0.26eb
Eugenia jambolana bark	19.2 ± 0.38ba	14.1 ± 0.56db	2.81 ± 0.39ec	13.5 ± 0.40db
Acacia nilotica bark	23.4 ± 0.47ab	31.6 ± 0.95aa	13.1 ± 0.52bc	15.7 ± 0.32cc
Azadirachta indica bark	10.7 ± 0.22dc	13.8 ± 0.55dc	37.2 ± 0.74aa	25.0 ± 0.53bb
Terminalia arjuna bark	22.5 ± 0.67ab	23.3 ± 0.45bb	34.5 ± 0.44aa	37.2 ± 0.46aa
Ficus religiosa fruit	18.9 ± 0.76bb	26.4 ± 0.52ba	16.9 ± 0.67bb	19.7 ± 0.39cb
Aloe barbadensis leaves	15.6 ± 0.62cb	17.8 ± 0.36ca	10.8 ± 0.43dc	15.2 ± 0.68cb
	Extraction by reflux
Moringa oleifera leaves	16.6 ± 0.33db	21.1 ± 0.84ca	12.2 ± 0.37cc	17.2 ± 0.35cb
Moringa oleifera root	5.12 ± 0.21ebc	8.97 ± 0.36da	4.86 ± 0.21dc	6.27 ± 0.26db
Eugenia jambolana bark	25.6 ± 0.51ba	16.9 ± 0.33cc	15.3 ± 0.37bcc	19.5 ± 0.46cb
Acacia nilotica bark	26.2 ± 0.78bb	32.8 ± 0.65aa	18.2 ± 0.55bc	20.2 ± 0.61cc
Azadirachta indica bark	14.2 ± 0.29dc	17.8 ± 0.53cc	42.4 ± 0.64aa	31.9 ± 0.63bb
Terminalia arjuna bark	28.6 ± 0.46ab	24.9 ± 0.49bb	40.7 ± 0.86aa	46.6 ± 083aa
Ficus religiosa fruit	21.3 ± 0.64cc	29.2 ± 0.88aa	19.5 ± 0.58bc	22.8 ± 0.91cc
Aloe barbadensis leaves	17.5 ± 0.73db	20.3 ± 0.41ca	13.2 ± 0.52cc	18.1 ± 0.72cb

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same column indicate significant (P< 0.05) differences of means within the plant materials.

Our findings are in agreement with previous investigation of Chatha et al. [8], who reported that maximum extract yield (g/100g) from rice bran was obtained with aqueous methanol.

The differences in the extract yields from the tested plant materials in the present analysis might be ascribed to the different availability of extractable components, resulting from the varied chemical composition of plants [14]. The amount of the antioxidant components that can be extracted from a plant material is mainly affected by the vigor of the extraction procedure, which may probably vary from sample to sample. Amongst other contributing factors, efficiency of the extracting solvent to dissolve endogenous compounds might also be very important [9,15].

For the effectiveness of extracting technique, the results showed that yields of the extract were better when extraction was done under reflux, regardless of the plant material and solvent used. This indicates that hot solvent systems under reflux state are more efficient for the recovery of antioxidant components, thus offering higher extract yields. This is in agreement with the findings of Shon et al. [16] who investigated that methanol and hot water are more efficient to extract antioxidant compounds from Phellinus baumii.

Effects of extracting solvent/technique on the total phenolic contents of different plant materials

Total phenolic contents (TPC) of different plant materials, using four solvent systems: absolute and aqueous methanol and absolute and aqueous ethanol and two extracting techniques (shaker and reflux) are presented in Table 2. Among the different medicinal plant materials, aqueous ethanolic extract of Acacia nilotica bark offered the highest TPC (16.5 g GAE/100g of DW), followed by aqueous ethanolic extract (aq. EE) of Terminalia arjuna bark (12.8 %), aq. ME of Moringa oleifera leaves (12.2%), aq. EE of Azadirachta indica bark (12.0%), aq. ME of Aloe barbadensis leaves (10.3%), aq. EE of Eugenia jambolana bark (9.03%), aq. ME of Ficus religiosa fruit (5.34%), and aq. ME of Moringa oleifera roots (0.31%).

Table 2

Effects of extracting solvent/technique on the total phenolic contents (GAE g/100 g of DW) of medicinal plants materials.

