Bioactivity Studies of Datura metel, Aegle marmelos, Annona reticulata and Saraca indica and their Green Synthesized Silver Nanoparticle

In this study, various plants like Datura metel, Aegle marmelos, Annona reticulata and Saraca indica were collected and subjected for extraction using various solvents, namely Water, Chloroform and Ethanol. The extracts were done with TLC bioautography for Antioxidant activity and Antibacterial activity, Minimum Inhibitory Concentration, Antibacterial activity, Antioxidant activity (DPPH and FRAP) and Phytochemical analysis. Plants used in this study showed antibacterial and antioxidant activity. These extracts were further utilized for Silver nanoparticle production and were characterized. Silver nanoparticles were utilized for In vitro antibacterial activity, where they did not show any antibacterial


INTRODUCTION
In the recent days, the awareness on the treasury that is the plant kingdom, which is flooded with such potential drugs has increased 1 . Bacteria have developed multiple defense mechanisms against antimicrobial drugs where a few are becoming multidrug resistant as well 2 . Hence novel methods must be simultaneously developed to eradicate such bacteria. Medicinal plants are known for their tremendous application in the medicinal world since long ago. These plants not only act as antimicrobial compounds but also suppress peroxidation by reacting with free radicals expressing anti-oxidant activity. The plants and their parts are known to contain certain bioactive compounds such as tannins, saponins, essential oils and more other metabolites 3 . These plants do also help in the production of nanoparticles [4][5][6] .
Nano biotechnology is the fast-rising sector in the present era. Metal nanoparticles are known for their structural flexibility and functionality 7 . Hence these nanoparticles find application in medicine, catalysts, contrasting agent etc 8 . Silver has been known for treating burns and wounds to prevent infections for centuries 9 . There are reports stating that silver nanoparticles are non-toxic to humankind but lethal to microorganisms 10 . There are a lot of methods exploited to synthesize silver nanoparticles viz., chemical, radiation, electrochemical and photochemical methods 11 . Although out of these methods, the chemical method produces a high yield for low cost, they have their own drawbacks such as contamination due to precursors, utilization of toxic solvents and generation of toxic byproducts. Hence biological approaches are adopted for the synthesis due to its abilities like cost effectiveness, non-toxic nature, ability to produce high yield, energy efficiency and environmental friendly 12 . The biological methods of nanoparticle production involve the action of plants, enzymes and fungi. But out of these the plant based nanoparticles are found to be efficient and safe for human therapeutic usage 13 . Plant metabolites are responsible for the formation of silver ions by reducing silver nitrate 4 . With these evidences, the present investigation focuses on the understanding of the bioactivity of selected four medicinal plants viz., Datura metel, Aegle marmelos, Annona reticulata and Saraca indica, the utilization of extracts of the same for silver nanoparticle production, the characterization of the nanoparticles produced and their antibacterial effect.

Sample collection
Datura metel, Aegle marmelos, Annona reticulata and Saraca indica leaves were chosen and procured from different localities of Chennai, Tamil Nadu. The healthy leaves were rinsed in water, shade dried, powdered and then stored for extraction.

Preparation of the extract
Plant powder was extracted with three solvents, namely Water, Ethanol and Chloroform. 10g plant powder was taken in 100mL of respective solvent (water, ethanol and chloroform). Plants leaves were macerated, filtered and concentrated using oven at 50°C.

Phytochemical analysis
Phytochemicals were screened for all the four plants 14 .

Antibacterial activity
Briefly, Pseudomonas aeruginosa was spread over nutrient agar under aseptic conditions and wells were filled with different concentrations (2µg, 4µg, 8µg and 16µg) of the crude extracts and incubated at 37°C for 24h. Inhibition zone was noted. Ciproflaxacin was exploited as positive control and their respective solvents as negative control 15 .

TLC and bioautography analysis
Each solvent extract was subjected to TLC bioautography (Merck, F245). The TLC plates were cut into required sizes and were solvent vapor-saturated in TLC glass chamber 16 . Sample was loaded onto TLC plate using capillary tube and run against the mobile phase i.e. Chloroform: Methanol (8:2). The Retention factor (Rf) was noted after exposing them to iodine. Antioxidant and antibacterial activity were detected on TLC plate as mentioned earlier 17,18 . The Rf values of the samples were calculated.

Antioxidant assays
Various antioxidant assays were performed for the metabolites namely FRAP assay 19 , DPPH assay 20 .

Synthesis of silver nanoparticles
The four various plant leaf extracts were subjected for silver nanoparticles as described by earlier studies, 21 except for chloroform extracts of the plants since they are immiscible in water.

Antibacterial activity
The antibacterial activity of silver nanoparticles was evaluated against the gramnegative bacterial species Pseudomonas aeruginosa by following Saravanakumar et al. 25 .

