Antioxidant Activity, Phenolic, Flavonoid and Tannin Content of Piper Betle and Leucosyke Capitella

Piper betle and Leucosyke capitella are plants that are commonly used as traditional medicine. In this study, antioxidant Activity, Phenolic, Flavonoids and Tannin content of these plants were evaluated. The plants were extracted using petroleum ether, acetone and methanol. The total phenolic, flavonoid and tannin extract of both plants is in the order of acetone > methanol > petroleum ether. The result shows that the total phenolic, flavonoid content for Piper Betle and Leucosyke Capitella is in the range of 31.25 to 47.48 mg/g and 1.68 to 7.19 mg/g, respectively. For Piper betle, the total flavonoid content is in the range of 29.58 to 46.08 mg/g and for Leucosyke capitella is in the range of 1.08 to 6.83 mg/g. It is also found that Piper betle extracted with methanol has higher antioxidant activity than vitamin E, butylated hydroxytoluene (BHT) and catechin but lower than quercetin. | antioxidant Activity | Phenolic | Flavonoids | Leucosyke Capitella | Piper Betle | ® 2012 Ibnu Sina Institute. All rights reserved. http://dx.doi.org/10.11113/mjfas.v8n1.115


INTRODUCTION
Antioxidant compounds play an important role in limiting the damaging effect of free radicals and retard the process of many chronic diseases as well as lipid oxidative rancidity in foods [1].Plants exhibit different antioxidant capacities according to their polyphenol content, vitamin C, vitamin E, carotenoids and flavonoids [2].Phenolics compounds are a large diverse group of secondary plant metabolites including phenolic acids, flavonoids, and tannins [3].Flavonoids are polyphenolic compounds that are the most abundant of the plant phenolics [3].Tannins are phenolic compounds that shown potential antiviral, antibacterial and anticancer effects [4].Many of these phytochemicals are found in plant that function with nutrients and dietary fiber and act as natural antioxidant.
Piper betle and Leucosyke capitella are plants that commonly used by indigenous people of Borneo for medicinal purpose.Piper betle also known as betel the leaf of a vine belonging to the Piperaceae family, while Leucosyke capitella is belonging to the Urticaceae family.In the traditional medicinal systems, the leaf of Piper betle has been advocated for the treatment of wounds, boils, bites of insects and for enhancing digestion [5].Locally, betel leaves are used for chewing.The extract are also found to possess both superoxide and hydroxyl free radical scavenging action [5].
Leucosyke capitella or also known locally as Mandahasi grows at Borneo area (Sabah and Kalimantan, Indonesia).This plant is used by communities in Kalimantan to cure diarrhoea [6].The leaf decoction is also used to treat hypertension and diabetes by Kadazandusun communities around Crocker Range [7].
Based on the traditional use of these plants in medicine, this research was conducted to determine its antioxidant activity, phenolic, flavonoid and tannin content.The relationship between antioxidant activity with phenolic, flavonoid and tannin content were also investigated.

Plant Materials
Piper betle was collected from Mentakab, Pahang while Leucosyke capitella was collected from Ranau, Sabah in November, 2008 and identified their scientific name by botanist Mr. Baharuddin Sulaiman, in the School of Biological Sciences at University Science of Malaysia.The whole both plants were dried for a few weeks under room temperature.

Plant Extracts
Dried leaves of Piper betle were ground into a fine powder with a grinder.Three solvents (petroleum ether, acetone and methanol) with distinct polarity were used for the preparation of plant extracts.100 g of powdered leaves were extracted with 250 mL of petroleum ether in 500 mL conical flask for 2 days and this procedure was repeated with the same solvent once again.The aliquot were pooled and filtered.Then, the filtrate was evaporated down to dryness under vacumm using rotary evaporator at 40 °C.This petroleum ether extract was weighed and transferred to a freezer.This process was repeated with acetone and methanol using the plant residue obtained from petroleum ether extraction.The whole process was repeated for the next sample which is the dried stem of Leucosyke capitella.

Preparation of Stock Solution
0.05 g samples or standards were dissolved in 50 mL volumetric flask with solvent to prepare 1000 ppm of stock solution.The solvent used depends on the samples or the standards (Table 1).