Medicinal plant organs	Extraction by shaker
	Absolute methanol	Aqueous (80%) methanol	Absolute ethanol	Aqueous (80%) ethanol
Moringa oleifera leaves	10.3 ± 0.41abab	12.2 ± 0.28aa	9.72 ± 0.21ab	11.6 ± 0.21bab
Moringa oleifera root	0.22 ± 0.07db	0.31± 0.06ea	0.14 ± 0.01ec	0.27 ± 0.08fab
Eugenia jambolana bark	10.1 ± 0.39aba	8.30 ± 0.49bb	8.12 ± 0.35bb	9.03 ± 0.45cab
Acacia nilotica bark	12.7 ± 0.28ab	11.2 ± 0.33abb	11.2 ± 0.31ab	16.5 ± 0.66aa
Azadirachta indica bark	11.1 ± 0.66abab	9.34 ± 0.37abab	8.48 ± 0.26bb	12.0 ± 0.36ba
Terminalia arjuna bark	12.2 ± 0.57aa	7.80 ± 0.39cb	10.2 ± 0.39aab	12.8 ± 0.26ba
Ficus religiosa Fruit	3.13 ± 0.19cab	5.34 ± 0.36da	2.67 ± 0.16db	4.11 ± 0.18eab
Aloe barbadensis leaves	8.25 ± 0.28bab	10.3 ± 0.28aba	6.53 ± 0.38cb	7.93 ± 0.31dab
	Extraction by reflux
Moringa oleifera leaves	9.63 ± 0.28bab	10.7 ± 0.31aa	6.16 ± 0.26cb	8.21 ± 0.36cab
Moringa oleifera root	0.17 ± 0.02ec	0.27 ± 0.04ea	0.12 ± 0.03ed	0.23 ± 0.06fb
Eugenia jambolana bark	8.91 ± 0.39ca	8.14 ± 0.33ba	7.94 ± 0.31ba	8.64 ± 0.27ca
Acacia nilotica bark	12.22 ± 0.21aab	10.7 ± 0.24ab	10.8 ± 0.28ab	14.6 ± 0.29aa
Azadirachta indica bark	9.72 ± 0.33ba	7.91 ± 0.39bb	7.23 ± 0.23bb	10.8 ± 0.38ba
Terminalia arjuna bark	11.63 ± 0.29aba	6.25 ± 0.30cb	9.67 ± 0.38aab	11.9 ± 0.46ba
Ficus religiosa Fruit	2.12 ± 0.09db	4.93 ± 0.28da	2.26 ± 0.10db	4.13 ± 0.21ea
Aloe barbadensis leaves	7.29 ± 0.27cb	9.24 ± 0.26aba	6.44 ± 0.29cb	6.94 ± 0.27df

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same column indicate significant (P< 0.05) differences of means within the plant materials.

Results of the present study showed that among all the solvent extracts; the aqueous methanol and aqueous ethanol extracts had the highest TPC. This may be due to the fact that phenolics are often extracted in higher amounts in more polar solvents such as aqueous methanol/ethanol as compared with absolute methanol/ethanol [9,10,15].

The determined amounts of total phenolics (TP) from the tree barks investigated in the present study were lower than that reported for Acacia confusa bark [17]. Except for Eugenia jambolana, the barks of the other three plants offered greater amount of total phenolics than those of pine bark (11.4 g GCE/100g DW) [18]. TPC of Moringa oleifera leaves investigated in the present analysis are in agreement with previous reports [19]. The amount of TP of Moringa oleifera roots were found to be lower than those of Chinese herbal roots of kudzu vine (1.37 g GAE/100g) and dahurian (1.2 g GAE/100g) [20]. The levels of TP determined in the present analysis of Ficus religiosa fruit were found to be lower than those reported in Ficus microcarpa fruit (17.9 g GAE/100g) [21].

In contrast to the trends noted for extraction yields, the TPC of all medicinal plant materials extracted using the reflux technique decreased, regardless of the nature of the extracting solvent used. The decrease in the amounts of TP of these plant material extracts, prepared under reflux might have been due to the thermal decomposition of some phenolic antioxidants at the higher temperatures used for reflux extraction.

It has been reported that thermal processing conditions might result in the loss of natural antioxidants because heat may accelerate their oxidation and other degenerative reactions. Thus, heating temperature is of much consideration during processing. An accelerated shelf-life test at 80 °C for 4 days resulted in 20-40% decrease of the antioxidant activity of the apple juice [22]. Cheng et al. [23] reported that antioxidant activity of wheat bran decreased up to 61% by heating at 100 °C for 9 days. On the other hand, Dutra et al. [24] reported that among different extraction techniques (reflux, maceration, ultrasound, heating plate), extraction made under reflux using ethanol/water (70:30, v/v) offered the highest polyphenol levels in Vogel seeds. This might be attributed to an effective extraction under reflux conditions leading to higher release of some bound phenolics [2].

