Antioxidant Activity , Total Phenolic Content and Total Flavonoid Content of Water and Methanol Extracts of Phyllanthus species from Malaysia

Aims: The effects of 2 types of solvents, water and methanol were investigated to determine the presence of antioxidant activity, total phenolic content (TPC) and total flavonoid content (TFC) from three Phyllanthus species namely, Phyllanthus urinaria, Phyllanthus niruri andPhyllanthus debilis. Material and Methods: The antioxidant activities were measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. The chemical contents of the Phyllanthus sp. were presented as total phenolic content (TPC) and total flavonoid content (TFC). Statistical analysis: All statistical analysis was conducted using SPSS for Windows, Version 22. All data were presented as mean ± standard deviation. Results: Our result showed that P. urinaria showed higher TPC, followed by P. debilis and P. niruri for both methanol and water extracts. Similarly, P. urinaria showed higher TFC than P. debilis and P. niruri. The antioxidant activity by using 2,2-diphenyl1-picrylhydrazyl (DPPH) assay showed EC50 of samples ranged from 15.8 to 29.3 μg/mL for methanol extract and 33.5 to 73.0 μg/mL for water extract. The 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay showed EC50 ranged from 11.2 to 26.0 μg/mL for methanol extract and 13.5 to 37.4 μg/mL for water extract. Conclusion: Methanol extract showed higher TPC, TFC value and lower EC50 values for antioxidant activities when compared to water extract. In both methanol and water extracts, P. urinaria had higher TPC and TFC value and lower EC50 for both DPPH and ABTS assay followed by P. debilis and P. niruri.

plant or parts of it were used for the treatment of kidney stones, intestinal infections, diabetes, hepatitis B and neonatal jaundice. 5,6There are 3 common Phyllanthus species that were traditionally used for treatment in Malaysia.The species are Phyllanthus niruri, Phyllanthus debilis and Phyllanthus urinaria.P. niruri is the most common Phyllanthus species used as medicine in most of tropical countries. 7It has diverse medicinal qualities where it has been used for treatment ranging from kidney problems, diarrhoea, fever, diabetes, colic, anti-lipid and analgesic. 8,91][12][13][14] Bioactive compounds such as lignans and terpenes were shown to have an excellent hepatoprotective effects, anti-cancer and anti-microbial properties. 11Other active compounds, niruriside has antiviral activity that can inhibit the replication of HIV virus. 15. urinaria has similar uses as P. niruri in traditional practises.It is commonly used for treatment of fever, improving eyesight, urinary problems, and liver diseases and to detoxify poison from the body.9,8 In China, P. urinaria was mainly used for treatment of cancer.16,17 Some compounds such as 7'-hydroxy-3' ,4' ,5,9,9'-pentamethoxy-3,4-methylene dioxy lignan isolated from P.urinaria extracts was shown to exhibit anticancer activity.18 Other bioactive compounds found in the P. urinaria extract such as coumarin, ellagitannin and sterol can act as antioxidant, antiviral and anti-inflammatory.19 P. debilis, which is a less known Phyllanthus species, is usually used as substitute to other popular Phyllanthus species such as P. amarus.8 P. debilis is commonly used for treatment such as jaundice, diarrhea, ulcers, sores, ringworms and scabies.20 Although known as a substitute for P. amarus, P. debilis has been found to have a better hepatoprotective activity than P. amarus.21 It is also showing a strong anti-inflammatory 22 and antihyperglycemic properties.20 Bioactive compound found in P. debilis such as debelalactone acts as antihepatotoxic whereas phytoesterols such as β-sitosterol act as analgesic and anti-inflammatory.19 This study was aimed to compare physicochemical profiles (TPC, TFC and antioxidant activity) of methanol and water extracts in 3 common Malaysian Phyllanthus species.

Herbal specimen
Three Phyllanthus species were collected from local collection at Tasek Gelugor, Penang, Malaysia.These species were identified by a botanist, Dr. Rahmad Zakaria from the School of Biological Sciences, Universiti Sains Malaysia.The voucher specimens (Phyllanthus debilis: 11623, Phyllanthus niruri: 11624 and Phyllanthus urinaria: 11625) were deposited at the USM herbarium.

Sample extraction Water/methanol extraction
The sample extraction method was done according to Huang et al.,  (2008)  23 with slight modification.Briefly, the sample was dried in the oven of 50°C for 3 days.Dried samples were ground and prepared in powder form.Five gram of sample was extracted with 100 mL of deionized water or methanol (Fisher Chemical) in ultrasonic bath (Power Sonic 405) for 20 min and then filtered.The process was repeated twice with the remaining residual extract.The extracts were dried using freeze dryer for water extract and rotary evaporator (Buchi Rotary Evaporator RII) for methanol extract.Dried extracts were stored at -20°C prior to use.

