INTRODUCTION:

Phenolic compounds are essential metabolites for physiology and cellular metabolism of plants. Those compounds gained much attention, the grace of their positive activity for human health, as antioxidants, antimicrobial, antiviral, anticancer, et cetera. Fruits and vegetables contain many phenolic, mostly in free or soluble form, where <24% in bound-form¹.

It makes fruits and vegetables as a source of bioactive phenolic, including Red Fruit (Pandanus conoideus Lam.).

The Red Fruit (RF) is one of the indigenous plants in the Papua region – Indonesia. The Papuan used this fruit as part of the daily meal as a source of calorie and health supplements². The extract of this fruit, especially the oil, had the potency as an antioxidant^3,4, but RF oil was more pro-oxidant than antioxidant⁵. The large variety of this species also made controversies. Until now, there are 36 cultivars of RF, which economically classified into four types (Short-Red, Long-Red, Long-Yellow, and Brown-Red)^2,6,7,8. Nutritional composition, physicochemical properties, and bioactive profile of RF were significantly affected by the type of cultivars and the origin district where the RF grew^9,10,11. The long-reds are dominant than short-red cultivars. The RF oil extracted from the high-land cultivar showed higher antioxidant activity than the one from low-land cultivar¹².

The antioxidant assay usually used in RF was the DPPH scavenging assay. There is no universal method for antioxidant activity evaluation; that is why researches did in antioxidants usually used several methods^13,14,15. The methods were different in principle and reaction; then, a single method will not accurately express all antioxidant activities^13,16. It was suggested to use at least two methods¹³.

No research comprehensively studies the activity of the RF-extracts instead of RF-oil, and also the relationship between total phenol and antioxidant activity. It needs to evaluate the antioxidant activity of RF extracts by various assay. We need to evaluate further the relationship between total phenol and antioxidant activities and also the effectiveness of total phenol for antioxidant capacity.

MATERIAL AND METHODS:

Chemicals and reagents:

The sample is Red Fruit/RF (Pandanus conoideus Lam.) Short-Red cultivar (local name: Monsor), was obtained from the botanical garden Laboratory of Papua State University (UNIPA), Manokwari, West Papua, Indonesia. Molecular Genetic Laboratory, Fishery Faculty, UNIPA Manokwari, Indonesia, made the specimen identification. All chemicals for extraction and partition (methanol/MeOH, hexane, and ethyl acetate/EtOAc) were analytical grade. Gallic acid/GA (Sigma-Aldrich 16654) is a standard for total phenolic analysis, whereas Quercetin/Q (Sigma-Aldrich Q4951) is a positive control for antioxidant activity analysis.

Red Fruit Sample Preparation:

The RF was ground manually to separate the grains (drupe) from the fruit (cepallum). The RF grains (10Kg) were subjected to methanol maceration (20ml, 2x) at room temperature for six days³. After the macerate filtration, this filtrate was evaporated at 40ºC (named as methanol extract/ME)^3,17. The ME (400g) was multilevel partitioned using 1200 ml hexane:water (2:1, v/v, 4x), then ethyl acetic (2:1, v/v, 6x). The extract was evaporated at 40ºC to yield ethyl acetic extract (EE). Each extract was dissolved in methanol and or water to get sample stock solutions (500μg/ml). Those solutions were diluted to obtain sample concentration 30, 100, and 300 μg/ml. These solutions were subjected to total phenolic content and antioxidant activity analysis.

Total Phenolic Content/TPC Analysis:

TPC of RF extracts was determined using the Folin-Ciocalteau method³. The ME and EE were dissolved in 1ml methanol absolute, then mixed with 0.4ml Folin Ciocalteau reagent, followed by incubation at room temperature for 5-8 minutes. Sodium carbonate 7% (4 ml) was added into each extract, before setting up the volume 10ml using distilled water. The solutions were incubated again at room temperature in the dark condition for two hours, before recording the absorbance at λ = 725nm. The calibration curve was done using Gallic Acid. The phenolic content was stated as mg Gallic Acid Equivalent per gram extract (mg GAE/g).

