Curcumin loaded chitin-glucan quercetin conjugate: Synthesis, characterization, antioxidant, in vitro release study, and anticancer activity

Accepted Manuscript Title: CURCUMIN LOADED CHITIN-GLUCAN QUERCETIN CONJUGATE: SYNTHESIS, CHARACTERIZATION, ANTIOXIDANT, IN VITRO RELEASE STUDY, AND ANTICANCER ACTIVITY Authors: Anu Singh, Lavkush, Amit Kumar Kureel, P.K. Dutta, Sushil Kumar, Ambak Kumar Rai PII: DOI: Reference:

S0141-8130(17)32811-8 https://doi.org/10.1016/j.ijbiomac.2017.11.002 BIOMAC 8482

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

30-7-2017 15-10-2017 1-11-2017

Please cite this article as: Anu Singh, Lavkush, Amit Kumar Kureel, P.K.Dutta, Sushil Kumar, Ambak Kumar Rai, CURCUMIN LOADED CHITIN-GLUCAN QUERCETIN CONJUGATE: SYNTHESIS, CHARACTERIZATION, ANTIOXIDANT, IN VITRO RELEASE STUDY, AND ANTICANCER ACTIVITY, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

CURCUMIN LOADED CHITIN-GLUCAN QUERCETIN CONJUGATE: SYNTHESIS, CHARACTERIZATION, ANTIOXIDANT, IN VITRO RELEASE STUDY, AND

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ANTICANCER ACTIVITY

Anu Singha, Lavkusha,b, Amit Kumar Kureelc, P.K. Duttaa,*, Sushil Kumarb, Ambak Kumar Raic

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Polymer Research laboratory, Department of Chemistry,

Department of Chemical

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Engineering, and c Department of Biotechnology

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*e-mail: [email protected] (PKDutta)

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Motilal Nehru National Institute of Technology, Allahabad 211004, India

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ABSTRACT

In this study, we have synthesized chitin-glucan quercetin conjugate (ChGCQ) by an easy and

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facile free radical grafting reaction. The structure of ChGCQ was confirmed by proton nuclear

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magnetic resonance (1H-NMR) and Fourier transforms infrared spectroscopy (FT-IR). Curcumin was loaded into ChGCQ to study its anti-cancer efficiency. The biocompatibility of ChGCQ and

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curcumin loaded ChGCQ (Cu-ChGCQ) were analysed by different assays in Peripheral blood mononuclear cells (PBMCs) and cytotoxicity test was performed in a macrophage cancer cell line (J774). The result shows tremendous biocompatibility of ChGCQ and Cu-ChGCQ in peripheral blood mononuclear cells and excellent cytotoxity in macrophage cancer cell line (J774). Chitin-glucan complex (ChGC), ChGCQ and Cu-ChGCQ showed 51%, 66% and 74% of 1

DPPH radical-scavenging activity at 1 mg/ml respectively, which are much higher than that of ChGC and in ABTS*+ assay 58%, 71% and 83% show radical-scavenging activity at 1 mg/ml. Antioxidant assay of Cu-ChGCQ conjugate expressed much higher antioxidant activity than

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ChGCQ and ChGC. In vitro drug release study of Cu-ChGCQ conjuagate showed faster drug release in acidic medium in comparison to PBS of physiological pH and anticancer activity in vitro assay showed more anticancer activity of Cu-ChGCQ in comparison to ChGCQ conjugate. Key words: chitin-glucan quercetin; conjugate; drug release; anticancer activity; antioxidant

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1. Introduction

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ChGC is a fungal polysaccharides found in most of the cell walls of fungi and yeasts such as

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Aspergillus niger [1], Schizophyllum commune [2], Saccharomyces cerevisiae [3], Gongronella

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butleri [4], Candida albicans [5], Armillariella mellea and Morchella esculenta [6], Agaricus bisporus [7] and Komagataella pastoris (Pichia pastoris) [8]. ChGC is made up of chitin

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(composed of long chain of N-acetyl D-glucosamine units) and β-glucan (polysaccharides of D-

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glucose monomer), linked by a glycosidic bond [9-10]. The main function of this complex is to

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protect the cell walls of fungi and yeasts. ChGC is a non-toxic, insoluble in water with a high water absorption capacity. It is a biocompatible and biodegradable biopolymer that has