RESULT AND DISCUSSIONS Phytochemical Analysis
Aqueous, ethanol and chloroform extracts of Datura metel, Annona reticulata, Aegle marmelos, Saraca indica leaves were quantitatively analyzed (Table 1). Screening confirmed Alkaloids, Saponins and Tannins presence in all extracts. Carbohydrates were noticed to be present in all three solvents of Saraca indica and aqueous extract of Datura metel. Phytosterols were absent in almost all extracts except aqueous extract of Datura metel and Saraca indica whereas phenols were found in chloroform extracts of Aegle marmelos and Saraca indica. Datura metel was reported to possess saponins and other phytochemicals 26 . Biologically active compounds can play a vital role in the pharmaceutical industries possessing pharmacological activities. Each of the phytochemicals have different bioactivities like antibiotic, analgesic etc 27 .

Antibacterial Activity
Datura metel's ethanolic extract was having antibacterial activity (Table 2), which is on par with the earlier report of Akharaiyi 26 . Chloroform extract of Datura metel was found to be non-effective against P.aeruginosa used in this study, which is contradictory to the result of Vadlapudi and Kaladhar 28 , where they found chloroform extract of Datura metel to have antibacterial activity against Erwinia caratovara and Pseudomonas syringae. Aqueous extract of Annona reticulata shows maximum zone of inhibition followed by ethanol extract of Aegle marmelos against the bacteria Pseudomonas aeruginosa.

Thin Layer Chromatography Thin Layer Chromatography Exposed to Iodine
Separation of compounds was done using thin layer chromatography and the TLC plates were exposed to iodine in iodine chamber 18 . Ethanol and chloroform extracts of Datura metel has shown the maximum of 3 bands (Fig. 1). The separated compounds were seen under ultra violet ray for the appearance of any extra bands, but no extra bands were found under ultra violet ray (Fig. 2), which is commonly used for observing other metabolites 29 .
Most of the extracts showed antioxidant activity, 3 bands of Chloroform extract of Datura metel has shown anti-oxidant activity, even chloroform extract of Aegle marmelos showed antioxidant activity (Fig. 3). TLC bioautography for antioxidant activity was identified in Aegle marmelos 30 .
TLC bioautography for antibacterial activity helps in identifying the compound which shows antibacterial activity in TLC plate itself. Antibacterial activity of Punica granatum, Acacia senegal and Mangifera indica 18,31, 32 has been reported by TLC bioautography. Ethanolic and chloroform extracts exhibited antibacterial activity in TLC plate itself (Fig. 4).

Assay for Antioxidant activity
All the plants showed antioxidant activity ( Fig. 5 and 6). In a study, it is reported by FRAP assay that leaf of Datura metel has more antioxidant activity than bark 26 . Chloroform extract of Annona reticulata possessed more scavenging activity (Fig.  6). The antioxidant activity was even comparable with ascorbic acid 33,34 . Reducing powers and scavenging activity of various medicinal plants were reported earlier 35,36 .

Characterization of silver nanoparticles UV-Visible Spectroscopy
Phytochemical compounds do reduce the Ag + to Ag 0 , but it also imparts stability of nanoparticles 37 . Silver nanoparticles produced using ethanol extracts showed peaks near 420nm (Fig. 7) but water extract mediated silver nanoparticles were showing peak nearing 200-250nm. For the Ethanol extract of Saraca indica, Annona reticulata and Aegle marmelos, the peaks range at 600-630nm. Efforts were made to produce silver nanoparticles using Annona muricata and the particles were found to show absorbance at 420nm 38 . In this study, silver nanoparticle produced using ethanol extract of Datura metel was found to show absorbance near 420nm to 440nm, where it is on par with the absorbance of silver nanoparticles produced 39,40 .

Scanning Electron Microscopy
Size ranging from 45 to 120nm was found for silver nanoparticles produced. Nanoparticles produced using Aegle marmelos ranged from 47 to 52nm (Fig. 8h) and was recorded to be the smallest in the group where Saraca indica induced nanoparticles were ranging from 90 to 110nm recorded to be the largest nanoparticle in the group. (Fig. 8a). 35nm -60nm sized particles were produced using Aegle marmelos 41,39 . Using methanolic extract of Annona muricata, 22nm sized nanoparticles were made 38 .

Antibacterial activity
There was no activity against the bacteria used (Table 3), which is contradictory to earlier reports 42 , where it was found that nanoparticles produced using Aegle marmelos to have antibiofilm activity. There are more evidences that silver nanoparticles synthesized using plant extracts have antibacterial activity 43 . Extract of Annona squamosa has been reported to produce silver nanoparticles with anti-microbial activity 44 .