Phytochemical and antioxidant properties of Diodia sarmentosa swartz leaves

This research studied proximate, phytochemical and antioxidant properties of Diodia sarmentosa leaves. The ethanol and aqueous extracts of the leaves significantly inhibited 2,2-diphenyl-1-picrylhydrazyl (DPPHo) radical formation with IC50 values of 10.994 and 10.121 μg/mL respectively, compared to the ascorbic acid standard (IC50 value = 17.916 μg/mL). The aqueous extract exhibited more inhibitory effect on thiobarbituric acid-reactive species (TBARS) with IC50 values of 2.657 μg/mL while the ethanol extract had an IC50 value of 8.53 μg/mL compared to butylated hydroxytoluene standard (IC50 = 2.142 μg/mL). For the total antioxidant capacity assay, the aqueous extract had higher ascorbic acid equivalent values than the ethanol extract. However, the two solvent extracts showed antioxidant activity. Diodia sarmentosa leaves possess useful phytochemicals which are indicative of its antioxidant properties.


Plant collection and identification:
Fresh samples of D. sarmentosa leaves were collected from farmlands and from natural vegetation within Federal University of Technology, Owerri (FUTO) premises. It was identified by Prof. Iloegbulam I.I. of the Department of Crop Science. A picture of a portion of D. sarmentosa plant is displayed in Figure 1.

