1. Introduction

Stevia rebaudiana (Bertoni) is a small perennial herb belonging to the family Asteraceae ^[1]. The plant is native to Paraguay and Brazil, and produces high potency low calorie sweeteners, stevioside and rebaudioside which are about 250-300 times sweeter than sucrose ^{[2, 3, 4, 5]}. These natural sweeteners are gaining very high popularity amongst all type of sweetener users as the most ideal substitute for sugar ^[6]. Other than, its sweetening property stevia is also known for its therapeutic properties including antidiabetic ^[7] antimicrobial ^[8], antiviral ^[9], antifungal ^[10], anti-hypertensive ^{[11, 12, 13]}, anti-hyperglycaemic ^{[14, 15]}, anti-tumour ^[8], anti-human rota-virus activities ^{[16, 17]}, anti-HIV ^[18], hepatoprotective ^[19] and immune-modulatory effects ^{[20, 21]}. Even though there are many information are available on sweetening capacity, information on health benefits due to the presence of therapeutically active secondary metabolites are scattered. However, phytochemical distribution in different parts of the plant and different leaf positions play an important role on therapeutic potential of the plant. Therefore, present study was undertaken to determine the Total Antioxidant Capacity (TAC), Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of different parts (leaves, stems, roots, flowers and branches) of S. rebaudiana and different leaf positions of the plant (1^st leaf, 2^nd leaf, 3^rd leaf, 4^th leaf and 5^th leaf).

2. Materials and Methods

Folin-Ciocalteu reagent, Gallic acid, Rutin, 2, 4, 6-trypyridyl-2-try-azine (TPTZ), 6-hydroxy-2, 5, 7, 8-tetramethy-chroman-2-carboxilic acid (Trolox) and ferric chloride (FeCl₃.6H₂O) were purchased from sigma Aldrich Chemical Co. (St. Louis, Mo). All other chemicals used were of analytical grade.

Samples of leaves, flowers, roots, stems parts and leaves from different leaf positions (1^st leaf, 2^nd leaf, 3^rd leaf, 4^th leaf and 5^th leaf) of S. rebaudiana were collected from same aged, similarly maintained plants grown under same soil and climatic conditions (Low Country Intermediate Zone (IL_1a), at an elevation of 25 m above mean sea level with soil type of red yellow podosolic at experimental plots of Wayamba University of Sri Lanka, Makandura, Gonawila (NWP).

Collected samples were cut into small pieces and air dried for three days at room temperature (28±2°C). Then samples (Three samples from each category) were powdered using motor and pestle and sieved with 0.25 mm mesh. Powdered samples (0.1 g) were mixed with 5 mL of 80% methanol and vortexed for 15 min. Then it was placed in a water bath at 60 °C for 40 min and vortex procedure was repeated at 10 min interval. After centrifugation at 4,000 rpm for 5 min, the supernatant was decanted into a 15 mL centrifuge tube and the remaining was re-extracted with 5 mL of 80% methanol. Supernatants were pooled and stored at -20 °C prior to analysis.

Total phenolic content was quantified using a modified Folin-Ciocalteu method. Briefly, 4 mL of distilled water and 0.5 mL of properly diluted leaf extract were mixed with 0.5 mL of 0.5 N Folin-Ciocalteu reagent (FCR) and allowed to react for 3 min. Then 1 mL saturated sodium carbonate solution was mixed and incubated in a water bath for 2 h at 30°C. The absorbance was measured at 760 nm using UV visible spectrophotometer (Shimadzu, UV Mini 1240, Japan). Gallic acid was used as the standard and data were expressed as mg of Gallic Acid Equivalent (GAE) per gram of dry weights.

Total flavonoid content (TFC) was determined by a colorimetric method, with slight modifications ^[22]. Briefly, 0.5 mL of the plant extract was diluted with 3.5 mL of distilled water. Then 0.3 mL of 5% NaNO₂ solution was added to the mixture. After 6 min, 0.3 mL of a 10% Al(NO₃)₃.6H₂O solution was added, and the mixture was allowed to stand for another 6 min. Then 2 mL of 2 M NaOH was added, and top up to 8 mL with distilled water. After thoroughly mixing, the absorbance was measured at 510 nm using UV visible spectrophotometer (Shimadzu UV-160, Japan). Rutin was used as the standard and data were expressed as mg of Rutin Equivalent (RE) /g DW.

