|Year : 2017 | Volume
| Issue : 1 | Page : 15-19
Total phenolic content and anti-oxidant potential of Ficus deltoidea using green and non-green solvents
Mun Hui Sin, Awang Soh Mamat, Muhammad Shahzad Aslam, Muhammad Syarhabil Ahmad
School of Bioprocess Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian, Jejawi, Arau, Perlis, Malaysia
|Date of Web Publication||21-Apr-2017|
Muhammad Shahzad Aslam
School of Bioprocess Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian, Jejawi, Arau, Perlis
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The aim of the present study is to determine the total phenolic content (TPC) and anti-oxidant activity of green and non-green solvent extracts. Solvents have an important role in chemical or pharmaceutical industry. The usage of non-green solvent may cause detrimental effects on human safety and health. Therefore, green solvents are encouraged to be utilized because they are environmental friendly and easily acquired during the agricultural crops processing. For this study, the green solvent used in the extraction of Ficus deltoidea's leaves was water whereas non-green solvents used were ethanol and methanol. Folin-Ciocalteu method was used for measuring the presence and amount of phenolic content in each of the sample. The results showed that aqueous extract had the highest TPC (368.42 ± 6.37 mg GAE/g), followed by methanolic (295.03 ± 16.65 mg GAE/g) and ethanolic (263.45 ± 5.28 mg GAE/g) extracts. Furthermore, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was used in determining the anti-oxidant activity of the extracts. The lowest IC50 value based on the graph of percentage inhibition against concentration of sample was shown by ethanolic extract (16.5 µg/mL), followed by methanolic extract (22.0 µg/mL), and aqueous extract (23.5 µg/mL). For the three samples, the correlations between TPC and IC50 were negative and weak. Hence, the anti-oxidant activity of an extract cannot be predicted based on its TPC. The present study has shown that F. deltoidea considerably has the potential as a source of natural anti-oxidants.
Keywords: Anti-oxidant activity, Ficus deltoidea, green solvent, maceration, Mas Cotek, total phenolics
|How to cite this article:|
Sin MH, Mamat AS, Aslam MS, Ahmad MS. Total phenolic content and anti-oxidant potential of Ficus deltoidea using green and non-green solvents. J Pharm Negative Results 2017;8:15-9
|How to cite this URL:|
Sin MH, Mamat AS, Aslam MS, Ahmad MS. Total phenolic content and anti-oxidant potential of Ficus deltoidea using green and non-green solvents. J Pharm Negative Results [serial online] 2017 [cited 2017 Sep 22];8:15-9. Available from: http://www.pnrjournal.com/text.asp?2017/8/1/15/204913
| Introduction|| |
Ficus deltoidea has the name of mistletoe fig or known as “Mas Cotek” locally. F. deltoidea is grown in a huge amount in Malaysia and it is a kind of medicinal plant. There are two types of plants in F. deltoidea which are male and female plants. Male and female plants can be recognized through the dimension of morphology, such as observing the shape of leaves. For the male plants, the shape of leaves is fine and small, while the shape of leaves is thick and big for the female plants. Apart from that, spot color beneath the F. deltoidea leaves is another characteristic to be identified as red spots for male plants and female plants' leaves have black spots. Furthermore, there are researches which stated that the efficiency of the extracts from the leaves of female plants was higher than the male plants. The is because the female plants have higher potential of anti-oxidants than the male plants and assist to scavenge more free radicals.
The leaves of F. deltoidea contain polyphenols which are a type of phytochemicals that reveal the biological activity, for instance, anti-microbial, anti-nociceptive, anti-inflammatory, anti-oxidant, anti-allergic, anti-viral, and stimulate the immune system. According to Ramamurthy et al., Malays cure rheumatism and wound traditionally by using the leaves of F. deltoidea due to the fact that F. deltoidea leaves contain many types of bioactive compounds and one of them which is flavonoids, especially vitexin and isovitexin showed a lot of pharmacological activities. Anti-oxidant is a stable molecule with a low molecular weight and it will release an electron to react with a molecule in the body that consists of extra electron, which is also described as rampaging free radical. Free radicals will be neutralized and yet decrease the capacity of damage of those vital molecules. Furthermore, F. deltoidea leaf extracts consist of a large amount of total polyphenols, tannins, and flavonoids that revealed good anti-oxidant activity.