LC-MS Analysis:

Sample and Standard Preparation:

Each RF extract (13-40mg) was dissolved in 1ml of methanol then sonicated for 5 minutes. Each extract was added by 100μl of lead(II) acetate solution (0.06 g/100ml). Centrifugation was done for 10 minutes at 4000rpm. Lead (II) acetate precipitated the unwanted compound that may disturb the flavonoid identification in the extract. Each supernatant then was passed through membrane filter 0.22μm, before LCMS analysis. The external standards were quercetin/Q, taxifolin/T, catechin gallate/CG, quercetin-3-glucoside/Q3G, and procyanidin B2/PB2. Those were prepared as standard solution 1000ppm, then diluted become several concentrations to make a calibration curve of each standard.

The LC-MS system used consisted of UHPLC ACCELLA type 1250 (ThermoScientific, San Jose, CA, USA) system and TSQ Quantum Quadrupole Mass Spectrometer ACCESS MAX (Thermo Finnigan, San Jose, CA, USA).

HPLC Analysis:

The UHPLC control is the Xcalibur ver 2.1 software package.

Chromatographic separations performed by Hypersil Gold C-18 column (50mm x 2.1mm; i.d: 1.9μm). The mobile phase was 0.1% formic acid in water (v/v) (A) and 0.1% formic acid in acetonitrile (v/v) (B). The gradient elution was performed by 10% (B) for 0 to 0.60 minute and 0.60 to 5 minute; followed by 55% (B) for 5 to 5.50 minute and 5.50 to 5.75 minute; then returned to 10% (B) for 5.75 to 7.50 minute; at flow rate of 250 μL/min. The injection volume was 2.0μL.The temperature setting was 30°C for columns and 20°C for autosampler.

MS Analysis:

The Mass Spectrophotometer used Electrospray Ionization (ESI) in negative mode. Each standard or RF extract was injected into MS by ESI source. ESI performance was: spray voltage at 3kV, evaporation temperature at 250°C, the capillary temperature at 300 °C, sheath gas (Nitrogen) pressure at 40 psi, and auxiliary gas (Argon) pressure at ten psi. The ESI control is TSQ Tune software. Selected Reaction Monitoring (SRM) method determined the concentration of compounds. The parent-to-product ion pairs setting for SRM of Q, Q3G, T, CG, and PB2 were 301→179, 463→300, 303→284, 441→289, and 577→426 (m/z), respectively. The collision energy was 20V, for each.

Total Antioxidant Activity/TAA determination:

This analysis used the phosphomolybdate assay¹⁸. This assay based on the capacity of the sample to decolorize phosphomolybdate. The ME or EE (1ml) was mixed with phosphomolybdate reagent (1ml), then incubated for one hour in 95ºC. Absolute methanol was added until volume 5.0ml. The absorbance was measured at λ=695 nm. The antioxidant activity was calculated using Quercetin standard curve and expressed as μg Quercetin Equivalent per g extract (μg QE/g)

Radical scavenging activity determination:

DPPH Radical Scavenging Assay:

DPPH (2.2‑Diphenyl‑1‑picrylhydrazyl) radical scavenging activity can be evaluated by the capacity of RF extract to change the DPPH solution from purple to yellow^19,20. The reaction mixture (5.0ml) consists of 3.0 ml of DPPH in methanol (0.3mM), 1.0ml of each RF extract in methanol, and 1.0ml of methanol. The solution was mixed well then incubated in the dark for 10 min. The absorbance was measured at 517nm. The activity was named as a percentage of scavenging activity that calculated based on this formula: % Scavenging Activity = [(Ac – As)/Ac] x 100; where Ac is the negative control solution absorbance and As was the absorbance of the ME or EE or positive control (Quercetin).