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demonstrated to combine antioxidant, antibacterial, anti-inflammatory properties and also useful for cosmetics, food packaging and medicine. In biomedical areas, these complexes have found

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valuable places in wound healing, antimicrobial and anticancer activities [2, 11-13]. It also possesses the advantage of being a non-animal chitin source. In wound dressing, ChGC have been used in different forms such as film and fibrous. One of the important tests is a biocompatibility and it is compulsory before wound dressing, anticancer tests and any

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biomedical application. It can be done in-vitro by using fibroblast cell in order to assure that the material is safe for medical usage and able to promote the cell proliferation for new skin regeneration. Many researchers nowadays use chitin and its derivatives as those are safe

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biomaterial for various biomedical functioning. Moreover, recent review articles are focused on biomedical sciences [14,15], engineering sciences [16,17] and in pharmaceutical sciences [18].

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a single bioflavonoid (studied by researcher over the past 30 years) occurs in natural substance such as fruits, tea, vegetables, bark roots, grains, stem, beverages, wine [19] and nutritional supplement products. These natural products contain

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phenolic compounds and it is beneficial for health purpose and bioactive compound i.e. possible

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to both treat and prevent cancer. More than 4000 varieties of flavonoids have been discovered,

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many of flavonoids are responsible for their attractive colors of flowers, fruits and leaves [20].

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Quercetin (Q) shows many medicinal properties such as anti-inflammatory, anti-oxidants, anti-

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allergic, antimicrobial, anti-ulcer, anticancer activities, anti-proliferative, anti-fibrotic [21] [22],

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anti-coagulative [23], anti-atherogenic [24] [25], anti-hypertensive [26] [27] and [28] [29] antiviral properties. It is available in conjugated isoforms bound to sugars and alcohols [30]. These

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isoforms get hydrolyzed in the gastrointestinal tract and are absorbed and metabolized into

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quercetin aglycone and its other derivatives [31] Curcumin (Cu) is a flavonoid derived from the rhizome of Curcuma longa also popularly known

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as turmeric. It is a polyphenol and used as a spice in dishes, Indian and Chinese traditional medicine, and a natural colouring agent. Cu is beneficial for health purpose and show many medicinal properties such as antioxidant [32], anti-atherosclerotic, and anti-inflammatory. It inhibits scarring, gallstone formation, cataract, liver injury, angiogenesis, and kidney toxicity. Cu is effective for heart disease, cancer, and metabolic syndrome [33-37]. In vitro and in vivo study 3

using cultured cells prove that Cu has helpful for the treatment of pancreatic cancer, liver cancer, colon cancer, breast cancer, thymus cancer, and prostate cancer. In recent years, chitin based polymer and bioflavonoid-polymer conjugates have shown many

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biomedical applications such as antioxidant, antidiabetic, anti-inflammatory, anticancer activity, wound dressing, etc. In order to increase the biological property of polymer and free bioflavonoid compound, a free radical coupling between a polymer and flavonoid was propelled in the presence of redox pair such as hydrogen peroxide and ascorbic acid. Lot of reports are available on synthesis of polymer-polyphenol conjugates based on various polymers including

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chitosan, dextran, and gelatin derivatives with polyphenols like gallic acid, caffeic acid and many

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others flavonoids. A recent report focused on Caffeic acid-conjugated chitosan derivatives has

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been evaluated for cancer treatment [38]. In this paper cytotoxicity tested against tumor cells and

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also a positive linear relationship between dose and cytotoxicity were found. But cytotoxicity

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against cancer cell lines was not good enough; very high dose was needed for desirable results.

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In the present paper, first time we have reported the preparation of ChGCQ conjugate and CuChGCQ conjugate to increase the anticancer activity in comparison to free quercetin. For

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polymer-polyphenol conjugate synthesis, we have chosen ChGC as polymer backbone since it

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has cationic character, along with the presence of reactive functional groups in ChGC providing sites for conjugation with many phenolic compounds. Q was chosen as a flavonoid for

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conjugation with ChGC regarding its wide number of biomedical applications. ChGC conjugation with phenolic compounds was done by redox system, resulting in a robust complex with unprecedented antioxidant and anticancer activity promted synergistically both by ChGC and polyphenol. Ascorbic acid and hydrogen peroxide were used to produce free radical sites on ChGC backbones. ChGCQ conjugate expressed enhanced anticancer activity, antioxidant, 4

antimicrobial properties, and those were confirmed by antioxidant, drug release study and cytotoxicity test performed on macrophage cancer cell lines. 2. Materials and methods

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2.1. Materials

ChGC was isolates from agaricus bisporous. Curcumin was purchased from Sigma Aldrich, India. Quercetin, and Folin-Ciocalteu reagent were purchased from SRL, India. Ascorbic acid, hydrogen peroxide, formic acid was purchased from Sisco Research Laboratories Pvt. Ltd, India.

