INTRODUCTION:

There was an estimated availability of approximately 10 billion tons of Chitin and Chitosan annually. This biopolymer considered an essential renewable source due to its high availability as waste.^1-2 The versatility of these biopolymers saves us from severe environmental pollution—the Chitosan isolation from the chitin of aquatic/marine species such as fishes and shrimps remarkably used in various fields. Chitin is an n-acetyl-d-glucosamine derivative of glucose, a linear polymer joined by b (1-4) glycosidic linkage.³ Chitin is a hard, nonflexible extracted from exoskeleton and endoskeleton such as crabs, fish, shrimps, lobsters, and prawns, insects, along with fungi spices. The chitin diversity is and considers to be the second most abundant biopolymer after cellulose.⁴ CS is a biodegradable polysaccharide, and it derived from the deacetylation of chitin.⁵

The Chitosan is biocompatible, biodegradable, and non-toxic; antimicrobial, anti-fungal, and anti-viral. These specific bioactivities; includes making this natural polymer desirable in various fields from food preservation, agriculture, cosmetics to health care and disease treatment.⁶ The antibacterial properties of chitin derivatives added value to its various applications in the pharmaceutical field.^7-9

Chitosan's antibacterial mechanisms of action depend upon various physicochemical properties, including pH and presence of functional group (C-3 and C-6 position) on Chitosan. Its believe that the molecular weight of polymer also regulates antimicrobial bioactivity. As In the case of higher molecular weight (MW) Chitosan, the effectiveness seen to be not efficient. However, low molecular weight (MW) enhance the killing of microbes. Low MW can penetrate the bacterial cell wall/ cells. Also, the metal chelating ability helps prevent the uptake of nutrients by cells by making them not available to cells. Also, Due to the permeability of Low MW chitosan, the inhibition of RNA, protein synthesis could be noticed.^10-12

However, Studies to exploit biomaterials as enhance drug therapeutics are still unfulfilled. Biopolymers always get attraction in biomedicine, control drug release formulation. The controlled release drug delivery holds a remarkable position in disease treatment. The biocompatible, biodegradable and effective drug carriers process a crucial role in the success of drug therapeutics. In our drug discovery studies, as well as another researcher study, natural products showed remarkable bioactivity. Similarly, in our previous study few synthetic complexes also display bioactivity, which is in the pipeline for antibacterial activity.^13-38 However, naked active compounds that are nonspecific, highly unstable, and prone to degradation must convert to control nanoformulation to give desirable results. As many potent anticancer drugs used in conventional chemotherapy, cisplatin and tamoxifen showed promising nanoformulation results. Despite having potential as a lead compound in drug discovery, these compound faces some limitation which needs various carrier/ delivery systems. These limitation includes poor solubility, increase toxicity due to—nonspecific targeting or absence of targeted drug delivery system, instability, early degradation in the biological environment. Various polymers successfully used in the various formulation and several in the trial. Another versatile polymer, hyaluronic acid, is also used in various targeted drug formulation to deliver anticancer drugs via various drug routes.

Similarly, poly(lactic-co-glycolic acid) PLGA also FDA approved and displayed promising results. Similarly, dendrimers, along with various other polymers, explored in the journey of drug discovery. Therefore, biopolymer such as chitosan and chitosan derivatives unprecedented investment in the emerging biomedical industries such as drug development and wound healing-related applications and wastewater treatments, biodegradable fertilizers and organic farming.

This study attempted to optimize chitosan extraction from Tilapia (Oreochromis sp.) fish scales. The Physico-chemical characterization and antimicrobial activities of the Chitosan evaluated. This valuable information could aid in applying Chitosan and its derivatives as antimicrobial and future use of Chitosan and other polymers as drug delivery system in various fields, especially food and biomedicine.

MATERIALS AND METHOD:

Isolation of Chitosan and Chito-oligosaccharides from fish scales:

Isolation of Chitosan performed by using the modified method.³⁹ Firstly for the demineralization process, fish scales were soaked in 2% HCl at ambient room temperature with a solid to solvent ratio of 1:5 (w/v) for 24 hours. The residues formed on the bottom of the bottle were collected and washed with distilled water. The precipitates were then placed on a petri dish and left to dry in a 37ºC incubator for 3 hours. Secondly, for the deproteinization process, the dried residues were treated with 4% NaOH at ambient room temperature with a solid ratio of 1:5 (w/v) for 24 hours. The residue formed on the bottom of the flask was collected and washed with distilled water. The residues were then placed on a petri dish and let dry in a 37ºC incubator for 3hours. The flaked residue, which is chitin, was then grounded and stored in an airtight sterilized bottle. Thirdly for the deacetylation process to obtain Chitosan, the chitin was treated with 80% NaOH at a solid/liquid ratio of 1:15 (g/ml) at 120ºC for 6 hours.