ABTS Radical Scavenging Assay:

The ability of the RF extracts to scavenge ABTS (2.2′‑Azino‑bis(3‑ethylbenzothiazoline‑6‑sulfonic acid) radical were shown by the decrease of absorbance, which caused by decolorization of ABTS radical working solution^19,20. The RF extracts in methanol, for each 200μL in a test tube, were mixed well with 3mL ABTS radical working solution, then incubated at 37ºC in the dark for 10 minutes. The absorbance was directly measured at λ= 734nm. The scavenging activity was determined using the formula: % Scavenging Activity = [(Ac – As)/Ac] x 100, where As and Ac are the absorbance of the sample (ME or EE) and control, respectively. The positive control is Quercetin that was prepared at some concentrations (ppm). The RF sample with 200μL methanol as a negative control.

Reducing power determination:

Ferric Reduction Assayevaluated the analysis of RF extracts reducing ability¹⁹. ME or EE (1ml) was mixed with 2.5ml phosphate buffer of 200mM (pH 6.6) and 2.5ml potassium ferricyanide (30mM). This mixture then was incubated in water-bath at 50ºC for 20 minutes. Trichloroacetic Acid 2.5ml (600mM) was added after cooling the mixture, then centrifuged at 3000rpm for 10 minutes. The 2.5ml the sample was taken out and placed in another glass tube, then added with 2.5ml deionized water and 0.5ml FeCl₃ (6mM). After mixing this solution, the absorbances were recorded at λ= 700nm. The standard curve was made using Quercetin. The power of ferric reduction was calculated as μg Quercetin Equivalent per gram extract (μg QE/ml extract).

RACI and PAC calculation of RF extracts:

RACI (Relative Activity Antioxidant Capacity Index) was calculated using the standard score^21,22. First, the extracts were ranked by the mean value (x) for its every antioxidant parameter and TPC, followed by calculates the mean value (μ) and standard deviation (σ) for all extracts of each antioxidant assay or TPC. The standard scores then were calculated by subtracting the difference between raw mean value (x) and the mean of each antioxidant or TPC (μ) with the standard deviation (σ). The mean of all standard scores for each extract is its RACI. Whereas, PAC (Phenolic Antioxidant Coefficient) for each RF extract was the ratio between the mean value of specific antioxidant capacity and TPC²³.

Data Analysis:

Data were shown as means+SD from at least three replication for each sample. One-way Analysis of variance that followed by Least Significant Difference at α= 0.05 was done using the Statistical Analysis System (SAS) ver. 9.3. The correlation between TPC and antioxidant activities was counted by Pearson Correlation using Minitab 16, where the significant correlation was shown by P-value < 0.05.

RESULT AND DISCUSSION:

Total phenolic content and antioxidant activities of RF extracts:

The ME and EE of short-red cultivar RF in this research contained total phenolic 9.88 and 20.11μg GAE/ml extract, respectively (Table 1). Extraction using ethyl acetic successfully brought out the phenolic compounds from RF than methanol. Other researchers found similar results (80.27 and 627.52mg GAE/g extract; 5.94 and 62.52% GAE, w/w, respectively)^3,4 even though in different concentrations that may be caused by the RF variety used and extraction process.

The extraction procedure is an essential step in sample pretreatment and extract or compound quantification and identification^1,24,25. We used a liquid-liquid extraction system that separated phenolic compounds between two immiscible phases. The fat content of RF was high, 50.8-55.58% (db), depending on the variety¹¹, but it may interfere with phenolic extraction. Fruits have phenolic in bound form; 24% of total phenolic¹. Partition the ME used a non-polar solvent like chloroform^3,4 or hexane before semi-polar solvent (ethyl acetic) effectively separated phenolic from the fat and another non-polar compound of RF. That is why EE had higher total phenolic content than ME.