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Solvents (ethanol, acetone, isopropanol, methanol, DMSO) were purchased from Merck, India.

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Macrophage cancer cell lines (J774) was purchased from NCCS Pune and Peripheral blood

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mononuclear cell (PBMCs) was isolate from blood withdrawn from healthy volunteers into the

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syringes. Culture flask was purchased from thermo fisher and Dulbecco’s modified eagle medium: nutrient mixture Ham’s F-12 powder (DMEM F12) was purchased from Invitrogen

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Corporation. Antibiotic antimycotic solution (penicillin, streptomycin and amphotericin6 B),

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dimethylthiazol-2-yl)-2,

5-diphenyl

tetrazolium

bromide

(MTT)

dye,

2,7-

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(4,

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Trypsin–EDTA and FBS were purchased from Gibco (Life Technologies AG, Switzerland). (3-

dichlorofluorescein diacetate (DCFDA) were purchased from Sigma Aldrich. Propidium iodide

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(PI) was purchased from molecular probes by life technologies. Sodium carbonate and potassium per sulphate were purchased from CDH. 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2, 2-

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azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) ABTS were purchased from SRL. Dialysis membrane was purchased from Hi-media and Milli-Q was obtained from our laboratory. 2.2. Preparation of chitin-glucan quercetin conjugate (ChGCQ)

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ChGCQ was formed by free radical grafting method [39,40]. According to this method differentdifferent concentration of ChGC was dissolved in 20 mL 2% formic acid after complete solutions add 1 mL of 5.0 M H2O2 and 0.25g of ascorbic acid. The mixture was maintained at

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25° C under atmospheric air. After 2 h different- different amount of quercetin 50 mg, 100 mg, 200 mg were added in a reaction flask and the reaction mixture were maintained at 25°C for 24 h under atmospheric air. The resulting polymer subsequently dialyzed with dialysis membrane against milli-Q water to remove salt and reactant for 72 h. The dialyzed polymer was lyophilized (in -50ºC, 3 days) to obtain yellow porous powder materials for (0.13 g - 0.398 g) shown in Fig.1

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and lyophilized material (ChGCQ) was stored at 4ºC until required.

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2.3. Preparation of drug loaded chitin-glucan quercetin conjugate (Cu-ChGCQ)

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Drug loaded ChGCQ conjugate was prepared by simple methods. First we dissolved different concentration of ChGCQ in 2% formic acid, after complete homogeneous solutions we added

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known weight of curcumin (2mg/ml) with continuous stirring for 24 h under room temperature

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and solution of colour changes confirms entrapment of drugs [41]. The prepared solution was put in sonicator to remove solvents. Then prepared material was centrifuged, the pallet was washed

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with milli-Q water to removes excess solvent and lyophilized in -50ºC for 3 days.

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2.4. Characterization of chitin-glucan quercetin conjugate (ChGCQ) and drug loaded chitin-

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glucan quercetin conjugate (Cu-ChGCQ) Proton nuclear magnetic resonance (1H-NMR) spectrum was performed in the range of 0-10 ppm and DMSO-d6 was used as a standard solvent. Fourier transform infrared (FT-IR) was done by Perkin Elmer Spectrum. Samples were prepared in KBr pellets and the result performed infrared range between 400-4500 cm-1 with 8 scans and resolution of 8 cm-1. The crystalline phase was 6

investigated by X-ray diffraction (XRD) patterns of samples by using a Rigaku Smart lab diffractometer in the range of 5°–80° at a scan rate of 4°min−1. The surface morphology of prepared conjugates was done by scanning electron microscopy (SEM) by using Zeiss.

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UV/visible absorption spectra (200 to 600 nm) were recorded by a Shimadzu UV-2450 spectrophotometer using slit width of 2 nm.

