Citrullus colocynthis (Bitter cucumber) belongs to the family of cucurbitaceae, which are abundantly grown along the arid soils of Southeast coast of Tamil Nadu. It has a large, fleshy perennial root, which sends out slender, tough, angular, scabrid vine-like stems. The therapeutic potentials viz., antimicrobial 1 , anti inflammatory 2 , anti diabetic 3 and anti oxidant 4 effect of Citrullus colocynthis have reported in our laboratory. For conservation of this potent medicinal plant we have micro propagated and transplanted to the coastal region of Parangipettai.

Nanoparticles usually referred as particles with a size up to 100 nm. Nanoparticles exhibit completely new properties based on specific characteristics such as size, distribution and morphology. As specific surface area of nanoparticles is increased, their biological effectiveness can increase in surface energy 5 . Silver has long been recognized as having an inhibitory effect towards many bacterial strains and micro organisms commonly present in medical and industrial processes 6 . The most widely used and known applications of silver and silver nanoparticles are include topical ointments and creams containing silver to prevent infection of burns and open wounds 7 . Production of nanoparticles can be achieved through different methods. Chemical approaches are the most popular methods for the production of nanoparticles. However, some chemical methods cannot avoid the use of toxic chemicals in the synthesis protocol. Biological methods of nanoparticles synthesis using micro organisms 8 , enzyme 9 , and plant or plant extract have been suggested as possible ecofriendly alternatives to chemical and physical methods. Using plant for nanoparticles can be advantageous over other biological processes by eliminating the elaborate process of maintaining cell culture 10 . If biological synthesis of nanoparticles can compete with chemical methods, there is a need to achieve faster synthesis rates. The exact mechanism of silver nanoparticles synthesis by plant extracts is not yet fully understood. Only participation of phenolics, proteins and reducing agents in their synthesis has been speculated. Recently nano-encapsulated therapeutic agents such as antineoplastic drugs had been used to selectively targeting anti tumor agents and obtaining higher drug concentration at the tumour site 11 . Nanotechnology could be very helpful in regenerating the injured nerves. For biological and clinical applications, the ability to control and manipulate the accumulation of nanoparticles for an extended period of time inside a cell can lead to improvements in diagnostic sensitivity and therapeutic efficiency. This when revealed completely would eliminate the use of expensive drugs for cancer treatment 12 . The callus and leaf extract of Citrullus colocynthis reported moderate antimicrobial activity against biofilm forming bacteria 13 and harmful human pathogens 14 . Therefore the present study, we evaluated, biomedical potential of silver nanopaticles synthesized from calli extract of Citrullus colocynthis on Human epidermoid larynx carcinoma (HEp -2) cell line.

The cumulative work on plant tissue culture revealed the maximum number of calli induction was achieved from stem explants of C. colocynthis on MS medium enriched with 0.5 mg L-1 IAA, 2, 4-D and 1 ppm of 6-BA which yielded morphogenic compact hard greenish white calli at a frequency of 80%. The appearance of brown colour in the reaction mixture indicates the synthesis of silver nanoparticles form stem derived callus extract with 1 mM silver nitrate solution (Figure 1). Our findings showed resemblance to the results already reported by in the case of callus extract of Carcia papaya 15 , leaf extract of Capsicum annum 16 and in case of extract of Aloe Vera 17 . The shape of the SNP synthesized by stem derived callus extract was spherical and was found to be in the range 31 nm by AFM (Figure 2).

Number of absorption spectrum of the nanoparticles obtained in the present study as shown in (Figure 3). Among them, the absorption peak at 1020 cm^-1 can be assigned a absorption peaks of C-O-C- or -C-O-, also the peak at 1020-1091 cm^-1 corresponds to C-N stretching vibrations of aliphatic amines or to alcohols or phenols representing the presence of polyphenols 18 . The absorbance peak at 1265 and 1384 - 1460 cm^-1 correspond to the amide III and II group respectively. The peak at 1624 cm^-1 is associated with stretch vibration of -C = C-and is assigned to the amide 1 bonds of proteins. The absorption at about 1384 cm^-1 is notably enhanced indicating residual amount of NO₃ in the solution 19 . The peak at 1539 cm^-1 may be assigned to symmetric stretching vibrations of -COO- (carboxyl ate ion) groups of amino acid residues with free carboxyl ate groups in the protein 20 . The peak at 3427 cm^-1 indicates polyphenolic OH group along with the peak of 882 cm^-1 which represents the aromatic ring C-H vibrations, indicate the involvement of free catechin 21 . This suggests the attachment of some polyphenolic components on to silver nanoparticles. This means the polyphenols attached to silver nano particles may have atleast one aromatic ring. The peaks at 1000-1200 cm^-1 indicate C-O single bond and peaks at 1620-1636 cm^-1 represent carbonyl groups (C = O) from polyphenols such as catechin gallate, epicatechin gallate and theaflavin 22 . Result suggests that molecules attached with silver nanoparticles have free and bound amide group. These amide groups may also be in the aromatic rings. This concludes that the compounds attached with silver nanoparticles could be polyphenols with aromatic ring and bound amide region. In our results showed that the average number of atoms per nanoparticles are N = 914047.97. The molar concentration of the silver nanoparticles solution in our present study is 1100 nM/10 mL.

In conclusions, plant based sliver nanoparticles possess considerable anticancer effect compared with commercial nanosilver. The reduction of the metal ions through the callus extracts leading to the formation of silver nanoparticles of fairly well defined dimensions. Use of AgNPs should emerge as one of the novel approaches in cancer therapy and, when the molecular mechanism of targeting is better understood, the applications of AgNPs are likely to expand further 28 .

A patent application will be filed with the content of this article, through the Annamalai University. The authors declare that they have no competing interests.

All authors read and approved the final manuscript.

KS and SG developed the concept and designed experiments. TR was research guide of this experimental study. SG and KS performed plant collection, micropropagation, nanoparticles synthesis, characterization and cell line studies. TR & TB provided chemicals, Instrumental studies and advised on experimental part.