Total phenolic content
Total phenolic concentration in the extracts was determined based on the method of Sahu and Saxena, (2013). 24with slight modification.Briefly, 2 mg/mL of sample was diluted in methanol or ultrapure water.Fifty microliters of Folin-Ciocalteu's reagent (50% v/v) (Merck) was then added to 10 µL of sample solution and incubated for 5 min incubation at room temperature.Forty microliters of 20% (w/v) sodium carbonate (Bio-basic) was added to the mixture which was then incubated for 30 min.The absorbance was read at 765 nm using microplate reader (FLUOstar omega).The total phenolic was expressed in gallic acid equivalent (GAE) per g dry weight (DW) of extract (mg GAE/g DW).
The standard curve of gallic acid was constructed using seven serial concentrations (1.563 to 50 µg/mL).All samples were assays in triplicate.Water and methanol were used as negative control.

Total flavonoid content
Total flavonoid content (TFC) of Phyllanthus samples was determined based on the method of Sahu and Saxena, (2013), 24 with some modifications.Briefly, sample solution was prepared using 2 mg/ml of extract, diluted using methanol or ultrapure water.Hundred µL of sample solution (2 mg/mL) was then mixed with 100 µL of 2% aluminium chloride (AlCl 3 ) (Merck) in 96 microwell plate.A flavonoid-aluminium complex was formed after 10 min of incubation time at 25°C.The formation of the complex was measured at 415 nm using microplate reader (FLUOstar omega).Total flavonoid content was measured as quercetin equivalent (QE) per g dry weight (DW) of extract (mg QE/g DW).The standard curve of quercetin was constructed using seven serial concentrations (1.563 to 50 µg/mL).All assays were carried out in triplicate.Water and methanol were used as blank and negative control.

Antioxidant activity: DPPH free radical scavenging assay
The antioxidant activity was measured based on the free radical scavenging activity of the Phyllanthus extracts against the stable 2, 2-diphenyl-1-picrylhydrazil (DPPH) free radical. 25A total 250 μL of a reaction mixture was prepared in 96 microwell plates.The reaction mixture consisted of 50 μL of extracts and 200 μL of 0.2 mM DPPH (Sigma-Aldrich).The solution was incubated at room temperature for 30 min before measuring the absorbance value at 517 nm using microplate reader (FLUOstar omega).Different concentrations of the extracts were measured and EC 50 concentration (concentration required to inhibit 50% of DPPH radicals) was determined.All assays were carried out in triplicate.Water and methanol were used as negative controls.Obtained absorbance value was converted into the percentage of radical scavenging activity using the following equation: Where AS refers to: absorbance of the sample; AC: absorbance of the negative control (methanol/ water).

Antioxidant activity: ABTS scavenging activity
Antioxidant activity was also measured using ABTS 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) (Roche Diagnostic).The ABTS radical cation ABTS + solution was prepared through the reaction of 7 mM ABTS with 2.45 mM potassium persulphate (Sigma-Aldrich).The mixture was incubated at room temperature for 16 hours in the dark.The ABTS + solution was then diluted with 99% ethanol (R&M Chemical) to obtain an absorbance of 0.7 ± 0.02 at 734nm.A total of 200µl of reaction mixture was prepared in 96 well plates.
The reaction mixture consists of 10µL of extracts and 190µL of diluted ABTS + .The mixture was incubated for 6 min at the room temperature before measuring the absorbance value at 734 nm using microplate reader (Powerwave XS bio-tek).Different concentrations of the extracts were measured and EC 50 concentration (concentration required to inhibit 50% of ABTS) was determined.All assays were carried out in triplicate.Water and methanol were used as negative controls.
Obtained absorbance value was converted into the percentage of radical scavenging activity using the following equation: Where AS: absorbance of the sample; AC: absorbance of the negative control (methanol/ water).

Statistical analysis
All statistical analysis was conducted using SPSS for Windows, Version 22.All data were presented as mean ± standard deviation.