Green solvents are known as bio-solvents because they are environmental friendly and easily acquired during the agricultural crops processing. Green solvents are known for their low toxicity, low miscibility in water, biodegradable, high boiling point, and easy for recovery. In addition, green solvents showed the least disposal or pollution problems, whereas those solvents that showed more of the problem were brown.
The objectives of this article are to produce the F. deltoidea extract using green (water) and non-green (methanol and ethanol) solvents and compare the difference of total phenolic content (TPC) and anti-oxidant potential of the extracts.
| Materials and Methods|| |
Preparation of Plant Material
The female leaves of F. deltoidea were collected from Uni MAP Agrotechnology Research Station, Sungai Chuchuh, Perlis, Malaysia. The leaves were then cleaned, washed, and oven-dried at 35-40°C for 48 h by using Binder FD115 heating oven (Fischer Scientific Sdn. Bhd., Malaysia). The dried leaves were ground into smaller size particles by using EBR2601 blender (Electrolux Sdn. Bhd., PRC). The uniform particle size was obtained from the powdered raw material by passing through a 250-µm mesh sieve using AS 200 sieve shaker (RetschGmBH, Haan, Germany). The uniform and smaller size of particles could increase the efficiency of the extraction.
Preparation of three types of extracts was done by using different types of solvents. The solvents used are green solvent (distilled water) and non-green solvents [methanol (HmbG Chemicals, Germany)] and ethanol [(HmbG Chemicals, Germany)]. About 10 g of powdered form of F. deltoidea leaves was weighed and soaked in 100 mL of each of the solvents in closed container at room temperature for 24 h with occasional shaking or stirring. The miscella was filtered by using What man filter paper and the remaining plant material was macerated again with the same volume of solvent. The procedures were repeated for three times in the dark and the total time required for the maceration is 72 h.
Then all the samples were vacuum dried in an N-1200A rotary evaporator (Fischer Scientific Sdn. Bhd., Selangor, Malaysia) at 40°C until the solvent was evaporated and left only dark green or dark-yellow viscous mass which is known as crude extract. Three different solvent extract products were stored and cooled at 4°C for further analysis.
Determination of Total Phenolic Content
Folin-Ciocalteu's method was used in measuring the TPC in the extract in term of milligram gallic acid equivalent (GAE) per gram dry weight of plant. Sodium carbonate (HmbG Chemicals, Germany) with concentration of 20% w/v was prepared by dissolving 20 g of solid to form sodium carbonate in 100 mL of water. The heat was required for the sodium carbonate to be dissolved in the water. For the test extract preparation, 1-mg/mL stock solution was prepared by dissolving 0.1 g of the test extract in 100 mL of ethanol. From this stock solution, serial dilutions were prepared up to 2 mL, where the concentration is 200 µg/mL. The reaction mixture consisted of 0.5-mL extract, 0.5-mL Folin–Ciocalteu's reagent (Merck KGaA, Darmstadt, Germany), and 8-mL distilled water. After mixing for 5 min, 1-mL sodium carbonate (20%) was added and the reaction mixture was vortexed for another 10-15 s and then allowed to stand at ambient temperature for a further 60 min. The absorbance of the sample was measured at 725 nm by using Genesys 20 spectrophotometer (Thermo Electron Corporation, Madison, USA) against ethanol as a blank. Gallic acid (Merck Schuchardt OHG, Hohenbrunn, Germany) was used as a standard reference. The TPC of the samples were calculated in milligram GAE per gram dry weight of plant from the calibration curve of gallic acid (y = mx + c) as shown in Eq 2.1 (9).
where y is the absorbance of sample at 725 nm, x the concentration of the sample.