Antibacterial Assay:

The antibacterial activity of gram-positive bacteria; (i) Staphylococcus aureus, (ii) Streptococcus agalactiea; and (iii) Bacillus cereus); and gram-negative bacteria (i) Escherichia coli, (ii) Pseudomonas aeruginosa, (iii) Salmonella typhii performed. By using aseptic techniques, colonies picked from 24hr old cultures grown on nutrient agar. The inoculum then suspended in sterile solution (saline) set to an optical density (OD) reading of 0.1 at wavelength 600nm. After this, 0.1mL of this bacterial suspension was then transferred to 9.9mL of sterile broth (Luria Bertani-LB Miller, Merck) at a dilution factor of 1:100 to achieve a final concentration of 1× 10⁶ CFUml^-1.

MIC and MBC Assay:

The minimum inhibitory concentration (MIC) of Chitosan determined according to the method stated.⁴⁰ The 100µL the bacteria suspension undergoes two-fold serial dilution to achieve final concentration ranging from 0.039mg/mL – 5mg/mL. The plates incubated for 24hours at 37ºC under aerobic conditions. The visible signs of growth checked using a microplate reader (Shimadzu UV-1800) at 630nm. The lowest concentration of Chitosan that can inhibit the growth of selected bacterial strains considered the minimum inhibitory concentration (MIC). Then, 100uL was drawn out onto a petri dish with nutrient agar, spread evenly via hockey stick. Then, the plates incubated for 24hours at 37ºC under aerobic conditions.

Mechanisms of Antibiosis Determination (MIC Index):

The mechanism of antibiosis of our Chitosan elucidates by using the given method.⁴¹ The MBC/MIC ratio > 4 values represent the bactericidal, whereas MBC/MIC≤ 4 is bacteriostatic.

Time-Kill Assay:

In this study, we employed the standard protocols of M26-A by CLSI and ASTM-2315. The Chitosan prepared at MIC value (4X). An inoculum size of 1.0 × 10⁶ CFU/mL of each bacterial strain was added and incubated at 37ºC on an orbital shake for 6 hr. At every 30 mins interval, the turbidity of the aliquots observed at an optical density (OD) at 630nm using a spectrophotometer (Shimadzu UV-1800). A blank control test performed with bacterial broth without the addition of Chitosan. The results value display in a graph as OD vs time.

Statistical Analysis:

The results display as the mean±Standard deviation (SD). The two-way analysis of variance (ANOVA) applied by using Origin 8 SR4. The significant data were determined (P-value < 0.05).

RESULTS:

Yield:

The chitin and chitosan yield presented in table 1. The 100grams of dried fish scales yielded Chitin 32.74%, whereas 62.13% chitosan yielded.

Chitosan (COS) exhibited inhibitory and killing activity against Gram-positive as well as gram-negative stains. The MIC values obtained in the range of 0.313 to 0.625 mg/mL, which was lower than the MBC value (Table 2), show that COS minimal concentration of 0.313mg/mL can inhibit the progression in cell growth of Gram-positive bacterial; Staphylococcus aureus and Streptococcus agalactiea. In comparison, inhibition was noticed at 0.625mg/mL in Bacillus cereus and remaining gram-negative stains. Similarly, the MBC values of Chitosan ranged from 1.25 to 5.0mg/mL. The study revealed that MIC and MBC more significant against gram-positive bacteria than gram-negative bacteria against Chitosan.