RF extracts generally showed good antioxidant activity in several mechanisms, not only in radical scavenging but also in ferric ion reduction (Table 1). Those activities were showed higher by EE than ME, significantly. RF extracts gave stronger scavenging activity than reducing power. EE at concentration 100-300μg/ml could scavenge 50% of DPPH radical, whereas ME need more than 300μg/ml to make the same effect. This effect was lower than other scavenging activity of RF ethyl acetic extract found by other study^3,4, where the IC50 was given at 10.35μg/ml and 10.41μg/ml respectively. The extracts of Short-Red RF show better scavenging activity than Mimosa pudica leave (64.48% inhibition at 1000 μg/ml)²⁶, but less potent than Alternanthera sessilis (51.7% inhibition at 20μg/ml)²⁷ or Mimosa pudica aerial part (IC50= 20.51 μg/ml)²⁸.

As far as we know that antioxidant research in RF, only used DPPH^3,4 or plus reducing power and metal-ion chelating assay³, whereas total antioxidant and ABTS scavenging assay never were used. DPPH assay was a popular method for scavenging activity evaluation of plant extract²⁹. Table 1 shows that RF extracts could scavenge cation radicals like ABTS• higher than DPPH radical significantly, even almost 100% at 300μg/ml. Based on this, RF extracts are a potent scavenger for cation radical. This activity was better than ethanol extract of Azima tetracantha leaves and methanol extract of Cissus quadrangualris that inhibited ABTS• radical but at higher concentration (IC50= 500mg/ml extract and 179.6 μg/ml respectively)^30,31. Cation radical is essential for hydrocarbon carcinogenesis, for example, PAH (polycyclic aromatic hydrocarbon)³². RF may play as an anticancer through its cation radical scavenging activity.

The reducing power was determined by the ability to donate a hydrogen atom by the extract in the reduction process of the ferricyanide complex become ferrocyanide. The ferrocyanide then makes a ferric-ferrous complex with ferric chloride that was measured at 700nm^{3,19,33,34,35}. Table 1 showed that EE had a higher reducing power than ME, which was three times higher at 300 μg/ml. This was also found in other RF study³.

TPC and Antioxidant Activity Correlation, RACI, and PAC of RF Extracts:

We observed the relationship between TPC and antioxidant activity and among the antioxidant activity of each RF extract by correlation, RACI, and PAC analysis. Extracts that had a difference in phenolic content and compositions will have different antioxidant activity, too^13,14,15. Table 2 showed that TPC contributed to the antioxidant activity. The correlation between TPC and different antioxidant capacity for EE (R²= 0.962-1.000) was higher than ME (R²= 0.551-0.999) (Table 2.).

Table 2: Pearson correlation between phenolic content and antioxidant activities of methanol and ethyl acetic extracts

Extract	Parameter	TPC	TAA	DPPH Scav.	ABTS Scav.	Reducing Power
Methanol	TPC	1.000
	TAA	0.995	1.000
	DPPH Scav.	0.999*	0.999*	1.000
	ABTS Scav.	0.915	0.949	0.935	1.000
	RP	0.551	0.628	0.594	0.841	1.000
Ethyl Acetic	TPC	1.000
	TAA	0.999*	1.000
	DPPH Scav.	0.996	0.998*	1.000
	ABTS Scav.	0.928	0.940	0.957	1.000
	RP	0.994	0.997*	1.000*	0.962	1.000

*= Significant at p<0.05

The ME had an only positive and significant strong correlation between TPC and DPPH scavenging (R²= 0.999) and between TAA and DPPH scavenging (R²= 0.999). Those mean that radical scavenging is the primary antioxidant capacity in ME. Whereas the EE had significantly strong capacity in radical scavenging (R²= 0.998) and ferric ion reduction capacity (R²= 0.997) as main antioxidant activities. This correlation reveals the role of phenolic compounds in RF antioxidant actitivies³⁶. This role mainly due to the redox properties of phenolic³⁷.

