Novel wound dressing material derived from crosslinked tamarind seeds polysaccharide: in vitro characterization and wound healing activity.
Abstract: In the present study, novel wound dressing films derived from Tamarind seed polysaccharide (TSP) were fabricated by crosslinking with epichlorohydrin and evaluated for wound healing activity in albino rats. The required film properties for the successful wound dressings, such as film elongation (elasticity), tensile strength, water uptake and water vapor transmission rates (WVTR) were examined. Results indicated that TSP films showed good elasticity (38.09-44.95 %) and showed acceptable tensile strengths (7.33-9.83 N) which were found to be depending on the thickness and extent of crosslinking of the films. Films showed water uptake of 157 to 189 % w/w and WVTR were found in the range between 8.24 x [10.sup.-3] to 0.24 x [10.sup.-3]g.[cm.sup.2]/day TSP films with an ideal characteristic were loaded with Povidone iodine (PI) solution by soaking method and were evaluated for antimicrobial and wound healing activity on excision wound model. Results indicated that PI loaded films showed significant antibacterial activity against both gram positive and gram negative selected bacteria and good wound healing property as compared to control group with faster epithelialization and greater rates of wound contraction.
Article Type: Report
Subject: Wound healing (Research)
Surgical dressings (Composition)
Polysaccharides (Properties)
Polysaccharides (Health aspects)
Proteins (Crosslinking)
Proteins (Research)
Authors: Patil, Basavaraj S.
Mastiholimath, Vinayak
Kulkarni, Anandrao R.
Pub Date: 10/01/2011
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: Oct, 2011 Source Volume: 25 Source Issue: 4
Topic: Event Code: 310 Science & research
Product: Product Code: 3842120 Surgical Dressings & Sutures; 2860440 Polysaccharides NAICS Code: 339113 Surgical Appliance and Supplies Manufacturing; 325199 All Other Basic Organic Chemical Manufacturing SIC Code: 3842 Surgical appliances and supplies
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 304842731
Full Text: Introduction

Wound dressing materials (WDMs) based on hydrogels were effectively used for healing of the diversity of the wounds as these materials protect the wounds from environmental, microbial attack and also to promote the healing process by providing an optimum microenvironment, removing any excess wound exudate, and allowing continuous tissue reconstruction [1-3]. WDMs also helps in the regeneration of vascular tissue by allowing gaseous exchange and promotes the epithelialization [4]. Wound disturbance during removal of WDMs is to be minimized in order to achieve an effective management of wounds. Hydrogel based WDM turns gel upon contact with wound exudates become smooth thus reduces wound disturbances while removal of the WDMs. The effectiveness of these hydrogel as wound dressing material is further increased when they exhibit an inherent anti-bacterial activity or by incorporating antibacterial agent [5]. In this direction many efforts have been made to identify the naturally occurring hydrogels possessing both antibacterial and film forming capacity.

Tamarindus indica L. commonly known as tamarind tree is one of the most important multipurpose tree species in the Indian sub-continent[6]. The tamarind fruit pulp has been an important culinary ingredient in India for a very long time. Almost all parts of the tree find uses in food, chemical, pharmaceutical and textile industries [7]. A tamarind seed polysaccharide (TSP) is obtained from the seeds contains about 65 % polysaccharides. TSP is a neutral hydrophilic and mucoadhesive polymer belongs to xyloglucan family consisting mainly of D-mannose and D-galactose units. TSP has a high capacity of water absorption and can retain a stable viscosity over a broad pH range and has been used as a pharmaceutical aid [8].

In this research, TSP was successfully isolated by hot water extraction process and films were fabricated by crosslinking with epichlorhydrin. Selected TSP films were loaded with Povidone iodine. The Povidone-iodine (PI) has well-known antiseptic properties and is effective against a wide spectrum of pathogens [9]. Aqueous PI has been used as a topical antiseptic and surgical scrub for more than 40 years and microbial resistance has not yet been reported[10]. Thus, the prepared TSP films were loaded with PI and were evaluated for various physicochemical parameters, required for an effective WDM. Selected PI loaded TSP films were also evaluated for wound healing activity in rats and the results were compared with control group as well as with standard group treated with Band aid[R] marketed product.

