Bio-Medical Applications of Chitosan Linked Rosmarinus Ocinalis Leaf Extract Nanopolymer

Nanotechnology has numerous applications in science and technology. The nanomaterial which are plant based have drawn more attention due to its immense application in various elds because of their physico-chemical properties. Physical, chemical, and biological processes are used to create nanomaterials. Physiochemical processes are costly and hazardous to the environment, whereas biological processes involving nanoparticles are thought to be environmentally friendly. In recent times considerable research interest has evolved using chitosan nanoparticles as they have emerged as one of the most exciting tools due to their increased surface-volume ratio and are of great interest for nanomedicine and development of new therapeutic drug release systems with improved bioavailability, increased specicity and sensitivity, and reduced pharmacological toxicity. In the present investigation, we have synthesized chitosan nanoparticles using Rosmarinus ocinalis leaf extract by the ionic gelation technique and was characterized using UV spectrophotometer and FTIR. The optical density of the broad peak of Chitosan-Rosemary Leaf nanopolymer was found to be at 0.587. The FTIR results recorded the absorption peaks at 3381 cm(cid:0) 1 , and 1666 cm(cid:0) 1 which represented the O-H stretch and C=C bond stretching respectively. Thus, there was a stretching from higher wavelength region to lower wavelength region demonstrating the interaction of tripolyphosphate with chitosan and biomolecules by reduced hydrogen bonding. Further, the study was designed to determine the cytoprotective and DNA protection activity. The results of our study lend pharmacological credence to the bio-medical applications, ethno medical use of this plant in traditional system of medicine.


Introduction
Nanobiotechnology is the application of nanotechnology in biological elds. Nanotechnology is a multidisciplinary eld that utilizes both conventional and advanced engineering, physics, chemistry, and biology technologies and facilities [1]. Nanobiotechnology has great potential in a wide range of life science elds. It also has potential applications in nanotechnology elds, such as nanomedicine and nano-biopharmaceuticals [2].
Chitosan is a polycation, soluble in acidic medium (pKa 6.5) in which the negatively charged TPP interacts with the positively charged groups of amino acids of Chitosan forming inter or intramolecular structure [3]. Chitosan is a partial N-deacetylated chitin (1→4)-2-amino-2-deoxy-D-glucan. The nanoparticulate forms of chitosan demonstrated improved solubility and bioavailability, resulting in more e cient drug delivery [4]. Chitosan is famous for anti-properties like antimicrobial, antioxidant, and chelating effects, as well as its nontoxic and biocompatible existence [5].
Green synthesis is an emerging area of bio-nanotechnology where plants are employed to produce environmentally friendly nanoparticles rather than the chemical technologies. This is supported by the fact that plant-derived products have lesser negative effects than many synthetics. Plants can be used to make nanoparticles instead of other biological processes because they reduce the time-consuming process of maintaining cell cultures and can be ramped up for large-scale synthesis of nanoparticles in a non-aseptic environment. In addition, it has quality role in the eld of high sensitivity bio-molecular detection, catalysis, biosensors, and medicine [6,7].
Rosmarinus o cinalis, more commonly known as rosemary, is a Mediterranean herb that belongs to the Lamiaceae family of mints. It's an annual aromatic plant with shrub-like branches brimming with leaves. R. o cinalis is used in cooking as a spice, as a natural preservative in the food industry, and as an ornamental and medicinal plant. Rosmarinus o cinalis shows adequate antioxidant activity due to the presence of phenolic acids like caffeic, rosmarinic acids and phenolic diterpenes like carnosol, carnosic acid and rosmanol. It also possesses antimicrobial, anti-in ammatory, antitumor, antifungal, antiviral activities. [8,9,10,11,12] The present investigation aims at the synthesis and characterization of Chitosan linked nanopolymer using Rosmarinus o cinalis leaf extract to identify DNA protection activity, DNA inhibition activity and cytotoxicity of chitosan Rosemary leaf nanopolymer (CNP(L)).  [13] Characterization of Chitosan nanopolymer:

Material And Methods
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 nanopolymer, 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 re ectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR). Sample spectra were recorded within the range of 4000 cm ¹ to 400 cm ¹ 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. [14] 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 2ṃ L 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 rst treatment, (treatment 1) reaction mixture was prepared with DNA and FeSO incubated for 5 minutes at 37 C, and then addition of the chitosan rosemary leaf nanopolymer (CNP(L)) extract which was again incubated for 5 minutes at 37 C.
In the second treatment (treatment 2), reaction mixture constituted DNA and (CNP(L)) was incubated for 5 minutes at 37 C followed by addition of FeSO . This mixture was incubated for 5 minutes at 37 C. 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 nally 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 [17].

