MATERIALS AND METHODS

Plant material: The seeds and leaves and of Sauropus androgynus were collected from the local area of Thanjavur district, India, during the month of May-June, 2017. Accurately, 1.0 kg of shade-dried coarse powder of the plant material was extracted with 80% v/v aqueous ethanol by maceration at room temperature for 72 h. After the completion of each extraction, the extracts were filtered, concentrated to dryness in a rotavapor under reduced pressure and controlled temperature (40-50°C). The residues were stored in a vacuum desiccator for further use.

Table 1:	Vitamin composition of leaves of Sauropus androgynus
Values are Means±SD calculated as mg/100 g dry weight (DW) for Sauropus androgynus analyzed individually in triplicate

Preparation of extract: Different concentrations of leaf and seed extracts (20, 40, 60 and 80 μg mL^–1) were chosen for in vitro antioxidant activity. L-ascorbic acid (20, 40, 60 and 80 μg mL^–1) was used as the reference standard.

Estimation of vitamins: Vitamins like ascorbic acid, riboflavin and thiamine were estimated according to the method by Koche⁴ (Table 1).

GC-MS analysis: Gas chromatography mass spectrum analysis was carried out using GCMS- QP 2010 PLUS SHIMADZU JAPAN with injector temperature of 270°C, carrier gas Helium (5.5 lb/in2) and carrier gas pressure of 11.6 kpa. RestekRtx- 5MS capillary column used, the column length was 30.0 m with a diameter of 0.32 mm and the flow rate of 41 mL min^–1. The mass spectrum was also equipped with a computer fed mass spectra data bank.

DPPH radical-scavenging activity: DPPH radical-scavenging activity was determined by the method of Shimada et al.⁵.

Procedure: Briefly, a 2 mL aliquot of DPPH ethanol solution (25 μg mL^–1) was added to 0.5 mL sample solution at different concentrations. The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 30 min. Then the absorbance was measured at 517 nm in a spectrophotometer. Then the values of absorbance are placed within the formula given below to obtain the scavenging activity of Sauropus androgynus phytocomponents:

Statistical analysis: The results were presented as Mean±SD. Data were statistically analyzed using student t-test. For the calculation of IC₅₀, linear regression analysis was done using Graph Pad prism statistical software.

RESULTS AND DISCUSSION

Chemical composition by GC-MS analysis: Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST). The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST Library. In the GC-MS analysis, 20 bioactive phytochemical compounds were identified in the ethanol extract of leaves (Fig. 1 and Table 2) and 10 bioactive compounds were identified in the ethanol extract of seeds (Fig. 2 and Table 3). Biological activity of some identified components listed in Table 4.

Twenty compounds were identified in the GCMS analysis of leaves of Sauropus androgynus. The Chromatogram (Fig. 1) showed 20 prominent peaks. The largest peak (RT 19.564) is due to the presence of l-(+)-Ascorbic acid 2,6-Dihexadecanoate. The dominant component (peak height 27.82) of this plant L-(+)-Ascorbic acid 2,6-Dihexadecanoate has been reported to have antioxidant, anti-inflammatory and anti-nociceptive properties^6,7. The1-(+)-Ascorbic acid 2,6-Dihexadecanoate which is vitamin C fatty acid conjugate, indicating the presence of vitamin C. The n-Hexadecanoic acid-palmitic acid found in seed extract (RT 19.611), possesses an antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant and antiandrogenic activities⁸.

Table 2:	Compounds identified in the leaves of Sauropus androgynus

Fig. 1:	GCMS chromatogram of leaves of Sauropus androgynus

Fig. 2:	GCMS chromatogram of seeds of Sauropus androgynus

Table 3:	Compounds identified in the seeds of Sauropus androgynus

Table 4:	Biological activity of some identified compounds

cis-Vaccenic acid, is an omega-7 fatty acid, is found in leaves extract. Its IUPAC name is (Z)-11-Octadecenoic acid and its lipid shorthand name is 18:1cis-11.

A well known antibiotic 2,4-Imidazolidinedione, 1-[{(5-nitro-2-furanyl)methylene}amino] available in leaves extract, its general name Nitrofurantoin, is an antibiotic that fights bacteria in the body. Nitrofurantoin is used to treaturinary tract infections. Nitrofurantoin, sold under the trade name Macrobid among others, is an antibiotic used to treat bladder infections. Nitrofurantoin is sometimes described as a urinary antiseptic, owing to its value in clearing infections of the urinary tract caused by Gram-negative pathogens such as Pseudomonas aeruginosa, Serratia marcescens and Proteus mirabilis⁹.