Table 1: Yield of chitin and Chitosan derived from fish scales

Chitin			Chitosan
Sample weight (g)	Yield weight (g)	Yield (%)	Sample weight (g)	Yield weight (g)	Yield (%)
100	32.74	32.74	100	62.13	62.13

MIC and MBC of Chitosan:

Table 2: Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Chitosan

Bacterial Stains	Chitosan
Bacterial Stains	M.I.C. (mg/mL)	M.B.C. (mg/mL)
Staphylococcus aureus	0.313	1.25
Bacillus cereus	0.313	1.25
Streptococcus agalactiea	0.625	2.5
Escherichia coli	0.625	2.5
Pseudomonas aeruginosa	0.625	5.0
Salmonella typhii	0.625	5.0

Mechanism of Antibiosis and Antibacterial Activity:

Table 3 shows the mechanism of antibiosis or MIC_index of Chitosan. The ratio of MBC/MIC determination reveals the bacteriostatic effect of Chitosan, against all tested bacterial strains.

Growth Inhibitory and Time Kill Assay:

The antibacterial activity on the growth of bacterial strains has done by growth inhibitory and Time kill assay (Figure 1, 2A and 2B). The increase in growth inhibition of Staphylococcus aureus, Bacillus cereus, Streptococcus agalactiea, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi. towards increase concentration of chitosan (0mg/mL – 5mg/mL).

Table 3: Comparative MBC/MIC ratios of Chitosan

Bacterial Stains	Chitosan	Antibacterial effects
Staphylococcus aureus	4.03	Bacteriostatic
Bacillus cereus	4.03	Bacteriostatic
Streptococcus agalactiea	4.03	Bacteriostatic
Escherichia coli	4.03	Bacteriostatic
Pseudomonas aeruginosa	8	Bacteriostatic
Salmonella typhii	8	Bacteriostatic

The horizontal axis shows the concentration of Chitosan, and the vertical axis displays the cell growth in terms of OD values for all selected strains. Gram-positive (Staphylococcus aureus, Bacillus cereus, Streptococcus agalactiea) show more sensitivity towards Chitosan as given concentration than gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi) bacterial stains. Results showed that the dose depends on inhibition in cell growth. Interestingly, 75% has noticed in S.aureus, B. cereus and S agalactiea at concentration 0.156. 0,313 and > 0.313mg/mL respectively.

Figure 1: Effect of different concentration of Chitosan against the growth of S. aureus, S.agalactiea, B. cereus, S. typhii, P.aeruginosa and E.coli

DISCUSSION:

The Chitosan started shown antibacterial activity at the concentrations 0.039mg/mL, which remarkably increased by an increase in concentration up to 5 mg/mL. We were quite familiar with two different bacterial strains terms as Gram-positive (+ve) and Gram-negative (-ve). The basic difference between the two types is due to the difference in the chemistry of their membrane. In Gram-positive, the membrane is enriched with peptidoglycans, whereas Gram-negative contains lipopolysaccharide molecules.^42-43 Interestingly, both types show the difference in response towards antibacterial agents/drugs due to this difference in membrane chemistry. The molecules present on the membrane mark them negative charge as well. In our study, Chitosan derived from Tilapia Fish Scales possess strong antibacterial activity against gram-negative stains; Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi. The results supported the previous study, chitosan ability to induce antibacterial activity in both strains, while chitin possesses lower activity levels against these two types of bacteria. The previously proposed mechanism behind the gram-negative antibacterial activity, due to the attachment of phosphorylated groups to enriched lipopolysaccharide molecules, makes this type negatively charge. The presence of charge on categorized bacteria type makes them more susceptible. In previous studies, Chitosan shows more promising results in antibacterial activity due to the effective compatibility of negative charge bacteria and cationic Chitosan. However, the efficiency remarkably regulates by various physio-chemical conditions like pH (acidic).^44-46

Figure 2A (Left column A,B, and C): Time kills assay of Chitosan at 4x the MIC value against gram-positive bacteria; (A) Staphylococcus aureus, (B) Bacillus cereus; (C) Streptococcus agalactiea. Figure 2B (Right column A,B and C): Time kill assay of Chitosan at 4x the MIC value against gram-negative bacteria; (A) Salmonella typhii, (B) Escherichia coli, (C) Pseudomonas aeruginosa.

