- Email: [email protected]
Synthesis, characterisation and biomedical applications of curcumin conjugated chitosan microspheres
G Model
ARTICLE IN PRESS
BIOMAC-8705; No. of Pages 7
International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis, characterisation and biomedical applications of curcumin conjugated chitosan microspheres T.S. Saranya a , V.K. Rajan b , Raja Biswas b , R. Jayakumar b,∗ , S. Sathianarayanan a,∗ a b
Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Kochi, 682041, Kerala, India Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi, 682041, India
a r t i c l e
i n f o
Article history: Received 7 October 2017 Received in revised form 16 November 2017 Accepted 6 December 2017 Available online xxx Keywords: Chitosan curcumin conjugates Microspheres Anti-inflammatory Anti-oxidant Anti-microbial
a b s t r a c t Curcumin is a diaryl heptanoid of curcuminoids class obtained from Curcuma longa. It possesses various biological activities like anti-inflammatory, hypoglycemic, antioxidant, wound-healing, and antimicrobial activities. Chitosan is a biocompatible, biodegradable and non–toxic natural polymer which enhances the adhesive property of the skin. Chemical conjugation will leads to sustained release action and to enhance the bioavailability. This study aims to synthesis and characterize biocompatible curcumin conjugated chitosan microspheres for bio-medical applications. The Schiff base reaction was carried out for the preparation of curcumin conjugated chitosan by microwave method and it was characterised using FTIR and NMR. Curcumin conjugated chitosan microspheres (CCCMs) were prepared by wet milling solvent evaporation method. SEM analysis showed these CCCMs were 2–5 m spherical particles. The antibacterial activities of the prepared CCCMs were studied against Staphylococcus aureus and Escherichia coli, the zone of inhibition was 28 mm and 23 mm respectively. Antioxidant activity of the prepared CCCMs was also studied by DPPH and H2 O2 method it showed IC50 esteem value of 216 g/ml and 228 g/ml, and anti-inflammatory activity results showed that CCCMs having IC50 value of 45 g/ml. The results conclude that the CCCMs having a good antibacterial, antioxidant and anti-inflammatory activities. This, the prepared CCCMs have potential application in preventing skin infections. © 2017 Published by Elsevier B.V.
1. Introduction Curcumin is a standout amongst the most flexible mixes acquired from Curcuma longa. Chemically, curcumin is (1,7bis(4-hydroxy 3-methoxyphenyl)- 1, 6-heptadiene-3, 5-dione) a characteristic polyphenol, which expo the keto-enol tautomerism having a ubiquitous keto structure in acidic medium and stable enol frame in antacid medium [1]. Curcumin have a large continuum of pharmacological activity especially antimicrobial, antiinflammatory, antioxidant, wound healing and cytotoxic activity against cancer cell lines etc. Several studies proved that curcumin have a potent activity against multi drug resistant microbes [2–5]. The real hindrance in the therapeutic utilization of curcumin is its solubility and plasma level concentration. The chemical conjuncation with biopolymer will enhance the bioavilabitity and solubility of the lipophillic drugs [6–8]. Chitosan is a biocompatible, biodegrad-
∗ Corresponding authors. E-mail addresses: [email protected], [email protected] (R. Jayakumar), [email protected], [email protected] (S. Sathianarayanan).
able polymer which improves adhesive property of the skin [9,10], chitosan has responsive amino and hydroxyl group whereas the amino gathering prompts to the likelihood of a few concoction modifications. The present study aimed to develop curcumin conjugated chitosan microspheres and its antioxidant, antimicrobial and anti-inflammatory activities were studied. The main advantages of this study was the synthesised curcumin derivatives consist of free phenolic hyrdroxyl group it is essential for pharmacological action especially antimicrobial, antiinflammatory and antioxidant activity [11]. The chemical conjugation of curcumin with chitosan may enhances its solubility and adhesive property. 2. Materials and methods All the commercial reagents procured were of GR/AR grade and the reactions were carried out in dried borosil glass vessels. The synthesized compound was characterized using TLC, IR, NMR. Curcumin (C21 H20 O6 ) has been purchased from Merck, Germany. TLC was performed on silica gel G F254 (Merck aluminium plate) as stationary phase and ethyl acetate: methanol: acetic acid (6:3:1) as a mobile phase. Iodine chamber was used for detection of the spots. All microwave syntheses were carried out in CatalystTM
https://doi.org/10.1016/j.ijbiomac.2017.12.044 0141-8130/© 2017 Published by Elsevier B.V.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7 2
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Systems CATA 2 R Scientific Microwave Synthesizer with ten different power settings. The IR spectra of the synthesized compounds were recorded on a Shimadzu-FTIR spectrometer (IR Affinity 1) using potassium bromide pellet technique. 1 H and 13 C NMR of the compounds were recorded using Bruker Spectrospin 400 MHz spectrometer with tetramethylsilane (TMS) as internal standard. Chemical shift was expressed as delta values relative to TMS in units of ppm. NIH 3T3 (Mouse embryonic fibroblast) cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM) and Trysin were obtained from Sigma Aldrich Co, St. Louis, USA; EDTA, glucose and antibiotics from Hi-Media Laboratories Ltd., Mumbai, India and DPPH, dimethyl sulfoxide (DMSO), propanol and other chemicals and medias were procured from E. Merck Ltd., Mumbai, India. 2.1. Synthesis of curcumin conjugated chitosan Curcumin conjugated chitosan (CCC) was synthesised by imine formation (Schiff base reaction) method. Curcumin (184 mg of (1 mol)) was dissolved in 15 mL of ethanol and mixed with Chitosan (255 mg of 2 mol) in 0.1 N acetic acid [12]. The mixture was stirred for 30 min continuously at 800 rpm and subsequently placed in a microwave oven (90 W) for 10 min at 60 ◦ C. TLC analysis was performed to ensure the completion of reaction. The reaction mixture was cooled at 4 ◦ C, filtered and recrystallized with ethanol and characterised by spectral analysis. (Fig. 1). 2.2. Synthesis of curcumin conjugated chitosan microspheres (CCCMs) CCCMs were prepared by wet milling solvent evaporating technique. 10 mg of CCC was dissolved with 5 mL of ethanol. From this 1 mL of CCC was added drop wise to deionised water (50 mL) with a flow rate of 0.1 mL/min under steady blending (800 rpm). Probe sonication was performed using a vibra cell sonicator with a working force of 130 W, and a recurrence of 20 KHz. Sonication was carried out for 20 min at room temperature until a turbid was acquired. It demonstrates that the curcumin conjugated chitosan microspheres are framed in milli-Q water. The prepared microspheres were lyophilized [13]. 2.3. Physiochemical characterization of prepared CCCMs 2.3.1. Particle size analysis and stability studies Measure appropriation of CCCMs was investigated by dynamic light scattering (DLS utilizing DLS-ZP/molecule sizer NicompTM 380 ZLS). Size and surface morphology of the microspheres were further affirmed by SEM (JEOLJSM- 6490 LA). The CCCMs suspension was weakened with water and a drop was set on the metallic stub, air dried and examined the surface charge and particle size of CCCMs was measured using a zeta potential analyser [14]. 2.3.2. FT-IR analysis FT-IR spectra of curcumin and CCCMs were recorded by KBr pelleting method (1% w/w of item in KBr) Transmittance was measured from 4000 to 400 cm−1 . This analysis has been performed in order to test the degradation or chemical interaction of formulation. 2.3.3. In vitro curcumin drug release In vitro curcumin release of CCCMs determined by resuspending 22 mg of CCCMs in 10 mL citrate buffer (pH 5) and incubated in 37 ◦ C shaking incubator. At different time intervals, 3 mL of the supernatant solution have been taken and centrifuged at 12000 rpm for 10 min. The released Curcumin has been evaluated at 429 nm using