RESULTS AND DISCUSSION
The total phenolic content of the whole plant of P. niruri, P. urinaria and P. debilis was presented in Table 1.For methanol extract, P. urinaria had a higher total phenolic content (308.71 ± 0.04 mg GAE/g DW) followed by P. debilis (197.09± 0.03 mg GAE/g DW) and P. niruri (159.13 ± 0.02 mg GAE/g DW).Similarly, for water extract, P. urinaria showed the highest phenolic content (219.83 ± 0.01 mg GAE/g DW), followed by P. debilis (172.09±0.01 mg GAE/g DW) and P. niruri (107.09± 0.01 mg GAE/g DW).The plant phenolic compounds constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators.This is due to their redox properties, which play an important role in adsorbing and neutralising free radicals, quenching singlet or triplet oxygen and decomposing peroxides. 26Our results showed that the methanol extract of P. urinaria exhibited the highest TPC, followed by P. debilis and P. niruri.Similar observation was previously reported 27 which found that P. urinaria had higher TPC content compared to P. debilis and P. amarus.However, TPC for methanol extract of P. urinaria in our study was higher than TPC content in methanol extract of P. urinaria found in Eldeen et al. (2011) 27 study (308.0 ± 0.01 mg GAE/g DW vs 205.0 ± 21.3 mg GAE/g DW).The water extract of P. urinaria showed lower TPC content when being compared to methanol extract.When being compared with the study by Cheah and Radu, (2011), 28 the water extracts of P. urinaria and P. niruri from our samples were showing higher TPC values (219.83 ± 0.01 mg GAE/g DW vs 41.19 ± 0.689 mg GAE/g DW for P. urinaria and 107.09 ± 0.01 mg GAE/g DW vs 55.38 ± 0.496 mg GAE/g DW for P. niruri).The higher TPC value found in our samples may be due to different drying process in which our samples were dried at 50°C whereas in Eldeen et al., (2011), 27 their samples were dried in an oven at 60°C.This may suggest that the drying temperature of more than 50°C may not be suitable for extracting the phenolic compounds.Exposing the samples to direct sunlight or relevant high temperature may cause some phenolic compounds to degrade rapidly. 29e total flavonoid content of three Phyllanthus species is presented in Table 2.For methanol extract, P. urinaria had a higher total flavonoid content (35.86 ± 0.04 mg QE/g DW) followed by P. debilis (25.64 ± 0.16 mg QE/g DW) and P. niruri (22.08 ± 0.04 mg QE/g DW).For water extracts, P. urinaria shows the highest total flavonoid content (17.27 ± 0.01 mg QE/g DW), followed by P. debilis (8.19 ± 0.01 mg QE/g DW) and P. niruri (7.07 ± 0.01 mg QE/g DW).Flavonoids are commonly found in natural products and one of the most important natural phenolics. 7For methanol and water extracts, P. urinaria exhibited the highest TFC compared to P. debilis and P. niruri.Methanol extracts showed higher TFC value when being compared to water extract.Our result was in agreement with Kumaran and Karunakaran (2007), 7 which showed that P. urinaria exhibited higher TFC value compared with other Phyllanthus species such as P. debilis and P. amarus.
For the antioxidant activity using ABTS scavenging activity, it was determined by the decolourisation of the ABTS by measuring the percentage of the reduction of absorbance at 734 nm and the results were presented as EC 50 concentration.Similar to what was observed in antioxidant activity of DPPH assay, P. urinaria exhibited the lowest EC 50 , followed by P. debilis and P. niruri in both methanol and water extracts.
Our data showed that the chemical contents and antioxidant activity of the methanol extract was consistently higher compared to water extract.We believed that the difference between methanol and water extracts was due to the solvent polarity difference.Lim and Murtijaya (2007) 29 had shown that methanol is better than water to degrade the cell wall, which results in greater amount of endocellular materials extracted.Furthermore, methanol is preferred than water to extract phenolic compounds in Phyllanthus species where major components, which are active hydrolysable tannins can be efficiently extracted using semi polar compounds. 31A study by Cheah and Radu, (2011) 28 also showed that methanol extract had a better TPC and DPPH activity when compared to water extract.Plant antioxidant activity however, also depends on other non-polyphenolic compound such as vitamins, minerals and carotenoids.These non-polyphenolic compounds may exert synergistic effect with TPC and TFC, which could further enhance the antioxidants activities. 7

CONCLUSION
Phyllanthus species originated from Malaysia are high in antioxidant activities, total phenolic and flavonoid contents with possibilities of having high medicinal properties that can be potentially developed as medicine in future.Phyllanthus species especially P. urinaria showed higher amount of total phenolic and flavonoid contents and antioxidant activities when compared to P. debilis and P. niruri.Further studies are warranted for the isolation and identification of individual phenolic and flavonoid compounds and for better understanding of their biological mechanism.