HPTLC study to determine the antioxidant activity of dried leaves of Portulaca oleracea L

This present study involves the assessment of the anti-oxidant activity studyof the sample which was obtained from the methanolic extracts of dried leaves of Portulaca Oleracea L.(common name Purslane). Purslane is a rich source of Vitamin A, Vitamin-C and some other B-complex vitamins like ribo lavin, niacin, pyridoxine and carotenoids which are known powerful natural antioxidants. Anti-oxidants are compounds that inhibit oxidation. This methanolic extract of leaves was evaluated for the determination of its anti-oxidant ef iciency by using 1,1–diphenyl-2-picryl-hydrazyl (DPPH) by using Silica TLC plates on CamagHigh-Performance Thin Layer Chromatography (HPTLC) system using visionCATS software. Densitograms and chromatographs obtained show the presence of anti-oxidant activity. It is a rapid, inexpensive and straightforward method to measure anti-oxidant properties of substances after separation byHPTLC. It involves the use of the free radical, 2, 2-Diphenyl1picrylhydrazyl (DPPH)which iswidely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and to evaluate antioxidant activity. When Anti-oxidants substances react with DPPH, which is a stable free radical becomes paired off in the presence of a hydrogen donor (e.g., a free radical scavenging anti-oxidant) and is reduced to the DPPHH. As a consequence, the absorbance’s decreased from the DPPH.


INTRODUCTION
Portulaca oleracea L. (Purslane) has wide cosmopolitan distribution around the globe as it can be seen in ields, gardens, vineyards, lawns, driveways, dunes and river banks. It is better known as wild plants but also an edible vegetable rich in many bene icial nutrients for human consumption (Alam et al., 2014). It contains more omega-3 fatty acids and alpha-linolenic acid (both are good anti-oxidants) in than any other leafy vegetable plant. (Chowdhary, 2013), and is a nutritious food rich in omega-3 fatty acids and anti-oxidants (Simopoulos et al., 1992). It possesses many anti-oxidant properties due to the excellent value contents of vitamins, minerals, essential fatty acids and other compounds and having rich medicinal properties (Rahimi, 2018), possess potent pharmacological activities as anti-oxidant (Naeem and Khan, 2013).
Portulaca oleracea L extracts showed the highest amount of total phenolics and revealed signi icant anti-oxidant activities (Nile, 2017) and are one of the richest green plant sources of omega-3 fatty acid (Uddin et al., 2014). Purslane is a nutritious vegetable used for human consumption (Dkhil et al., 2011) and having anti oxidative property. Phytochemical investigations revealed that this plant has a wide range of secondary metabolites including alkaloids, terpenoids, lavonoids and organic acids (Iranshahy et al., 2017). Anti-oxidants are the compounds that inhibit oxidation and oxidation is a chemical reaction that can produce free radicals (Sicari et al., 2018), thereby leading to chain reactions that may damage the cells of organisms. Many anti-oxidants like thiols or ascorbic acid (vitamin C) terminate these chain DPPH (2,2 diphenyl-1-picrylhydrazyl) is a free radical which produces a violet solution dissolved in Methanol (Erkan, 2012). It is stable at room temperature and ambient environmental conditions. Antioxidants present in the extract reacts with the DPPH free radicals and breaks the chain reaction and further formation of free radicals (Sanja et al., 2009) this causes a colour change in DPPH to yellow. The formula of DPPH is C18H12N5O6 and its Molecular Weight is 394.32 g/mol. Structure of DPPH (Fig-ure 1). High-Performance Thin Layer Chromatography (HPTLC) is a powerful and versatile chromatographic technique for the separation and analysis of natural products as compared to other techniques such as HPLC, spectrophotometry, titrimetric. HPTLC system of Camag brand consists of following components such as Plate Developing Chambers (Twin trough), TLC Applicator (Linomet 5), TLC plate heater, Derivatier Sprayer, Detection component (Scanner 4), Evaluation is done by software (visionCATS) and documentation by TLC Visualizer.

MATERIALS AND METHODS
The whole plant of Portulaca oleracea along with leaves on which study is performed was authenticated by Raw Materials Herbarium & Museum, Delhi (RHMD), National Institute of Science Communication & Information Resources (CSIR-NISCAIR) New Delhi and requisite certi icates are obtained.

Reagent Preparation
Exactly 7.89 mg of DPPH reagent is weighed by analytical balance and diluted to 100 ml by 99.5 % ethanol. The concentration of this solution is 0.2 mM. It is kept in an amber-coloured bottle which is then wrapped with aluminium foil as DPPH activity is light sensitive.

Sample preparation
One gram of ine powder made from dried leaves of Portulaca oleracea (Purslane) is precisely weighed and dissolved in 10 ml of Methanol and then kept in Ultrasonic bath for 15 minutes after its supernatant is taken with the help of a pipette. After that, it is centrifuged at 3000 rpm for 10 minutes in a centrifuge machine, and the inal extract is taken for the analysis.

PROCEDURE
The study is performed in a dark environment only as the DPPH activity is light sensitive, DPPH reagent is dissolved in ethanol and dissolved. After application of the sample on stationary phase HPTLC plates silica gel 60 F 254 of size 10 cm x10 cm the and standard are run in the mobile phase which contains Ethyl Acetate: Methyl ethyl ketone: Formic Acid: Water in a ratio of 5:3:1:1 volume/volume.     After the development of standard and sample, the plate is then derivatized with DPPH reagent. A working program is prepared in visionCATS software. A speci ic volume of sample 2µl, 4µl, 6µl, 8µl and 10µl is taken by using 100 µl syringe and applied through sample applicator. After application, the plate is air-dried for 10 minutes. For the development of chromatogram, a ilter paper lined in the Twin Trough Chamber for uniform saturation chamber is used. Chamber saturation is done for 20 minutes (according to USP chapter 202). Then the applied plate is placed in a twin trough chamber layer facing towards the paper. The plate is a run-up to the marked position, i.e. 70 mm and then removed and air-dried for 5 minutes. The dip tank is used for derivatization with DPPH reagent. It is kept in the dark place for 10 min for development of proper intensity of colour. Then images are taken in visible light if anti-oxidant is present it will show the yellow colour spot.

RESULTS AND DISCUSSION
Images of HPTLC plate after application of sample of methanolic extract applied in a volume of by microsyringe in 2µl, 4µl, 6µl, 8µl and 10µl concentrations is obtained under normal white light ( Figure 6).
The developed plate is then derivatized by using freshly prepared DPPH reagents and images are observed under white light (Figure 7) and at 366 nm ( Figure 8).

Evaluation of results
Area, Area % and Rf values of the peaks detected during densitometry analysis of extract are given in Table 1 for ive chromatographs are obtained at 254 nm on applying 2µl, 4µl, 6µl, 8µl and 10µl consequently. (Figure 9) and in Table 2 for the other ive chromatographs are obtained at 366 nm on applying the same concentrations as above consequently ( Figure 10). TLC visualize used for imaging and documentation in which yellow colour bands can be seen at 0.18, 0.78 and 0.87 Rf values (Figure 11), and detailed results can be seen as in Table 3.

CONCLUSION
This study concludes that when ive tracks of different concentrations, i.e. 2µl,4 µl,6 µl, 8 µl and 10 µl were applied and studied at 254 nm and 366 nm and again studied at after derivatization by DPPH reagent, it is found that the yellow colour band present at Rf 0.18, Rf 0.78 and Rf 0.87 shows the presence of anti-oxidant activity present in the methanolic extract of Portulaca oleracea dried leaves.