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Compounds from plants can be separated based on their relative solubilitites by using several of plant extraction method such as maceration. Firstly, the plant samples were put inside oven until there is no changes in weight to allow homogenous drying. The container of the samples inside the oven was wrapped with aluminium foil to prevent the exposure to sunlight. This will reduce the chemical transformation that might occur due to exposure to UV radiation. The plant samples were then ground to make the sample more homogenous as well as to increase the surface area of the sample so that the solvent will have a better diffusion property (Sarker, Latif, & Gray, 2006). Besides this, the objective of powdering the plant material is to rupture its organ, tissue and cell structures so that the antioxidant compounds are exposed to the extraction solvent (Sukhdev, Suman, Gennaro, & Dev, 2008).
The extraction solution were chosed based on their increasing polarity property i.e. n-hexane, chloroform, acetone, methanol to ascertain the extraction of plant metabolite since the plants matrices are complex with a range of secondary metabolites. After extraction, the solvent was separated from the residual plant through filtration of cheese cloth and filter paper to obtain more concentrated of plant liquid that contain the antioxidant property (Sarker et.al., 2006). The filtrate solvent is then was evaporated under pressure to concentrate the active metabolites that present inside the filtrate solvent.
The evaporated extract was further diluted with acetone, methanol, chloroform and n-hexane for each plant and were left in the oven under approprite temperature to remove the residual solvent. Before put inside the oven, these extracts were suspended and diluted in extraction solvent to bring the characteristic of water-solubility to the extracts as well as to make sure that they are more stable at pH media. These properties are important in most of the in vitro assays (Liu, 2008).
5.2 Extracts yields
Extraction yield is a quantitative representation of the efficiency of the extraction solvent to extract compound from the plant (Silla, Arnau & Tunon, 2001). Extraction yield depends on solvent, temperature, and time as well as the chemical nature of the sample. The same condition of time and temperature, the solvent used and the chemical property of sample are the most vital factors. The extraction solvent itself such as methanol, acetone, water or mixture are another important aspect affecting both extraction yield and antioxidant activity of extracts. The extraction yields of five samples were shown in Table 4.1. The ranking of these five samples in four different solvents are different. The behaviour of compound material towards solvent were different from each other due to the complexities of both the chemical properties of solvents and the diverse structure and composition of the plant compounds. No single solvent could extract all the antioxidants of different polarity and solubility in a single plant. (Yang, Wang, Ke, Jiang & Ying, 2007).
The yield of the extracts was calculated based on a dry weight basis to eliminate the influence of the moisture content of the plant (Silla et al., 2001). From the four extracts, methanol extraction produced the highest amount of extractable compounds followed by acetone extraction and the least are extracted by n-hexane extract. The extraction ability of methanol and acetone were very close whereas the n-hexane extract was only small in relative compare to other solvents. This proved that methanol is an effective solvent with good recovery (Yao, Jiang, Datta, Singanusong, Liu, Duan, et al., 2004). The higher yield of methanol and acetone show that the soluble compounds in the five plants can be categorised into intermediate and high polarity.
5.3 Assessment of Antioxidant activity
5.3.1 Preliminary evaluation using TLC-DPPH analysis.
All the plant extract compounds except Brassica oleracea in methanol were found to exhibit DPPH free radicals scavenging activity at different levels in the rapid screening. The purple colour of DPPH rapidly faded when it was in contact with antioxidant compounds extracted by different solvents. It took 40 to 90 seconds for the extracted plant antioxidant compounds to fully bleached the DPPH purple colour background respectively. The ââ‚¬Ëœtailingââ‚¬â„¢ appearance on TLC plate is due to the continous loading of sample on the same location that made the crude extract concentrated, and made it to move like a tail during the process. It could be safe to conclude that all the extracted plants could serve as good antioxidant compounds due to their ability to scavenge the DPPH free radicals. The Brassica oleracea in methanol cannot be determined on the TLC plate due to the relatively high concentration of compounds within the plant. High concentration make the plant sample to possess a high molecular weight that disable the compound constituent to travel further and become significant under the short or long UV light. On the other hand, the failure of Brassica oleracea compounds in methanol on TLC plate is due to its high volatile chareteristic that made it to evaporate in a short duaration of time. DPPH test on TLC, allow to observe a yellow-pale stain that is classified as zones of antioxidant activity and brown grayish under purple bottom. This situation can be concluded that the grayish brown stains are those that correspond to the alkaloids that can be identified as antioxidant compounds and are inactivated with regard to the DPPH (Janat, Koffi, Yves, Marc, DjiÃ©, Tra, et al., 2008).