Similarly, our study showed more profound antibacterial activity against gram-positive stains; Staphylococcus aureus, Bacillus cereus, Streptococcus agalactiea. The activity might be due to electrostatic interaction present in gram-positive stains. Previous studies elucidate negative charge (i.e., Teichoic acids) in Gram-positive type for interaction.^47-48 However, few studies show the activation of resistive mechanism in gram-positive stains that could acquire resistance towards Chitosan. The resistance is mainly due to the downregulation of Teichoic acids, crucial in electrostatic attraction and killing the bacteria. The above acquiring resistance noticeable in Gram-positive stain, S aureus, shows that the resistive mechanism is a huge hurdle in susceptibility. Nevertheless, the potential biopolymers -adhesive nature aids in the adsorbtion of essential nutrient necessary for bacterial growth.⁴⁹ The minimal inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) ratio revealed the effective bacteriostatic nature of Chitosan towards gram-positive bacteria than gram-negative. The bacteriostatic activity well supported by previously reported research. Irreversible binding of Chitosan towards microbial cells proves that Chitosan is a bacteriostatic agent.^50-51 Previous studies revealed that chitosan activity is mostly growth-inhibitory,⁵² where bacterial cells can revive due to physiological adaptations of cells to chitosan stress. The growth curves were similar to the blank samples, which represented bacteria growth rate without the additions of any inhibitors—the ability of bacteria to regain rapid growth after the separation of chitosan solution via membrane filtration. The regrowth of bacterial cell treated with Chitosan and the irreversible binding of Chitosan to microbial cells, leading to inactivity against the remaining unbound microorganisms, proves Chitosan's notion of a bacteriostatic agent.^50-51 Moreover, other proposed mechanism of antibacterial activity besides the electrostatic interactions. The role of MW in inducing the sensitivity in bacterial stains regardless of acquire resistance by simply deletion of charge molecules. As we know, Chitosan is a polysaccharides polymer and could be available in various molecular weights. The heavy molecular weight (≤50 kDa) aid susceptibility in Gram-positive only via electrostatic interaction between thick membrane and chitosan molecule. However, in Gram-negative, the low MW (≤5 kDa) Chitosan remarkably induced transcription inhibition via direct bind with genetic material.^52-53 Our pipeline study might well support this. The use of oligosaccharides depicted the bacterial death mechanisms using COS. The low MW and a higher degree of deacetylated COS permeable to the membrane targeted the genetic material and fulfilled the mechanism.

CONCLUSION:

In conclusion, the antibacterial activity of Chitosan derived from fish scales noticeable against both gram-positive and negative bacterial strains. The antibacterial, biodegradable, cationic nature of Chitosan, mucoadhesive property, improving permeability, and efflux pump inhibitory quality mark chitosan as potential biopolymer various fields. Due to the presence of cationic groups, Chitosan triggers the remarkable potential for polymer-drug conjugation. The control release system with various drugs holds its place in nanoparticle drug delivery. However, using the benefit of chitosan derivatization and control release quality might show Chitosan as a successful candidate in biomedicines, especially drug therapeutics development.

CONFLICT OF INTEREST:

The authors have no conflict of interest.

ETHICAL STATEMENT:

This article does not contain any animal or human study.

REFERENCES:

1. Argar V, Asghari M, Dashti A. A review on chitin and chitosan polymers: Structure, chemistry, solubility, derivatives, and applications. Chembioeng Rev. 2015;2: 204–226. doi.org/10.1002/cben.201400025.

2. Yan N, Chen X. Sustainability: Don’t waste seafood waste. Nat News 2015;524:155. doi: 10.1038/524155a.

3. Dutta PK, Ravikumar MNV, Dutta J. Chitin and chitosan for versatile applications. Polym 2002;42:307–54.

4. Wan ACA, Tai BCU. CHITIN-A promising biomaterial for tissue engineering and stem cell technologies. Biotechnol Adv 2013;31: 1776–85. doi.org/10.1016/j.biotechadv.2013.09.007.

5. Kurita K, Chitin and chitosan: functional biopolymers from marine crustaceans. Journal of Marine Biotechnology 2005;8(3): 203–226. doi: 10.1007/s10126-005-0097-5.

6. Caprile MD. Obtencion y utilizacion de quitina y quitosano a partir de desechos de crustaceos. Congreso Mundial ISW: Haciaunsistema integral de residuossolidosurbanos, Argentina; 2005.

7. Chellat F, Grandjean-Laquerriere A, Naour RLe, et al., Metalloproteinase and cytokine production by THP-1 macrophages following exposure to chitosan-DNA nanoparticles. Biomaterials 2005;26(9): 961–970. DOI: 10.1016/j.biomaterials.2004.04.006.