Effects of extracting solvent/technique on the total flavonoids of different plant materials

Total flavonoid contents (TFC) of various plant materials, extracted with four different solvent systems, using shaker and reflux extracting techniques, are given in Table 3. TFC were determined as catechin equivalents (CE). Among medicinal plant materials, aq. ME of Moringa oleifera leaves offered the highest TFC (8.66 g CE/100 g of DW) followed by aq. EE of Acacia nilotica bark (4.93), aq. ME of Aloe barbadensis leaves (2.28), aq. ME of Ficus religiosa fruit (3.77), aq. EE of Terminalia arjuna bark (3.49), aq. EE of Azadirachta indica bark (3.14), aq. ME of Moringa oleifera root(2.94), and ab. ME of Eugenia jambolana bark (2.63). Amount of TF in all the medicinal plant extracts generally decreased when reflux technique employed for their preparation. However, TFC of Aloe barbadensis leaves increased from 4.28 to 4.66 g CE/100 g of DW, when extracted with aqueous methanol using the reflux technique. Ficus religiosa fruits also contained higher TFC using the reflux technique with absolute and aqueous ethanol. TFC (1.47-3.77g/100g) of Ficus religiosa fruit in our analysis were found to be higher than that reported for Ficus microcarpa fruit (0.6 g/100 g dry weight) [21]. On the other hand, TFC in Terminalia arjuna bark (1.52-3.49 g/100g) determined in our work were lower than those (5.70 g/100 g dry weight) investigated by Dwivedi [12].

Table 3

Effects of extracting solvent/technique on the total flavonoid contents (CE g/100 g of DW) of medicinal plants materials.

Medicinal plant organs	Extraction by shaker
	Absolute methanol	Aqueous (80%) methanol	Absolute ethanol	Aqueous (80%) ethanol
Moringa oleifera leaves	6.06 ± 0.12ab	8.66 ± 0.21aa	5.33 ± 0.13ab	6.21 ± 0.11ab
Moringa oleifera root	1.68 ± 0.06db	2.94 ± 0.08ca	1.22 ± 0.04eb	1.59 ± 0.03db
Eugenia jambolana bark	2.63 ± 0.04ca	1.72 ± 0.07dab	1.68 ± 0.06db	2.10 ± 0.06cdab
Acacia nilotica bark	4.86 ± 0.09ba	3.21 ± 0.12bcb	3.15 ± 0.14bb	4.93 ± 0.15ba
Azadirachta indica bark	2.93 ± 0.10ca	3.31 ± 0.16bca	2.68 ± 0.12ca	3.14 ± 0.09ca
Terminalia arjuna bark	3.01 ± 0.13cab	2.13 ± 0.13cb	2.64 ± 0.09cab	3.49 ± 0.11ca
Ficus religiosa fruit	2.16 ± 0.08cb	3.77 ±0.10bca	1.28 ± 0.04ec	2.03 ± 0.06cdb
Aloe barbadensis leaves	2.91 ± 0.16cb	4.28 ± 0.17ba	1.68 ± 0.02dc	2.96 ± 0.04cb
	Extraction by reflux
Moringa oleifera leaves	5.90 ± 0.16ab	7.29 ± 0.18aa	4.19 ± 0.09ac	5.31 ± 0.19ab
Moringa oleifera root	1.02 ± 0.03eb	2.86 ± 0.13ca	0.89 ± 0.07eb	1.21 ± 0.07db
Eugenia jambolana bark	1.99 ± 0.06da	0.83 ± 0.02ebc	1.06 ± 0.04dec	1.55 ± 0.05db
Acacia nilotica bark	3.92 ± 0.12ba	2.52 ± 0.06cb	3.00 ± 0.13bb	4.19 ± 0.11ba
Azadirachta indica bark	2.16 ± 0.08db	2.90 ± 0.04ca	1.66 ± 0.06dc	2.99 ± 0.13ca
Terminalia arjuna bark	1.78 ± 0.06dab	1.52 ± 0.05db	2.11 ± 0.11cab	2.63 ± 0.08ca
Ficus religiosa fruit	1.97 ± 0.05dc	3.56 ± 0.11bca	1.47 ± 0.05dc	2.86 ± 0.12cb
Aloe barbadensis leaves	2.90 ± 0.07cb	4.66 ± 0.09ba	1.39 ± 0.07dc	2.55 ± 0.09cb

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same column indicate significant (P< 0.05) differences of means within the plant materials.