2.5. Determination of Quercetin Content by Folin-Ciocalteu Method

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The amount of quercetin in ChGCQ conjugate was measured using Folin-Ciocalteu reagent

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method with some modifications [42]. Briefly, 10 mg of quercetin conjugate was dissolved in 10

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ml of slightly acidic solution. Take 1ml of this solution in vials and diluted to 10 times. After

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dissolution 0.5 ml of freshly prepared Folin-Ciocalteu reagent (10N) was added and shakes the mixture. After 3 min 2 ml of Na2CO3 (10%) was added and the mixture was allowed to stand for

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2 h in a dark with intermittent shaking. The absorbance was measured at 760 nm with UV-vis

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spectrophotometer (UV-2450, Shimadzu, India) against a control solution prepared using the

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blank polymer under the same reaction conditions. The quercetin content in this conjugate was expressed in mg per g of dry ChGCQ conjugate by using the equation of the free gallic acid

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calibration curve, recorded by use ten different gallic acid standard solutions.

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2.6. Determination of antioxidant activity in vitro 2.6.1. DPPH radical-scavenging activity assay The antioxidant properties of ChGC and ChGCQ were evaluated by determination of scavenging effect on DPPH radicals. According to Gong et al., [43] the conjugates were reacting with a 7

stable free radical DPPH, with slight modification. In this assay ten different-different concentrations of conjugates (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1) mg/ml were mixed with 1 ml of 0.25mM DPPH (in methanol solution). Then the mixture was stored in dark condition for

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30 min at 25° C and the absorbance of the residual DPPH concentration was determined colorimetrically at 517 nm using a spectrophotometer (UV-2450, Shimadzu, India) , the same reaction condition were applied for the blank and standard solution. The decrease in absorbance of the DPPH was used to calculate the following equation: A0  A1 100 ) A0

(1)

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Inhibition  %  

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and A1 is the absorbance of the polymeric samples.

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Where A0 is the absorbance of the standard (Ascorbic Acid) solution or without any polymers,

2.6.2. ABTS•+ scavenging activity assay

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The ABTS•+ scavenging activity was estimated according to Siddhuraju et al [44]. according to

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this method 7mM of ABTS and 2.45mM of potassium persulphate were mixed in flask and

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incubated in a dark condition at room temperature for 12-16 h to generate the free radical of ABTS, then 1 ml of ABTS•+ diluted with 50ml distilled water to obtained an absorbance of

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0.70±0.02 at 734 nm. Then different-different concentration of polymeric samples (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1) mg/ml were added to 3 ml of ABTS•+ solution and the reaction

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mixture was incubated at room temperature for 1 h, and the absorbance at 734 nm was then measured using a spectrophotometer (UV-2450, Shimadzu, India). The scavenging activity was calculated according to equation 1. 2.7. Entrapment efficiency 8

Entrapment efficiency of Cu-ChGCQ conjugate was measured by pelletizing [45] the sample at 10000 rpm for 20 min. The resulting sample was lyophilized and known weight of sample was dissolve in 10 ml ethanol, then resulting solution was sonicate and centrifuge at 10000 rpm for

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20 min. The supernatant was collected and amount of drug within the supernatant was determined by UV-Vis spectrophotometer at absorption of 420 nm, which assign to the peak absorption of curcumin. Drug entrapment efficiency was determined by the following equation

EE  %  

Total amount of curcumin with in pellet 100 Initial amount of curcumin taken for loading studies

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2.8. In vitro drug release studies

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In vitro drug release of Cu-ChGCQ conjugate was check on phosphate buffer saline (PBS) and

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acidic pH [41]. In brief, known quantity of Cu-ChGCQ conjugate was dispersed in 10 ml PBS buffer solution and in acidic pH with continues stirring at 37° C. Then different-different time

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intervals 3 ml of PBS buffer solution and acid taken off and added same volume of PBS solution

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and acid. Then the release of drug was determined by UV-Vis spectrophotometer (λmax = 420 nm). Then plot the curve and calculate the cumulative drug release (%) with the help of

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calibration curve.