8. Zhang L, Liu JN, Li L, Xia WH. Dietary chitosan improves hypercholesterolemia in rats fed high-fat diets.” Nutrition Research, 2008;28(6): 383–390. doi.org/10.1016/j.nutres.2007.12.013.

9. K. V. Harish Prashanth and R. N. Tharanathan, “Chitin/chitosan: modifications and their unlimited application potential—an overview,” Trends in Food Science and Technology 2007;18(3): 117–131. doi.org/10.1016/j.tifs.2006.10.022.

10. Kravanja G, Primozic M, Knez Z, Leitgeb M. Chitosan-based (Nano) materials for novel biomedical applications. Molecules 2019;24: 1960. DOI: 10.3390/molecules24101960.

11. Rabea EI, Badawy MET, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: Applications and mode of action. Biomacromolecules 2003;4: 1457–1465. doi.org/10.1021/bm034130m.

12. Kong M, Chen XG, Xing K, Park HJ. Antimicrobial properties of chitosan and mode of action: A state of the art review. Int J Food Microbiol 2010;144: 51–63. doi: 10.1016/j.ijfoodmicro.2010.09.012.

13. Gul-e-Saba, Abdah Akim, Hyaluronan-mediated CD44 Receptor Cancer Cells Progression and the Application of Controlled Drug-delivery System. Int J Curr Chem 2010;1(4): 245-265.

14. Chaudhry GS, Akim A, Zafar MN, Sung YY, Muhammad TST. Understanding HA-CD44 interaction, HA-CD44 activated potential targets in cancer progression and future therapeutics. Adv Pharm Bull Adv Pharm Bull 2021;11(3): 426-438. doi: 10.34172/apb.2021.050.

15. Chaudhry GS, Jan R, Zafar MN, Habsah M, Muhammad TST Vitex rotundifolia fractions induced apoptosis in human breast cancer T-47D cell line via activation of extrinsic and intrinsic pathway. Asian Pac J Cancer Prev 2019b: 3555-3562. doi: 10.31557/APJCP.2019.20.12.3555.

16. Gul-e-Saba, Murni Ismail, Noraznawati Ismail, Habsah, Tengku Sifzizul Tengku Muhammad. Induction of Apoptosis by Aaptos sp., fractions in human breast cancer. Int J Res Pharm Sci 20189(2), 328-237.

17. Hudaya T, Gul-e-Saba, Taib M, Ismail N, Mohammad TST. Methanol extract of four selected marine sponges induces apoptosis in human breast cancer cell line, MCF-7. Int J Res Pharm Sci 2017;8(3): 667-675.

18. Chaudhry GS, Rahman NH, Vigneswari S, Aziz A, et al. Cytotoxicity Effect and Cell Death Mechanism of Bruguiera gymnorrhiza Extracts on Human Breast Cancer Cell Line (MCF-7). J Adv Pharm Technol Res 2020;11(4): 233-237. doi: 10.4103/japtr.JAPTR_81_20.

19. Chaudhry GE, Sohimi NKA, Mohamad H, Zafar MN, Ahmed A, et al. Xylocarpus moluccensis induces cytotoxicity in human hepatocellular carcinoma HepG2 cell line via activation of the extrinsic pathway. Asian Pac J Cancer Prev 2021;1;22(S1): 17-24. doi: 10.31557/APJCP.2021.22.S1.17.

20. Chaudhry GE, Zafar MN, Yeong Yik Sung, Muhammad TST. Phytochemistry and Biological activity of Vitex rotundifolia L., Research J Pharm and Tech 2020;13(11): 5534-5538. doi: 10.5958/0974-360X.2020.00966.X.

21. Chaudhry GE, Nur Khairina Ahmed Sohimi, Zafar MN, Habsah M, Yeong Yik Sung, Muhammad TST. Induction of apoptosis by selected Xylocarpus sp., fractions in the human cervical cancer cell line, HeLa. Int J Res Pharm Sci, 2020;11(2): 2332-2339. doi.org/10.26452/ijrps.v11i2.2210

22. Chaudhry GE, Murni NIK, Zafar MN, Habsah M, Yosie A, et al. Induction of apoptosis by Stichopus chloronotus and Holothuria nobilis fractions in human cervical cancer cell line, HeLa. Int J Res Pharm Sci, 2020;11(1): 1238-1247. doi.org/10.26452/ijrps.v11i1.1964.