Effects of extracting solvent/technique on the reducing power of different plant materials

The results showing the effects of extracting solvent/techniques on the reducing potential of extracts of different plant materials at concentration of 10 mg/mL, are shown in Table 4. The reducing power of the medicinal plant extracts increased in a concentration dependent manner (data not shown). The values of absorbance for the tested extract solutions at concentration of 10 mg/mL determined in this assay, ranged from 0.09 to 2.88 and followed the order of effectiveness as: aq. ME of Moringa oleifera leaves (2.88)> aq. ME of Aloe barbadensis leaves (2.81) > aq. EE of Acacia nilotica bark (1.87)> aq. EE of Azadirachta indica bark (1.71) > aq. ME of Terminalia arjuna bark (1.66) > aq. EE of Eugenia jambolana bark (1.60) > aq. ME of Ficus religisa fruit (1.36) > aq. ME of Moringa oleifera roots (0.14).

Table 4

Effects of extracting solvent/technique on reducing power (expressed as absorbance values at 700 nm) of different medicinal plant materials.

Medicinal plant organs	Extraction by shaker
	Absolute methanol	Aqueous (80%) methanol	Absolute ethanol	Aqueous (80%) ethanol
Moringa oleifera leaves	2.45 ± 0.05ab	2.88 ± 0.03aa	1.53 ± 0.04ac	2.50 ± 0.06ab
Moringa oleifera root	0.09 ± 0.01eb	0.14 ± 0.02ca	0.09 ± 0.01cb	0.12 ± 0.02da
Eugenia jambolana bark	1.06 ± 0.04dc	1.48 ± 0.04bb	0.98 ± 0.02bc	1.60 ± 0.03bca
Acacia nilotica bark	1.68 ± 0.05cab	1.52 ± 0.04bab	1.45 ± 0.06ab	1.87 ± 0.05ba
Azadirachta indica bark	1.55 ± 0.03cb	1.46 ± 0.05bb	1.05 ± 0.05bc	1.71 ± 0.03ba
Terminalia arjuna bark	1.26 ± 0.02cdab	1.66 ± 0.04ba	1.12 ± 0.02bb	1.34 ±0.04bcab
Ficus religiosa Fruit	1.06 ± 0.04db	1.36 ± 0.07ba	0.92 ± 0.06bb	0.99 ± 0.07cdb
Aloe barbadensis leaves	2.01 ± 0.03bb	2.81 ± 0.05aa	1.56 ± 0.04ac	2.16 ± 0.04ab
	Extraction by reflux
Moringa oleifera leaves	1.25 ± 0.05bb	1.78 ± 0.03ba	0.94 ± 0.04bcc	0.95 ± 0.04cc
Moringa oleifera root	0.06 ± 0.01db	0.13 ± 0.02da	0.09 ± 0.03db	0.11 ± 0.02da
Eugenia jambolana bark	0.80 ± 0.04cb	1.26 ± 0.05cda	0.61 ± 0.05cb	1.39 ± 0.07bca
Acacia nilotica bark	1.25 ± 0.06bab	1.13 ± 0.04db	1.05 ± 0.07bcb	1.62 ± 0.05ba
Azadirachta indica bark	1.16 ± 0.03bb	1.10 ± 0.07db	0.79 ± 0.02cb	1.56 ± 0.06ba
Terminalia arjuna bark	1.11 ± 0.05bb	1.46 ± 0.02ca	0.62 ± 0.03cc	0.99 ± 0.03cb
Ficus religiosa Fruit	1.13 ± 0.02bb	1.22 ± 0.05cda	1.26 ± 0.06ba	1.32 ± 0.02bca
Aloe barbadensis leaves	2.18 ± 0.04ab	2.96 ± 0.08aa	1.72 ± 0.04ac	1.88 ± 0.04ac

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same column indicate significant (P< 0.05) differences of means within the plant materials.

In general, the aqueous organic solvent extracts of the tested plant materials, exhibiting greater TPC, also depicted good reducing power in the present analysis. The reducing potential of antioxidant components is very much associated with their TPC. The plant extracts with higher levels of total phenolics also exhibit greater reducing power [23,9,15].