Anti-oxidant Potential Activity
The anti-oxidant potential activity was carried out in determining the significance of the free radical scavenging activity of the extracts by using free radical scavenging assay (DPPH) method. About 0.1 mM DPPH (Sigma-Aldrich Chemie GmbH, Riedstr, Steinheim) solution was prepaed by dissolving 3.94 mg of DPPH in 100-mL methanol. For the test extract preparation, 1-mg/mL stock solution was prepared by dissolving 0.1 g of the test extract in 100 mL of methanol. From this stock solution, serial dilutions with methanol were prepared up to 2 mL where the range of concentration was between 1 and 100 µg/mL. The reaction mixture consisted of 2 mL of 0.1 mM DPPH and 2 mL of the test extracts. The absorbances of the mixtures were taken after 30 min at 517 nm against methanol as a blank. Each sample will be measured in triplicate and averaged. Ascorbic acid (HmbG Chemicals, Germany) at the final concentration within 1–100 µg/mL will be used as a reference. The percentage of inhibition of each sample was calculated as follows:
The ability of anti-oxidant activity of the plant extract can be evaluated by comparing the IC50 values (according to the percentage of DPPH reduction) of the plant extracts in DPPH assay with the standard.
All analyses were done in triplicate and the data were presented as means ± standard deviation using Microsoft Excel.
| Results and Discussion|| |
Total Phenolic Content
Folin-Ciocalteu reagent is a combination of acids which comprises of phosphotungstic (H3PW12O40) and phosphomolybdic (H3PMo12O40) acids. The oxidation of phenolic compounds will lead to the reduction of phosphomolybdic–phosphotungstic acid (Folin–Ciocalteu reagent) and then transformed to blue oxides of tungsten (W8O23) and molybdene (Mo8O23) under alkaline condition in which sodium carbonate is added to the mixture. The intensity of the blue color depends on the concentration of phenolic compounds where the darker the blue color represented the higher the TPC. TPC of extracts were predicted in the unit of milligram GAE/gram dry extract by comparing with the calibration curve of standard gallic acid.
[Figure 1] showed the mean TPC of different solvents used for F. deltoidea's leaves extraction that measured by using the GAE equation of y = 0.0057x + 0.025 (R2 = 0.9973), whereby y = absorbance at 725 nm and x = concentration of total phenolic compounds in µg/mL of the extract. Among the solvent extracts, aqueous was the most extractible solvent of phenols (368.42 ± 6.37 mg GAE/g), followed by methanolic extract (295.03 ± 16.65 mg GAE/g), and ethanolic extract (263.45 ± 5.28 mg/g). ANOVA analysis illustrated significant differences which is p = 0.00018 (p < 0.05) between TPCs of the samples. The results suggested that the TPC varied significantly from one solvent extract to another.
|Figure 1: Total phenolic content of different solvent extracts. (p < 0.05)|
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Likewise, Pushpanathan and Nithyanandam (2015) reported that for F. deltoidea plant, 80% ethanolic extract had the highest total phenolic compounds (298.832 mg/g) compared to 80% methanolic extract (287.476 mg/g) and aqueous extract (275.822 mg/g). Besides, the research from Seo et al. (2014) revealed that the TPC in aqueous extract was higher than that of the pure ethanol and pure methanol extracts.
Almey et al. (2010) stated that the different levels of TPC might be attributed to the different plants, extraction methods, procedures, and standards used to express the TPCs; the color measurement of Folin-Ciocalteu reagent which was non-specific on phenol, and perhaps there were other components that can react with Folin-Ciocalteu reagent, such as ascorbic acid. Different solvents will be used to isolate anti-oxidants contained in the plants because different anti-oxidant compounds have different polarities. The common solvents used for the extraction are water, methanol, ethanol, and acetone. The anti-oxidant activity of the plant extracts and the yield of extract depend on the selected solvent.
DPPH Radical Scavenging Activity Assay
DPPH is one of the radical scavenging activity methods for determining the ability of the extract to donate hydrogen or scavenge free radicals. DPPH radical is a stable free radical and it will be reduced to diphenyl picryl hydrazine when it reacts with an anti-oxidant which can donate hydrogen. Spectrophotometer is used to measure the intensity of color changes from deep violet to light yellow.