UV/VIS spectrophotometer. These procedures have been continued until 100% drug release has been obtained. After withdrawn of sample has been replaced with 3 mL of the buffer solution. Data obtained from in vitro release studies were fitted to various kinetic models to find the mechanism of drug release. The kinetic models used were Zero order, First order, Higuchi model and KorsemeyerPeppas Kinetic models [15]. 2.4. Biological evaluations 2.4.1. In vitro antimicrobial studies The antimicrobial action of curcumin and CCCMs were tested against Staphylococus aureus (S. aureus) and Escherichia coli (E.coli) by disc diffusion and serial dilution method. About 100 L of overnight S. aureus and E.coli cultures were spread over Muller Hinton agar plates (Composition- 2 g of beef extract, 17 g of casein hydrolysate, 1.5 g starch, 17 g agar and make upto 1000 mL with water and adjust the pH for 7.2.), respectively. 0.5 cm filter paper discs containing 50, 100, 150, 200, 250 and 300 g of CCCMs and curcumin were located on the agar media. Following which, the plates were incubated overnight in an vertical position at 37◦ C and the zone of inhibition was measured. [16,17] The minimum inhibitory concentration (MIC) of CCCMs and curcumin was determined in accordance with the Clinical and Laboratory Standards Institute guidelines. Serial two-fold dilutions of CCCMs and curcumin were prepared in triplicate followed by addition of a standard S. aureus and E. coli suspension of 1–5 × 105 CFU/ml. After 24 h at 37 ◦ C, the opacity were determined using a microplate reader (BioTek) at 578 nm. The MIC was determined as the lowest concentration of CCCMs and curcumin with where no visible bacterial growth was observed. 2.4.2. Estimation of antioxidant activity by DPPH and hydrogen peroxide method The antioxidant action of the were measured by 2,2−diphenyl2-picryl-hydrazyl (DPPH) free radical scavenging method [18]. Various concentrations of curcumin, CCCMs and standard ascorbic acid (100, 200, 300, 400 and 500 g/ml) were taken and mixed with 5 mL of 0.01 mM methanolic solution of DPPH was added, shaken well and blend was kept at 37 ◦ C for 30 min. Absorbance was measured at 517 nm using an UV/Vis spectrophotometer. Absorbance of DPPH was taken as control. The investigation was done in triplicate. The half maximal inhibitory concentration (IC50 ) values calculated. About 0.04 mM Hydrogen peroxide solution (0.6 mL) in phosphate buffer was mixed with different concentration of CCCMs. The above solution was stand for 10 min and the absorbance was measured at 230 nm in UV spectrophotometer, phosphate buffer used as the blank and the percentage scavenging activity of H2 O2 were determined and the IC50 Values were calculated [19]. Percentage scavenging effect =
Absorbance of the sample × 100 Absorbance of the control
2.4.3. In vitro anti −inflammatory studies Curcumin and CCCMs were screened for anti-inflammatory activity utilizing hindrance of albumin denaturation strategy The response blend comprises of 1 mL of 1% aqueous arrangement of bovine serum albumin division, 1 mL of PBS at pH 6.4 and 1 mL of different concentration of curcumin and CCCMs were arranged (25, 50, 100, 250, 500, 750 and 1000 g/ml) utilizing DMSO and distilled water individually. 10 g/ml diclofenac sodium has been used as the standard drug and distilled water as the control. Then the mixtures were incubated at (37 ± 2) ◦ C for 15 min and then heated at 70 ◦ C for 5 min. After bring down the temperature of the mixture, the absorbance was measured at 660 nm. The experiment was
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model
ARTICLE IN PRESS
BIOMAC-8705; No. of Pages 7
T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
3
Fig. 1. Schematic representation of synthesis of curcumin conjugated chitosan.
performed in triplicate. Percent inhibition of protein conjugated curcumin with chitosan denaturation was calculated [20].
3. Results and discussion 3.1. Synthesis and characterisation of curcumin conjugated chitosan (CCC)
2.4.4. In vitro cytotoxic study The viability of NIH 3T3 (Mouse embryonic fibroblast) cells in the presence of different concentrations of curcumin and sample were quantified. The nanoparticles were resuspended in complete DMEM, which is used as stock for the assay. 20,000 cells were seeded in each well of 24 well plates. Different concentration of CCCMs were added to the well plate and incubated at 37 ◦ C in CO2 incubator for 24 and 48 h. Cells with complete DMEM was used as positive control. After predetermined time points the medium from the wells were removed and washed twice with PBS. 100 L of 10% (v/v) alamar blue solution in DMEM were added to each well and incubated in CO2 incubator at 37 ◦ C for 4 h. Using a micro plate spectrophotometer the color changes in the alamar blue solution was quantified by measuring the absorbance (O.D) using at 570 nm and at 600 nm as reference wavelength and the percentage of viability was calculated from the absorbance using following equation. All values were taken as triplicate and the results were expressed as mean ± SD value [21]. Cell viability(%) =
T 570 - T 600 × 100 P 570 - P 600
Where, Where, T 570 = O.D of test at 570 nm T 600 = O.D of test at 600 nm P 570 = O.D of positive control (Triton X-100 treated) at 570 nm P 600 = O.D of positive control (Triton X-100 treated) at 600 nm
2.4.5. Hemo-compatibility assay The hemolysis assay was performed to measure the extent of hemolysis caused by CCCMs when it comes into contact with human blood. 100 L of diluted blood (1:9 in PBS) was taken in several 1.5 mL vials and different concentrations of prepared CCMs (1,0.7, and0.5 mg/ml) were added and diluted by using PBS, The tubes were incubated in 37 ◦ C for 24 h. Triton X-100 (10%) (v/v) treated blood sample was taken as positive control. Followed by incubation, samples were centrifuged for 10 mins at 700 rcf in 4 ◦ C, supernatants were collected and by using micro titter plate reader, the absorbance was measured at 540 nm. The hemolysis was calculated using the following equation Hemolysis (%) =
Optical Density (T- N) × 100 Optical Density (P- N)
Where T = Test, N = Negative control and P = Positive control
The CCC compound was synthesized by the Shiff base reaction (Fig. 2), characterized by standard spectroscopic techniques and their purity ascertained by routine TLC analysis along with melting point determination. In this synthesis curcumin undergoes an acid catalyzed addition in presence of acetic acid got an positive charge electrophilic carbonyl carbon, the neucleophilic nitrogen atom of the amino group from the chitosan will attack the carbonyl carbon to form carbon- nitrogen double bond (imine group) [21,22]. The overall yield of CCC was 67% and the melting point of the compound is 168 ◦ C −171 ◦ C and the TLC results showed the Rf value was 0.63. The max was found at 429 nm by using UV Spectroscopy. IR spectral data showed the curcumin C O group at 1723 cm−1 but after the imine formation the band shifted to 1627 cm−1 shifting of this peak was confirmed that the formation of CCC. 13 C and proton NMR data’s proved that the reaction completion, the 13 C NMR showed a peak at 163 ppm it confirmed the formation of imine (C N) at the position of J in Fig. 3, all other curcumin and chitosan peaks were present in the 1 H NMR data. (Fig. 3A & B). Further CCC was formulated as microspheres and it was characterised. The surface morphology of the prepared CCCMs has been performed using SEM and to be spherical in shape with diameter 2–4 m (Fig. 3C). The image of the CCCMs showed highly monodispersed and distinct spherical particles. [23]. The Z.P was found to be −31 mV [19]. This value indicates the enhancement of stability for the prepared CCCMs. The presence of negative zeta potential may be due the presence of −OH functionalities in the prepared microspheres as well conjucation to chitosan and gives negatively charge of the CCCMs. [24,25]. 3.1.1. In-vitro curcumin release The cumulative release studies have been performed until up to 100% release. The percentage drug releases have been plotted against time, and it was showed that the maximum release of curcumin derivative was found to be at the 48th h (Fig. 4A) [26] and more than 50% of drug release was observed at 24th h,The amino group of chitosan will enhance swelling property due to its protonation.The release data were fitted in to four important kinetic models. The linearity (R2 ) and slope (n) were determined to study the release mechanism. The higher linearity of first order kinetics indicates that, the drug release from the microsphers is depending on the concentration of drug(Fig. 4B & C). The drug release mechanism was further investigated with the help of Higuchi and Korsmeyer- Peppas model(Fig. 4D&E). The higher linearity of Korsmeyer- Peppas model for proves the diffusion dependency of drug release. From this graph we can conclude the release pat-
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model
ARTICLE IN PRESS
BIOMAC-8705; No. of Pages 7 4
T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 2. Convergent synthesis of curcumin conjugated chitosan.
