Bangladesh Journal of Pharmacology Research Article Modified and unmodified arabinoxy- lans from Plantago ovata husk: Novel excipients with antimicrobial potential BJP Introduction Among natural polymeric materials, polysaccharides and their derivatives are the most abundantly used in pharmaceutical formulations. Polysaccharides are consi- dered to be most versatile polymers because they have structural diversity and create many linkages between monomer units. Chemical modification of polysaccha- rides results in improved process ability and generates industrially important derivatives (Lowman, 2000). Plantago ovata is a hairy soft annual herb of 12-18 inches in height and belongs to the Plantaginaceae family. The polysaccharides extracted from the husk of P. ovata have been chemically characterized to contain a high proportion of hemicellulose which is the alkali soluble fraction of the husk. It consists of highly branched acidic arabinoxylan comprising of xylan backbone chain with xylose and arabinose forming the side chains (Kennedy et al., 1979). Chemical modification of arabino- xylan can easily be performed because some xylp residues of the chain carry a single xylp moiety at position 2 with hydroxyl groups at position 2, 3, and 4. The ethylation and carboxymethylation of arabinoxy- lan, isolated from P. ovata seed husk, were carried out (Saghir et al., 2008). Antimicrobial activity of ethanolic and methanolic extract of P. ovata has been reported against Gram positive and Gram negative bacteria (Motamedi et al., 2010). In our previous studies, we evaluated arabinoxylan as potential pharmaceutical excipient (Sajid et al., 2014; Alia et al., 2014). During these studies, formulation containing arabinoxylan as excipient was not prone to microbial contamination and was assumed that it inhibits microbial growth. So, in present study attempt was made to evaluate antibacterial activity of arabinoxylan and its chemically modified forms. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 765-769 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index; ISSN: 1991-0088 Abstract Arabinoxylan is a polysaccharide of Plantago ovata, an indigenous plant of Pakistan and several European countries. In the present investigation arabinoxylan isolated from P. ovata husk was chemically modified i.e., carboxymethylated and was converted into its sodium salt. Physicochemical characterization of modified arabinoxylans was done. The antimicrobial assay of arabinoxylan and modified arabinoxylans was done by Kirby-Bauer disk diffusion method against Gram negative and Gram positive bacterial strains. Physicochemical properties of modified arabinoxylans were different from arabinoxylan yet were satisfactory for their use as pharmaceutical excipients. Arabinoxylan showed strong antimicrobial activity against Gram positive strains. However, it showed no antimicrobial activity against Gram negative strains. Carboxymethyl arabinoxylan showed maximum activity against S. epidermidis. It also showed activity against S. aureus and P. aeruginosa. Arabinoxylan Na showed concentration-dependent antimicrobial activity. This study provides the first report on the antibacterial properties of arabinoxylan and its modified forms. Article Info Received: 21 June 2015 Accepted: 9 July 2015 Available Online: 24 September 2015 DOI: 10.3329/bjp.v10i4.23788 Cite this article: Erum A, Bashir S, Saghir S. Modified and unmodified arabinoxylans from Plantago ovata husk: Novel excipients with antimicrobial potential . Bangladesh J Pharmacol. 2015; 10: 765 -69. Modified and unmodified arabinoxylans from Plantago ovata husk: Novel excipients with antimicrobial potential Alia Erum1, Sajid Bashir1 and Shazia Saghir2 1Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan; 2Department of Chemistry, Government Jinnah Degree College for Women, Mozang, Lahore, Pakistan. