Chemicals : Ethanol, Ferric chloride, Sodium hydroxide and Hydrochloric acid, glacial acetic acid, Chloroform, Sulphuric acid, Dragendroff’s reagent, n-butanol, Ethyl acetate, L- Ascorbic acid, Ascorbic acid, Ferrous sulphate, Aluminum chloride, Potassium Acetate obtained from Fischer Scientific, Quercetin, Chitosan, Sodium Tripolyphosphate (TPP), Salmon Milt DNA were analytical grade chemicals from HiMedia, Karnataka, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), Thin layer chromatography Silica gel plate was of GF₂₅₄ Merck.
Plant source: The Fresh Rose Mary (Rosmarinus officinalis) plant was collected from Lal Bagh Botanical garden and Kaval Bysandra, Bangalore through random selection.
Preparation of plant extract: The plant extract was prepared from leaves of Rose Mary (Rosmarinus officinalis). The leaves were washed properly under tap water to remove the dust particles and other impurities from the plant. These leaves were then oven dried overnight at 60°C and was made into a coarse powder. A 5% aqueous and ethanolic (50% ethanol) extracts of the leaves of the Rosemary plant was prepared. The weighed amount of the coarse powder was mixed with the solvent system and placed on a magnetic stirrer for 30 minutes. After which the solution was centrifuged at 8,000 rpm for 18 mins at 25°C. The supernatant was then separately collected and used for further experimental analysis.
Synthesis of chitosan nanopolymer: The ionic gelation method was employed for the synthesis of chitosan nanoparticle using sodium tripolyphosphate (TPP) as a cross linking agent . About 1% (w/v) of the plant extract was mixed with 0.5% (w/v) of TPP and the solution was added drop wise into the chitosan solution containing 0.5% (w/v) chitosan and 1% (v/v) acetic acid under gentle magnetic stirring of REMI 2-MLH. The solution was incubated for 20 mins and used for characterization 
Characterization of Chitosan nanopolymer:
a. Ultra-Visible spectrophotometer: The chitosan and the target compound interaction were monitored by measuring the UV- Vis spectrum of the nano-polymer suspension. The absorbance spectrum of the nano-polymer suspension was recorded immediately after the synthesis and a reference of de-ionized water was recorded before the actual sample analysis. To check the stability of nano-polymer, the absorption spectrum was recorded for pure chitosan, Rosemary extract, TPP, TPP –Rosemary extract, Chitosan –TPP, Chitosan-Rosemary extract and Chitosan-TPP-Rosemary extract. The spectrophotometer used was UV Scan 2600 (Thermo Fisher) and the software was spectrum TM version 6.87. Absorbance spectra were recorded over the range of 220-400 nm. Wavelength of peak absorbance and λ max was calculated.
b. Fourier Transform Infrared Spectroscopy analysis: The biomolecules stacked chitosan nanoparticles were freeze dried and the powdered test was used for FTIR spectroscopy studies. The FTIR examination of chitosan nanoparticles sample was performed with a2 technologies portable attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR). Sample spectra were recorded within the range of 4000 cmˉ¹ to 400 cmˉ¹ with a resolution of 4cm in the absorbance mode for 10 scans at room temperature. FTIR spectra of chitosan nanoparticles were obtained by placing 1mg of test on the sensor of the instrument and spectrum was then compared with the spectrum of Chitosan standard.
Biomedical applications of chitosan nanopolymer: The characterized nanopolymer was used to carry out the following assays:
Detection of DNA protection by chitosan nanopolymer using Ultra Visible spectrophotometer: UV spectroscopy was used to compare the level of DNA protection provided by synthesized chitosan Rosemary leaf nanopolymer to that provided by the standard antioxidant (ascorbic acid). Standard antioxidant Ascorbic acid (10 mM) and standard oxidant - ferrous sulphate (10 mM) were used in the reaction mixture with Salmon milt DNA (200g/mL). TAE buffer was used as a reference standard and the absorbance was estimated at 220-400 nm. Different reaction mixtures were prepared as follows and the absorbance of each was recorded:- DNA, DNA + buffer, DNA +ascorbic acid+ buffer, DNA+ ferrous sulphate + buffer, DNA + ferrous sulphate + ascorbic acid, DNA + ascorbic acid + ferrous sulphate, DNA + nanopolymer + buffer, DNA + ferrous sulphate + nanopolymer and DNA + nanopolymer + ferrous sulphate with 5 min intervals. 
DNA inhibition assay using agarose gel electrophoresis: The ability of Chitosan nanopolymer to protect Salmon milt double stranded DNA from devastating effects of free radicals generated was assessed by DNA damage inhibition assay. The reaction mixture contained 2ṃL DNA (200µg/ mL), 1mL 10mM ferrous sulphate and 1 mL 10mM ascorbic acid or 1mL of 1% nanopolymer rosemary (leaf) extract. The DNA was processed and checked in two different treatments. In the first treatment, (treatment 1) reaction mixture was prepared with DNA and FeSO₄ incubated for 5 minutes at 37⁰C, and then addition of the chitosan rosemary leaf nanopolymer (CNP(L)) extract which was again incubated for 5 minutes at 37⁰C. In the second treatment (treatment 2), reaction mixture constituted DNA and (CNP(L)) was incubated for 5 minutes at 37⁰C followed by addition of FeSO₄. This mixture was incubated for 5 minutes at 37⁰C. The samples were then read under UV spectrophotometer from 320-520. 20µL of each reaction mixture were loaded on 0.8% agarose gel. Electrophoresis was carried out at 100V and 120A for 1 hour. The gel was then visualized under Gel Documentation for the appearance of bands [15, 16].
Cytotoxicity studies by MTT assay: MTT (3-[4,5-dimethylthiazole]-2,5-diphenyltetrazolium bromide) assay was performed to check cell viability and cell toxicity. MTT is a yellow colour solution which gets reduced by the action of dehydrogenases and other reducing agents produced by metabolically active cell. This reduction of MTT finally yields a violet-coloured water insoluble product called formazan which indicated the non-viability of the cells. The dead or non-viable cells will not cleave MTT, so this assay is very sensitive to the presence of living cells .
0.25 g of liver tissues were weighed and placed in cell culture plate. 1 ml of DMEM (Dulbecco’s Modified Eagle Medium) containing 1% penicillin was added to each of the wells containing the liver tissues. A comparative study was performed. 50µl, 100µl and 150µl of the Chitosan linked Rosemary leaf extract nanopolymer were added into a cell culture plate containing the weighed liver tissues. The same aliquots of the 5% ethanolic Rosemary leaf extract was added into another cell culture plate containing the liver tissues. The plates were then incubated at 37°C for 24 hours in an incubator. After incubation, 20µl of MTT (0.5 mg/ml) was added to each well and again incubated for 4 hours at 37°C in an incubator. To stop this reaction, 1 ml of 0.04 N HCl in isopropanol was added and the reaction mixture was centrifuged at 3000 rpm for 10 mins at room temperature. The wells 4 and 5 of both the cell culture plates had none of the contents added to it. The absorbance of the supernatant was read at 570nm in UV-Vis spectrophotometer