Materials and methods

Tamarind seeds were collected locally and washed thoroughly with water and completely dried seeds were used for extraction of the polymer. Acetone, alcohol, hydrochloric acid and sodium hydroxide were purchased from S. D. Fine chemicals, Mumbai, India. Nutrient agar medium was purchased from Himedia Laboratories, Pvt. Ltd, Mumbai, India. Gram positive and gram negative bacteria culture samples were obtained from Karnataka Institute of Medical Sciences, Hubli, India. Albino wistar rats were purchased from Venkateshwara enterprises, Bangalore, India. Animals were maintained under conventional laboratory conditions, at temperature 25 [+ or -] 2[degrees]C, and a 12 h natural light period. Commercial pellet diet (Lipton India) and drinking water were provided ad libitum. For the present study, clearance from institutional animal ethical committee was obtained prior to the animal activity.

Extraction of polymer

TPS was extracted as per the method explained in the literature with an appropriate modification [11]. Dried seeds were soaked in distilled water for 24 hours in a beaker. After complete swelling, seeds were crushed in mortar and pestle for size reduction. Weighed quantity of fine powder of tamarind seeds was mixed with distilled water to get slurry. The slurry was poured into sufficient quantity of boiling distilled water and kept to cool to room temperature. The supernatant was separated by centrifugation and the residue was successively washed with petroleum ether, diethyl ether and acetone. Further, precipitate was dried at 50-60[degrees]C under vacuum to remove volatiles and moister. The dried material was further grounded and sieved to get uniform sized particles.

FTIR spectroscopy studies

Polymeric sample was ground with potassium bromide (KBr), and pellets were made by applying 6 tons of hydraulic pressure. The FTIR spectra were obtained on Nicolet, Model Impact 410, USA at USIC, Karnatak University, Dharwad, India in the wavelength region between 400 and 4000 [cm.sup.-1].

Fabrication of films

TSP films were prepared by pouring know volume of TSP solution (4 % w/v) on a glass Petridish placed at a constant level. The rate of evaporation was controlled by inverting a cut fennel over the petridish. After 3 days the dried films were taken out and boiled in hydroalcoholic solution containing 5 % sodium sulfate and 1 % epichlorohydrin. Crosslinked films were washed with deionized (DI) water for several time and dried and stored in descicator at room temperature. Povidone iodine (PI) loaded films were prepared by soaking films in iodine solution for 12 hours. After a complete soaking PI loaded films were washed with DI water and dried in descicator and used for screening the wound healing activity.

Scanning electronic microscopy

Polymer samples were cut in to small pieces of 5 mm circle and were fixed on a brass holder. SEM photographs were taken with a JSM 6400 Scanning Microscope (Japan) at the required magnification at room temperature. The working distance of 39 mm was maintained and the acceleration voltage applied was 5 KV, with a secondary electron image (SEI) as a detector.

Determination of tensile strength and percentage of elongation

Tensile strength and percentage of elongation were determined using a universal testing machine (Shimadzu). Films of 10 mm width and 80mm length were cut and fixed to the machine jaws. Then load on the film was increased gradually to a maximum and the tensile strength and percentage of elongation was noted.

Equilibrium swelling studies

TSP films of 10 mm diameters were made and put in watch glass containing DI water. After definite intervals of time films were taken out and blotted with filter paper and weighed on an electronic balance with 0.1 mg sensitivity.

Water Vapor Transmission Rate (WVTR)

Water vapor transmission studies were carried out using pre-weighed glass vials of 5ml containing 1 ml distilled water. TSP films were fixed on the brim of the vials with a adhesive and stored in a desiccators containing anhydrous calcium chloride at room for seven days. The weight of the vials was noted down at every 24 hours to calculate weight loss per day and WVTR were calculated as mentioned in earlier methods using equation 1 [12].