Results And Discussion
Rosemary plant has various pharmacological, ethnobotanical and other miscellaneous uses. Rosemary plant has enormous bene ts in medical eld and chitosan linked nanopolymer will be more effective than its original form which is proved in the present study.
CHARACTERISATION OF NANOPOLYMER-UV Spectrophotometer analysis : A UV-Vis spectrophotometer was used for the preliminary characterisation of the prepared Chitosan-Rosemary nanopolymer. The maximum optical density of the Chitosan-Rosemary nanopolymer was found to be at 0.587 while the pure chitosan showed an optical density of 0.352. The bonding of pure chitosan with Rosemary leaf extract resulted in an increase in the optical density. This revealed the formation of chitosan plant nanopolymer. Our current study correlated with the revelation of the broad peak for chitosan nanoparticle at the range of 220-322 nm [19].
FTIR Analysis : The capability of the ionic gelation protocol to form the plant-chitosan nanoparticles loaded with biomolecules was assessed by FTIR with transmission values on the Y-axis and wavelength on the X-axis. The spectral analysis of pure chitosan is depicted in g 2(a) and g 2(b) shows the spectral analysis for the chitosan rosemary leaf nanopolymer loaded with bioactive molecules. The IR spectra of pure chitosan nanoparticles (CNPs) showed 2 major peaks at 3333 cm ¹ and 1637 cm ¹, while the FTIR spectra of the Chitosan-Rosemary leaf nanoparticle revealed two major absorption peaks at 3381 cm ¹ and 1666 cm ¹.The presence of O-H bonds was con rmed by the spectra observed at 3381 cm ¹ for the synthesised nanoparticle. For the biomolecules loaded chitosan nanoparticles, the isolated alkene groups of the C=C stretch were observed at 1666 cm ¹ [20].
Detection of DNA protection assay: Reactive oxygen species are thought to cause severe oxidative damage to various macromolecules, including DNA, RNA, and protein [21]. In our study Salmon milt DNA which is double stranded has a molecular mass of 1.3 x 10 Da (~2,000 bp). The ability of the CNP(L) to protect the DNA strands from the oxidative damage caused by the oxidant was by two treatments. The absorbance value higher than the standard DNA indicating strand breakage. Treatment 1-was the pretreatment of DNA with the CNP(L) followed by treatment with the oxidant to analyze the DNA protection activity and Treatment 2-was the pre-treatment of the DNA with oxidant-FeSO , followed by the treatment with the synthesized chitosan plant nanopolymer to assess the DNA repair activity. In g (3), the addition of FeSO to the pure DNA resulted in an increase in the absorbance at 260 nm. The addition of the CNP(L) showed a relatively less increase in absorbance, indicating that the nanopolymer does not cause DNA damage.
The results indicated that chitosan-rosemary leaf nanopolymer contemplated higher DNA protection activity than DNA repair activity. The oxidative damage caused by the oxidant, FeSO , lead to denaturation of the double stranded DNA which gave a high optical density value than tthe standard DNA. The reduction in the optical density value on treatment with the low concentrations of the Chitosanrosemary nanopolymer indicated the signi cant DNA protection potential of the nanopolymer.
DNA INHIBITION ASSAY USING AGAROSE GEL ELECTROPHORESIS: DNA damage can also be caused by alkylating agents. Reactive carbonyl species are some of the chemical agents that results the DNA damage. These highly reactive oxygen species attack the bases or the deoxyribosyl backbone of DNA leading to an interruption in the normal structure of the DNA causing single or double stranded breaks. [22] In the g 4 a), DNA revealed the least absorbance serving as the control for the assay. A slight increase was observed in the absorbance when DNA was incubated with the oxidant, FeSO . This increase in absorbance is due to the formation of free radicals by FeSO that led to the DNA damage. There was a small decrease in optical density when DNA was treated with an antioxidant (ascorbic acid), indicating the release of oxidative stress on DNA. In contrast, when the DNA was treated with the antioxidant followed by the oxidant (FeSO ) an optical density was noted in between the antioxidant and oxidant treated. This indicated that the stress was caused by the addition of FeSO to DNA but at the same, the antioxidant ascorbic acid suppressed the additive effects of FeSO and protected the DNA from any further damage. Furthermore, when the pre-treated DNA and CNP(L) mixture was treated with the oxidant, the optical density was found to be similar to that of DNA+ Ascorbic acid+ FeSO optical density values.
This indicated that plant nanopolymer was found to have antioxidant activity and DNA damage inhibition properties alike the standard antioxidant (ascorbic acid).
The DNA inhibition activity was con rmed on an agarose gel. The mixtures used for the UV spectral analysis of DNA inhibition assay was mixed with small aliquots of EtBr and loaded on to an 0.8% agarose gel. Ethidium bromide aids in the visualization of DNA bands as well as the size determination of separated DNA fragments [23].
In gure 4 b), the Lane 1 which consisted of DNA (control) showed an orange-uorescent band as the DNA existed in its supercoiled form itself. Lane 2 showed a much lighter uorescent band due the oxidative stress caused by the free radicals on addition of FeSO . Lane 3 indicated the DNA protection by the addition of ascorbic acid with orange-uorescent bands which was in same lines with the control. Lane 4 indicated DNA protection by ascorbic from the oxidative stress caused by FeSO . In Lane 5, the DNA was pre-treated with the Chitosan-Rosemary nanopolymer followed by addition of FeSO . A single orange bright uorescent band was observed which indicated a potent inhibition effect towards DNA damage which could be caused by FeSO . This proved that CNP(L) has signi cant DNA damage inhibition activity.
The thinning of bands were observed in all the wells indicating the complete degradation of DNA. DNA molecules are divided in a pattern based on their size, with the distance travelled being inversely proportional to the log of their molecular weight. These ndings were in accordance with the UV spectrophotometric analysis.
Cytotoxicity studies by MTT assay: Cytotoxicity of the CNP(L) was studied using liver cells by MTT assay. The MTT assay is a colorimetric assay in which NAD(P)H-dependent cellular oxido-reductase enzymes, under de ned conditions, re ect the number of viable cells present. These enzymes can reduce the tetrazolium dye MTT, a yellow-coloured solution, to its insoluble purple coloured formazan.. According to the study, the liver cells were viable even after 24 hours of incubation with DMEM containing 1% penicillin with different concentrations of the Chitosan plant nanopolymer. When MTT was treated with CNP(L), no change in colour from yellow to purple was detected. This revealed that the plant nanopolymer is non-toxic and keeps the liver cells viable.
A negative result was observed when the liver cells were treated with different aliquots of the plant extracts under the same conditions. The liver cells lost their viability after the incubation period and all the wells in the cell culture plate turned purple indicating toxicity.
The picture above shows the results when liver tissues were being treated with the chitosan nanopolymer and the negative control. The picture below shows the results when the liver tissues were treated with just plant extract.