The used multiple assays often give difficulty in comparing the results of antioxidant capacity among the assays^21,22. Although the correlation analysis above shown the strong relationship, each assay has its reaction mechanism and unit or dimension. The use of multiple assays needs a standard to evaluate antioxidant capacity. Relative Antioxidant Capacity Index (RACI) integrated antioxidant capacity data obtained by different assays²¹. RACI is dimensionless. EE showed a positive and high RACI value that was opposite ME (Figure 1.). It emphasizes that EE has an antioxidant capacity higher than ME. PAC value enables a comparison of the effectiveness of phenolics present in analyzed samples and also provides specific insight into differences between applied assays²³. The PAC values in Figure 1 show the vital role of the presence of phenol compounds in the extract against the antioxidant activity. PAC of all antioxidant assays applied has a positive value. It means phenolic presented in both RF extracts effectively play as an antioxidant in each assay. PAC-ABTS was higher than PAC-DPPH. It may RF phenolic scavenges ABTS (cation) radical, more efficient than DPPH (anion) radical.

Fig 1: RACI and PAC of Methanol/ME and Ethyl Acetic/EE Extract of RF

Flavonoid identification of RF extracts by LCMS:

LCMS analysis proved that ME and EE contained quercetin 3-glucoside, taxifolin, and quercetin. EE contained those flavonoids higher than ME (Table 3.). This identification strengthens the discovery of RF flavonoid by the Chemistry Department - Padjajaran University, Bandung, Indonesia research team³⁸. They isolated eight flavonoids, including the three flavonoids above, from ethyl acetic extract of RF long-red cultivar. We can guess that those flavonoids are dominant in RF since they used long-red cultivar instead of the short-red cultivar.

Table 3: Flavonoids detected in methanol and ethyl acetic extract of Pandanus conoideus Lam. using LC-MS in negative ionization mode

No	RT (minute)	[M-H]^- m/z	Concentration (μg/ml extract)		Identified Compounds
No	RT (minute)	[M-H]^- m/z	ME	EE	Identified Compounds
1	3.38	463	148.52	311.26	Quercetin-3-Glucoside
2	3.43	303	245.1	931.17	Taxifolin
3	4.45	301	166.15	428.62	Quercetin

RT: Retention Time, ME: Methanol Extract, EE: Ethyl Acetic Extract

Taxifolin, quercetin, and quercetin-3-glucoside have the same number and position of the hydroxyl group at A ring (C5 and C-7) and B ring (C-4’ and C-5’)³⁸. The presence of those compounds in EE that higher than ME (Table 3.) may cause the higher scavenging capacity of EE (Table 1-2) and PAC-DPPH and PAC-ABTS (Figure 1.).

Taxifolin is an efficient DPPH and ABTS radical scavenger. It has two aromatic rings, where two phenolic groups attached at meta- and para- positions at A and B ring. Para positions are essential for radical scavenging^39,40. The ortho position in the B ring is essential for electron delocalization. It enhances the stability of taxifolin radicals that formed after reacted with DPPH or ABTS radical^39,40,41. Hydroxyl group at C-5 and C-7 in A ring also determine antioxidant activity⁴¹.

EE and ME showed the reducing capacity (Table 3), but the PAC for this activity was lower than for scavenging activity (Fig. 1). Taxifolin has reducing activity against Fe³⁺ and Cu²⁺ions³⁹. Taxifolin can also chelate three Fe²⁺ ions by involving the hydroxyl group in all rings (at C-3, 5, 4', 5') and carbonyl group in C ring (at C-4). This chelation after the ferric reduction by EE or ME was determined well at λ=700 nm. The chelation makes a stabile complex that will stabilize and terminate radical chain reaction^33,34.

CONCLUSION:

The ME and EE of short-red RF had strong significant radical scavenging capacity as the primary role in antioxidant activity. The phenolic present in the extracts, especially taxifolin, quercetin, and quercetin-3-glucoside, contributed to the radicals scavenging and also ferric ion reducing activity. Short-Red RF extracts can be a potential scavenger for not only anion radical but also cation radical.