WVTR = WL/S (1)

Where, W is weight loss, L is thickness and S is the surface area of the film.

Antimicrobial Study

Circular PI Loaded TSP films and standard medicated WDM were cut with a diameter of 10 mm and placed on nutrient agar medium containing standard bacterial inoculum. Anti-microbial activity was carried out against Escherischia coli, Shigella dysentrae, Pseudomonas aeruginosa and Bacillus subtilis. The anti-microbial activity was measured by measuring the zone of inhibition using a standard technique.

Wound Healing Studies

Wistar albino rats were purchased from Venkateshwara enterprises, Bangalore, India. The animals were maintained under conventional laboratory conditions, at temperature 25 [+ or -] 5[degrees]C, and a 12 h natural light period. Commercial pellet diet (Lipton India) and drinking water were provided ad libitum. Animal experiments were carried out after obtaining the clearance from the institutional animal ethical committee. Albino male rats weighing in between 175-250 grams were selected for the study and divided into three groups of five animals each. On the 0th day, animals were anaesthetized and secured to operation table on its natural position. An ink impression was made on the dorsal thoracic central region 5mm away from the ears by using round seal of 2.5 cm diameter as described by Morton and Malone. [13] The skin of the impressed area was excised to the full thickness to obtain a wound area of about 500 [mm.sup.2]. Wounds of the animals in the control groups were kept open without any treatment whereas wounds of the animals in standard groups were applied with a Band-aid[R] and wounds of the animals in test groups were applied with PI loaded TSP films. The physical attribute of healing which mainly contributes for wound closure in the first three weeks were studied by tracing the raw wound area on the tracing paper till complete epithelialization occurred.

Tissue tensile strength and collagen estimation

The regenerated tissue after a complete epithelialization was collected from the excision wounds and tissue tensile strength was measured by the method of Lee [13]. For determination of collagen content regenerated tissue of the known area was cut into pieces. They were washed with 0.5M sodium acetate and then suspended in to 10 parts w/v of 0.5M acetic acid and stirred intermittently for 48 hrs. The solution was centrifuged at 5000g for 2hrs. Then sodium chloride (5%w/v) solution was added to precipitate the collagen. The collagen so precipitated was filtered using a preweighed Whatman filter paper. The weight of the collagen precipitate obtained was calculated by taking difference between the initial and the final weights of the filter paper [14].

Results and Discussion

TSP was successfully isolated from the kernel using the method adopted in the present research. The average yield was found to be 40.18 % w/w of dry weight of the kernel powder of tamarind seeds and TSP was completely soluble in the water. Preliminary qualitative tests indicated that the polymer isolated is carbohydrate polymer and free from soluble constituents such as protein, phenol, tannin and alkaloid. A 4 % w/v solution of TSP was prepared in water and the films were fabricated by casting TSP solution on a plain Petridish. After complete drying, the films were crosslinked with epichlorohydrin in the presence of sodium sulfate. Hydroxyl groups present in the TSP undergo reaction with epichlorohydrin. The possible chemical reaction in between TSP and epichlorohydrin is shown in the scheme 1 [15]. Crosslinking reactions were confirmed by FTIR spectral studies.

[FORMULA NOT REPRODUCIBLE IN ASCII]

FTIR spectrum peaks of TSP and crosslinked TSP are shown in Figure 1. The results indicated that the characteristic peaks of TSP at 1000-1200 [cm.sup.-1] correspond to the presence of alcoholic group mostly secondary alcohols. Peaks at 1260-1400 [cm.sup.-1] are due to O-H bending which were altered after crosslinking. Peaks at around 1639 [cm.sup.-1] are due xyloglucan polysaccharide of TSP [16].

Films characterization

Results of thickness, % elongation, tensile strength, swelling and WVTR of various TSP films are shown in Table 1. TSP films produced were transparent, smooth and flexible. Surface properties of all the three different films were studied and evaluated by scanning electronic microscopy at various magnifications. SEM photographs of F1, F2 and F3 TSP films are shown in figure 2a, 2b and 2c respectively. The results indicated that the films were having smooth surfaces without any pores indication uniform dispersion.