23. Chaudhry GE, Murni Islamiah, Muhammad Naveed Zafar, Habsah Mohamad, et al. Induction of apoptosis by Acanthaster planci sp., and Diadema setosum sp., fractions in human cervical cancer cell line, HeLa 2021;1(22(5)):1365-1373. doi: 10.31557/APJCP.2021.22.5.1365.

24. Chaudhry GS, Jan R, Habsah M, Mohammad T.S.T (2019a). Vitex rotundifolia fractions induce apoptosis in the human breast cancer cell line, MCF-7, via extrinsic and intrinsic pathways. Res Pharma Sci;14(3): 273-285. doi: 10.4103/1735-5362.258496.

25. Yunus U, Chaudhry GS, Sung YY, et al. Targeted Drug Delivery Systems: Synthesis and In-Vitro Bioactivity and Apoptosis Studies of Gemcitabine-Carbon Dots Conjugates Biomed Mater 2020;26(15(6): 065004. doi: 10.1088/1748-605X/ab95e1.

26. Imran M, Rehman ZU, Hogarth G, Tocher DA, Chaudhry GS, et al. Two new monofunctional platinum(II) dithiocarbamate complexes: phenanthriplatin-type axial protection, equatorial-axial conformational isomerism, anticancer and DNA binding studies Dalton Transacrions 2020;49: 15385-15396. doi.org/10.1039/D0DT03018J.

27. Mahar J, Saeed A, Gul-e-Saba Chaudhry et al. Synthesis, characterization and cytotoxic studies of novel 1,2,4-triazole-azomethine conjugates. J Iran Chem Soc (2020) doi:10.1007/s13738-019-01826-9.

28. Zafar MN, Masood S, Chaudhry GS, Muhammad TST, et al. Synthesis, characterization and anti-cancer properties of water-soluble bis(PYE) pro-ligands and derived palladium(ii) complexes. Dalton Trans 2019;48: 15408-15418. doi.org/10.1039/C9DT01923E.

29. Pookashree, Anitha R. In-vitro Antibacterial activity of Ethyl Acetate extract of Sesbania grandiflora leaf against E. faecalis – A root Canal threat. Research J Pharm and Tech 2016;9(12): 2147-2149. DOI: 10.5958/0974-360X.

30. Kanagavalli U, Sadiq AM, Sathishkumar, Rajeshkumar S. Plant Assisted Synthesis of Silver Nanoparticles Using Boerhaavia diffusa Leaves Extract and Evolution of Antibacterial Activity. Research J. Pharm. and Tech 2016;9(8): 1064-1068. DOI: 10.5958/0974-360X.

31. Kumari R, Singh S, Pradhan N, Chandni S, Karthik L, Kumar G, Bhaskara KV, Rao RSM. Optimized Media to Increase the Antibacterial Activity of Wild and Mutated Strain of Nocardiopsis VITSRTB. Research J Pharm and Tech 2014;7(2): 213-220.

32. Disha MD, Shahare HV, Gedam SS, Bhoyar PK, RO Ganjiwale. Aging of Honey Enhances Its Antibacterial Activity. Research J Pharm and Tech 2009;2(4): 872-873. DOI: 10.5958/0974-360X.

33. Aravind KS, Lakshmi T, Arun AV. Invitro Antibacterial Activity of Acacia catechu ethanolic leaf extract against selected acidogenic oral bacteria. Research J Pharm and Tech 2012;5(3): 333-336. DOI: 10.5958/0974-360X.

34. Pooja K, Umakant GS. Synthesis and Antibacterial activity of some newer Benzimidazole derivatives. Research J Pharm and Tech 2020;13(6): 2597-2600. DOI: 10.5958/0974-360X.2020.00462.X.

35. Shruthi C, Geetha RV. Antibacterial Activity of the Three Essential Oils on Oral Pathogens- An In-vitro Study. Research J Pharm and Tech 2014;7(10): 1128-1129.