As far as the effects of extraction techniques on the antioxidant activity is concerned, apart from the leaves of Aloe vera and fruit of Ficus religiosa, reducing powers of all the medicinal plant materials extracts were adversely affected by reflux extracting technique, regardless of the solvent used. However, each material tested retained the same efficacy order as displayed in the case of shaker extraction.

Effects of extracting solvent/technique on the DPPH. Scavenging activity (% DPPH. remaining) of different plant materials

DPPH. scavenging activity of different plant materials as affected by extracting methods is depicted in Table 5. Absorbance in this assay was recorded at 0.5 to 10 min time intervals from initiation of the reaction. Observed scavenging activity was similar at the beginning of the reaction and changed with increase in the reaction time until it stabilized by the 10th min. Significant (p< 0.05) differences of DPPH. scavenging capacities among extracts were observed at 5th minute of the reaction. The DPPH. scavenging ability of the sample extracts was reported as the percent of DPPH. scavenged (% DPPH. scavenging). As expected, a higher percent of DPPH. scavenging is correlated to a higher antioxidant activity [15,20].

Table 5

Effects of extracting solvent/technique the DPPH. scavenging activity (%) of different medicinal plant materials.

Medicinal plant organs	Extraction by shaker
	Absolute methanol	Aqueous (80%) methanol	Absolute ethanol	Aqueous (80%) ethanol
Moringa oleifera leaves	82.8 ± 1.7aa	86.3 ± 1.8aa	70.9 ± 2.2bb	85.2 ± 1.7aa
Moringa oleifera root	51.4 ± 2.2cdb	62.9 ± 1.6ba	56.6 ± 1.2cdab	61.8 ± 2.3ca
Eugenia jambolana bark	48.8 ± 1.4db	52.7 ± 1.3bca	50.4 ± 1.1dab	53.9 ± 1.9da
Acacia nilotica bark	56.3 ± 1.6cb	60.9 ± 1.4bb	82.5 ± 1.7aa	86.6 ± 1.5aa
Azadirachta indica bark	53.2 ± 1.5cdb	57.4 ± 1.3bab	57.3 ± 1.4cdab	60.8 ± 1.8ca
Terminalia arjuna bark	37.7 ± 1.3ec	48.5 ± 1.5cb	62.9 ± 1.5ca	67.4 ± 1.4ba
Ficus religiosa fruit	57.2 ± 1.7cb	63.4 ± 1.1ba	55.3 ± 1.3cdb	60.1 ± 1.9ca
Aloe barbadensis leaves	73.7 ± 1.3bb	80.1 ± 2.3aa	67.2 ± 1.9bc	70.7 ± 1.2bb
	Extraction by reflux
Moringa oleifera leaves	81.6 ± 1.9aa	79.4 ± 1.6ab	69.2 ± 1.3bc	80.6 ± 1.8aa
Moringa oleifera root	53.9 ± 1.5cb	64.8 ± 1.2ba	58.8 ± 1.6cab	62.7 ± 1.9ca
Eugenia jambolana bark	48.1 ± 1.7cb	51.4 ± 1.4ca	49.2 ± 1.2dab	52.7 ± 1.5da
Acacia nilotica bark	55.8 ± 1.3cc	60.7 ± 1.6bb	81.9 ± 1.7aa	81.1 ± 1.8aa
Azadirachta indica bark	51.6 ± 1.2cb	54.4 ± 1.4cab	57.7 ± 1.3ca	58.4 ± 1.7ca
Terminalia arjuna bark	37.2 ± 1.1dc	47.3 ± 1.3cb	61.6 ± 1.3ca	65.9 ± 1.4ca
Ficus religiosa fruit	55.9 ± 1.4cb	62.9 ± 1.7ba	56.2 ± 1.4cb	63.8 ± 1.6ca
Aloe barbadensis leaves	72.9 ± 1.5bab	77.6 ± 1.9aa	68.0 ± 1.3bb	71.9 ± 1.2bab

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same column indicate significant (P< 0.05) differences of means within the plant materials.