[FIGURE 1 OMITTED]

An ideal WDM should possess good elasticity and enough tensile strength, in order to avoid wear and tear WDM during dressings of wounds. TSP films showed good elasticity of 38.09 to 44.95 % of elongation and elasticity increase with increase in thickness. Tensile strengths were in the range of 7.33 and 9.83 N/[mm.sup.2] and increase with the increase in thickness of the films.

Hydrogel based WDMs absorb the exudates from the wound thereby keep the wound dry, promotes the healing process. In order to know the swelling capacity of prepared TSP films, equilibrium swelling studies were conducted to predict the absorption capacity of the films. Results of equilibrium swelling studies indicated that all formulations showed good swelling i.e., more than 100 % and at the same time films were found to be intact throughout the experiments without dispersing in swelling media (soluble).

The WVTR data through the TSP films were measured to understand the permeation characteristics of the TSP films. Results indicated that all the films were permeable to water vapor which is one of important parameter for WDM for better wound healing property. WVTR were found in the range of 8.24 x [10.sup.-3] and 0.24 x [10.sup.-3]g.[cm.sup.2]/day and rate of permeation found to be increasing with the decrease in thickness of the films. Since the films prepared in this research are hydrophilic, water transport through these films depends on the rigidity of the polymer backbone in addition to the extent of cross-linking.

Antimicrobial studies

The antimicrobial activity of PI Loaded TSP film was investigated with four different pathogenic bacteria usually found in wounds. P. aeruginosa is one of the causes of delayed healing and infection in both acute and chronic wounds. Results of antibacterial activities of PI Loaded TSP film are summarized in Table 2. The antibacterial PI Loaded TSP film was compared with standard medicated WDMs. It was observed that the zone of inhibition for PI Loaded TSP films were 25, 30, 26 and 25 mm where as for the band-aid[R] films were 22, 20, 21 and 23 mm respectively for Escherischia coli, Shigella dysentrae, Pseudomonas aeruginosa and Bacillus subtilis. The antibacterial activity in both cases may be due to the fact that the antibacterial agent present in the respective patches were easily released in to agar media and produced zone of inhibition depending upon their sensitivity. The diffusive permeability of a polymer is one of important parameters for WDMs which is present in the prepared TSP films.

[FIGURE 2 OMITTED]

Wound Healing Studies

Excision wounds were created on 0th day and the average diameters for different groups are noted. Percentage closure of original wound area was calculated in [mm.sup.2] at different time intervals which are shown in Table 3. In the control group the percentage wound closer at 0, 4th, 8th, 12th, 16th and 20th day were 0, 31.96, 54.3, 70, 84.4 and 95% respectively. In the PI loaded TSP films treated group the percentage wound closer at 0, 4th, 8th, 12th, 16th and 20th day were 0, 30, 89, 91, 99 and 100 % respectively. In the Band aid[R] treated group the percentage wound closer at 0, 4th, 8th, 12th, 16th and 20th day were 0, 32, 91, 94.2, 99.2 and 100% respectively. Results clearly indicate that the percentage wound closer and epithelialization for the PI loaded TSP films treated group were comparable with Band aid[R] treated group.

Results of collagen content and tensile strength of regenerated tissue in PI loaded TSP films treated (test) and Band aid[R] treated groups (standard) are shown in Table 4. The collagen content in test as well as standard groups was comparatively higher than the control group.

Similarly, tensile strength of the tissue in test as well as standard groups was also comparatively higher than the control group. Healing process involves collagen synthesis which is the formation of a precursor polypeptide such as proline and lysine residues. Therefore, remodeling of tissue occurs by faster inter and intermolecular cross linking of collagen. The present finding suggests that wounds treated with novel TSP films are comparable to those treated with standard similar material selected in the study.