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2.9. Mononuclear cell isolation

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Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by ficoll hypaque gradiant centrifugation, washed twice with PBS and finally suspended in complete RPMI-1640 supplemented with 10% FCS, cytotoxicity assay was set in 96 well flat-bottom culture plates. 2.9.1. Cell Culture 9

A macrophage cancer cell line (J774) was purchased from NCCS pune and cells were maintained in Dulbecco’s modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were maintained in an incubator at 37°C with 5% CO2. The cells were detached

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with trypsin (0.5 ml) and then add 1 ml DMEM media with 10% FBS to quench activity of trypsin. This cell suspension was centrifuge at 1000 rpm for 5 min and then resuspended in growth medium for further study.

2.9.2. Flow cytometric analysis (Cytotoxicity assay by flow cytometry)

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In this analysis we analyze cytotoxicity assay of Peripheral blood mononuclear cell (PBMCs)

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and macrophage cell line (J774) by using propidium iodide fluorescent dye. In briefly, Peripheral

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blood mononuclear cell (PBMCs) and macrophage cell line were seeded in 96 well plates and

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treatment in macrophage cell line was started after 4h to allow for surface adhesion. After adhesion cells were treated with different concentration (50 µg and 100 µg) of ChGCQ and Cu-

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ChGCQ dissolved in 0.1% DMSO solvent. Peripheral blood mononuclear cell (PBMCs) were

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treated with 12 h and 24 h in macrophage cancer cell lines (J774) then added 10 μl PI (10 μg/ml) in a dark condition, further incubated for 20 min. Then cells were acquired in flow cytometer

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(Accuri C6 flow cytometer, Becton Dickenson Biosciences, San Jose, CA, USA) and the data

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was analyzed with Flow Jo analysis software, measure the Mean Fluorescence Intensity of PI positive cells. All experiments were performed in duplicates well plates for every sample and

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control.

2.9.3. Cell viability: MTT Assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) assay is a

colorimetric assay for assessing cell metabolic activity. In this assay, the mitochondrial 10

dehydrogenase of viable cells reduces the water-soluble MTT to water-insoluble formazan crystals [38]. Briefly, cells were grown in 96 well plates and add different concentration (50 µg and 100 µg) of ChGCQ and Cu-ChGCQ in Peripheral blood mononuclear cell (PBMCs) for 12 h

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and in macrophage cancer cell line (J774) incubate for 24 h. Then we add 10 µl MTT and incubate for 4 h to form formazan crystal. After, incubation add DMSO in 1:1 ratio in each well plate and incubated for 20 min to solubilize formazan crystals and absorbance was measured at 570 nm. All experiments were performed in duplicates well plates for every sample and control.

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2.9.4. Estimation of reactive oxygen species (ROS)

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In this assay 2,7-dichlorofluorescein diacetate (DCFDA) was used to determine the intracellular

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ROS generation. [46] Briefly, 0.1 millions cells per well were seeded in 96 well plates after the

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surface adhesion of cells add different concentration (50 µg and 100 µg) of ChGCQ and CuChGCQ for 24 h in macrophage cancer cell lines. After completion of time, 20 µM of DCFDA

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was added in each well plate and incubated for 30 min. then cells were acquired in flow

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cytometer (Accuri C6 flow cytometer, Becton Dickenson Biosciences, San Jose, CA, USA), in FL1 channel and the data was analyzed with analysis software Flow Jo, to measured the ROS (%

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control.

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of Control). All experiments were performed in duplicates well plates for every sample and

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2.9.5. Statistical analysis Statistical analysis of the data was analyzed by analysis software Prism 5.0 (GraphPad Software, CA, USA). All the data were expressed as unpaired t test and measure P value in all experiments.

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3. Results and Discussion 3.1. Characterization of ChGCQ and Cu-ChGCQ We have to confirm that product was conjugated or not by chemical and instrumental methods.

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60% of quercetin conjugated to ChGC determined by Folin–Ciocalteu method.

H-NMR spectrum of ChGCQ shown in Fig. 3 and conjugate was dissolved in a DMSO-d6

solvent. The spectrum showed a peak at 1.9 ppm for the methyl protons of acetylated glucosamine residues [47] indicates conjugation. The peak at 2.3 ppm show a CH2 group and the

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peak found at around 6.2 ppm, 6.4 ppm and 6.9 ppm in the 1H-NMR spectrum of ChGCQ assign

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the aromatic proton of quercetin and peak around 7.5-7.7 ppm assign the phenolic proton of

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quercetin. In quercetin no peak observed around 1-3 ppm, this spectra clearly indicates that

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ChGC successfully grafted with Q.