The extracts of all the tested medicinal plant materials possessed free radical scavenging properties, but to varying degrees, ranging from 37.2 to 86.6% DPPH. scavenging. Using the shaker extraction technique, generally aq. EE and aq. ME showed better DPPH. scavenging activity. A maximum scavenging activity was offered by aq. EE of Acacia nilotica bark (86.6 %), followed by aq. ME of Moringa oleifera leaves (86.3%), aq. ME of Aloe barbadensis leaves (80.1%), aq. EE Terminalia arjuna bark (67.4%), aq. ME of Ficus religiosa fruit (63.4%), aq. ME of Moringa oleifera root (62.9%), aq. EE of Azadirachta indica bark (60.8%), and aq. EE of Eugenia jambolana bark (53.9%). The DPPH. scavenging activity seen in the different barks investigated in the present study was found to be lower than that of pine bark (95.1%) [17], while methanolic extract of Aloe barbadensis presented DPPH. scavenging activity (72.19%) comparable with that of earlier findings of Hu et al. [11].

The ethanolic extracts of roots of Moringa oleifera, leaves of Aloe barbadensis, and fruit of Ficus religiosa prepared by the reflux technique showed better scavenging activity as compared with those of prepared by the shaking technique. These results are in good agreement with the previous findings of Dutra et al. [23] who found that among different extraction techniques (reflux, maceration, ultrasound, heating plate) used, extraction made under reflux using ethanol/water (70:30, v/v) exhibited highest DPPH. scavenging activity. The rest of the medicinal plant materials extracts, prepared using shaker extracting method exhibited better scavenging activity than their corresponding extracts, obtained by reflux technique. It has well established that free radical scavenging activity of plant extracts is mainly due to phenolic compounds. This reduction in the radical scavenging activity of the extracts, obtained by the reflux technique might be ascribed to the thermal decomposition of phenolics [23].

Effects of extracting solvent/technique on the percent inhibition of linoleic acid peroxidation of different plant materials

Inhibition of linoleic acid oxidation determined for extracts of different plant materials as affected by extracting schemes are shown in Table 6.

Table 6

Effects of extracting solvent/technique on the percent inhibition of linoleic acid peroxidation of different medicinal plant materials.

Medicinal plant organs	Extraction by shaker
	Absolute methanol	Aqueous (80%) methanol	Absolute ethanol	Aqueous (80%) ethanol
Moringa oleifera leaves	79.9 ± 2.4bab	86.2 ±2.6aa	73.3 ± 1.5bb	82.9 ± 1.6ba
Moringa oleifera root	47.6 ± 1.9eb	66.7 ±2.4da	45.3 ± 1.8eb	65.2 ± 1.3ca
Eugenia jambolana bark	85.2 ± 2.6aab	80.4 ± 1.6bb	90.6 ± 2.7aa	90.2 ± 1.8aa
Acacia nilotica bark	85.2 ± 1.8aa	78.2 ± 2.3bb	86.2 ± 1.6aa	69.2 ± 2.7cc
Azadirachta indica bark	65.0 ± 2.6cb	71.4 ± 2.8ca	47.8 ± 1.8ed	55.1 ± 1.6dc
Terminalia arjuna bark	31.1 ± 1.2fc	44.4 ±1.7ec	61.8 ± 2.4cb	66.0 ± 2.6ca
Ficus religiosa fruit	59.2 ± 1.7db	67.4 ±2.1da	54.9 ± 2.1ddc	60.8 ± 2.4db
Aloe barbadensis leaves	66.2 ± 1.3ca	68.3 ±1.4da	63.7 ± 1.9cb	65.9 ± 1.9cab
	Extraction by reflux
Moringa oleifera leaves	68.2 ± 2.0bb	82.6 ± 1.6aa	68.7 ± 2.0bb	80.5 ± 1.7ba
Moringa oleifera root	40.8 ± 1.6db	64.8 ± 2.5ca	43.2 ± 1.7db	63.6 ± 1.9cda
Eugenia jambolana bark	83.3 ± 2.4aa	78.1 ± 2.3ab	88.9 ± 2.6aa	87.1 ± 1.8aa
Acacia nilotica bark	84.1 ± 1.5aa	72.1 ± 1.4bb	85.3 ± 2.5ba	66.3 ± 1.6cc
Azadirachta indica bark	63.2 ± 1.2bb	69.1 ± 2.0bca	44.3 ± 1.7dd	50.8 ± 2.0ec
Terminalia arjuna bark	59.4 ± 1.7ca	36.2 ± 1.4db	61.4 ± 1.2ba	63.2 ± 2.5cda
Ficus religiosa fruit	59.6 ± 2.3cb	67.1 ± 2.0bca	55.2 ± 1.6cc	61.9 ± 2.4db
Aloe barbadensis leaves	64.3 ± 1.9bb	67.9 ± 1.3bca	66.2 ± 1.3ba	67.3 ± 2.1ca

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same column indicate significant (P< 0.05) differences of means within the plant materials.