Conclusion

Tamarind seed polysaccharide based wound dressing materials were prepared in the form of thin, transparent and flexible films. The films showed all the properties for the successful wound dressings with antibacterial activity. Povidone iodine loaded TSP films showed significant wound healing property and it is comparable to standard medicated wound dressing material available in the market. TSP films treated groups showed faster epithelialization and grater rates of wound contraction with significant increased collagen content and tensile strengths of the regenerated tissue as compared to the control group.

References

[1.] A.R. Kulkarni, V. H. Kulkarni, J. Keshavayya, V. I. Hukkeri, H.W. Sung, Anti-microbial Activity and Film Characterization of Thiazolidinone Derivatives of Chitosan, Macromol. Biosci. 5, 490-493 (2005).

[2.] R. Muzzarelli, M. Weckx, O. Fillippini, C. Lough, Characteristic properties of N-carboxybutyl chitosan, Carbohydrate Polym. 11, 307-320. (1989).

[3.] R. Muzzarelli, R. Tarsi, O. Filippini, E. Giovanetti, G. Biagini, P. E. Varaldo, Antimicrobial properties of N-carboxybutyl chitosan. Antimicrob Agents Chemother Antimicrob. Agents Chemother. 34, 2019-2023 (1990).

[4.] J.S. Boateng, K.H. Matthews, H.N. Stevens, G.M. Eccleston, Wound healing dressings and drug delivery systems: a review, J Pharm Sci. 97, 2892-2893 (2008).

[5.] H. Sashiwa, S. Aiba, Chemically modified chitin and chitosan as biomaterials, Prog. Polym. Sci., 29, 887-908 (2002).

[6.] ANON. (1976), 'Tamarind'. In The Wealth of India (Publications and Informations Directorate, CSIR Vol X, 114-22.

[7.] J.C.Dagar, G.Singh, N.T.Singht, Evaluation of Crops in agroforestry with teak, (Tectonia grandis) Maharuk (Ailanthus excelsa) and Tamarind (Tamarindus indica L.) on reclaimed salt affected soil. J Tropical Forest Sci., 7, 623-624 (1995).

[8.] S. Sumathi, A. R. Ray, Release behavior of drugs from tamarind seed polysaccharide tablets. J Pharm Pharmaceut Sci,, 5, 12-18 (2002).

[9.] E.T. Houang, O.J.A. Gilmore, C. Reid, E.J. Shaw, Absence of bacterial resistance to povidone-iodine. J Clin Pathol., 29, 752-755 (1976).

[10.] T.J. Simmons, S.H. Lee, T.J. Park, D.P. Hashim, P.M. Ajayan, R.J. Linhardt, Antiseptic single wall carbon nanotube bandages. Carbon, 47, 1561-1564 (2009).

[11.] S Sumathi. and A.R. Ray. Role of modulating factors on release of caffeine from. Tamarind seed polysaccharide tablets. Trends in Biomaterials & Artificial Organs, 17, 41-46 (2003).

[12.] M. D. Kurkuri, A. R. Kulkarni, M. Y. Kariduraganavar, T. M. Aminabhavi, In-vitro release study of veerpmyl hydrochloride through sodium alginate interpenetrating network monolithic membrane. Drug Dev. Ind. Pharm., 27, 1107-1114 (2001).

[13.] KH Lee, TG Tong, Study on the mechanism of action of salicylates, retardation of wound healing by aspirin. J Pharm Sci., 57, 1042-1046 (1968).

[14.] J. J. P. Morton, M.H. Malone, Evaluation of vulnerary activity by an open procedure in rats. Arch. Int. Pharmacodyn., 196, 117-126 (1972).

[15.] S. Sumathi, A.R.Ray, Release behavior of drugs from tamarind seed polysaccharide tablets. J Pharm Pharmaceut Sci. 5, 12-18 (2002).

[16.] G.Kaur, S. Jain, A.K. Tiwary, Chitosan-Carboxymethyl Tamarind Kernel Powder Interpolymer Complexation: Investigations for Colon Drug Delivery. Sci Pharm, 78, 57-78 (2010).