36. Jobin J, Dhidhin R, Prashanth N. Microspheres - Novel Drug Delivery Carrier for Plant Extracts for Antibacterial Activity. Research J Pharm and Tech 2018;11(4):1681-1684. DOI : 10.5958/0974-360X.2018.00313.X

37. Kumar S, Mazumder A, Vanitha J, Ganesh M, Venkateshwaran K, Saravanan VS, Sivakumar T. Antibacterial Activity of Methanolic Extract of Sesbania Grandiflora (Fabaceae). Research J. Pharm. and Tech 2008;1(1): Page 59-60. DOI: 10.5958/0974-360X

38. El-Sayed MA, Kamel MM, El-Raei MA, Osman SM, Gamil L, Abbas Hisham A. Study of Antibacterial Activity of Some Plant Extracts Against Enterohemorrhagic Escherichia coli O157:H7. Research J Pharm and Tech 2013;6(8): 916-919. DOI: 10.5958/0974-360X

39. Toan NV. Production of Chitin and Chitosan from Partially Autolyzed Shrimp Shell Materials. The Open Biomaterials Journal 2009;1: 21-24. DOI: 10.2174/1876502500901010021.

40. Qi L, Xu Z, Jiang X, Hu C, Zou X. Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate Research 2014;339(16): 2693–2700. doi.org/10.1016/j.carres.2004.09.007.

41. Gnanamani A, Priya KS, Radhakrishnan N, Babu M. Antibacterial activity of two plant extracts on eight burn pathogens. J Ethnopharmacol 2003;86: 59-61. doi: 10.1016/s0378-8741(03)00044-8.

42. HanpanichOWongkongkatep P, Pongtharangkul T, Wongkongkatep J. Turn hydrophilic bacteria into bio renewable hydrophobic material with potential antimicrobial activity via interaction with chitosan. Bioresour Technol 2017;230: 97 102. doi.org/10.1016/j.biortech.2017.01.047.

43. Ma ZX, Garrido-Maestu A, Jeong KC. Application, mode of action, and in vivo activity of chitosan and its micro and nanoparticles as antimicrobial agents: A review Carbohyd Polym 2017;176: 257–265. doi.org/10.1016/j.carbpol.2017.08.082.

44. Kumar MNVR. A review of chitin and chitosan applications. React Funct Polym 2000; 46: 1–27. doi.org/10.1016/S1381-5148(00)00038-9.

45. Kraus, D, Peschel A. Molecular mechanisms of bacterial resistance to antimicrobial peptides. Curr Top Microbiol Immunol 2006;306: 231–250. doi: 10.1007/3-540-29916-5_9.

46. Chung YC, Su YP, Chen CC, Jia G, Wang HI, Wu JCG, Lin JG. Relationship between antibacterial activity of chitosan and surface characteristics of cell wall. Acta Pharmacol Sin 2004;25: 932–936.

47. PasquinaLemonche, L, Burns J, Turner RD, Kumar S. et al. The architecture of the gram-positive bacterial cell wall. Nature 2020;582: 294–297. doi.org/10.1038/s41586-020-2236-6.

48. Rohde M. The gram-positive bacterial cell wall. Microbiol Spectr 2019;7. doi: 10.1128/microbiolspec.GPP3-0044-2018.

49. Tsai JL, Chentsova-Dutton Y, Friere-Bebeau L, Przymus DE. Emotional expression and physiology in European Americans and Hmong Americans. Emotion. 2002;2: 380-397. doi: 10.1037/1528-3542.2.4.380.

50. Rhoades J, Roller S. Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods. Appl Environ Microbiol 2000;66: 80-86. doi: 10.1128/aem.66.1.80-86.2000.

51. Jarry C, Chaput C, Chenite A, Renaud MA, Buschmann M, Leroux JC. Effects of steam sterilization on thermogelling chitosan-based gels. J Biomed Mater Res 2001;58: 127-135. doi: 10.1002/1097-4636(2001)58:1<127:aid-jbm190>3.0.co;2-g.

52. Raafat D, Von Bargen K, has A, Sahl HG. Insights into the mode of action of chitosan as an antibacterial compound. Appl Environ Microbiol 2008;74: 3764-3773. doi: 10.1128/AEM.00453-08.

53. Kravanja G, Primozic M, Knez Z, Leitgeb M. Chitosan-based (Nano) materials for novel biomedical applications. Molecules 2019;24: 1960. doi: 10.3390/molecules24101960.

Received on 18.05.2021 Modified on 26.10.2021

Research J. Pharm. and Tech 2022; 15(10):4627-4632.

DOI: 10.52711/0974-360X.2022.00776