The present data were also compared with that of the synthetic antioxidant BHT (reference compound), which exhibited inhibition of linoleic acid oxidation at a level of 81.3 %. Among medicinal plant materials, maximum inhibition was noted by ab. EE of Eugenia jambolana bark (90.6%), followed byab. EE of Acacia nilotica bark= aq. ME of Moringa oleifera leaves (86.2%), aq. ME Azadirachta indica bark (71.4%), aq. ME of Aloe barbadensis leaves (68.3%), aq. ME of Ficus religiosa fruits (67.4%), aq. ME of Moringa oleifera roots (66.7%), and aq. EE of Terminalia arjuna bark (66.0%). The present level of percent inhibition exhibited by Moringa oleifera leaf extract (86.2%) was slightly lower than the values (89.7-92.0%) reported by Siddhuraju and Becker [9]. The extents of percent inhibition of linoleic acid (31.1-90.6%) exhibited by tree barks in the present investigation were found to be lower than the values (95.1%) offered by pine bark [17]. The present data revealed that, regardless of the solvent used, the extracts of all medicinal plant materials, prepared using the shaker extracting technique, exhibited higher levels of inhibition of linoleic acid oxidation than those obtained by the reflux method.

Conclusions

The results of the present investigation revealed that aqueous solvent (80% methanol, 80% ethanol) extracts of plant materials, prepared by both the shaker and reflux extraction techniques, exhibited better antioxidant activities and higher phenolic contents. Moreover, higher antioxidant extracts yields from the tested plant materials were obtained using the reflux extraction technique than by shaker, regardless of the solvent system used. Contrarily to extraction yield, the total phenolic contents and antioxidant activities of the tested plant materials decreased when these were extracted using the reflux technique. The present data would certainly help to ascertain the potency of the tested medicinal plant materials as potential source of natural antioxidants to be used for nutraceutical and functional food applications. However, further research is needed to identify individual components forming antioxidative system and develop their applications for food and pharmaceutical industries.

Experimental

Plant material

The selection of the plant materials in the present study was based on their potential medicinal uses. Medicinal plant parts i.e. barks of Azadirachta indica (Neem), Acacia nilotica (Desi kiker), Eugenia jambolana (Jaman), Terminalia arjuna (Arjun), leaves and roots of Moringa oleifera (Sohanjana), fruit of Ficus religiosa (Peepal), and leaves of Aloe barbadensis (Aloe vera) were collected from plants in the vicinity of the University of Agriculture, Faisalabad, Pakistan. The subject plant material specimens were further authenticated by a taxonomist, Dr. Mansoor Hameed, Department of Botany, University of Agriculture, Faisalabad, Pakistan.

Chemicals and reagents

Analytical grade, Merck, Sigma and Fluka brand chemicals and reagents were used for the entire experimentation. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPHSigma, 90.0 %), linoleic acid, food grade synthetic antioxidant butylated hydroxytoluene (BHT, 99.0 %), Folin-Ciocalteu reagent (2 N), and gallic acid were purchased from Sigma Chemicals Co (St, Louis, MO, USA). All other chemicals (analytical grade) i.e. sodium hydroxide, sodium nitrite, ferrous chloride, ammonium thiocyanate, aluminum chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, used in this study were purchased from Merck (Darmstadt, Germany), unless stated otherwise.

Extraction of phenolic antioxidants

The air-dried ground (80 mesh) plant material (20 g for each sample) was extracted with each of the solvents – absolute ethanol, absolute methanol, aqueous ethanol (ethanol: water, 80:20 v/v) and aqueous methanol (methanol: water, 80:20 v/v) (200 mL) – for 6 hours at room temperature in an orbital shaker (Gallenkamp, UK), or under reflux on a water bath in separate experiments. The extracts were separated from the residues by filtering through Whatman No. 1 filter paper. The residues were extracted twice with the same fresh solvent and extracts combined. The combined extracts were concentrated and freed of solvent under reduced pressure at 45 °C, using a rotary evaporator (EYELA, SB-651, Rikakikai Co. Ltd. Tokyo, Japan). The dried crude concentrated extracts were weighed to calculate the yield and stored in a refrigerator (- 4 oC), until used for analyses.