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Materials and Methods P. ovata husk was purchased from the local market in Pakistan. Other chemicals used were sodium hydroxide (Merck), ethanol (Riedel-de-Haen), sodium monochloroacetate (BDH Analar) and dimethylsulfoxide (Panreac). Isolation of arabinoxylan Arabinoxylan was isolated from the P. ovata husk by the method described elsewhere (Saghir et al., 2008) with slight modification. Percentage yield of gel was calculated by equation Percentage yield = (Weight of gel/weight of husk) x 100 Modification of arabinoxylan Carboxymethylation Arabinoxylan (1.0 g) was suspended in 72 mL ethanol. About 3.6 mL of 25% aqueous NaOH solution was added to the above reaction mixture and was vigorous- ly stirred at room temperature. After 1 hour, sodium monochloroacetate (2.64 g) was added and the tempera- ture of mixture was raised to 55°C. The stirring of mix- ture was continued for 5 hours. The mixture was then filtered and suspended in 80% (v/v) aqueous methanol. pH of solution was neutralized with dilute acetic acid and was filtered. The filtered product was washed with 20 mL ethanol five times and then dried for 2 days at 60°C (Sagir et al., 2008). Arabinoxylan Na salt formation Arabinoxylan (1.0 g) was dissolved in 50 mL of aqueous NaOH solution (2.5%) to yield 2% solution. The solu- tion was continuously stirred for 24 hours and was dried at 40°C in an oven under vacuum (James, 1939). Physicochemical characterization Physicochemical properties of modified arabinoxylans were determined such as their solubility, swelling index, tapped density, bulk density, angle of repose, compressibility index and pH (Sajid et al., 2014). Antimicrobial assay The antimicrobial assay of arabinoxylan, carboxymethyl arabinoxylan and arabinoxylan-Na against different Gram positive and Gram negative bacterial strains were conducted by Kirby-Bauer disk diffusion method. Preparation of stock solution for antimicrobial assay Stock solution (5.0 mL) of arabinoxylan (5 mg/mL) was prepared in DMSO. Arabinoxylan (25 mg) was added in 5 mL DMSO and stirred at 80°C for one hour until it was completely dissolved. Stock solutions of carboxy- methyl arabinoxylan, A1 (5 mg/mL) and A2 (7.5 mg/ mL) were prepared in distilled water. Arabinoxylan-Na stock solutions were made in 2.5% NaOH containing (5 mg/mL), (10 mg/mL), and 20 mg/mL arabinoxylan-Na for solutions A, B and C respectively. Microorganisms used The test organisms used were Gram positive strains such as Staphylococcus aureus (ATCC 25923), Actinomyces odontolyticus (ATCC 17929), Bacillus subtilis (ATCC 6633) and Staphylococcus epidermidis (ATCC 12228), and Gram negative strains such as Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Kirby Bauer disc diffusion method About 0.1 mL of bacterial inoculums having turbidity adjusted according to McFarland’s 1 as standard was spread uniformly on Mueller Hinton agar plates. Arabi- noxylan, carboxymethyl arabinoxylan and arabinoxylan- Na loaded 6 mm discs of Whatman No. 1 filter paper were placed on the surface of medium and plates were incubated for 24 hours at 37ºC. Ciprofloxacin loaded disc containing 0.1 µg/disc was used as positive control while DMSO loaded disc was used as a negative control. In case of arabinoxylan-Na and carboxymethyl arabinoxylan, distilled water dipped disc was used as solvent control. At the end of incubation period, inhibition zones were measured in millimeters. These studies were performed in triplicate. Results and Discussion Arabinoxylan gel was successfully isolated from husk (Figure 1) and its yield was about 45% which was rated as good. Modification of polysaccharide yield structurally and functionally altered derivatives having potential to be explored for their use in different fields. The polysaccha- rides which have undergone modification include starch (Chen et al., 2007), cellulose (Hon, 1996), chitosan (Rinaudo, 2006), psyllium (Singh and Sharma, 2010) and guar-gum (Dodi et al., 2011). Carboxymethylation of arabinoxylan with sodium monochloroacetate resulted in replacement of – OH group with carboxymethyl group. 