tern of CCCMs as evidence for sustained release and confirmed the diffusion of CCCMs from the delivery system in Fickian diffusion [27]. 3.2. Biological evaluation of CCCMs 3.2.1. In vitro Antibacterial Activity Antibacterial activity has been tested against S. aureus and E. coli. In agar diffusion assays E. coli showed the zone of inhibition was 28 mm for CCCMs and 23 mm for Curcumin, the zone of inhibition of S. aureus showed that 33 mm and 31 mm for CCCMs and curcumin respectively. Broth dilution method has been employed for testing antibacterial activity of samples [28]. Minimum Inhibitory Concentration (MIC) of CCCMs and Curcumin for E. coli and S. Auerus was determined, the results were indicate that the antibacterial activity of the CCCMs were greater than curcumin alone. From the graph plotted we can also conclude that as concentration of sample increases the bacterial colony decreases (Fig. 5A and B). The results confirmed that the CCCMs are effective against both organisms [29]. 3.2.2. Antioxidant activity 3.2.2.1. DPPH assay. One of the most economic methods to find out the antioxidant activity is the DPPH method. DPPH is a free radical that is stable at room temperature. It produces a violet colour, when it is blend with organic solvents. DPPH is a free radical that usually has the capacity to combine with hydrogen donors. When it combines with hydrogen donor DPPH gets converted into
DPPHH, which is no longer a free radical. Conversion of DPPH to DPPHH will result in decrease in the absorbance value. Decolourization will depend on the number of electrons captured. Degree of decolourization will decide the antioxidant strength of the compound under investigation. From the results obtained it is clear that the absorbance of the DPPH in sample solution has been decreased as compared to control curcumin solution and standard ascorbic acid. These obtained results suggests that curcumin conjugated chitosan microspheres are having higher antioxidant property than curcumin solution alone, which might be due to higher solubility and dissolution rate of CCCMs as compared to curcumin.& CCCMs showed better antioxidant potential than standard ascorbic acid by DPPH scavenging test strategy. The absorbance at 429 nm by UV spectrophotometer and IC50 esteem acquired were as 216 g/ml and 232 g/ml (Fig. 6B) for same ascorbic acid and CCCMs individually. It implies CCCMs at higher fixation captured all the free radicals produced by DPPH which resulted in decrease in absorbance and increment in IC50 esteem. Hydrogen Peroxide is one of the important oxidative species which destroys the cell through DNA damage. Peroxidation dominates to oxidative degradation. The oxygen atom in the hydrogen peroxide ends up with oxidation. It may cause production of freeradical. In this study to evaluate the scavenging activity of hyrogenperoxide by CCCMs the IC50 value was 228 g/ml for CCCMS and 220 g/ml for curcumin (Fig. 6A). The antioxidant activity was proved and it is more or less equal to the standard curcumin.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
5
Fig. 3. (A) 1 H NMR of CCC, (B) 13 C NMR of CCC, (D) FTIR of CCCMs and (C) SEM images of CCCMs.
Fig. 4. (A) In vitro curcumin release of prepared microspheres. Drug release kinetics data analysis of CCCMs (B) First order, (C)Zero order (D) Higuchi and (E) Korsmeyer-pappas kinetic.
3.2.3. Anti-inflammatory activity (in-vitro) In this study the anti-inflammatory potential has been examined by denaturation of Bovine Serum Albumin Method. The results indicate higher percentage inhibition when concentration of the CCCMs was increased. The curcumin was used as reference stan-
dard also exhibit concentrate dependent inhibition. It is clear that the phenolic OH group of curcumin moiety play a major role in antiinflammatory property. The results proved that CCCMs is about equal percentage of inhibition when compare to standard curcumin. IC50 value is considered as the amount of sample that is
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7 6
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 5. Anti-microbial activity of CCCMs by serial dilution method (A) Percentage inhibition for E.coli and (B) Percentage inhibition for S. auerus.
Fig. 6. (A) Anti-oxidant activity of CCCMs by H2 O2 method, (B) Anti-oxidant activity of CCCMs by DPPH method, (C) Anti-inflammatory activity by Albumin denaturation method of curcumin and CCCMS and (D) Cell viability study of CCCMs against NIH3T3 (Mouse embryonic fibroblast) cell.
required to inhibit half of the radicals produced. The IC50 value was 45 g/ml for curcumin and 44 g/ml for CCCMs (Fig. 6C). 3.2.4. In-vitro cytotoxic study against normal cell line Cell Viability study for chitosan, curcumin and the CCCMs is shown in Fig. 6D. From the results it is evident that the percentage viability of CCCMs is higher as compared to chitosan and curcumin alone. It showed more than 70% of the cells were viable after 24 h and 48 h (Fig. 6D). Which point out that the prepared microspheres did not show any toxicity in normal cell lines and it is useful for topical application. 3.2.5. Hemo-compatibility assay The developed CCCMs were subjected to hemo-compatibility test. It shows significance in percentage hemolysis of microspheres treated blood samples when compared with the Triton X-100 (positive control) treated blood samples which show complete hemolysis (Fig. 7). PBS was taken as the negative control. The results
showed that the developed system were hemocompatible, The data indicates that the integrity of the erythrocytes was not affected by the prepared system. Since it is blood compatible and can be possible to administer directly to blood circulation [30].
4. Conclusion In this study we have prepared curcumin conjugated chitosan by shiff base reaction in presence of acidic condition and it was characterized and proved by using FTIR, 1 H NMR and 13 C NMR. The CCCMs was also prepared and characterised. The size of the prepared CCCMs is 2–5 m. CCCMs was capable to release the curcumin in a sustained manner and a shown anti-microbial activity was proved by agar diffusion method and serial dilution method, anti-inflammatory and anti-oxidant property also confirmed by albumin denaturation, DPPH and hydrogen peroxide method. The prepared CCCMs were cyto and hemo-compatible. These prepared CCCMs have potential application in the biomedical field.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
7
Fig. 7. Hemolysis analysis of different CCCMs concentrations.
