ANTIMICROBIAL ACTIVITY OF CHITOSAN, MORINGA OLEIFERA , CROTON ZAMBESICUS AND THEIR SYNERGISTIC ACTIVITY AS AN IMMUNE BOOSTER

The plants used for this study were selected from a collection of medicinal herbs collected from traditional healers during a prior ethnobotanical investigation. Chitosan derived from chitin is effective as an antibacterial agent in pharmaceutics. The Mauline, 2017 approach, which incorporates processing/maceration, demineralization,

The plants used for this study were selected from a collection of medicinal herbs collected from traditional healers during a prior ethnobotanical investigation. Chitosan derived from chitin is effective as an antibacterial agent in pharmaceutics. The Mauline, 2017 approach, which incorporates processing/maceration, demineralization, deproteinization, and deacetylation, was used to synthesize chitosan from crayfish and prawns. Moringaoleifera and Croton zambesicuswere chosen for their phytochemical capabilities, including antibacterial, anti-inflammatory, and antioxidant properties. These plants were acquired locally, and combined with the various chitosan extracts in powdered form. They were tested against selected clinically resistant bacterial and fungal isolates. Chitosan extracted from crayfish and the synergized extracts of Chitosan sources with Croton zambesicuswere effective against organisms like Candida albicans, Vibrio cholerae, Enterobacter cloacaeand Salmonella typhiwith 30mm, 13mm and 12mm zones of inhibition respectively. In Table 2, water and diethylether extracts of Croton zambesicus, synergized with one (1%) Chitosan extracted from crayfish were only effective against Bacillus cereus and Eschericha coli. During the experimental stages, both bacteriostatic and bacteriocidal effects were observed in some media cultures; however, the overall results from the synergy between Chitosan (crayfish and prawn extracts), Moringa, and Croton zambesicusunderscores the possibilities of exploring these combinations in novel drug designs.