Preparation of plant extract and extraction:
Fresh leaves of D. sarmentosa were rinsed using distilled H 2 O, dried at room temperature and then in a laboratory oven at 40 °C. The dried plant material was grinded into fine powder and 160 g was weighed, soaked in ethanol (800 mL) for 48 h and placed on an orbital shaker. Filtration was done using Whatman No.1 filter paper placed inside a funnel, then dried at 40 °C using a rotary evaporator. Proximate analysis of sample: Determination of moisture content was carried out using a laboratory oven at 105 °C. Ash was determined using an electric furnace at 550 °C. Soxhlet apparatus was used in the determination of the crude fat. Crude protein was determined using Kjeldahl method. Percentage carbohydrate was done by difference. These were all done using AOAC method [11]. Qualitative phytochemical screening: The test sample (0.2 g) was placed in separate test tubes to assay for phytochemicals as follows: Saponin: Frothing test was used in the detection of saponin, which was confirmed by constant foaming. Flavonoid: Ammonium test method was used in the determination of flavonoid. A yellowish colouration confirmed the presence of flavonoid. Carbohydrate: Molisch's method was used in the determination of carbohydrate. This was confirmed by the formation of a brown ring. Phenol: Ferric chloride test was used in the determination of phenol. A greenish colouration was observed and this confirmed the presence of phenol. Reducing Sugar: Reducing sugar was confirmed by a reddish colouration using Fehling's method. Glycoside: Fehling's method was used in glycoside determination. Glycoside was confirmed by a reddish colouration. Gallic acid was used as standard. Folin-Ciocalteu reagent was used in oxidizing D. sarmentosa leaves extract. This was then neutralized using Na 2 CO 3 . A portion (100 μL) of the different concentrations (15.63, 31.25, 62.5 µg/mL) of this extract was put into a mixture of 0.5 mL Folin-Ciocalteau reagent (0.1 dilution) and 1.5 mL sodium carbonate 2 % (w/v), then incubated for about 15 min at 25 °C. The absorbance of the blue coloured solution was read at 765 nm and results expressed in mg of gallic acid equivalent (GAE)/100 g of dry weight of plant powder. Determination of tannins: The leaves extract (1 mL) of D. sarmentosa was put into a solution containing 0.5 mL Folin-Ciocalteau reagent, 1 mL sodium carbonate and 8 mL of distilled water. This was kept at 25 °C for about 30 min. It was then centrifuged to get a supernatant and absorbance read at 765 nm. The results were expressed as mg tannic acid/100 g of dry weight of plant powder. Determination of flavonoid: Zhishen colorimetric method was used in the determination of flavonoid [12]. The diluted sample solution (0.5 mL) was put into a mixture containing 2 mL of distilled H 2 O and 0.15 mL of sodium nitrate (5 %) solution. After a short while, 0.15 mL of 10 % AlCl 3 (aq) was put into the solution and kept for about 6 min. Diluted sodium hydroxide (4 %) solution (2 mL) was added to bring the total volume to 5 mL. The absorbance was taken after 15 min at 510 nm against water blank. The results were expressed as mg/100 g of dry weight of plant powder. Determination of terpenoids: A portion of D. sarmentosa leaves (1 g) was extracted using ethanol (50 mL). The filtrate (2.5 mL) was mixed with 5% aqueous phosphomolybdic acid (2.5 mL) and 2.5 mL concentrated tetraoxosulphate (VI). After that, the volume of the solution was made up to 12.5 mL after 30 min using ethanol. The absorbance was read at 700 nm. The results were expressed as mg/100 g of dry weight of plant powder. Determination of steroids: A portion of D. sarmentosa leaves (1 g) was extracted with 20 mL of ethanol. The filtrate (2 mL) was mixed with 2 mL of chromogen solution and kept at room temperature (25 °C) for about 30 min. Then the absorbance was read at 550 nm. The results were expressed as mg/100 g of dry weight of plant powder. Determination of saponin: Extraction of the sample (1 g) was done using 10 mL petroleum ether. After that, an additional 10 mL of petroleum ether was mixed with the solution and then subjected to dryness by evaporation. The residue obtained after dryness was dissolved in 6 mL of ethanol. Then, 2 mL of the solution was mixed with 2 mL of chromogen and left at room temperature for about 30 min. Absorbance of the solution was read at 550 nm. The results were expressed as mg/100 g of dry weight of plant powder. Determination of glycosides: A sample of D. sarmentosa leaves (1 g) was extracted with 50 mL of distilled water. Alkaline picrate (4 mL) solution was mixed with 1 mL of the sample filtrate and heated for about 5 min. Absorbance was read at a wavelength of 490 nm. The results were expressed as mg/100 g of dry weight of plant powder. Reducing sugar: Extraction of the sample (1 g) was carried out using 20 mL of distilled water. Alkaline copper reagent (1 mL) was mixed with the filtrate (1 mL) and heated for about 5 min. Then 1 mL of phosphomolybdic acid reagent and 2 mL of distilled water was mixed with the resultant solution and absorbance read at 420 nm. The results were expressed as mg/100 g of dry weight of plant powder. Determination of soluble carbohydrates: Extraction of the sample (1 g) was done using 50 mL of distilled water. The filtrate (I mL) was mixed with picric acid solution and the absorbance read at 580 nm. The results were expressed as mg/100 g of dry weight of plant powder.

In vitro screening for antioxidant activities:
Quantitative DPPH o radical scavenging assay: Quantitative DPPHº was determined according to a modified Gyamfi method [13]. The test was performed in triplicates. The sample extracts (1 mL each) were diluted 2-fold in 10 mL of water mixed with 0.5 mL of 0.076 mM DPPHº. After that, it was mixed properly and kept away from sunlight at 25 °C for about 25 min. An aqueous solution (1 ml) of 0.076 mM DPPHº was used as a negative control while the positive control was L-Ascorbic acid. Absorbance was read at 517 nm. Thiobarbituric acid-reactive species (TBARS): Thiobarbituric acid-reactive species assay was carried out by a modified method of Banerjee [14]. This aimed at quantifying the amount of lipid peroxide formed. Egg yolk homogenate served as lipid-rich media [15]. The sample (100 μL) was mixed with egg homogenate (500 μL) in a test tube and distilled water was used to make up the volume to 1.0 mL. A mixture of 0.075 M FeSO 4 (50 μl) and 0.1 M L-Ascorbic acid (20 μL) were added to the solution, and kept at 37 °C for about an hour. Then 0.2 mL EDTA (0.1 M) and 1.5 mL of TBA reagent were put into each sample and heated for 15 min at 100 °C. After cooling, the samples were centrifuged for 10 min at 3000 rpm. The absorbance was read at 532 nm. Butylated hydroxytoluene (BHT) was used as the assay standard.  MoO 4 ). The test tubes were heated in a water bath at 95 °C for 90 min. After cooling, the absorbance was read at 765 nm. The reagent solution (1 mL) was used as blank and ascorbic acid was used as standard. Statistical analysis: Pearson's correlation coefficient at 95% confidence interval was used in the determination of correlation coefficient between analytes. One way analysis of variance (ANOVA) was used in determining the standard deviation between means of values.