Acknowledgment The authors are thankful to Dr. Sabitha M for continuous support and providing the facilities for this study, the authors also grateful to Ms Elbi Saju, Ms Sandhya Murali, Ms. Namy George and Shammika P for their valuable help during our study.
References [1] Wing-Hin Lee, Ching-Yee Loo, Mary Bebawy, Frederick Luk, Rebecca S Mason, Ramin Rohanizadeh, Curcumin and its derivatives: their application in neuropharmacology and neuroscience in the 21st century, Curr. Neuropharmacol. 11 (2013) 338–378. [2] J.W. Betts, A.S. Sharili, R.M. La Ragione, D.W. Wareham, In vitro antibacterial activity of curcumin-polymyxin B combinations against multidrug-resistant bacteria associated with traumatic wound infections, J. Nat. Prod. 6 (2016) 1702–1706. [3] Xiao-Qing Tang, B. Hu, Jian-Qiang Feng, Jian-Guo Cao, Effect of curcumin on multidrug resistance in resistant human gastric carcinoma cell line SGC7901/VCR, Acta Pharmacol. Sin. 26 (2005) 1009–1016. [4] Xia Xue, Jin-Long Yu, De-Qing Sun, Wen Zou, Feng Kong, Jing Wu, Hui-ping Liu, Xian-jun Qu, Rong-Mei Wang, Curcumin as a multidrug resistance modulator −A quick review, Biomed. Prevent. Nutr. 3 (2013) 173–176. [5] S.Y. Teow, K. Liew, A.S.B. AliS.A. Khoo, S.C. Peh, Antibacterial action of curcumin-against Staphylococcus aureus: a Brief review, J. Trop. Med. 13 (2016) 1–10. [6] S. Wei, D. Sukanta, R. Samar, H.T. Afshan Hussain, A. Saadyah, William L’Amoreaux, Abdeslem E. Idrissi, B. Probal, R. Krishnaswami, Synthesis of monofunctional curcumin derivatives, clicked curcumin dimer, and a pamam dendrimer curcumin conjugate for therapeutic applications, Org. Lett. 9 (2007) 5461–5464. [7] M. Prabaharan, J.J. Grailer, D.A. Steeber, S. Gong, Stimuli-responsive chitosan-graft-poly (N vinylcaprolactam) as a promising material for controlled hydrophobic drug deliver, Macromol. Biosci. 8 (2008) 843–851. [8] P. Mani, G. Shaoqin, Novel thiolated carboxymethyl chitosan-g--cyclodextrin amucoadhesive hydrophobic drug delivery carriers, Carbohydr. Polym. 73 (2008) 117–125. [9] V. Jaleh, J. Fariba, K. Sara, Development of bioadhesive chitosan gels for topical delivery of lidocaine, Sci. Pharm. 74 (2006) 209–223. [10] M. Prabaharan, Chitosan derivatives as promising materials for controlled drug delivery, J. Biomater. Appl 23 (2008) 5–36. [11] An-Na Li, Li Sha, Yu-Jie Zhang, Xiang-Rong Xu, Yu-Ming Chen, Hua-Bin Li, resources and biological activities of natural polyphenols, Nutrients 6 (2014) 6020–6047. [12] X. Jin, J. Wang, J. Bai, Synthesis and antimicrobial activity of the Schiff base from chitosan and citral, Carbohydr. Res. 344 (2009) 825–829.
[13] T.R. Arunraj, N.S. Rejinold, S. Mangalathillam, S. Saroj, R. Biswas, R. Jayakumar, Synthesis, characterization and biological activities of curcumin nanospheres, J. Biomed. Nanotechnol. 10 (2014) 238–250. [14] S. Selvaraj, J. Karthikeya, N. Saravanakumar, Chitoasan loaded microspheres as an ocular delivery system for acyclovir, Int. J. Pharm. Sci. 4 (2012) 125–132. [15] S. Sathianarayanan, R. Gandimathi, A. Saravanakumar, B. Lima, A. Suja, J. Asha, antimicrobial activity of various extracts of justicatranqurbariensis, Adv. Pharmacol. Toxicol. 10 (2009) 81–84. [16] J.L. Rios, M.C. Reico, Medicinal plants and antimicrobial activity, J. Ethnopharmacol. 100 (2005) 80–84. [17] A.Z. Beg Ahmad, Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi drug resistant human pathogens, J. Ethnopharmacol. 74 (2001) 113–123. [18] J. Asha, C. Amrutha, S. Sathianarayanan, T. Saranya, K. Prem, Synthesis and characterization of quinazolinone derivative against mammary carcinoma, J. Pharm. Res. 6 (2013) 933–938. [19] S. Sathianarayanan, J. Asha, B. Ranganathan, T. Diana, B. Ashitha, A. Rajasekaran, Alcoholic extract of Wrightiatinctoria leaves and isolates thereof afford DNA protection in post irradiated Sprague dawley rats, J. Young Pharm. 8 (2016) 4–8. [20] I. Hiba, P. Vishakh, S. Athul, B. Chandrika, Synthesis, anti-inflammatory activity of ring A-monosubstituted chalcone derivative, Med. Chem. Res. 23 (2014) 4383–4394. [21] S. Sathianarayanan, B.C. Amrutha, J. Asha, B. Ranganathan, T.S. Saranya, M. Asha Asokan, Utility of isatin semicarbazones in mammary carcinoma cells − a proof of concept study, J. Young Pharm. 9 (2017) 218–223. [22] E.H. Cordes, W.P. Jencks, On the mechanism of Schiff base formation and hydrolysis, J. Am. Chem. Soc. 84 (1962) 832–837. [23] J. Zhang, L. Xue, Y. Han, Fabrication gradient surfaces by changing polystyrene microsphere topography, Langmuir 21 (2005) 5–8. [24] S. Honary, F. Zahir, Effect of zeta potential on the properties of nano-drug delivery systems- a review (part 2), Trop. J. Pharm. Res. 12 (2013) 265–273. [25] C. He, Y. Hu, L. Yin, C. Tang, C. Yin, Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles, Biomaterials 31 (2010) 3657–3666. [26] C. Huabing, K. Chalermchai, Y. Xiangliang, C. Xueling, G. Jinming, Nanonization strategies for poorly water-soluble drugs, Drug Discov. Today 16 (2011) 354–360. [27] A.A. Noyes, W.R. Whitney, The rate of solution of solid substances in their own solutions, J. Am. Chem. Soc. 19 (1897) 930–934. [28] I. Wiegand, K. Hilpert, H.E.W. Robert, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163–175. [29] K.W. Jung, C. Hye, W. Kim, S. Sook, Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli, Appl. Environ. Microbiol. 74 (2008) 2171–2178. [30] S. Elbi, T.R. Nimal, V.K. Rajan, Gaurav Baranwal, Raja Biswas, R. Jayakumar, S. Sathianarayanan, Fucoidan coated ciprofloxacin loaded chitosan nanoparticles for the treatment of intracellular and biofilm infections of Salmonella, Colloids Surf. B Biointerfaces 160 (2017) 40–47.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
ARTICLE IN PRESS
BIOMAC-8705; No. of Pages 7
International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis, characterisation and biomedical applications of curcumin conjugated chitosan microspheres T.S. Saranya a , V.K. Rajan b , Raja Biswas b , R. Jayakumar b,∗ , S. Sathianarayanan a,∗ a b
Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, Kochi, 682041, Kerala, India Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi, 682041, India
a r t i c l e
i n f o
Article history: Received 7 October 2017 Received in revised form 16 November 2017 Accepted 6 December 2017 Available online xxx Keywords: Chitosan curcumin conjugates Microspheres Anti-inflammatory Anti-oxidant Anti-microbial
a b s t r a c t Curcumin is a diaryl heptanoid of curcuminoids class obtained from Curcuma longa. It possesses various biological activities like anti-inflammatory, hypoglycemic, antioxidant, wound-healing, and antimicrobial activities. Chitosan is a biocompatible, biodegradable and non–toxic natural polymer which enhances the adhesive property of the skin. Chemical conjugation will leads to sustained release action and to enhance the bioavailability. This study aims to synthesis and characterize biocompatible curcumin conjugated chitosan microspheres for bio-medical applications. The Schiff base reaction was carried out for the preparation of curcumin conjugated chitosan by microwave method and it was characterised using FTIR and NMR. Curcumin conjugated chitosan microspheres (CCCMs) were prepared by wet milling solvent evaporation method. SEM analysis showed these CCCMs were 2–5 m spherical particles. The antibacterial activities of the prepared CCCMs were studied against Staphylococcus aureus and Escherichia coli, the zone of inhibition was 28 mm and 23 mm respectively. Antioxidant activity of the prepared CCCMs was also studied by DPPH and H2 O2 method it showed IC50 esteem value of 216 g/ml and 228 g/ml, and anti-inflammatory activity results showed that CCCMs having IC50 value of 45 g/ml. The results conclude that the CCCMs having a good antibacterial, antioxidant and anti-inflammatory activities. This, the prepared CCCMs have potential application in preventing skin infections. © 2017 Published by Elsevier B.V.