Total Phenolic Assay
Total phenolic content was determined using the modified Folin-Ciocalteu method.Eight concentrations (0, 5, 10, 20, 30, 40, 50 and 100 ppm) of catechin (standard) were prepared to obtained catechin standard calibration curve and 100 ppm of each sample was prepared to determine total phenolic content in catechin equivalent.0.25 mL of samples or standards was diluted with 3.75 mL of distilled water followed by the addition of 0.25 mL of Folin-Ciocalteau reagent and left for 3 minutes.1.25 mL of 20 % (w/v) sodium carbonate, Na 2 CO 3 was added to the mixture, and then incubated for 40 minutes at 40 °C.The absorbance was measured at 685 nm using UV-Vis spectrometer.

Total Flavonoid Assay
Total flavonoid content was determined using the method of Quettier-Deleu.Eight concentrations (0, 5, 10, 20, 30, 40, 50 and 100 ppm) of quercetin (standard) were prepared to obtained quercetin standard calibration curve and 100 ppm of each sample was prepared to determine total flavonoid content in quercetin equivalent.5 mL of samples or standards were mixed with 5 mL of 2 % (w/v) of aluminium chloride hexahydrate, AlCl 3 .6H 2 O.After 10 minutes, the absorbance was measured at 415 nm using UV-Vis spectrometer.

Total Tannin Assay
Total tannin content was determined using vanillin assay.Eight concentrations (0, 5, 10, 20, 30, 40, 50 and 100 ppm) of catechin (standard) were prepared to obtained catechin standard calibration curve and 100 ppm of each sample was prepared to determine total tannin content in catechin equivalent.1.0 mL of samples or standards was mixed with 3.0 mL of 4 % (w/v) vanillin followed by addition of 1.5 mL of 1 M hydrochloric acid, HCl, then incubated in the dark for 5 minutes and the absorbance was measured at 500 nm using UV-Vis spectrometer.

Total Phenolic Content
The Follin-Ciocalteu's assay is one of the oldest methods designed to determine the total content of phenolics in food or medicinal plantsm [8].Phenolic compound react with Follin-Ciocalteu'Reagent (FCR) only under basic conditions.Dissociation of a phenolic proton in basic medium leads to a phenolate anion, which is capable of reducing FCR in which the molybdate in testing system is reduced forming a blue coloured molybdenum oxide with maximum absorption near 685 nm.The intensity of blue calouration produced is proportional to the total to the total quantity of phenolic compounds present in the testing samples [8,9].
The total phenolic content (TPC) of Piper betle ranged from 31.25 to 47.48 mg/g (Table 2) was determined at 100 ppm from standard calibration curve of catechin (Figure 1).The Piper betle extracted using acetone had the highest total phenolic content of 47.48 mg/g, followed by methanol of 34.29 mg/g and petroleum ether of 31.25 mg/g.The total phenolic content of Leucosyke capitella ranged from 1.68 to 7.19 mg/g (Table 2).The Leucosyke capitella extracted using acetone had the highest total phenolic content of 7.19 mg/g, followed by methanol of 2.26 mg/g and petroleum ether 1.68 mg/g.
From the results, it shows that acetone extract more phenolic compound compares to methanol and petroleum ether.Besides, it also shows that acetone is more effective than methanol to extract polar phenolic compound for Piper betle and Leucosyke capitella.It is also noted that Piper betle contain more phenolic compound than Leucosyke capitella.

Total Flavonoid Content
The total flavonoid content (TFC) of Piper betle ranged from 29.58 to 46.08 mg/g (Table 2) was determined at 100 ppm from standard calibration curve of quercetin (Figure 2).The Piper betle extracted with acetone of 46.08 mg/g contain significantly higher flavonoid than the corresponding Piper betle extracted with methanol of 31.08 mg/g and petroleum ether of 29.58 mg/g.The total flavonoid content of Leucosyke capitella ranged from 1.08 to 6.83 mg/g (Table 2).The Leucosyke capitella extracted with acetone of 6.83 mg/g contain significantly higher flavonoid than the corresponding Leucosyke capitella extracted with methanol of 1.83 mg/g and petroleum ether of 1.08 mg/g.This shows that acetone extracted more flavonoid compound compare to methanol and petroleum ether.These again suggested that acetone is more competitive than methanol to extract polar flavonoid compound for Piper betle and Leucosyke capitella.Result shows that at any specific types of solvent used in extraction, Piper Betle has higher amount of flavonoid compound as compared to Leucosyke capitella.

