Characterization and Antimicrobial Characteristics of Chitosan Modified Hibiscus Sabdariffal.Extract

Samples of Chitosan (Cs) natural polymer containing successive amount of Hibiscus sabdariffaLextract were sucssesfuly synthesized using traditional simple casting route. Prepared thin films were characterized using fourier transform infrared (FTIR). FT-IR of synthesized thin films reveals maintenance of the characteristic bands of chitosan in addition to the appearance of two new sharp intense bands at 1782 and 954 cm-1 intensified with increasing plant extract content and assigned to the interaction between NH2 of polymer skeleton with falvanoids present in the extract. Obtained data poit out to a formation of homogenous composite structure. X-ray diffraction data (XRD) reval no prounounced band indicating the amorphous structure of synthesized final polymeric product. In vitro antimicrobial studies were performed using both gram negative and positive bacteria in addition to Fungul and Yeast activity using simple minimum inhibition zone (MIZ) standeredroutain.


Introduction
Recently biodegradable, biocompatible polymers modified with different fillers are studied by different researchers [1-3] for their versatile uses in different fields of application. Chitosan is one of the most important and low cost natural biodegradable polymers used as a carrier for pharacuticals active ingredients as stabilizing agent to enhance their characteristics.
Chitosan represent a class of linear polysaccharide prepared through acetylation process of chitin and their monomer consists of a combination of N-acetyle-D-glucosamine (acetyled) and D-glucosamine (deacetyled) groups whose percent depends on the deactylation ratio [4]. Chitosan usually obtained from the hard outer skeleton of shellfish and owe a huge medical uses especially in the obesity and high cholesterol disease treatments [5].
Chitosan shows a promising biological activities against pathogenic grams so its film is unique in the application of food preservation through extension of time interval and reduction of the growth rate of microorganisms as reported by Han [6] in addition to their uses as chelating agent and heavy metal trapper and also in water treatment [7,8].
Hibiscus sabdariffaL. usually used in foods,wines while their leaves and tender stem can be added to salads and chutney. As all plants, the compositions of Hibiscus sabdariffaL, contains polyphenolic acids, flavonoids, and anthocyanins [9] which owes as manymedicinal applications including treatment of hypertension, pyrexia, liver damage and leukaemia due to its high content of protocatechuic acid [10].In addition to these characteristics Hibiscus sabdariffaL. extract can also usedas an antioxidant for protection against low density lipoprotein (LDL)-oxidation and has hypolipidemic effects in vivo [11].
Present work aims to evaluate the role of Hibiscus sabdariffa L. addatives for anatural degradable polymer (chitosan) in the antimicrobial activity and to correlate change in the activity index with the concentration and type of interaction between composite constituent material.

Materials and Methods
Chitosan of low molecular weight supplied by Sigma Aldrich Co. Hibiscus sabdariffa L. obtained from Egyptian markets. The studied pristinechitosan thin films and other samples containing successive amount of plant extract were synthesized via casting technique with labled in Table (1). Bi-distiled water containing 2% acetic acid were used a common solvent. Starting materials was vigorously stirred at room temperature until a clear transparent viscous liquid is obtaine. Calculated amounts of extract were added to the polymer solution and poured in a plastic Petri dishes. Dishes are incubated in an oven at 50 up to two days for drying. Obtained films was peeled from the Petri dishes and kept in desiccator until use. XRD diffraction (XRD) was used to identifythe degree of crystallinity throughout the samples and to investigate complexation behavior between polymeric matrices and Hibiscus sabdariffaL..XRD diffraction data plotted as Bragg's angle (2) versus intensity using PANalyticalX`Pert PRO adopting Cu Ktargetusing wavelength  = 1.540 Å and tube operating at 45 kV-40 mA within the Bragg's angle (2) ranging between 5-80. FT-IR spectral data collected using Nicolet iS10 spectrophotometer adopted 32 runs at room temperature within the spectral range 4000-400 cm -1 . UV/vis. spectral data collected using Jasco 570 double beam spectrometer within the range 190-650 nm.
Biological activities were evaluated against Candida albicans fungi, Pseudomonas aeuroginosa, Escherichia coli gram negative bacteria and Bacillus subtilis, Staphylococcus aureusgram positive bacteria via minimum inhibition zone (MIZ) route previously discuused [12].Antibacterial activity of a known standard antibiotic (ampicillin) and antifungal (Colitrimazole) was also measured to calculate the percent activity index of synthesized samples using the formula:

Figure (3) Functional groups in the structure of flavonoids
Chitosan represents a class of natural polymers that are mainly consists three functional groups, amino grous, primary and secondary alcohol. Figure (3) indicates FTIR spectral data of pure chitosan and other samples that contain variable amounts of the Hibiscus extract. All samples show the maintenance of the main constituent material (chitosan) with an obvious change in peak position and intensity with increasing filler content indicating notable change in their physicochemical characteristics. Table (2) list the observed band position and their assignments.

Antibacterial Studies
In vitro antimicrobial studies were performed using both gram negative and positive bacteria in addition to Yeast activity using simple minimum inhibition zone (MIZ) standered routain. Biological activities were evaluated against Pseudomonas aeuroginosa, Escherichia coli gram negative bacteria and Bacillus subtilis, Staphylococcus aureusgram positive bacteria in addition to Candida albicans yeast. Obtained data was also compared with a standard antibiotic to calculate the activity index.    Ampicillin  26  100  23  100  24  100  24  100  ---NA   Colitrimazole  NA  ----NA  ----NA  ----NA  ----27

Conclusions
Chitosan doped Hibiscus sabdariffa L extract were preperead via traditional solution casting route. FT-IR of synthesized reveals maintenance of the characteristic bands in addition to the appearance of two new bands at 1782 and 954 cm -1 whose intensity increases with further addition of plant extract indicating a formation of new vibrational mode resulting from interaction between NH2 of polymer skeleton with falvanoids present in the extract. All samples were tested for their in vitro antimicrobial behavior, their activity index calculated and compared to a standared drug via simple minimum inhibition zone (MIZ) standered routain. It was observed that increasing of Hibiscus sabdariffa L. content result in increase of the diameter zone and activity index in all cases as shown in Figure (5). Such effect can be attributed to the presence of OH group in the falvonoids structure which may react with free ions released during the interaction between NH 2 group of chitosan and other constituent of the extract.