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.

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.  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%).  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].

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).
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 10 th min. Significant (p< 0.05) differences of DPPH . scavenging capacities among extracts were observed at 5 th 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].  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. 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 by ab. 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.

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.