ACKNOWLEDGMENT:

This research was financially supported by Doctorate Research Grant of Directorate General Higher Education Indonesia (No. 1014/UN10.14/KU/2013) and Widya Mandala Catholic University Surabaya/WMCUS Grant. We also thank to (TEW Widyastuti, I Kuswardani, MMD Intan, and NA Tristanto (Agricultural Technology Faculty and Food and Nutrition Research Center, WMCUS, Surabaya, Indonesia), MC Al-Ayubi (Science and Technology Faculty, UIN Malang), and K Kalimullah (Chemistry Department of Politeknik Negeri Malang, Indonesia) for technical supports during laboratory analysis.

CONFLICT OF INTEREST:

We declare no conflict of interest.

REFERENCES:

1. Santos-Zea L, Villela-Castrejón J, and Gutriérrez-Uribe JA. Bound Phenolics in Foods. In Merillon JM and Ramawat KG. Bioactive molecules in food, reference series in phytochemistry. Switzerland AG: Springer Nature. 2019; pp. 973-989

2. Sadsoeitoeboen MJ. Pandanaceae: aspek botani dan etnobotani dalam kehidupan suku Arfak di Irian Jaya. (Master Thesis). Bogor, Indonesia: Program Pascasarjana, Institut Pertanian Bogor. 1999.

3. Rohman A, Riyanto S, Yuniarti N, Saputra WR, Utami R, and Mulatsih W. Antioxidant activity, total phenolic, and total flavonoid of extracts and fractions of red fruit (Pandanus conoideus Lam). International Food Research Journal. 2010; 17: 97-106

4. Sandhiutami NMD and Indrayani AAW. Uji aktivitas antioksidan, kandungan fenolik total, dan kandungan flavonoid total buah merah. Jurnal Ilmu Kefarmasian Indonesia. 2012; 10: 13-19

5. Handayani MDN, Soekarno PA, and Wanandi SI. Red fruit oil supplementation fails to prevent oxidative stress in rats. Universa Medicina. 2013; 32: 86-91

6. Nainggolan D. Aspek ekologis kultivar buah merah panjang (Pandanus conoideus Lam) di daerah dataran rendah Manokwari. (Research Report). Manokwari, Indonesia: Jurusan Kehutanan, Fakultas Pertanian, Universitas Cenderawasih. 2001.

7. Limbongan J, and Malik A. Peluang pengembangan buah merah (Pandanus conoideus Lamk.) di provinsi Papua. Jurnal Litbang Pertanian. 2009; 28: 134-141

8. Zebua LI, Supriatna J, Walujo EB, and Chikmawati T. Inventory of red fruit pandan (Pandanus conoideus Lamarck) in Papua, Indonesia. In Wickneswari Ratnam. Editor. New Trends and Challenges in Science and Technology: Proceedings the Second UKM-UI Joint Seminar Bangi, Selangor: Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia. 2009; pp. 360-363).

9. Murtiningrum, Sarungallo ZL, and Mawikere NL. The exploration and diversity of red fruit (Pandanus conoideus L.) from Papua based on its physical characteristics and chemical composition. Biodiversitas. 2012; 13: 124-129

10. Sarungallo ZL, Hariyadi P, Andarwulan N, and Purnomo EH. Characterization of chemical properties, lipid profile, total phenol, and tocopherol content of oils extracted from nine clones of red fruit (Pandanus conoideus). Kasetsart Journal (Natural Science). 2015; 49: 237-250

11. Sarungallo ZL, Murtiningrum, Santoso B, Roreng MK, and Latumahina. Nutrient content of three clones of red fruit (Pandanus conoideus) during the maturity development. International Food Research Journal. 2016; 23: 1217-1225

12. Taher A, and Repasi MS. Aktivitas antioksidan minyak buah merah dari kultivar Pandanus conoideus L yang berbeda. Jurnal Natural. 2009; 8: 58

13. Schlesier K, Harwat M, Bohm V, and Bitsch R. Assessment of antioxidant activity by using different in vitro Methods. Free Radical Research. 2002; 36: 177–187