Conclusions
The necessity of looking for alternative plant products to combat a variety of ailments linked to Reactive Oxygen Species has remained popular due to the lower toxicity of natural goods compared to synthetic solutions.
In the present research, the Chitosan Rosemary leaf nanopolymer was synthesized using the ionic gelation technique. The synthesized chitosan Rosemary nanopolymer was characterized using UV spectrophotometer and FTIR analysis. The characterized nanoparticles were then investigated for biomedical applications such as DNA protection, DNA inhibition and Cytotoxicity studies by MTT assay. From the results of UV spectrophotometric analysis for the DNA protection and repair assay it was observed that the synthesized Chitosan Rosemary nanopolymer showed signi cant DNA protection activity and moderate DNA repair activity. The invitro DNA inhibition activity of CNP(L) was evaluated by UV spectrophotometric analysis. It revealed that the CNP(L) prevented the degradation of DNA into small fragments and had convincing DNA damage inhibition properties. This was con rmed on an agarose gel which should a brighter orange uorescent band. In the MTT assay, cell viability and cytotoxicity was studied in response to the chitosan-plant nanopolymer. No colour change was observed when the cells were treated with MTT and CNP(L) indicating that they were still viable. This assay proved that the chitosan-plant nanopolymer synthesized does not affect the viability of the cells and is also non-toxic.
Since the biomolecules loaded chitosan nanopolymer conferred potential abilities, the nding of this study opens future applications in the eld of drug and gene delivery, tissue engineering and various other biomedical applications.  Cytotoxicity by MTT Assay