# Efficacy of Herbal Extracts-Based Nano-Formulations in Extending Guava Fruit Shelf-Life
## 2 Materials and Methods
### Preparation of Plant Extracts  
**Ajwain extract:** The ajwain seeds were purchased from the local market and air dried. The ajwain extracts were prepared in methanol: acetone (7:3) using hot extraction methods. In the hot extraction method, dried seeds in the ratio 0.1 g/mL of solvent were kept in a water bath for 20 min at 52 \({}^{\circ}\)C and then filtered through Whatman filter paper no. 1 and stored at 4 \({}^{\circ}\)C.  
**Giloy extract:** The leaves of the giloy plant were collected from the premises of CCS. Leaves and stems were thoroughly washed in the distilled water, cut into pieces, and dried at room temperature. Extraction was carried out in methanol using the Soxhlet extraction method.  
**Synthesis of herbal nano-formulation(s):** Alginate nanoparticles were prepared using cation induced controlled gelification of alginate with a slight modification. Firstly, sodium alginate solution (0.06% in water) was prepared and stirred at 800 rpm for 8 h and then ajwain and giloy extracts were added dropwise to the to 28.5 mL of sodium alginate solution at 1:1 ratio. Then, 1.5 mL of calcium chloride (36 mM) was added to the solution under constant stirring, which forms the crosslinks with alginate and entraps the herbal extracts inside. The mixture is stirred for about an hour. The resultant solution is a herbal nano-formulation (NF) of ajwain and giloy.  
**Antibacterial property:** The antibacterial activities were determined by modified agar well diffusion assay. Under aseptic conditions and in laminar air flow, 20 mL of nutrient agar medium was dispensed into pre-sterilized petri dishes. Once the media solidified, it was then inoculated with a micro-organism culture of _Escherichia coli_ (ATCC no. BAA-2523), _Pseudomonas aeruginosa_ (ATCC no. BAA-2796) and _Bacillus cereus_ (ATCC no. 7039). The media were then punched with 6 mm diameter holes and filled with NFs and herbal extracts at two concentrations, viz., 100 \(\mu\)L and 150 \(\mu\)L. Finally, the petri dishes were incubated for 48 h at 37 \({}^{\circ}\)C. The diameter of the zone of inhibition as indicated by a clear area which was devoid of growth of microbes was measured. Each experiment was done in triplicate.  
**Antioxidant activity**: Antioxidant activity was measured using stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as per the method described by [PERSON] et al. [20]. 3 mL dye (0.5 mM DPPH solution in methanol) (diluted) was mixed with 0.5 mL diluted herbal extracts and NFs solution and incubated in the dark for 20 min. The absorbance was read at 517 nm on a spectrophotometer. Dye mixed with 0.5 mL methanol was used as blank and the scavenging of DPPH was calculated using the following formula:  
\[\%\ \text{Scavenging capacity of DPPH}=[(\text{Ao}-\text{A}_{1})/\text{Ao}]\times 100\%\]  
where Ao = Absorbance of blank and A\({}_{1}\) = Absorbance of sample  
**Treatment of guava fruit with NFs:** Guava fruits were procured from the horticulture farm, CCS-HAU, Hisar. They were washed twice with distilled water and then extra water was removed under a fan. Washed guavas were dipped in NFs solution for 3-5 min and then air dried. The treated guava were kept at 25 \(\pm\) 2 \({}^{\circ}\)C and 32 \(\pm\) 2 \({}^{\circ}\)C temperature, maintained in labs. The treated fruits were analyzed till total decay on every alternate day. Uncoated fruits were treated as control and the three treatments were T1, T2 and T3.