14. Piluzza G, and Bullitta S. Correlations between phenolic content and antioxidant properties in twenty-four plant species of traditional ethnoveterinary use in the mediterranean area. Pharmaceutical Biology. 2011; 49: 240–247

15. Rebaya A, Belghith SI, Baghdikian B, Leddet VM, Mabrouki F, Olivier E, Cherif JK, and Ayadi MT. Total phenolic, total flavonoid, tannin content, and antioxidant capacity of Halimium halimifolium (Cistaceae). Journal of Applied Pharmaceutical Science. 2015; 5: 052-057

16. Büyüktuncel E, Porgali E, and Çolak C. Comparison of total phenolic content and total antioxidant activity in local red wines determined by spectrophotometric methods. Food and Nutrition Science. 2014; 5: 1660-1667

17. Achadiyani, Septiani L, Faried A, Bolly HMB, and Kurnia D. Role of the red fruit (Pandanus conoideus LAM) ethyl acetate fraction on the induction of apoptosis vs. downregulation of survival signaling pathways in cervical cancer cells. European Journal of Medicinal Plants. 2016; 13: 1-9

18. Salamah N, and Farahana L. Uji aktivitas antioksidan ekstrak etanol herba pegagan (Centella asiatica (L.) Urb) dengan metode fosfomolibdat. Pharmaçiana. 2014; 4: 23-30

19. Chanda S, and Dave R. In vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties: an overview. African Journal of Microbiology Research. 2009; 3: 981-996

20. Ahmed D, Khan MM, and Saeed R. Comparative analysis of phenolics, flavonoids, and antioxidant and antibacterial potential of methanolic, hexanic, and aqueous extracts from Adiantum caudatum leaves. Antioxidants. 2015; 4: 394-409

21. Sun T, and Tanumihardjo SA. An integrated approach to evaluate food antioxidant capacity. Journal of Food Science. 2007; 72: R159-R165

22. Gorjanović SẐ, Alvarez-Suarez JM, Novaković MM, Pastor FT, Pezo L, Battino M, and Sužnjevic DẐ. Comparative analysis of antioxidant activity of honey of different floral sources using recently developed polarographic and various spectrophotometric assays. Journal of Food Composition and Analysis. 2013; 30: 13-18

23. Petrović M, Sužnjević DẐ, Pastor F, Veljović M, Pezo L, Antić M, and Gorjanović SẐ. Antioxidant capacity determination of complex samples and individual phenolics - multilateral approach. Combinatorial Chemistry High Throughput Screening. 2016; 19: 58-65

24. Salas PG, Soto AM, Carretero AS, and Gutiérrez AF. Phenolic-compound-extraction systems for fruit and vegetable samples. Molecules. 2010; 15: 8813-8826

25. Xu CC, Wang B, Pu YQ, Tao JS, and Zhang T. Advances in extraction and analysis of phenolic compounds from plant materials. Chinese Journal of Natural Medicines. 2017; 15: 0721-0731

26. Rajendran R, Hemachander R, Ezhilarasan T, Keerthana C, Saroja DL, Saichand KV, Abdullah MG. Phytochemical analysis and in-vitro antioxidant activity of Mimosa pudica Lin. leaves. Research Journal of Pharmacy and Technology. 2010; 3: 551-555

27. Roy A, and Saraf S. Antioxidant and antiulcer activities of an ethnomedicine: Alternanthera sessilis. Research Journal of Pharmacy and Technology. 2008; 1: 75-79

28. Faruk MO, Saha D, Chowdhury S, Paul S, and Kabir MG. In vitro antioxidant activity of methanolic aerial part extract of Mimosa pudica. Research Journal of Pharmacology and Pharmacodynamics. 2012; 4: 202-205

29. Sravanthi J, and Gangadhar RS. Quantification of antioxidant-phytochemical studies in Vitis vinifera L. varieties. Asian Journal of Pharmaceutical and Clinical Research. 2015; 8: 295-301