…………………………………………………………………………………………………….... Introduction:-
Human health and the environment are inextricably linked. If the body is unfit owing to a weakened immune system (deficiency), infectious microorganisms such as viruses, bacteria, and fungi may readily attack, resulting in the emergence of numerous illnesses. Antibiotics or other synthetic medication treatments are often used to treat ISSN: 2320-5407 Int. J. Adv. Res. 9(10), 495-502 496 developing diseases in order to achieve an immediate healing effect. Another issue that will arise as a result of this practice is antibiotic and synthetic drug resistance. Furthermore, some medicines may cause adverse effects such as nausea, bone marrow destruction, thrombocytopenic purpura, and agranulocytosis, which can lead to other illnesses, necessitating the development of new therapies derived from natural sources (medicinal plants) (Sharififaret al., 2009). Modulating the immune system may help to prevent illnesses and free radicals from attacking the body. An immunomodulator is a substance that is able to modulate the function and activity of the immune system (Koruthuet al., 2011).
There is a dearth of information on the synergistic effect of these plants and their utility as an immune booster and in medication development. Antibiotic drug resistance in bacteria has forced a quest for new antimicrobial compounds from other sources, such as animal extracts and medicinal plants. Bacterial mechanisms have evolved their genetic capacity to gain drug resistance, particularly those used as therapeutic agents in the medical sector. For example, as many studies have shown, active efflux is a resistance mechanism evolved by most bacteria species against practically all antibiotics. Non-drug specific proteins, which make up the bulk of efflux systems, may recognize and export a wide range of chemically and structurally unrelated bacterial molecules without causing drug breakdown or modification (Kumar and Schweizer, 2005).
Chitosan is a biopolymer made from chitin, found in the exoskeletons of prawns, crabs, shrimp, lobsters, and crayfish. In the environment, these crustaceans can be found in both fresh and saltwater basins. Deacetylation of chitin in any of these crustaceans results in chitosan, a fibrous material. This fibre has strong antibacterial properties and can treat a wide range of microbial infections . Previous research has demonstrated that chitosan, with or without chemical modification, can be effective against microorganisms such as fungi and bacteria (Phaechamud, 2008).
Chitosan's antibacterial and anticancer characteristics are due to its structure, which encapsulates amine/acetamido and hydroxyl groups at 2, 3 and 6 carbon positions, respectively, in repeating units. In vivo and In vitro, chitosan's antibacterial action is efficient against many microorganisms (Rabea et al., 2003;Ganan et al., 2009). Chitosan can be used alone as an antibacterial agent (Islam et al., 2011). Also, it can be combined with ethnomedicinal plants to boost its antimicrobial effectiveness and use (Batista et al., 2003).Chitosan is available in three different types: alpha, beta, and gamma. The most common are alpha and beta, which have distinct molecular weights. The antibacterial activity of chitosan is influenced by the type of microbe, ambient circumstances, and the type of chitosan used (Hafdaniet al., 2011). Its poly-cationic characteristics can explain the antibacterial mechanism of chitosan. Chitosan's antimicrobial action method is based on the fact that it interacts with the pathogenic membrane's negatively charged phospholipids and inhibits its activity. It acts as a chelating agent, penetrates the cell wall to bind to DNA, changes membrane permeability, and leaks intracellular contents There is also a probability that the charged chitosan will interact with some essential minerals, reducing their potency and inhibiting microbial growth (Jiaet al., 2001). Chitosan's antibacterial efficacy improves as its concentration rises (Goy et al., 2016;Tayelet al., 2010).
The Moringa plant is grown for its leaves, fruits, roots, and seeds, which are used for various purposes, including food and medicine. Almost every component of the plant has nutritional value. The leaves and pods, on the other hand, are more commonly utilized as food or supplements. M. oleifera'syoung leaves are edible and are used in traditional diets in many areas where the tree grows. They are cooked or used as dried leaf powder in meals. Peanuts are made from seeds, and the oil is also edible. The treatment and prevention of malnutrition, particularly in children, is one of the most well-known applications of Moringa leaf powder. M. oleifera has a long history of medical usage in folklore. Plant parts other than the leaves, particularly the roots and seeds, are responsible for most of the plant's therapeutic applications.
Moringaoleifera Lam. is one of the species which is known for having the immunomodulatory activity to the immune system (Harriet, 2020; Gupta et al., 2010). This plant has a high value because almost all parts of the plant (leaf, roots, stems, flowers, fruit peel and seeds) can be used as highly nutritious food and also has been reported to have antimicrobial compound (Moyoet al., 2012). This plant also serves as an immune system builder and is used in some countries to overcome malnutrition and malaria (Thilzaet al., 2010). This herb was chosen due to its rich 497 phytochemical compound, which including saponins, carotenoids, phenolic compounds and flavonoids. Saponin and flavonoid can serve as natural immunomodulator that is expected to enhance lymphocytes cell development which is very important in the immune system (Wagner, 1999).
Croton zambesicusis is a medicinal plant that is primarily found in Africa. It is known by many names depending on where it is found. It is commonly known as "Ajekobale" in Ondo State, Nigeria, among other names. The leaves and twigs of the plant are used to extract the plant's therapeutic powers. The leaf decoction is used as an antihypertensive and antimicrobial (urinary infections) remedy in Benin (Adjanohounet al., 1989) and as an antidiabetic and malarial remedy in portions of Nigeria (Adjanohounet al., 1989). The Ibibios of Nigeria's Niger Delta region utilizes the roots as an antimalarial, febrifuge, and anti-diabetic . The root is also used in Sudan for menstruation pain and as aperients (El-Hamidi, 1970). Ngadjuiet al., 1999 and Boyomet al., 2002 investigated the composition of essential oils extracted from Croton zambesicusleaves, stems, and roots and discovered that the three types of oils were comparable in composition, with monoterpenes abundant in the leaves and stems and sesquiterpenes in the root bark. Spathulenol and linalool were revealed to be critical components of the root and stem bark oils, which were shown to be rich in oxygen-containing chemicals. Antiplasmodial, antidiabetic, anti-inflammatory, analgesic, and antipyretic activities have been reported for the ethanolic leaf extract Mcnulty, 2007), while the root extract has been reported to have antimalarial, anticonvulsant, and antiulcer activities. Ent-trachylobanediterpene, derived from dichloromethane extract of the leaves, shows the cytotoxic effect on Hela cells, according to Block et al. (2002). Antimicrobial properties of the leaf and stem have also been studied (Abo et al., 1999).