Basavaraj S. Patil (1), Vinayak Mastiholimath (2) and Anandrao R. Kulkarni (1) *

(1) SET's College of Pharmacy, S.R. Nagar, Dharwad 580 002 India

(2) KLE University's College of Pharmacy, Belgaum 590 010, India

* Corresponding author, (kulkarni73@yahoo.com) Dr. Anandrao R. Kulkarni

Received 30 April 2011; Accepted 6 September 2011; Available online 8 September 2011
Table 1: Results of thickness, % elongation, tensile strength,
swelling and WVTR of various TSP films

Film code     Volume of     Thickness *      Elongation *
             TSP solution   ([micro]m)           (%)

F1                10            210       38.09 [+ or -] 2.0
F2                15            284       41.25 [+ or -] 3.2
F3                25            326       44.95 [+ or -] 1.9

Film code    Tensile strength *   Swelling          WVTR
               (N/[mm.sup.2])       (%)      (g.[cm.sup.2]/day)

F1           7.33 [+ or -] 1.2      189      8.24 x [10.sup.-3]
F2           9.42 [+ or -] 0.8      164      7.01 x [10.sup.-3]
F3           9.83 [+ or -] 0.7      157      0.24 x [10.sup.-3]

* Average of three readings

Table 2: Antibacterial activity of PI Loaded TSP film and Band-
aid against various bacteria

Organism                  Zone of Inhibition in mm *

                          PI loaded TSP     Band-aid

Escherischia coli               25             22
Shigella dysentrae              30             20
Pseudomonas aeruginosa          26             21
Bacillus subtilis               25             23

* Average of three readings

Table 3: Results of wound area ([mm.sup.2]) and % wound closer of
excision wounds of various groups

                    Raw Wound Area# in [mm.sup.2] [+ or -]
                            SD at Day (% closer)

Group                      0                      4th

Control            535 [+ or -] 12.0      370.8 [+ or -] 4.5
                          (0)                   (31.96)

PI loaded TSP     514 [+ or -] 18.01     340.9 [+ or -] 2.2 *
Film treated              (0)                    (30)

Band aid[R]       510 [+ or -] 19.11     337.9 [+ or -] 7.1 *
treated                   (0)                    (32)

                    Raw Wound Area# in [mm.sup.2] [+ or -]
                            SD at Day (% closer)

Group                     8th                    12th

Control           249.06 [+ or -] 4.2     163.5 [+ or -] 2.4
                        (54.3)                   (70)

PI loaded TSP    53.57 [+ or -] 7.4 *    43.83 [+ or -] 5.7 *
Film treated             (89)                    (91)

Band aid[R]      44.73 [+ or -] 8.14 *   28.82 [+ or -] 8.17 *
treated                  (91)                   (94.2)

                    Raw Wound Area# in [mm.sup.2] [+ or -]
                            SD at Day (% closer)

Group                    16th                    20th

Control            85.3 [+ or -] 8.4      27.25 [+ or -] 5.3
                        (84.4)                   (95)

PI loaded TSP     4.87 [+ or -] 0.2 *            (100)
Film treated             (99)

Band aid[R]       3.97 [+ or -] 0.7 *            (100)
treated                 (99.2)

# Average of three readings * p <0.001 when compared by One way
ANOVA followed by Tukey's test

Table 4: Results collagen content and tensile strength of
regenerated tissue of various groups

                              Collagen content#    Tensile strength#
Group                              (mg/kg)              (grams)

Control                       41 [+ or -] 1.0     280 [+ or -] 11.3
PI loaded TSP film treated    58 [+ or -] 0.9 *   319 [+ or -] 9.1 **
Band aid[R] treated           61 [+ or -] 1.3 *   322 [+ or -] 8.4 **

# Average of three readings * p <0.001 and ** p <0.01 when compared
by One way ANOVA followed by Tukey's test
Gale Copyright: Copyright 2011 Gale, Cengage Learning. All rights reserved.