In vitro antioxidant activities of leaves extracts of D. sarmentosa: DPPH o assay:
A graph of inhibition (%) was plotted against DPPH o concentration (μg/mL) (Fig. 2). There was a significant (r = -0.955, p < 0.05) negative Pearson correlation between DPPH o aqueous and concentration while a significant (r = 0.704, p < 0.05) positive correlation was observed between DPPH o ethanol and concentration. DPPH o aqueous was negatively (r = -0.511, p < 0.05) correlated with the standard (ascorbic acid) while DPPH o ethanol was positively (r = 0.257, p < 0.05) correlated with the standard.

Total antioxidant capacity:
A graph of ascorbic acid equivalent was plotted against concentration (Fig. 4).
This reveals the nutritional composition of the leaves and suggest that it could be used as part of a healthy meal. Qualitative phytochemical screening of 30    D. sarmentosa leaves revealed the presence of saponin, flavonoid, terpenoid, phenol, reducing sugar, glycoside, tannin, steroid and carbohydrate except alkaloid which was absent ( Table 1). Presence of saponin indicates that the leaves can be useful in treating yeast and fungal infections [16]. This agrees with the findings of Umoh [2] who reported the ability of D. sarmentosa leaves in preventing inflammation.
Flavonoids are natural antioxidants [17] which boost immune function, protect against microbial infections and prevent the formation of free radicals [18]. Phenols prevent inflammation and also boost the body immune system [19,20]. This supports the findings of Umoh [2], who reported the anti-inflammatory properties of the leaves. Presence of glycosides suggest that the leaves extract have the ability to lower blood pressure [21]. Presence of tannins suggest that the leaves can be used in the treatment of wounds [22]. Results of this study validates that of Akah [5] regarding presence of tannins and of the antiulcer effect of D. sarmentosa leaves. DPPH o assay of the leaves extract of D. sarmentosa revealed significant antioxidant activity (Fig. 2). The IC 50 (inhibitory concentration at 50 %) value of aqueous and ethanol extracts of D. sarmentosa (10.121 and 10.994 μg/mL respectively) were significantly lower than the IC 50 value of ascorbic acid (17.916 μg/mL). Lower values reveal better antioxidant activity [23,24]. In the Thiobarbituric acid assay (TBARS) (Fig. 3), the aqueous extract showed a significantly higher percentage inhibition compared to the ethanol extract. The IC 50 values for the aqueous and ethanol extracts were 2.657 and 8.53 μg/mL respectively compared to IC 50 value of BHT standard, which was 2.142 μg/ mL. This suggest that water soluble phytochemicals possess a stronger potential to reduce malondialdehyde (MDA) formation. For the total antioxidant capacity assay (Fig. 4), the aqueous extract had higher ascorbic acid equivalent values compared to the ethanol extract. This is quite normal since the solubility of ascorbic acid increase with increasing polarity [25]. However, the two solvent extracts showed antioxidant activity.

CONCLUSIONS
The results of proximate assessment of D. sarmentosa leaves which revealed the edibility of the leaves, supports the use of the leaves in southern Nigeria for culinary purposes. Evidence from the qualitative and quantitative phytochemical evaluation as well as the different in vitro antioxidant assessments of D. sarmentosa leaves suggest that the plant has useful phytochemicals, indicative of its antioxidant properties.