30. Salomi S, Muthukumaran P, and Umamaheshwari R. In-vitro antioxidant activity of Azima tetracantha leaves. Research Journal of Science and Technology. 2012; 4: 148-151

31. Raj AJ, and Dorairaj S. Phytochemical screening and in-vitro antioxidant activity of Cissus quadrangualris. Asian Journal of Research in Chemistry. 2011; 4: 876-878

32. Pryor WA. Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity. Environmental Health Perspectives 1997; 105: S875-882

33. Elias J, Rajesh MG, Anish NP, Deepa P, and Jayan N. In vitro antioxidant activity of the methanolic extract of Simaruba glauca DC. Asian Journal of Research in Chemistry. 2010; 3: 312-315

34. Jayasri R, and Anuradha R. In vitro antioxidant and antibacterial activity of Boerhaavia diffusa. Research Journal Pharmacognosy and Phytochemistry. 2012; 4: 223-225

35. Suneetha B, Prasad KVSRG, Soumya BR, Nishantha PD, Kumar BS, and Rajaneekar D. Evaluation of in-vitro antioxidant activity of various extracts of Actinodaphne madraspatana leaves. Research Journal of Pharmacognosy and Phytochemistry. 2014; 6: 1-4

36. Mohite MS, Dr. Yadav AV, Raje VN, Mohite YG, and Thombre SA. Preliminary phytochemical investigation and in-vitro antioxidant activity of Borassus flabellifer Linn. Fruit Pulp. Research Journal of Pharmacognosy and Phytochemistry. 2012; 4: 326-329

37. Elias J, Rajesh MG, Anish NP, Manu MS, and Varkey IC. Terminalia chebula Retz. stem bark extract: a potent natural antioxidant. Asian Journal of Research in Chemistry. 2011; 4: 445-449

38. Suprijono MM, Sujuti H, Kurnia D, and Widjanarko SB. Computational study of antioxidant activity and bioavailability of Papua red fruit (Pandanus conoideus Lam.) flavonoids through docking toward human serum albumin. AIP Conference Proceeding 2019; 2108:020020 https://doi.org/10.1063/1.5109995

39. Topal F, Nar M, Gocer H, Kalin P, Kocyigit UM, Gülçin I, and Alwasel SH. Antioxidant activity of taxifolin: an activity-structure relationship. Journal of Enzyme Inhibition and Medicinal Chemistry. 2015; Early online: 1-10

40. Watak S, and Patil SS. Evaluation and Comparison of Antioxidant activity of Herbomineral Complex. Research Journal of Pharmacognosy and Phytochemistry. 2012; 4: 171-177

41. Shubina VS and Shatalin YV. Antioxidant and iron-chelating properties of taxifolin and its condensation product with glyoxylic acid. Journal of Food Science and Technology. 2017; 54: 1467-1475

Received on 31.10.2019 Modified on 28.12.2019

Research J. Pharm. and Tech 2020; 13(9):4158-4164.

DOI: 10.5958/0974-360X.2020.00734.9

Extract	Concentration (μg/ml)	TPC	TAA	DPPH Scav.	ABTS Scav.	Reducing Power
ME	30	3.45+0.38^a	2.06+0.08^a	7.52+0.45^a	12.64+0.72^a	(-)0.59+0.10^a
	100	5.63+0.40^c	10.73+0.27^c	13.48+0.21^b	47.31+1.17^c	7.87+0.45^d
	300	20.56+0.74^e	41.24+0.85^e	40.7+0.72^e	80.89+0.84^e	7.33+0.11^c
EE	30	4.82+0.27^b	4.66+0.18^b	14.9+0.52^c	23.34+0.41^b	2.07+0.31^b
	100	14.44+0.24^d	18.24+0.40^d	35.74+0.55^d	69.09+0.17^d	10.3+0.54^e
	300	41.07+0.34^f	50.46+1.35^f	74.92+1.33^f	99.23+0.16^f	24.77+0.64^f