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FTIR spectra were determined the functional group and composition present in ChGC, quercetin

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(Q), ChGCQ and Cu-ChGCQ. In Fig. 5 clearly shows that all spectra shows broad band around 3400-3200 cm-1 which may be attributed to –OH band, in ChGC [48] and ChGCQ conjugate the

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band appear at 2852-2958 cm-1 which assign to –NH2 band. The Q spectrum show C=C stretching vibration of phenyl ring at 1660 cm-1, peaks around 1260-1165 cm-1 and 1130-1012

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cm-1 show C=O=C anti-symmetrical and symmetrical stretching respectively [49]. In-plane C=O

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stretching vibration combined with the ring stretch of phenyl ring at 1316 cm-1. In the spectrum of ChGCQ conjugate new peaks are observed at 1260 cm-1 to 1003 cm-1 correspond to the typical stretching vibrations of quercetin. The change in wave number of the peaks in regions 1500-1000 cm-1 indicates that there is an interaction between the two groups. In the spectrum of Cu-ChGCQ shows a deformation of NH- group at 2852-2921 cm-1 and formation of keto group of the 12

curcumin around 1022 cm-1. From the FT-IR data confirmed that ChGC successfully grafted with quercetin and curcumin successfully loaded in ChGCQ by the interaction between keto group of curcumin and amine group of ChGC.

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The powder X-ray diffractogram of ChGC, Q, ChGCQ and Cu-ChGCQ are shown in Fig. 6 In X-ray diffraction we study nature of samples, in ChGC four peaks at 9.58°, 19.72°, 26.24° and 37.95° are observed and major peak exhibit around 19.72°. the X-ray diffraction pattern of pure quercetin show crystalline nature with characteristic peak at 10.61°, 12.26°, 15.64°, 15.94°, 23.70°, 24.24°, 26.43°, 27.23°, 29.49°, 31.75°, of 2θ respectively [49]. In ChGCQ conjugate, the

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typical peak of ChGC at 19.72° has been shifted to a lower 2θ value of 18.71°, peak become

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broad and intensity reduced [47]. The characteristic peaks of quercetin were observed in the

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ChGCQ with reduced intensity than that of pure quercetin. The spectrum of Cu-ChGCQ

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conjugate, the distinctive peaks observed at 6.42°, 8.64°, 12.12°, 17.05° and 27.15° with reduced

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intensity than that of others.

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The surface morphology of ChGC, ChGCQ and Cu-ChGCQ are observed by SEM. As shown in Fig.7 the SEM images shows a flaky nature after grafting queretin surface become change. The

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change in morphology of the conjugate and Cu loaded ChGCQ indicates that intramolecular and

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intermolecular hydrogen bonds of ChGC have been decreased during the grafting and Cu loaded process

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The UV-Vis spectra of Cu, ChGCQ, and Cu-ChGCQ were shown in Fig. 8. All conjugates were dissolved in DMSO solvent. In ChGCQ peaks are obtained at 225 nm assign to aromatic region [50] and other peak observed at 367 nm this peaks corresponds to the interaction of quercetin and in Cu-ChGCQ peaks are observed at 224 nm, 333 nm and 426 nm this new peaks corresponds to

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the interaction of curcumin. After loaded curcumin drug, the peaks are shifted and all peaks are lower wavelength in comparison to ChGCQ. 3.2. Biological Evaluation

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3.2.1. Antioxidant activity of ChGC, ChGCQ,, and Cu-ChGCQ

The antioxidant activities were determined by two stable free radical the hydrophobic radical DPPH and the hydrophilic radical ABTS [51].

DPPH is a free radical compound used for the determination of free radical-scavenging activity.

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In this assay methanolic DPPH solution reduced to DPPH-H in the presence of H-donating

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antioxidant sample and colour of solution changes to a yellow, indicates that sample is

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antioxidant. The reducing power of all samples determined by the decreasing the absorbance

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value at 517 nm, the concentration of sample increase absorbance value decrease. In Fig. 9

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shows the % inhibition of DPPH radical.