1. Introduction Curcumin is a standout amongst the most flexible mixes acquired from Curcuma longa. Chemically, curcumin is (1,7bis(4-hydroxy 3-methoxyphenyl)- 1, 6-heptadiene-3, 5-dione) a characteristic polyphenol, which expo the keto-enol tautomerism having a ubiquitous keto structure in acidic medium and stable enol frame in antacid medium [1]. Curcumin have a large continuum of pharmacological activity especially antimicrobial, antiinflammatory, antioxidant, wound healing and cytotoxic activity against cancer cell lines etc. Several studies proved that curcumin have a potent activity against multi drug resistant microbes [2–5]. The real hindrance in the therapeutic utilization of curcumin is its solubility and plasma level concentration. The chemical conjuncation with biopolymer will enhance the bioavilabitity and solubility of the lipophillic drugs [6–8]. Chitosan is a biocompatible, biodegrad-
∗ Corresponding authors. E-mail addresses: [email protected], [email protected] (R. Jayakumar), [email protected], [email protected] (S. Sathianarayanan).
able polymer which improves adhesive property of the skin [9,10], chitosan has responsive amino and hydroxyl group whereas the amino gathering prompts to the likelihood of a few concoction modifications. The present study aimed to develop curcumin conjugated chitosan microspheres and its antioxidant, antimicrobial and anti-inflammatory activities were studied. The main advantages of this study was the synthesised curcumin derivatives consist of free phenolic hyrdroxyl group it is essential for pharmacological action especially antimicrobial, antiinflammatory and antioxidant activity [11]. The chemical conjugation of curcumin with chitosan may enhances its solubility and adhesive property. 2. Materials and methods All the commercial reagents procured were of GR/AR grade and the reactions were carried out in dried borosil glass vessels. The synthesized compound was characterized using TLC, IR, NMR. Curcumin (C21 H20 O6 ) has been purchased from Merck, Germany. TLC was performed on silica gel G F254 (Merck aluminium plate) as stationary phase and ethyl acetate: methanol: acetic acid (6:3:1) as a mobile phase. Iodine chamber was used for detection of the spots. All microwave syntheses were carried out in CatalystTM
https://doi.org/10.1016/j.ijbiomac.2017.12.044 0141-8130/© 2017 Published by Elsevier B.V.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7 2
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Systems CATA 2 R Scientific Microwave Synthesizer with ten different power settings. The IR spectra of the synthesized compounds were recorded on a Shimadzu-FTIR spectrometer (IR Affinity 1) using potassium bromide pellet technique. 1 H and 13 C NMR of the compounds were recorded using Bruker Spectrospin 400 MHz spectrometer with tetramethylsilane (TMS) as internal standard. Chemical shift was expressed as delta values relative to TMS in units of ppm. NIH 3T3 (Mouse embryonic fibroblast) cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM) and Trysin were obtained from Sigma Aldrich Co, St. Louis, USA; EDTA, glucose and antibiotics from Hi-Media Laboratories Ltd., Mumbai, India and DPPH, dimethyl sulfoxide (DMSO), propanol and other chemicals and medias were procured from E. Merck Ltd., Mumbai, India. 2.1. Synthesis of curcumin conjugated chitosan Curcumin conjugated chitosan (CCC) was synthesised by imine formation (Schiff base reaction) method. Curcumin (184 mg of (1 mol)) was dissolved in 15 mL of ethanol and mixed with Chitosan (255 mg of 2 mol) in 0.1 N acetic acid [12]. The mixture was stirred for 30 min continuously at 800 rpm and subsequently placed in a microwave oven (90 W) for 10 min at 60 ◦ C. TLC analysis was performed to ensure the completion of reaction. The reaction mixture was cooled at 4 ◦ C, filtered and recrystallized with ethanol and characterised by spectral analysis. (Fig. 1). 2.2. Synthesis of curcumin conjugated chitosan microspheres (CCCMs) CCCMs were prepared by wet milling solvent evaporating technique. 10 mg of CCC was dissolved with 5 mL of ethanol. From this 1 mL of CCC was added drop wise to deionised water (50 mL) with a flow rate of 0.1 mL/min under steady blending (800 rpm). Probe sonication was performed using a vibra cell sonicator with a working force of 130 W, and a recurrence of 20 KHz. Sonication was carried out for 20 min at room temperature until a turbid was acquired. It demonstrates that the curcumin conjugated chitosan microspheres are framed in milli-Q water. The prepared microspheres were lyophilized [13]. 2.3. Physiochemical characterization of prepared CCCMs 2.3.1. Particle size analysis and stability studies Measure appropriation of CCCMs was investigated by dynamic light scattering (DLS utilizing DLS-ZP/molecule sizer NicompTM 380 ZLS). Size and surface morphology of the microspheres were further affirmed by SEM (JEOLJSM- 6490 LA). The CCCMs suspension was weakened with water and a drop was set on the metallic stub, air dried and examined the surface charge and particle size of CCCMs was measured using a zeta potential analyser [14]. 2.3.2. FT-IR analysis FT-IR spectra of curcumin and CCCMs were recorded by KBr pelleting method (1% w/w of item in KBr) Transmittance was measured from 4000 to 400 cm−1 . This analysis has been performed in order to test the degradation or chemical interaction of formulation. 2.3.3. In vitro curcumin drug release In vitro curcumin release of CCCMs determined by resuspending 22 mg of CCCMs in 10 mL citrate buffer (pH 5) and incubated in 37 ◦ C shaking incubator. At different time intervals, 3 mL of the supernatant solution have been taken and centrifuged at 12000 rpm for 10 min. The released Curcumin has been evaluated at 429 nm using
UV/VIS spectrophotometer. These procedures have been continued until 100% drug release has been obtained. After withdrawn of sample has been replaced with 3 mL of the buffer solution. Data obtained from in vitro release studies were fitted to various kinetic models to find the mechanism of drug release. The kinetic models used were Zero order, First order, Higuchi model and KorsemeyerPeppas Kinetic models [15]. 2.4. Biological evaluations 2.4.1. In vitro antimicrobial studies The antimicrobial action of curcumin and CCCMs were tested against Staphylococus aureus (S. aureus) and Escherichia coli (E.coli) by disc diffusion and serial dilution method. About 100 L of overnight S. aureus and E.coli cultures were spread over Muller Hinton agar plates (Composition- 2 g of beef extract, 17 g of casein hydrolysate, 1.5 g starch, 17 g agar and make upto 1000 mL with water and adjust the pH for 7.2.), respectively. 0.5 cm filter paper discs containing 50, 100, 150, 200, 250 and 300 g of CCCMs and curcumin were located on the agar media. Following which, the plates were incubated overnight in an vertical position at 37◦ C and the zone of inhibition was measured. [16,17] The minimum inhibitory concentration (MIC) of CCCMs and curcumin was determined in accordance with the Clinical and Laboratory Standards Institute guidelines. Serial two-fold dilutions of CCCMs and curcumin were prepared in triplicate followed by addition of a standard S. aureus and E. coli suspension of 1–5 × 105 CFU/ml. After 24 h at 37 ◦ C, the opacity were determined using a microplate reader (BioTek) at 578 nm. The MIC was determined as the lowest concentration of CCCMs and curcumin with where no visible bacterial growth was observed. 2.4.2. Estimation of antioxidant activity by DPPH and hydrogen peroxide method The antioxidant action of the were measured by 2,2−diphenyl2-picryl-hydrazyl (DPPH) free radical scavenging method [18]. Various concentrations of curcumin, CCCMs and standard ascorbic acid (100, 200, 300, 400 and 500 g/ml) were taken and mixed with 5 mL of 0.01 mM methanolic solution of DPPH was added, shaken well and blend was kept at 37 ◦ C for 30 min. Absorbance was measured at 517 nm using an UV/Vis spectrophotometer. Absorbance of DPPH was taken as control. The investigation was done in triplicate. The half maximal inhibitory concentration (IC50 ) values calculated. About 0.04 mM Hydrogen peroxide solution (0.6 mL) in phosphate buffer was mixed with different concentration of CCCMs. The above solution was stand for 10 min and the absorbance was measured at 230 nm in UV spectrophotometer, phosphate buffer used as the blank and the percentage scavenging activity of H2 O2 were determined and the IC50 Values were calculated [19]. Percentage scavenging effect =