5.3.2 DPPH Radical Scavenging Activity
According to Matsuthisakul et al. (2005), The DPPH method was used to measure the primary antioxidant activity of the plant extracts. This is because, DPPH method is one of the most efficient method for evaluating the concentration of radical scavenging compounds rapidly by a chain breaking mechanism. The reduction capability of DPPH is determined by the decrease in its absorbance at 517 nm due to the presence of antioxidant compounds. The decolourisation of the purple reaction solution to pale yellow hydrazine is stoichiometric with respect to number of free radical electrons captured (Norshazila et al., 2010).
In this study, five plants samples extracted with four different solvents were used to estimate the antioxidant activity by measuring the free radical scavenging activity of DPPH. In this machinery, ascorbic acid was used as positive control. The scavenging activity of the samples on DPPH was strongly dependant on the concentration of the extract (Sawadago, Meda, Lamien, Kiendrebeogo, Guissaou & Nacoulma, 2006). The antioxidant activity of each of the samples was expressed as IC50. If IC50 of a particular sample was close to the value of ascorbic acid, then the sample was considered to have a good radical scavenging activity. This is because the ascorbic acid is known as potent antioxidants. The DPPH radical scavenging activity can also be expressed as ascorbic acid equivalent to antioxidant capacity (AEAC) (Banerjee, Chakrabarti, Hazra, Banerjee & Mukherjee, 2008).
In this study, methanol extracts showed the highest antioxidant activity by DPPH assay followed by acetone. In the methanol extraction, Abelmoschus esculentus and Nelumbo nucifera extracts showed more than 80% free radical scavenging activities at all level of concentration suggesting their high potential as good free radical scavengers. On the other hand, the IC50 data in Figure 4.3 showed that most of the chloroform and hexane plant extracts except Moringa deifera and Brassica oleracea scavenged less than 50% of DPPH radicals even at the highest concentration. Polar solvent such as methanol and acetone are those that generally have higher antioxidant activity. The antioxidant activity shown by the more polar extraction solvent could be due to the presence of phenolic compounds and flavonoids because they contain an aromatic hydroxyl moiety (Chaifang & Shifeng, 2009). Generally phenolic compounds with an aromatic ring possess a stronger antioxidant activity than monophenolics (Gordana, Jasna, Sonja, Vesna, Sinisa & Dragoljub, 2007).
Consequently, methanol extracts was ranked as the best DPPH radical scavenger followed by acetone, then chloroform and finally n-hexane as the weakest radical scavenger. These data correlate well with the work of Norshaila et al. (2010) on Malaysian Tropical fruits which revealed the weak radical scavenging activity of n-hexane extracts and the highly potent antioxidant activity of methanol extracts. This is because compounds extracted in non-polar solvent such as n-hexane evidently lacked of hydrogen donating capability. This signifies that compounds with strongest radical activity in these five edible extracts are high polarity plants. However, there are some disadvantages in this method. The major disadvantage would be the use of non-physiological related radical, which does not resemble free radicals involved in biological system. Other than that, the absorbance of 517 nm used in this assay causes the interference of carotenoids. Lastly, this method is only applicable to lipophilic antioxidants (Yu, 2007).
5.3.3 Ferric Thiocyanate Method
The mechanism associated with this method are the same as those involve in the Ferrous Oxidation-Xylenol Orange (FOX) assay. The difference is that a ferric ion formed by an oxidant from a ferrous ion is monitored as a thiocyanate complex by a spectrophotometer at 500 nm. The inhibitory effect toward oxidation from ferrous ion to ferric ion by antioxidants is evaluated by monitoring the formation of ferric thiocyanate complex. This assay is simple and highly reproducible. One disadvantage of FTC is that if any chemical with UV absorption around 500 nm is present, the results are overestimated or not reliable. This is true for any other assay using a spectrophotometer (Joon & Takayuki, 2009).
FTC measures the amount of peroxide generated at the initial stage of linoleic acid emulsion during incubation. Generally, the absorbance will increase over time, which shows that the autoxidation of linoleic acid emulsion in the control or sample fraction increase in the peroxide formation. Significantly lower lipid peroxidation activity has been observed in ascorbic acid compare to other plant samples. Different plant extracts obtained from different extraction solvents exhibited different level of antioxidant activity within the range of 63-81% by the FTC method except for chloroform and hexane extracted samples which is less than 60%. This is may be due to the failure of the non-polar solvent to extract out the antioxidant property from the plant samples.