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KUMBHOJE S.R.1*, SHIROTE P.J.2, MAGDUM C.S.3
1Department of Pharmaceutical Chemistry, Ashokrao Mane College of Pharmacy, Peth-Vadgaon- 416 112, Kolhapur, MS, India.
2Department of Pharmaceutical Chemistry, Appasaheb Birnale College of Pharmacy, Sangli- 416 113, MS, India.
3Department of Pharmaceutical Chemistry, Rajarambapu Patil College of Pharmacy, Kasegaon, Sangli- 415 404, MS, India.
* Corresponding Author : sachin.kumbhoje@rediffmail.com
Received : 14-09-2012 Accepted : 27-10-2012 Published : 29-09-2012
Volume : 1 Issue : 1 Pages : 9 - 11
World Res J Org Chem 1.1 (2012):9-11
The present research work entails synthesis and pharmacological screening of chemically modified chitosan derivatives. Chitosan is an amino polysaccharide obtained from natural origin and it has attracted attention because of its unique physicochemical characteristics and biological activities. It has great pharmaceutical futuristic potential with unusual extent of possibilities for structural modifications to impart desired functions. This chemically modified chitosan can be used as absorption enhancers needed for controlled drug delivery in alkaline environment. Various aldehydes can be used for the chemical modification of chitosan, where aldehyde attached to the free amino group of chitosan polymer imparts different physicochemical properties, not exhibited before modification. An imine formation as an intermediate is then reduced to N-aryl derivative of chitosan. This chemical modification enhances solubility of chitosan in slightly acidic, alkaline and neutral media. Solubility property of chitosan is related to long alkyl chains attached to hydrophilic parts. The Schiff base and N-aryl derivative of chitosan were synthesized from chitosan and aromatic aldehyde. It was characterised by Fourier-transform Infrared (FTIR) and Nuclear magnetic Resonance (NMR). The antimicrobial assessment of chitosan derivatives has been carried out. It has been recognised for its antimicrobial activity, in that it is destructive towards the bacterium S. Aureus, Eschericia Coli, Candida albicans, and Asperagus Niger. The results showed that N-aryl chitosan had better inhibitory properties than chitosan, Schiff base of chitosan. The antimicrobial activity of chitosan is well known against a variety of bacteria and fungi coming from its polycationic nature. Thus, the chemically substituted chitosan was successfully synthesized.
Schiff base, Aldehyde, N-Aryl chitosan, Antimicrobial activity.
Chitosan is a one of the most naturally occurring biodegradable polymer which is extensively used in the controlled drug delivery. It has a broad range of application such as drug delivery, wound healing, food preservation, enzyme immobilization, tissue engineering etc. It has extensive biopharmaceutical characteristics such as biocompatibility, pH sensitivity and non toxicity. The property of biodegradability is due to its metabolism by certain human enzymes, especially lysozyme. Because of these properties, the interest in chitosan and its derivatives as excipients in drug delivery has been increased in decades. The idea of modified chitosan synthesis arises mainly from the fact that this natural polysaccharide allows the production of biocompatible and biodegradable drug delivery systems. Despite having mucoadhesive property it exhibits poor solubility at pH values above 6 that prevent enhancing effects at sites of absorption of drugs. Recently, many studies have reported the use of chitosan in the formation of gels, particles, and microspheres. However, it suffers from low solubility at a physiological pH of 7.4, which limits its application as absorption enhancer. In order to overcome these problems, a number of chemically modified chitosan derivatives have been synthesized and tested. Chemical modification of chitosan improves its solubility and widens its applications [1,2] .
Chitosan exhibits an antimicrobial activity against range of microorganisms. There are number of studies to suggest mechanism behind the antimicrobial activity. Chitosan is polycationic in nature and the target of its antimicrobial action is with the negatively charged cell wall of bacteria, where it binds and disrupts the normal functioning of the cell membrane. It inhibits the transport of nutrients into cells by promoting the release of intracellular components. Chemically modified chitosan have great utility in controlled release and targeting studies of almost all class of bioactive molecules. The aim is to focus on the different methods of synthesis, properties, and applications of the chemically modified chitosan designed for the association and delivery of drugs [1,2] .
The introduction of an alkyl chain offers several possibilities in molecular design of chitosan. Its highly reactive free amino group offer great potential for further derivatisation. The pathway of synthesis of N-substituted chitosan also involves the Schiff base formation. The assessment of biological activities of this derivative is necessary.
In this paper, the derivatives of chitosan, N-(2- hydroxyl naphthyl) chitosan (HNCS), N-N-dimethyl amino chitosan (DACS), 2, 4-dimethoxy benzyl chitosan (DBCS) are synthesized and evaluated. The chemical structures of newly formed derivatives were characterized by FTIR, 1H NMR. [1-4] .
The synthesis of substituted N-aryl derivatives of chitosan was carried out by following step:
Chitosan was dissolved in a solution of acetic acid in methanol. Aldehyde was added to the solution and stirred at room temperature for 4 Hrs. After standing the mixture for 3 Hrs, NaBH4 was added and stirred at room temperature for 24 Hrs. The solution was then made alkaline by addition of sodium hydroxide. The precipitate formed after addition of sodium hydroxide is separated by filtration, dried at 60ºC in oven for 24 Hrs.
Infrared (IR) and Proton Nuclear Magnetic Resonance (1H-NMR) were used to confirm the structures of all the synthesized compounds. IR spectra were recorded on a Jasco FTIR-410 spectrophotometer using KBr pellets. 1H-NMR spectra were recorded on varian mercury YH-300 using CF3COOD as solvent at 300 MHz. TMS was used as an internal reference standard for relative proton chemical shift.