Absorbance of the sample × 100 Absorbance of the control
2.4.3. In vitro anti −inflammatory studies Curcumin and CCCMs were screened for anti-inflammatory activity utilizing hindrance of albumin denaturation strategy The response blend comprises of 1 mL of 1% aqueous arrangement of bovine serum albumin division, 1 mL of PBS at pH 6.4 and 1 mL of different concentration of curcumin and CCCMs were arranged (25, 50, 100, 250, 500, 750 and 1000 g/ml) utilizing DMSO and distilled water individually. 10 g/ml diclofenac sodium has been used as the standard drug and distilled water as the control. Then the mixtures were incubated at (37 ± 2) ◦ C for 15 min and then heated at 70 ◦ C for 5 min. After bring down the temperature of the mixture, the absorbance was measured at 660 nm. The experiment was
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model
ARTICLE IN PRESS
BIOMAC-8705; No. of Pages 7
T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
3
Fig. 1. Schematic representation of synthesis of curcumin conjugated chitosan.
performed in triplicate. Percent inhibition of protein conjugated curcumin with chitosan denaturation was calculated [20].
3. Results and discussion 3.1. Synthesis and characterisation of curcumin conjugated chitosan (CCC)
2.4.4. In vitro cytotoxic study The viability of NIH 3T3 (Mouse embryonic fibroblast) cells in the presence of different concentrations of curcumin and sample were quantified. The nanoparticles were resuspended in complete DMEM, which is used as stock for the assay. 20,000 cells were seeded in each well of 24 well plates. Different concentration of CCCMs were added to the well plate and incubated at 37 ◦ C in CO2 incubator for 24 and 48 h. Cells with complete DMEM was used as positive control. After predetermined time points the medium from the wells were removed and washed twice with PBS. 100 L of 10% (v/v) alamar blue solution in DMEM were added to each well and incubated in CO2 incubator at 37 ◦ C for 4 h. Using a micro plate spectrophotometer the color changes in the alamar blue solution was quantified by measuring the absorbance (O.D) using at 570 nm and at 600 nm as reference wavelength and the percentage of viability was calculated from the absorbance using following equation. All values were taken as triplicate and the results were expressed as mean ± SD value [21]. Cell viability(%) =
T 570 - T 600 × 100 P 570 - P 600
Where, Where, T 570 = O.D of test at 570 nm T 600 = O.D of test at 600 nm P 570 = O.D of positive control (Triton X-100 treated) at 570 nm P 600 = O.D of positive control (Triton X-100 treated) at 600 nm
2.4.5. Hemo-compatibility assay The hemolysis assay was performed to measure the extent of hemolysis caused by CCCMs when it comes into contact with human blood. 100 L of diluted blood (1:9 in PBS) was taken in several 1.5 mL vials and different concentrations of prepared CCMs (1,0.7, and0.5 mg/ml) were added and diluted by using PBS, The tubes were incubated in 37 ◦ C for 24 h. Triton X-100 (10%) (v/v) treated blood sample was taken as positive control. Followed by incubation, samples were centrifuged for 10 mins at 700 rcf in 4 ◦ C, supernatants were collected and by using micro titter plate reader, the absorbance was measured at 540 nm. The hemolysis was calculated using the following equation Hemolysis (%) =
Optical Density (T- N) × 100 Optical Density (P- N)
Where T = Test, N = Negative control and P = Positive control
The CCC compound was synthesized by the Shiff base reaction (Fig. 2), characterized by standard spectroscopic techniques and their purity ascertained by routine TLC analysis along with melting point determination. In this synthesis curcumin undergoes an acid catalyzed addition in presence of acetic acid got an positive charge electrophilic carbonyl carbon, the neucleophilic nitrogen atom of the amino group from the chitosan will attack the carbonyl carbon to form carbon- nitrogen double bond (imine group) [21,22]. The overall yield of CCC was 67% and the melting point of the compound is 168 ◦ C −171 ◦ C and the TLC results showed the Rf value was 0.63. The max was found at 429 nm by using UV Spectroscopy. IR spectral data showed the curcumin C O group at 1723 cm−1 but after the imine formation the band shifted to 1627 cm−1 shifting of this peak was confirmed that the formation of CCC. 13 C and proton NMR data’s proved that the reaction completion, the 13 C NMR showed a peak at 163 ppm it confirmed the formation of imine (C N) at the position of J in Fig. 3, all other curcumin and chitosan peaks were present in the 1 H NMR data. (Fig. 3A & B). Further CCC was formulated as microspheres and it was characterised. The surface morphology of the prepared CCCMs has been performed using SEM and to be spherical in shape with diameter 2–4 m (Fig. 3C). The image of the CCCMs showed highly monodispersed and distinct spherical particles. [23]. The Z.P was found to be −31 mV [19]. This value indicates the enhancement of stability for the prepared CCCMs. The presence of negative zeta potential may be due the presence of −OH functionalities in the prepared microspheres as well conjucation to chitosan and gives negatively charge of the CCCMs. [24,25]. 3.1.1. In-vitro curcumin release The cumulative release studies have been performed until up to 100% release. The percentage drug releases have been plotted against time, and it was showed that the maximum release of curcumin derivative was found to be at the 48th h (Fig. 4A) [26] and more than 50% of drug release was observed at 24th h,The amino group of chitosan will enhance swelling property due to its protonation.The release data were fitted in to four important kinetic models. The linearity (R2 ) and slope (n) were determined to study the release mechanism. The higher linearity of first order kinetics indicates that, the drug release from the microsphers is depending on the concentration of drug(Fig. 4B & C). The drug release mechanism was further investigated with the help of Higuchi and Korsmeyer- Peppas model(Fig. 4D&E). The higher linearity of Korsmeyer- Peppas model for proves the diffusion dependency of drug release. From this graph we can conclude the release pat-
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model
ARTICLE IN PRESS
BIOMAC-8705; No. of Pages 7 4
T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 2. Convergent synthesis of curcumin conjugated chitosan.