Evaluation of antioxidant activity of plant materials/extracts

Determination of total phenolic content (TPC): Amount of TP were assessed using the Folin-Ciocalteu reagent [25]. Briefly, the crude extract (50 mg) was mixed with Folin-Ciocalteu reagent (0.5 mL) and deionized water (7.5 mL). The mixture was kept at room temperature for 10 min, and then 20% sodium carbonate (w/v, 1.5 mL) was added. The mixture was heated in a water bath at 40 oC for 20 min and then cooled in an ice bath; absorbance was read at 755 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). Amounts of TP were calculated using gallic acid calibration curve within range of 10-100 mgL-1(R2 = 0.9986). The results were expressed as gallic acid equivalents (GAE) g/100g of dry plant matter. All samples were analyzed thrice and the results averaged. The results are reported on dry weight basis (DW).

Determination of total flavonoid contents (TFC): The TFC were measured following a previously reported spectrophotometric method [26]. Briefly, extracts of each plant material (1 mL containing 0.1 mg/mL) were diluted with water (4 mL) in a 10 mL volumetric flask. Initially, 5% NaNO2 solution (0.3 mL) was added to each volumetric flask; at 5 min, 10% AlCl3 (0.3 mL) was added; and at 6 min, 1.0 M NaOH (2 mL) was added. Water (2.4 mL) was then added to the reaction flask and mixed well. Absorbance of the reaction mixture was read at 510 nm. TFC were determined as catechin equivalents (g/100g of dry weight). Three readings were taken for each sample and the results averaged.

Determination of reducing power: The reducing power of the extracts was determined according to the procedure described earlier [27], with a slight modification. Concentrated extract (2.5-10.0 mg) was mixed with sodium phosphate buffer (5.0 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the mixture was incubated at 50 oC for 20 min. Then 10% trichloroacetic acid (5 mL) was added and the mixture centrifuged at 980 g for 10 min at 5 °C in a refrigerated centrifuge (CHM-17; Kokusan Denki, Tokyo, Japan). The upper layer of the solution (5.0 mL) was decanted and diluted with 5.0 mL of distilled water and ferric chloride (1.0 mL, 0.1%), and absorbance read at 700 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). All samples were analyzed thrice and the results averaged.

DPPH: 1, 1’–diphenyl–2-picrylhydrazyl (DPPH) free radical scavenging activity of the extracts was assessed using the procedure reported earlier [28]. Briefly, to extract (1.0 mL) containing 25 μg/mL of dry matter in methanol, freshly prepared solution of DPPH (0.025 g/L, 5.0 mL) was added. Absorbance at 0, 0.5, 1, 2, 5 and 10 min was measured at 515 nm using a spectrophotometer. The scavenging amounts of DPPH radical (DPPH.) was calculated from a calibration curve. Absorbance read at the 5th min was used for comparison of radical scavenging activity of the extracts.

Determination of antioxidant activity in linoleic acid system: The antioxidant activity of the tested plant extracts was also determined by measuring the oxidation of linoleic acid [28]. Five mg of each plant extract were added separately to a solution of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 0.2 M sodium phosphate buffer (pH 7, 10 mL). The mixture was made up to 25 mL with distilled water and incubated at 40 oC up to 360 h. Extent of oxidation was measured by peroxide value applying thiocyanate method as described by Yen et al. [27]. Briefly, ethanol (75% v/v, 10 mL ), aqueous solution of ammonium thiocyanate (30% w/v, 0.2 mL), sample solution (0.2 mL) and ferrous chloride (FeCl2) solution (20 mM in 3.5% HCl; v/v, 0.2 mL) were added sequentially. After 3 min of stirring, the absorption was measured at 500 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). A control contained all reagents with exception of extracts. Synthetic antioxidants butylated hydroxytoluene (BHT) was used as a positive control. Percent inhibition of linoleic acid oxidation was calculated with the following equation: 100 – [(increase in absorbance of sample at 360 h / increase in absorbance of control at 360 h) × 100], to express antioxidant activity.

Statistical analysis

Three samples of each plant material were assayed. Each sample was analyzed individually in triplicate and data are reported as mean (n = 3 x 3 x1) ± SD (n = 3 x 3 x 1). Data were analyzed using a 2- way analysis of variance (ANOVA) using Minitab 2000 Version 13.2 statistical software (Minitab Inc. Pennsylvania, USA). A probability value of p ≤ 0.05 was considered to denote a statistically significance difference.