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The DPPH radical-scavenging activity of ChGC, ChGCQ and Cu-ChGCQ at concentration 1

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mg/ml are 50.84, 66.28, and 74.26 % respectively. In ChGC, DPPH radical-scavenging activity low in comparison to ChGCQ and Cu-ChGCQ it means that after grafting quercetin in ChGC

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and curcumin loaded in ChGCQ conjugate, the radical-scavenging activity enhanced. ABTS is a 2, 2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid), used by the food industry

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and agricultural researchers to measure the antioxidant activity. In this assay first we generate radical cation by the addition of potassium persulphate a blue/green colour form and the colour of the solution decolorize after added sample indicates that sample is antioxidant. The cation radical forms on the basis of spectrophotometric methods. ABTS•+ scavenging activity of ChGC,

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ChGCQ and Cu-ChGCQ shown in Fig. 10 Scavenging activity of Cu-ChGCQ much higher than the ChGCQ and ChGC shown in Table. 1 DPPH radical are less reactive in comparison to ABTS•+ radical and the reaction with DPPH radical involves H-atom transfer, the reaction with

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ABTS•+ radical involves electron process. 3.2.2. Entrapment efficiency and drug release study of Cu-ChGCQ conjugate

The entrapment efficiency of Cu-ChGCQ was found to be 77.32%. In vitro drug release studies of Cu-ChGCQ conjugate was done by direct dispersion method [41] at acidic medium and PBS

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medium shown in Fig. 11 The amount of drug release is faster in acidic medium than in PBS

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medium. In acidic medium the chitin-glucan polymer swells due to protonation of amine group,

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therefore drug release faster in acidic environment and Facilitating the faster drug elution.

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3.2.3. Cell Cytotoxicity test: PI Assay

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In this assay we analyzed cell death by Propidium iodide (or PI) staining [52]. It is a fluorescent

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intercalating agent that can be used as a DNA stain in flow cytometry to evaluate cell viability. It cannot cross the membrane of live cells, making it useful to differentiate necrotic, apoptotic and

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healthy cells. In Fig. 12 shows that concentration increases then cell death is significantly

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increases in macrophage cancer cell line (J774) and in Peripheral blood mononuclear cell (PBMCs) less effect non significant of concentration and time. In Cu-ChGCQ show more death

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in comparison to ChGCQ. 3.2.4. Cell Cytotoxicity test: MTT assay In this assay yellow tetrazolium salt reduced to a purple formazan crystals depends on the cellular metabolic activity due to NAD(P)H flux. The purple formazan crystal related to living

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cells, more formazan crystal formed that means more number of living cells [53]. In this assay cells were incubated in different concentration (50µg/ml, 100µg/ml) of compounds for 12 h in Peripheral blood mononuclear cells (PBMCs) and in macrophage cancer cell lines (J774)

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incubated for 24 h. In Fig. 13 shows that no sign of cell death in case of PBMCs whereas in macrophage cancer cell line cell death increase when concentration of sample increase. This results shows that living cells decrease in cancer cell line when concentration increase. In drug loaded ChGCQ i.e, Cu-ChGCQ show more cell death in comparison to others.

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3.1.1 Intracellular ROS generation:

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In this assay we have seen that introduction of ROS by compounds leading to cell death in

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various cells. First we add different concentration (50µg/ml, 100µg/ml) of ChGCQ and Cu-

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ChGCQ in macrophage cancer cell line (J774) for 24 h. After completion of time we add DCFDA and measured intracellular ROS generation shown in Fig. 14 in cancer cell line J774

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4. Conclusion

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significant ROS with increase of concentration.

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In this study, ChGC successfully grafted with quercetin by ascorbic acid/ hydrogen peroxide redox pair and curcumin was loaded on ChGCQ. 1H-NMR, FT-IR, XRD and UV-vis spectra

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confirmed that quercetin successfully grafted with ChGC and curcumin successfully loaded with

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ChGCQ. The amount of quercetin bonded per g of conjugate was determined by Folin-Ciocalteu reagent and those were found well in consistence with available reports. The results of the antioxidant, in vitro drug release study and anticancer activities of Cu-ChGCQ conjugate were much better than ChGCQ. The PI and MTT assays confirmed significant cytotoxicity against J774 cancer cell lines and non significant cytotoxicity against PBMCs. In future work we will 16

study the effects of ChGCQ and Cu-ChGCQ conjugates in healthy mice and in a cancerous mouse model to further confirm the in vivo stability and pharmacokinetics. Anticancer activity of the conjugate could be potentially useful in chemotherapy. Also it could be considered as an