tern of CCCMs as evidence for sustained release and confirmed the diffusion of CCCMs from the delivery system in Fickian diffusion [27]. 3.2. Biological evaluation of CCCMs 3.2.1. In vitro Antibacterial Activity Antibacterial activity has been tested against S. aureus and E. coli. In agar diffusion assays E. coli showed the zone of inhibition was 28 mm for CCCMs and 23 mm for Curcumin, the zone of inhibition of S. aureus showed that 33 mm and 31 mm for CCCMs and curcumin respectively. Broth dilution method has been employed for testing antibacterial activity of samples [28]. Minimum Inhibitory Concentration (MIC) of CCCMs and Curcumin for E. coli and S. Auerus was determined, the results were indicate that the antibacterial activity of the CCCMs were greater than curcumin alone. From the graph plotted we can also conclude that as concentration of sample increases the bacterial colony decreases (Fig. 5A and B). The results confirmed that the CCCMs are effective against both organisms [29]. 3.2.2. Antioxidant activity 3.2.2.1. DPPH assay. One of the most economic methods to find out the antioxidant activity is the DPPH method. DPPH is a free radical that is stable at room temperature. It produces a violet colour, when it is blend with organic solvents. DPPH is a free radical that usually has the capacity to combine with hydrogen donors. When it combines with hydrogen donor DPPH gets converted into
DPPHH, which is no longer a free radical. Conversion of DPPH to DPPHH will result in decrease in the absorbance value. Decolourization will depend on the number of electrons captured. Degree of decolourization will decide the antioxidant strength of the compound under investigation. From the results obtained it is clear that the absorbance of the DPPH in sample solution has been decreased as compared to control curcumin solution and standard ascorbic acid. These obtained results suggests that curcumin conjugated chitosan microspheres are having higher antioxidant property than curcumin solution alone, which might be due to higher solubility and dissolution rate of CCCMs as compared to curcumin.& CCCMs showed better antioxidant potential than standard ascorbic acid by DPPH scavenging test strategy. The absorbance at 429 nm by UV spectrophotometer and IC50 esteem acquired were as 216 g/ml and 232 g/ml (Fig. 6B) for same ascorbic acid and CCCMs individually. It implies CCCMs at higher fixation captured all the free radicals produced by DPPH which resulted in decrease in absorbance and increment in IC50 esteem. Hydrogen Peroxide is one of the important oxidative species which destroys the cell through DNA damage. Peroxidation dominates to oxidative degradation. The oxygen atom in the hydrogen peroxide ends up with oxidation. It may cause production of freeradical. In this study to evaluate the scavenging activity of hyrogenperoxide by CCCMs the IC50 value was 228 g/ml for CCCMS and 220 g/ml for curcumin (Fig. 6A). The antioxidant activity was proved and it is more or less equal to the standard curcumin.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
5
Fig. 3. (A) 1 H NMR of CCC, (B) 13 C NMR of CCC, (D) FTIR of CCCMs and (C) SEM images of CCCMs.
Fig. 4. (A) In vitro curcumin release of prepared microspheres. Drug release kinetics data analysis of CCCMs (B) First order, (C)Zero order (D) Higuchi and (E) Korsmeyer-pappas kinetic.
3.2.3. Anti-inflammatory activity (in-vitro) In this study the anti-inflammatory potential has been examined by denaturation of Bovine Serum Albumin Method. The results indicate higher percentage inhibition when concentration of the CCCMs was increased. The curcumin was used as reference stan-
dard also exhibit concentrate dependent inhibition. It is clear that the phenolic OH group of curcumin moiety play a major role in antiinflammatory property. The results proved that CCCMs is about equal percentage of inhibition when compare to standard curcumin. IC50 value is considered as the amount of sample that is
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7 6
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
Fig. 5. Anti-microbial activity of CCCMs by serial dilution method (A) Percentage inhibition for E.coli and (B) Percentage inhibition for S. auerus.
Fig. 6. (A) Anti-oxidant activity of CCCMs by H2 O2 method, (B) Anti-oxidant activity of CCCMs by DPPH method, (C) Anti-inflammatory activity by Albumin denaturation method of curcumin and CCCMS and (D) Cell viability study of CCCMs against NIH3T3 (Mouse embryonic fibroblast) cell.
required to inhibit half of the radicals produced. The IC50 value was 45 g/ml for curcumin and 44 g/ml for CCCMs (Fig. 6C). 3.2.4. In-vitro cytotoxic study against normal cell line Cell Viability study for chitosan, curcumin and the CCCMs is shown in Fig. 6D. From the results it is evident that the percentage viability of CCCMs is higher as compared to chitosan and curcumin alone. It showed more than 70% of the cells were viable after 24 h and 48 h (Fig. 6D). Which point out that the prepared microspheres did not show any toxicity in normal cell lines and it is useful for topical application. 3.2.5. Hemo-compatibility assay The developed CCCMs were subjected to hemo-compatibility test. It shows significance in percentage hemolysis of microspheres treated blood samples when compared with the Triton X-100 (positive control) treated blood samples which show complete hemolysis (Fig. 7). PBS was taken as the negative control. The results
showed that the developed system were hemocompatible, The data indicates that the integrity of the erythrocytes was not affected by the prepared system. Since it is blood compatible and can be possible to administer directly to blood circulation [30].
4. Conclusion In this study we have prepared curcumin conjugated chitosan by shiff base reaction in presence of acidic condition and it was characterized and proved by using FTIR, 1 H NMR and 13 C NMR. The CCCMs was also prepared and characterised. The size of the prepared CCCMs is 2–5 m. CCCMs was capable to release the curcumin in a sustained manner and a shown anti-microbial activity was proved by agar diffusion method and serial dilution method, anti-inflammatory and anti-oxidant property also confirmed by albumin denaturation, DPPH and hydrogen peroxide method. The prepared CCCMs were cyto and hemo-compatible. These prepared CCCMs have potential application in the biomedical field.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044
G Model BIOMAC-8705; No. of Pages 7
ARTICLE IN PRESS T.S. Saranya et al. / International Journal of Biological Macromolecules xxx (2017) xxx–xxx
7
Fig. 7. Hemolysis analysis of different CCCMs concentrations.