766 Bangladesh J Pharmacol 2015; 10: 765-769 Figure 1: Extracted gel from Plantago ovata husk Biomaterial AX-Na R=Na NaOH aq Stirring at room temperature Most polysaccharides such as alginic acid, astragulus gummifer etc. are available in a variety of mono and multivalent cation salt form with varying properties (Abdul et al., 2014). In arabinoxylan-Na, -OH group of arabinoxylan was replaced with sodium ion. Carboxymethylation of arabinoxylan yielded water solu- ble derivative, which is in accordance with the litera- ture as the solubility of gums is improved by carboxy- methylation (Olusola et al., 2014). Carboxymethyl arabinoxylan was also soluble in 0.1 N HCl and aqueous NaOH. Arabinoxylan-Na was partially soluble in water having aqueous solubility of 42.8% (Table I). It was also soluble in 60% ethanol on slight heating and aqueous NaOH while it was insoluble in 0.1 N HCl methanol and ethanol. Arabinoxylan-Na salt had reduced swelling than arabinoxylan with swelling index of 27 ± 1% in distilled water which was due to presence of water soluble fraction within it. The moisture content of carboxymethyl arabinoxylan and arabinoxylan-Na was low as calculated from its loss on drying. The low moisture values suggest their suitability as excipient in formulations of moisture sensitive drugs. The know- ledge of compressibility and flow indices is important for justifying the use of materials as excipients in a pharmaceutical formulation. So, according to values of bulk and tapped densities and their differences, compre -ssibility index was calculated (Table I). According to these parameters, carboxymethyl arabinoxylan was the freest flowing. Arabinoxylan-Na was also free flowing. The stability of most preparations is highly dependent on pH. Excipient pH influences the pH of formulation. Therefore, pH of 1% solution of modified Arabinoxylans were determined by pH meter (Luiz et al., 2005). The pH of carboxymethyl arabinoxylan was 7.5 and for arabinoxylan-Na was 7.61 (Table I), which are almost neutral. This implies that they can be used as excipients in all types of drugs i.e. acidic, basic and neutral. These properties of modified arabinoxylans are different from arabinoxylan (Sajid et al., 2014). Secondary metabolites of some plants showed antibac- terial activity. The medicinal importance of phytoche- mical constituents has been established in the treatment of various diseases (Yadav and Munin, 2011). Antimi- crobial activity of some polysaccharide gels such as gel from Durio zibethinus had been documented (Lipipun et al., 2002). Arabinoxylan showed strong antimicrobial activity against Gram positive strains, arabinoxylan showed maximum activity against B. subtilis with zone of inhibition 35 ± 2 (Table II). It also showed activity against S. mutans with zone of inhibition 12 ± 1.5, A. odontolyticus with zone of inhibition 10 ± 1 and S. aureus with zone of inhibition 10 ± 1. However, it showed no antimicrobial activity against Gram negative strains E. coli and P. aeruginosa. This was most probably due to different cell membrane structure of Gram negative bacteria and presence of enzymes in periplasmic space that cause destruction of foreign particles (Bassam et al., 2004). Modification of arabinoxylan resulted in products carboxymethyl arabinoxylan and arabinoxylan-Na with altered structures and properties. Antimicrobial activity of modified arabinoxylans was quiet different from arabinoxylan. Etherification of arabinoxylan not only changed its structure, its physicochemical properties but also changed its antimicrobial spectra which was most probably because antimicrobial agents showed structure activity based relationship (George and Eleftherios, 2012). In carboxymethyl arabinoxylan greater antimicrobial activity was shown by A1 than A2, which was most probably due to a very viscous solution that was unable Table I Physicochemical characterization of modified arabinoxylans Parameters Carboxymethyl arabinoxylan Arabinoxylan-Na salt Solubility Soluble in water on slight heating and in 0.1N HCl solution, aqueous NaOH, insoluble in ethanol and methanol Partially soluble in water 42.8%, 60% ethanol on slight heating, insoluble in 0.1N HCl and methanol Swelling index in distilled water - 27 ± 1 Loss on drying (%) 0.4 ± 0.0 0.4 ± 0.1 Bulk density (g/mL) 0.5 ± 0.0 0.8 ± 0.0 Tapped density (g/mL) 0.6 ± 0.0 1.0 ± 0.0 Compressibility index (%) 14.3 18.2 Angle of repose (º) 29.0 ± 1.1 32.0 ± 1.5 pH (1% solution) 7.5 ± 0.0 7.6 ± 0.0 Bangladesh J Pharmacol 2015; 10: 765-769 767 to penetrate the agar resulting in less zone of inhibition. Carboxymethyl arabinoxylan (A1) showed maximum activity against S. epidermidis with zone of inhibition 26 ± 2. It also showed activity against S. aureus with zone of inhibition 21 ± 2 as well as against Gram negative P. aeruginosa with zone of inhibition 20 ± 1. Carboxymethyl arabinoxylan (A2) was also effective against same strains as A1 but with less zone of inhibition (Table II). However, unlike A1 it was effective against B. subtilis with zone of inhibition 12 ± 3 and S. mutans with zone of inhibition 10 ± 1. Similar to carboxymethyl arabinoxylan, arabinoxylan-Na also showed different antimicrobial spectrum from arabinoxylan. In arabinoxylan-Na antimicrobial activity was observed to be concentration dependent. Solution A (100 µg/disc) showed maximum activity against P. aeruginosa with zone of inhibition 14 ± 3. It also showed activity against B. subtilis, S. epidermidis, A. odontolyticus, P. aeruginosa and S. mutans. Solution B (200 µg/disc) showed more activity as compared to solution A i.e., greater zones of inhibition against B. subtilis, S. epidermidis, A. odontolyticus, P. aeruginosa and S. mutans (Table II). In addition, it also showed activity against E. coli and S. aureus. However, solution C (400 µg/disc), highest tested strength of arabinoxylan-Na, showed least activity against all tested strains and no activity against E. coli. This was most probably due to highly concentrated gel that was unable to diffuse agar to inhibit bacterial growth. Conclusion Arabinoxylan, carboxymethyl arabinoxylan and arabinoxy- lan-Na possessed significant antibacterial activity. How- ever, difference in intensity and spectrum of antimicro- bial activity was due to variations in their structures. Maximum activity was shown by arabinoxylan-Na. References Abdul M, Nayyar P, Pramod KS. Novel polymers for muco- adhesive drug delivery. Int J Pharm. 2014; 4: 212-20. 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Table II Antimicrobial susceptibility testing of modified arabinoxylans against bacterial strains Positive control ciprofloxacin (0.1 µg/disc) Arabinoxylan (µg/disc) Carboxymethyl arabinoxylan (µg/disc) Arabinoxylan-Na (µg/disc) Strains 100 100 A1 200 A2 100 A 200 B 400 C Zone of inhibition (mm) E. coli 40 0 0 0 0 7 ± 0 0 S. aureus 36 10 ± 1 21 ± 2 19 ± 1.5 0 17 ± 2.5 2 ± 1.5 B. subtilis 40 35 ± 2 0 12 ± 3 3 ± 0 18 ± 2 12 ±1 S. epidermidis 34 0 26 ± 2 12 ± 1 5 ± 1.5 14 ± 2 3 ± 1 A. odontolyticus 40 10 ± 1 0 0 3 ± 1 19 ± 2 8 ± 2 P. aeruginosa 37 0 20 ± 1 20 ± 2 14 ± 3 19 ± 1.5 4 ± 1.5 S. mutans 38 12 ± 1.5 0 10 ± 1 12 ± 2 15 ± 1.4 5 ± 1 768 Bangladesh J Pharmacol 2015; 10: 765-769 Author Info Alia Erum (Principal contact) e-mail: alia_erum@hotmail.com Motamedi H, Darabpour E, Gholipour M, Nejad SMS. Antibac- terial effect of ethanolic and methanolic extracts of Plantago ovata and Oliveria decumbens endemic in Iran against some pathogenic bacteria. Int J Pharmacol. 2010; 6: 117-22. 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