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effective product for a pharmaceutical industry. The present synthetic strategy allows improving the properties of the natural polymer and gives an insight for many other applications. Acknowledgements

The authors are thankful to Director Prof. Rajeev Tripathi, MNNIT, Allahabad, for providing

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stipend to AS. The authors are thankful to Centre for Interdisciplinary Research (CIR), CMDR,

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and health centre MNNIT, Allahabad for XRD, UV-vis spectroscopy, and flow cytometry

A

respectively. The authors are also thankful to Material Research Centre (MRC) MNIT Jaipur for

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FTIR analysis, CDRI Lucknow for 1H-NMR analysis and IIT Kanpur for SEM analysis.

17

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Caption of Figures: Fig.1(a). Graphical representation of reaction scheme in ChGCQ Fig.1(b). (A) Chitin-glucan complex (ChGC) and (B) chitin-glucan quercetin conjugate

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(ChGCQ)

Fig.2. (A) curcumin (Cu) and (B) curcumin loaded chitin-glucan quercetin conjugate (Cu-ChGCQ)

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Fig.3. 1H-NMR spectrum of ChGCQ

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Fig. 4. Grafting of chitin-glucan complex with quercetin molecule

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Fig. 5. FTIR spectra of (A) ChGC, (B) quercetin (Q), (C) ChGCQ, and (D) Cu-ChGCQ Fig. 6. XRD spectra of (A) ChGC, (B) quercetin (Q), (C) ChGCQ, and (D) Cu-ChGCQ

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Fig. 7. SEM images of (A) ChGC, (B) quercetin (Q), (C) ChGCQ, and (D) Cu-ChGCQ Fig. 8. UV-vis spectra of Cu, ChGCQ and Cu-ChGCQ

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Fig.9. DPPH radical-scavenging activities of ChGC, ChGCQ and Cu-ChGCQ

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Fig. 10. ABTS assay of ChGC, ChGCQ and Cu-ChGCQ

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Fig. 11. Drug release profile of Cu-ChGCQ in acidic medium and PBS Fig. 12. In vitro cytotoxicity of ChGCQ and Cu-ChGCQ using PI assay in (a) Peripheral blood mononuclear cells (PBMCs) [12 h] and (b) Macrophage cancer cell line (J774) [24 h] and results are expressed as unpaired t test, from four independent experiments P = 0.0021, P = 0.0011, P = 0.0006, and P = 0.0045 in J774 and P = NS in PBMCs. 25

Fig. 13 In vitro cytotoxicity of ChGCQ and Cu-ChGCQ using MTT assay in (a) Peripheral blood mononuclear cells (PBMCs) [12 h] and (b) Macrophage cancer cell line (J774) [24 h] and results are expressed as unpaired t test, from four independent experiments P = 0.0047, P = 0.0055, P =

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0.0073, and P = 0.0029 in J774 and P = NS in PBMCs. Fig. 14. Detection of intracellular ROS in J774 after 24 h ChGCQ and Cu-ChGCQ and results represents the unpaired t test, from four independent experiments. P = 0.0021, P = 0.0011, P =

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0.0038, and P = 0.0001 denotes a significant difference from the control.

26

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Fig. 1(a)

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Fig.3

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Fig. 2

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Fig. 4

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A B

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Fig. 5

30

ChGC Q ChGCQ Cu loaded ChGCQ

12000

10000

6000

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Intensity (a.u.)

8000

4000

2000

0 10

20

30

40

50

60

Fig. 7

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Fig. 6

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2(degree)

31

70

80

Cu-ChGCQ ChGCQ Cu

1.8 1.6

1.2

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Absorbance

1.4

1.0 0.8 0.6 0.4

0.0 200

300

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0.2

400

500

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Wavelength (nm)

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Fig. 8

Fig.9

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600

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Fig. 11

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Fig. 12.

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Fig. 14.

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Table 1. Antioxidant activity of ChGC, ChGCQ and Cu-ChGCQ Sample

ABTS*+ scavenging activity

DPPH* scavenging activity (mg/ml)

(mg/ml)

ChGCQ

66.28

Cu-ChGCQ

74.26

58.21

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50.84

71.24 82.86

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EP

TE

D

M

A

N

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ChGC

36