Decamethylcyclopentasiloxane(D₅) found in leaves extract (RT 8.471), is an organosilicon compound with the formula [(CH₃)₂SiO]₅. It is becoming more common in hair conditioners as it makes the hair easier to brush without breakage. In Canada, among the volume used in consumer products approximately 70% were for antiperspirants and 20% for hair care products¹⁰.

Squalene (C₃₀H₅₀) available in seed extract at RT 26.544. Squalene has several beneficial properties. It is a natural antioxidant, serves in skin hydration¹¹ and has been used as emollient in adjuvant for vaccines¹². As a compound of olive oil, it also has a preventive effect on breast cancer, possesses tumor-protective and cardio-protective properties^13,14 and decreases the serum cholesterol level¹⁵. It has been reported that squalene emulsions given simultaneously with anti-cancer drugs provide favorable effects either directly or indirectly by enhancing efficacy of anti-cancer drugs^16-18.

10 components identified in seeds extract in which the major compound were fatty acid such as 9,12-Octadecadienoic acid (Z,Z)-, 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-, 9-Octadecenoic acid (Z)- and Hexadecenoic acid. Samad et al.¹⁹ also conducted GCMS analysis in the leaves extract of S. androgynus and identified major components were 9,12,15-Octadecatrienoic acid (Z,Z,Z)-, hexadecanoic acid.

Antioxidant activity: The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen-donating ability. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule²². Extracts of plants are allowed to react with the stable radical, DPPH, in methanol solution. The reduction capability of DPPH radicals is determined by the decrease in its absorbance at 517 nm, induced by an anti-oxidant (AH) after 30 min as follows:

Fig. 3:	DPPH Radical scavenging activity of leaf and seed extracts of Sauropus androgynus at different concentrations

DPPH+AH→DPPH-H+A^∙

The concentration of antioxidant needed to decrease the initial DPPH concentration by 50% (IC₅₀) is a parameter widely used to measure the antioxidant activity²³. The IC₅₀ value was obtained from a graph plotted between sample concentration and the absorbance of the DPPH radical. A lower IC₅₀ value corresponds with a higher antioxidant power. The leaf and seed extracts showed significant DPPH scavenging activity (IC₅₀ values are 49.62±2.52 and 52.47±2.21 μg mL^–1) compared with the values obtained for ascorbic acid standard (IC₅₀ value 48.82±1.80). DPPH Radical scavenging activity of leaf and seed extracts are shown in Fig. 3.

Previous study revealed that S. androgynus leaves possess a high total phenolic content and antioxidant activities . Based on the electron transfer reaction assays, S. androgynus leaves contained higher antioxidant activities than many other leafy vegetables of Indonesian origin²⁴. A previous study on antioxidant activities on aqueous extracts of 25 tropical plants showed that S. androgynus has a high polyphenol content, cupric ion chelating activities, free radical scavenging and reducing ferric ion antioxidant properties²⁵. The leaves have containing bioactive antioxidants, such as carotenoids, flavonoids and other phytochemicals. These phytochemicals can reduce oxidative stress, thus reduce the risk of some chronic diseases²⁴.

CONCLUSION

It was concluded that Sauropus androgynus extracts constitute a wide range of bioactive phytochemicals with high therapeutic values. The identified phytochemicals have several activities such as antioxidant, analgesic and anti-inflammatory, anti biotic, anti cancer, anti nociceptive, anti arthritic and hypoglycemic. The results revealed that Sauropus androgynus leaves were excellent source of vitamin C derivatives. Due to good antioxidant phytochemicals and vitamin composition, Sauropus androgynus would be an important plant in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.

SIGNIFICANCE STATEMENT

The present study conducted to evaluate the antioxidant activity and components of Sauropus androgynus seeds and leaves and found that about 20 phyto active components are obtain from the leaves having vitamin C as major component while seed contain 10 components having fatty acids as major components which confirmed its strong antioxidant activity whereas, antibiotic component nitrofurantoin also obtained from it. The study result helps the researchers in determining its strong antioxidant activity in vivo and also other active components of plant in future. Thus best theory on it may be arrived at.

ACKNOWLEDGMENT

The authors would like to acknowledge Periyar TBI, Periyar Maniammai University, Thanjavur for GCMS analysis.