Antimicrobial activity of chitosan, N-aryl derivative of chitosan was assessed against S. Aureus, Eschericia Coli, Candida albicans, and Asperagus Niger.
All chemicals and solvents were procured from commercial sources and purified and sterilized using standard procedures from literature whenever required.
MacConkey agar (M081) was procured from Himedia Laboratories Ltd. Mumbai. Nutrient Agar medium (Nutrient broth N011) was procured from Himedia Laboratories Ltd. Mumbai. Agar agar powder was purchased from S. D. Fine Chem. Ltd. Mumbai. Glacial acetic acid was purchased from Research Lab Fine Chem. Industries, Mumbai.
All the synthesized compounds i.e. Chitosan derivatives dissolved in aq. Acetic acid so as to get concentration of 500 µg/ml. [5,6] .
MacConkey agar (M081), Soybean casein digest medium (M011), Saboraud’s agar and normal saline solution were sterilized in autoclave at 15 lbs pressure (121ºC) for 15 mins. Petri plates, disc of whatman (41) paper, cotton swabs were sterilized in oven at 160ºC for 2 hrs.
206 mg of MacConkey agar was dissolved in 4ml of distilled water, boiled and then poured it in the test tube and the test tube was plugged with cotton and then sterilized in autoclave at 15 lbs pressure (121ºC) for 15 min. After the sterilization the tubes containing the MacConkey agar were kept in inclined position for ½ hrs. Then on the solid surface of these slants the pure culture of the test bacteria i.e. S. aureus and Escherichia coli were streaked in aseptic condition and then incubated at 37ºC for 24 hrs [5,6] .
112 mg of Nutrient agar medium and 100 mg of agar powder was dissolved in 4ml of distilled water boiled and then poured it in the test tube and the test tube was plugged with cotton and then sterilized in autoclave at 15 lbs pressure (121ºC) for 15mins. After sterilization the tube containing Nutrient Agar medium were kept in inclined position for 30 mins. Then on solid surface of these slants pure culture of test bacteria i.e. Staphylococcus aureus and Escherichia coli were streaked in aseptic condition and then incubated at 37ºC for 24 hrs [5,6] .
250 mg of Saboraud’s dextrose agar was dissolved in 4ml of distilled water, boiled and then poured it in the test tube and the test tube was plugged with cotton and then sterilized in autoclave at 15 lbs pressure (121ºC) for 15 min. After the sterilization the tubes containing the Saboraud’s agar were kept in inclined position for ½ hrs. Then on the solid surface of these slants the pure culture of the test fungi i.e. Candida albicans were streaked in aseptic condition and then incubated at 37ºC for 24 hrs [5,6] .
By using the 24 hrs old growth of the test bacteria/fungi from the slant, suspension of the bacteria/ fungi were made separately in sterile normal saline solution (0.85% NaCl in distilled water) in aseptic condition, to get moderate turbidity. The turbidity of each suspension was compared and adjusted with the turbidity of the solution resulting by mixing 0.5 ml of 1.175% of barium chloride and 99.5 ml of 0.36N, H2SO4 acid [5,6] .
Sterile, non-toxic cotton swab were dipped into the standardized inoculum (turbidity as adjusted as to obtained confluent growth on the Petri plate) and then the entire agar surface of the plate was streaked with the swab three times, turning the plate at 60o angle between streaking. Then the streaked inoculum was allowed to dry for 5-15 mins with lid in place. Microns loop the culture media. After 30 mins the plates were incubated at 37ºC [5,6] .
The derivative of chitosan with aromatic aldehyde was synthesized via the Schiff base intermediate. The reaction was carried by using homogeneous reaction between chitosan and aromatic aldehyde in methanolic acetic acid. The representation of Schiff base preparation reaction is presented in scheme: 1. the reaction mixture was reduced by sodium borohydride yielded N-aryl derivative [Scheme-1] .
The spectral data of IR shows main bands of chitosan: OH stretching at 3444.24 cm -1, N-H bending vibration at 1644.02 cm-1, CH3 symmetrical angular deformation 1384.64 cm-1, C-N amino axial deformation at 1020.16 cm-1 (fig.1). The IR spectra of Schiff base of chitosan presented a strong absorption band at 1636.3 cm-1 attributed to the C=N vibrations characteristics of imines, which is not observed in chitosan. [Graph-1]
A: Chitosan,
B: 2, 4-dimethoxybenzyl Chitosan,
C: 2, 4-dimethylamino benzyl Chitosan,
D: Hydroxy naphthyl chitosan
The antimicrobial activities of the synthesized derivatives were carried out. It shows inhibitory action against S. Aureus, Eschericia Coli, Candida albicans, and Asperagus Niger under experimental conditions. [Table-1]
All the values are in mg/L which represents MIC (Minimum Inhibitory Concentration).
Chemically modified chitosan derivatives were successfully synthesized by the reductive N-arylation under mild acidic conditions by using aromatic aldehyde. Intermediate Schiff base formation was achieved as well. These derivatives were water soluble at the neutral pH and displayed antimicrobial activity against S. Aureus, Eschericia Coli, Candida albicans, and Asperagus Niger as observed by using the minimum inhibitory concentration method.
We are really thankful to Prof. D.D. Chougule, Dr. C.S. Magdum, for their kind support and time to time encouragement. I also want to thank Dr. J.I. D’souza and Prof. Rajesh Patil for their kind support. We also pleased to thank department of pharmaceutical chemistry of the college.
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 | Graph 1- IR Spectra of Chitosan and Modified Chitosan Derivatives |
 | Scheme 1- General scheme for the synthesis of N-alkyl Chitosan derivative |
 | Table 1- Antimicrobial Activity of Synthesized Derivatives |