Total antioxidant capacity was determined using Ferric Reducing Antioxidant Power (FRAP) assay ^[23] with slight modifications. Methanolic extract of sample (100 μL) was mixed with 900 μL of freshly prepared FRAP reagent of pH 3.6 containing 2.5 mL of 10 mol/L, 2,4,6-Tripyridyl-s-Triazine (TPTZ) solution in 40 mmol/L, HCl plus 2.5 mL of 20 mmol/L FeCl₃ and 25 mL of 300 mol/L acetate buffer. Absorbance was measured at 593 nm using the spectrophotometer (Shimadzu, UV Mini 1240, Japan) after incubating for 4 min. Trolox was used as the standard solution and TAC was expressed as mg Trolox Equivalents (TE) /g DW.

Leaf area was measured using a leaf area meter (LI-3100C, USA). Samples were collected from 10 plants and ten leaves from each category (part from first to fifth leaves).

Samples were collected from 10 plants (part from first to fifth leaves) and fresh weight was measured then the samples were air dried. After three days dry weight of the samples were measured.

To verify the statistical significance of all parameters the values of means and ±SD were calculated. Statistical comparison of mean values was performed by General Linear Model (GLM) of ANOVA followed by Turkey Multiple Range Test using SAS (SAS institute, 1999). The P values of less than 0.05 were adopted as statistically significant.

3. Results and Discussion

In the present study attempts were made to determine economical yield, therapeutically important phytochemicals (phenolics and flavonoids) and Total Antioxidant Capacity (TAC) of different parts and different leaf positions of S. rebaudiana.

As demonstrated in Table 1, current study exhibited the presence of major phytochemical secondary metabolites (TPC and TFC) and TAC in all five main parts (leaves, stems branches, flowers and roots) of S. rebaudiana. Significantly higher TAC, TPC and TFC were reported in leaf extracts. Order of increase was leaves>flowers>stem>branches>roots. The higher content of TAC in leaf and flower extract and TPC and TFC in leaf extract might be due to presence of high content of phenolics, flavonoids and variety of other pigments in the leaf. Systematic investigation of distribution of important phytochemicals is important for the quantitative estimation and locating of pharmacologically active chemical compounds ^[24]. Presence of biologically active phytochemicals in all parts of the plant is important for the therapeutic activity of plant. Further, presence of higher content of investigated phytochemicals and antioxidant activity is in agreement with ^[25], who observed the higher antioxidant capacity in leaf extracts of S. rebaudiana. Further, the highest therapeutically active components presence in leaf extracts are in agreement with ^{[26, 27]} and ^[28], who demonstrated the presence of higher content of TAC in leaf extracts of Acmella oleraceae, and Withania somnifera respectively. Economical yield of any plant depend on the physical and chemical yield of the plant. As shown in Figure 1 and Table 2, in the current study, leaf fresh weight, leaf dry weight, fresh to dry weight ratio, leaf area, TPC, TFC and TAC of different leaf positions were investigated. The results revealed that TAC was decreased gradually from immature to mature leaf (1^st leaf>2^nd leaf>3^rd leaf>4^th leaf>5^th leaf). Further, all tested phytochemicals (TPC and TFC) were significantly higher in extracts prepared from the first leaf (Bud).

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Figure 1. Changes in area of leaves (A) and fresh weight, dry weight (B) at different leaf positions of Stevia rebaudiana

Table 1. Distribution of Total Antioxidant Capacity (TAC), Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) in different parts of Stevia rebaudiana

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Table 2. Total Antioxidant Capacity (TAC), Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) and fresh to dry weight ratio of leaves at different positions of Stevia rebaudiana

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4. Conclusions

Present study investigated Total Antioxidant Capacity (TAC), Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of different plant parts and different leaf positions of Stevia rebaudiana. Results indicated, all plant parts of S. rebaudiana possess TPC, TFC and TAC. The higher TPC, TFC and TAC were recorded in leaf extracts than other parts of plant. Moreover, all tested secondary metabolite contents (TPC and TFC) and antioxidant capacity were reduced with the leaf maturity. Strong significant correlations were observed between TAC and tested secondary metabolites (TFC, R²=0.85).

Acknowledgements

Authors wish to express their gratitude to all staff of the Department of Plantation Management, Wayamba university of Sri Lanka for their help during study period and Dr. J Ranatunga, who provided the planting materials for the establishment of research plot.

References

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