Total Tannin Content
The total tannin content (TTC) of Piper betle ranged from 13.33 to 29.33 mg/g (Table 2) was determined at 100 ppm from standard calibration curve of catechin.The Piper betle extracted using acetone had the highest total tannin content of 29.33 mg/g, followed by methanol of 18.67 mg/g and petroleum ether of 13.33 mg/g.The total tannin content of Leucosyke capitella ranged from 0.67 to 5.33 mg/g (Table 2).The Leucosyke capitella extracted using acetone had the highest total tannin content of 5.33 mg/g, followed by methanol of 1.33 mg/g and petroleum ether of 0.67 mg/g.Again, acetone seems to show better extraction capability compare to methanol and petroleum ether and Piper betle had more tannin compound than Leucosyke capitella.
The comparison of total phenolic, flavonoid and tannin contents of Piper betle and Leucosyke capitella at Figure 1.From that graph it is highly suggested that the best solvent to extract phenolic, flavonoid and tannin will be acetone.Also, the Piper betle contain more phytochemical than Leucosyke capitella.An ESR signal is directly proportional to the number of radicals present.The 0.25 mM DPPH radicals give a typical ESR spectrum as shown in Figure 1.0.The peaks height will be reduced when an antioxidant was added to the ethanolic DPPH solution.The radical scavenging activity of flavanones was expressed by means of SC 50 which represent the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%.
Based on Figure 4, Piper betle had higher antioxidant activity than Leucosyke capitella.These findings support the results obtained from the total phenolic, flavonoid and tannin content that Piper betle plants showed higher content than Leucosyke capitella plants.Therefore, the ability to scavenge the DPPH radical for Piper betle is better than Leucosyke capitella.However, the scavenging of radical efficiency of Piper betle plant is less than quercetin antioxidant.The half maximal inhibitory concentration or the concentration of substance that provides 50 % inhibition to certain reaction, IC 50 is a measure of the effectiveness of a compound in inhibiting biological or biochemical function.From Figure 5, the IC 50 of Piper betle and Leucosyke capitella extracted with petroleum ether can not be determined within 0 to 100 ppm concentration of sample.This possibly due to the polarity of solvent, which is the petroleum ether, is lower and only extracted non-polar compound.Thus, the non-polar compound which extracted using petroleum ether is not strong antioxidant because they did not reach steady state to react with DPPH or not enough ionization to donate hydrogen or electrons to quench DPPH radicals.The IC 50 of the samples decreased in the following order of Leucosyke capitella methanol > Leucosyke capitella acetone > Piper betle methanol > Piper betle acetone.Furthermore, the percentage of inhibition and total phenolic, flavonoid and tannin contents have similar trend except for the Piper betle in methanol, because of the total phenolic, flavonoid and tannin contents decreases but the percentage of inhibition increases compare with Piper betle in acetone (Figure 6, 7 and 8).

CONCLUSION
The results obtained in this study demonstrate that Piper betle and Leucosyke capitella contain phytochemical such as phenolic, flavonoid and tannin compounds.It also shows that petroleum ether, methanol and acetone extracts of Piper betle and Leucosyke capitella have the ability to quench free radicals but petroleum ether contain mild antioxidant since the IC 50 can not be determined between range 0 to 100 ppm.The extractions using acetone is more efficient to extract phenolic, flavonoid and tannin compounds than methanol and petroleum ether for these types of plants.It also found that Piper betle contain more antioxidant compound compare to Leucosyke capitella.The ability to scavenge radicals for Piper betle herbal plant is better than Leucosyke capitella plant but lower than quercetin antioxidant standard.

Fig. 1
Fig. 1 Total phenolic, flavonoid and tannin contents in Piper betle and Leucosyke capitellaIn this study, the ESR spectrometry assay has been used to measure the free radical scavenging activity of the antioxidant against DPPH radical.Vitamin C was used as a reference antioxidant and screened for comparison purposes

Table 1
The solvent used for samples

Table 2
Total phenolic, flavonoid and tannin contents in Piper betle and Leucosyke capitella