[Figure 2] showed that the standard (ascorbic acid) had the highest percentage of inhibition compared to other solvent extracts whereas both the alcoholic solvents, methanol and ethanol had relatively high percentage inhibition as compared to water.
|Figure 2: Scavenging activities of the different solvent extracts of F. deltodea leaves and standard on DPPH radicals. Ascorbic acid was used as the standard|
Click here to view
From [Figure 2], the trend of the DPPH radical scavenging activity for all solvents can be observed where the anti-oxidant activity increased as the concentration of the sample increased. The result of standard was excellent in which the radical activity rose sharply to more than 95%. For methanol and ethanol extracts, the percentage of inhibition increased gradually to around 90% before becoming plateau and close to each other. However, the scavenging activity of aqueous extract increased when the concentration of sample increased and reached only close to 80% which is significantly lower than the methanol and ethanol extracts. A large decrease in the absorbance of the reaction mixture represented the significant free radical scavenging activity of the sample. It depends on intrinsic anti-oxidant activity of anti-oxidant, as well as on the speed of reaction between DPPH and anti-oxidant.
IC50 of a compound is inversely related to its anti-oxidant capacity, as it shows the amount of anti-oxidant required to decrease the DPPH concentration by 50%, which can be obtained from the graph of percentage inhibition against concentration. A lower IC50 indicates a higher anti-oxidant activity of a compound. From [Table 1], the standard ascorbic acid showed the lowest IC50 indicated that it was the strongest anti-oxidant. It was observed that ethanolic, methanolic, as well as aqueous extract exhibited the anti-oxidant activity in decreasing order of 16.5, 22, and 23.5 µg/mL, respectively.
|Table 1: DPPH radical scavenging activity (IC50) of different plant extracts and standard|
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The variation in the anti-oxidant activity among the extracts was mainly attributed to the presence of different extent of flavonoids and alkaloids.
Correlation Between TPC and DPPH Assay
In the present study, linear regression analysis was used to study the correlation between the TPC and anti-oxidant activity of the three samples. As shown in [Figure 3], the correlation coefficient between total phenolics and radical scavenging activity (R2 = 0.6817, y = -0.0001x + 0.0955) was found to be medium, which is less than 0.9 and the relationship is negative.
From [Figure 1], the TPC of different solvents was in decreasing order: water, methanol, and ethanol but from [Table 1], the best solvent extract that demonstrated the best role as anti-oxidant in DPPH assay was ethanol, followed by methanol and water. From the previous study regarding the relationships between phenolic content and anti-oxidant activity, there should be a positive relationship, implying that the anti-oxidant activity of these extracts strongly depend on the amount of phenolic content in the extracts. However, the result showed from [Figure 1] and [Table 1] did not fulfill the statement. This situation is similar to some previous researches, for example, as reported by Almey et al. (2010), the correlations between TPC and anti-oxidant activity for both ethanolic and methanolic extracts were negative and weak. Furthermore, Norra (2011) also found that TPC and TFC in F. deltoidea accession MFD6 were higher than MFD4 but anti-oxidant activity of MFD4 was higher than MFD6 which meant that the relationship between TPC and anti-oxidant activity was also not found.
According to Almey et al. (2010), the Folin-Ciocalteu method is a crude estimation for the total phenolic compounds in an extract, while the DPPH assay is not only specific to polyphenols. In addition, phenolic compounds respond differently in different solvents during extraction and therefore their performance is also different in DPPH assay. It depends on the number of phenolic groups consisted in the solvent extracts. From the result of TPC determination, polyphenol compounds have higher affinity toward water molecules. Hence, it was dissolved in a polar solvent like water. Aqueous extract had much higher TPC than the others, for example, methanolic and ethanolic extract in which these solvents were dissolved were of medium polarity in nature.
| Conclusions|| |
In general, TPC and free radical scavenging activity of aqueous, methanolic, and ethanolic extracts were determined. It is discovered that the solvents used in plant extraction significantly affected both the TPC and anti-oxidant activities of the F. deltoidea leaves. Relatively, aqueous extract was determined to have the highest phenolic compounds but ethanolic extract was the strongest anti-oxidant among the three solvent extracts. Therefore, the correlation between total phenolics and radical scavenging activity was weak. These results indicated that the different solvents used in the extraction of F. deltoidea leaves had produced the extracts with different amounts of total phenolics and anti-radical activity. Hence, the anti-oxidant activity of an extract cannot be predicted on the basis of its TPC. However, the research showed that green solvent extract, aqueous extract had a high TPC and anti-oxidant activity, thus green solvent is recommended to be used as a solvent in the extraction of F. deltoidea.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]