Acknowledgment The authors are thankful to Dr. Sabitha M for continuous support and providing the facilities for this study, the authors also grateful to Ms Elbi Saju, Ms Sandhya Murali, Ms. Namy George and Shammika P for their valuable help during our study.
References [1] Wing-Hin Lee, Ching-Yee Loo, Mary Bebawy, Frederick Luk, Rebecca S Mason, Ramin Rohanizadeh, Curcumin and its derivatives: their application in neuropharmacology and neuroscience in the 21st century, Curr. Neuropharmacol. 11 (2013) 338–378. [2] J.W. Betts, A.S. Sharili, R.M. La Ragione, D.W. Wareham, In vitro antibacterial activity of curcumin-polymyxin B combinations against multidrug-resistant bacteria associated with traumatic wound infections, J. Nat. Prod. 6 (2016) 1702–1706. [3] Xiao-Qing Tang, B. Hu, Jian-Qiang Feng, Jian-Guo Cao, Effect of curcumin on multidrug resistance in resistant human gastric carcinoma cell line SGC7901/VCR, Acta Pharmacol. Sin. 26 (2005) 1009–1016. [4] Xia Xue, Jin-Long Yu, De-Qing Sun, Wen Zou, Feng Kong, Jing Wu, Hui-ping Liu, Xian-jun Qu, Rong-Mei Wang, Curcumin as a multidrug resistance modulator −A quick review, Biomed. Prevent. Nutr. 3 (2013) 173–176. [5] S.Y. Teow, K. Liew, A.S.B. AliS.A. Khoo, S.C. Peh, Antibacterial action of curcumin-against Staphylococcus aureus: a Brief review, J. Trop. Med. 13 (2016) 1–10. [6] S. Wei, D. Sukanta, R. Samar, H.T. Afshan Hussain, A. Saadyah, William L’Amoreaux, Abdeslem E. Idrissi, B. Probal, R. Krishnaswami, Synthesis of monofunctional curcumin derivatives, clicked curcumin dimer, and a pamam dendrimer curcumin conjugate for therapeutic applications, Org. Lett. 9 (2007) 5461–5464. [7] M. Prabaharan, J.J. Grailer, D.A. Steeber, S. Gong, Stimuli-responsive chitosan-graft-poly (N vinylcaprolactam) as a promising material for controlled hydrophobic drug deliver, Macromol. Biosci. 8 (2008) 843–851. [8] P. Mani, G. Shaoqin, Novel thiolated carboxymethyl chitosan-g--cyclodextrin amucoadhesive hydrophobic drug delivery carriers, Carbohydr. Polym. 73 (2008) 117–125. [9] V. Jaleh, J. Fariba, K. Sara, Development of bioadhesive chitosan gels for topical delivery of lidocaine, Sci. Pharm. 74 (2006) 209–223. [10] M. Prabaharan, Chitosan derivatives as promising materials for controlled drug delivery, J. Biomater. Appl 23 (2008) 5–36. [11] An-Na Li, Li Sha, Yu-Jie Zhang, Xiang-Rong Xu, Yu-Ming Chen, Hua-Bin Li, resources and biological activities of natural polyphenols, Nutrients 6 (2014) 6020–6047. [12] X. Jin, J. Wang, J. Bai, Synthesis and antimicrobial activity of the Schiff base from chitosan and citral, Carbohydr. Res. 344 (2009) 825–829.
[13] T.R. Arunraj, N.S. Rejinold, S. Mangalathillam, S. Saroj, R. Biswas, R. Jayakumar, Synthesis, characterization and biological activities of curcumin nanospheres, J. Biomed. Nanotechnol. 10 (2014) 238–250. [14] S. Selvaraj, J. Karthikeya, N. Saravanakumar, Chitoasan loaded microspheres as an ocular delivery system for acyclovir, Int. J. Pharm. Sci. 4 (2012) 125–132. [15] S. Sathianarayanan, R. Gandimathi, A. Saravanakumar, B. Lima, A. Suja, J. Asha, antimicrobial activity of various extracts of justicatranqurbariensis, Adv. Pharmacol. Toxicol. 10 (2009) 81–84. [16] J.L. Rios, M.C. Reico, Medicinal plants and antimicrobial activity, J. Ethnopharmacol. 100 (2005) 80–84. [17] A.Z. Beg Ahmad, Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi drug resistant human pathogens, J. Ethnopharmacol. 74 (2001) 113–123. [18] J. Asha, C. Amrutha, S. Sathianarayanan, T. Saranya, K. Prem, Synthesis and characterization of quinazolinone derivative against mammary carcinoma, J. Pharm. Res. 6 (2013) 933–938. [19] S. Sathianarayanan, J. Asha, B. Ranganathan, T. Diana, B. Ashitha, A. Rajasekaran, Alcoholic extract of Wrightiatinctoria leaves and isolates thereof afford DNA protection in post irradiated Sprague dawley rats, J. Young Pharm. 8 (2016) 4–8. [20] I. Hiba, P. Vishakh, S. Athul, B. Chandrika, Synthesis, anti-inflammatory activity of ring A-monosubstituted chalcone derivative, Med. Chem. Res. 23 (2014) 4383–4394. [21] S. Sathianarayanan, B.C. Amrutha, J. Asha, B. Ranganathan, T.S. Saranya, M. Asha Asokan, Utility of isatin semicarbazones in mammary carcinoma cells − a proof of concept study, J. Young Pharm. 9 (2017) 218–223. [22] E.H. Cordes, W.P. Jencks, On the mechanism of Schiff base formation and hydrolysis, J. Am. Chem. Soc. 84 (1962) 832–837. [23] J. Zhang, L. Xue, Y. Han, Fabrication gradient surfaces by changing polystyrene microsphere topography, Langmuir 21 (2005) 5–8. [24] S. Honary, F. Zahir, Effect of zeta potential on the properties of nano-drug delivery systems- a review (part 2), Trop. J. Pharm. Res. 12 (2013) 265–273. [25] C. He, Y. Hu, L. Yin, C. Tang, C. Yin, Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles, Biomaterials 31 (2010) 3657–3666. [26] C. Huabing, K. Chalermchai, Y. Xiangliang, C. Xueling, G. Jinming, Nanonization strategies for poorly water-soluble drugs, Drug Discov. Today 16 (2011) 354–360. [27] A.A. Noyes, W.R. Whitney, The rate of solution of solid substances in their own solutions, J. Am. Chem. Soc. 19 (1897) 930–934. [28] I. Wiegand, K. Hilpert, H.E.W. Robert, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163–175. [29] K.W. Jung, C. Hye, W. Kim, S. Sook, Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli, Appl. Environ. Microbiol. 74 (2008) 2171–2178. [30] S. Elbi, T.R. Nimal, V.K. Rajan, Gaurav Baranwal, Raja Biswas, R. Jayakumar, S. Sathianarayanan, Fucoidan coated ciprofloxacin loaded chitosan nanoparticles for the treatment of intracellular and biofilm infections of Salmonella, Colloids Surf. B Biointerfaces 160 (2017) 40–47.
Please cite this article in press as: T.S. Saranya, et al., Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.12.044