Ethno-medicinal surveys in Africa (Nigeria) have found that various plants can have beneficial therapeutic effects.
Moringaoleiferaand Croton zambesicusare among the two frequently cultivated and utilized plants that can be found in our habitats. Folk medicine practitioners and the Nigerian ethnomedicine for microbiological infections, antiinflammation, sexually transmitted diseases, malnutrition, and diarrhoea have also recognized the efficacy of these herbs in treating a variety of ailments. This study aims to extract, synthesize, and evaluate the antibacterial potential of Moringaoleifera, Croton zambesicus, and chitosan from crayfish in synergy to assess their antimicrobial efficacy and utility as an immune booster.

Sample preparation
Maulin's method (2017) was used for the synthesis and extraction of chitosan from two crustaceans: crayfish and prawn.

Plant preparation and extraction
Moringaoleiferaand Croton zambesicuswere acquired from the local market at Akungba-Akoko, Ondo State, Nigeria, and authenticated. The leaves were washed, dried and macerated to their powder form.

Raw materials (Prawn and Crayfish)
Benefitted shell Demineralized shell Chitin Chitosan 498 0.5g of Croton zambesicus, Moringa and chitosan powder/fibre (from crayfish and prawn) were weighed and solubilized in water. The extracts were diluted with DMSO and water in a ratio of 3:1. Moringa powder and chitosan from prawn was diluted with 5mL of water, while Croton zambesicusand chitosan extracted from crayfish were diluted with 7mL of water. The dilution factor is vital in order to get more extracts for the antimicrobial analysis.

Procedures for antibiotic susceptibilities screening
Cultures of Salmonella typhi, Pseudomonas aeraginosa, Enterobacter cloacae, Klebsiellaoxytoca, Vibrio cholerae, Staphylococcus aureus, and Candida albicans were used for this study. The plates were sterilized with an autoclave and divided into two groups; the first group was used to study the individual antimicrobial effect of the sample with these micro-organisms, while the second group was used to evaluate the synergistic antimicrobial effect of the samples on the micro-organism.
Mueller Hinton Agar (MHA) was prepared according tomanufacturer's specification and sterilisedto evaluate microbial growth and antimicrobial potential of the bacterial isolates. Using the direct pouring method, MHA was poured into the Petri dishes, was allowed to solidify, and then a cork borer with a diameter of 9mm was used to bore holes on the Petri plates. Five holes were bored, and the center was used as the control using a modified CLSI, (2016) method.
Broth culture needed for proper growth of the isolates was also prepared (3:10 w/v)this is to enable the microorganism viable in an artificial environment and evaluate its reaction and sensitivity. 3g of Nutrient Agar was dissolved in 100mL of water and sieved twice to extract the agar-agar, a solidifying agent for the media. The prepared broth was poured into 7 test tubes at an equal volume of 9mL, respectively, sterilized for 15 minutes using an autoclave. After sterilization, the broth was allowed to cool. After that, each test tubes containing the broth was inoculated with 0.5mL broth containing viable micro-organisms taken from an already prepared broth culture. The test tubes were labelled correctly accordingly, and then the streak method was used to introduce each of the microorganisms in respective plates.
The antibiotictetracycline was used as the positive control. Tetracycline is water-soluble; therefore, it was diluted with water to increase its surface area for better sensitivity. The syringe was used to introduce the control and the extracts into the plates.

Plant extracts and their antimicrobial activities
The Agar disc diffusion method (Mwitari et  Two different replicates of antimicrobial controls were used for each comparative analysis. A positive control named tetracycline was used in the microbial activities presented in first set of the assay, while water was used as a negative control for microbial activities presented in second set of the assay.
In the first antimicrobial analysis, the dilution ratio in 100mg/mL were; A: 0.09g of Chitosan from crayfish diluted in 0.9mL of solvent plus 1mL of Croton zambesicusdiethylether extracts. B: 1g of chitosan from prawn diluted in 10mL of solvent plus 1mL of Croton zambesicuswater extracts. C: 0.09g of Chitosan from crayfish diluted in 0.9mL of solvent plus 1mL of Croton zambesicusdiethylether extracts. D: 1g of Chitosan from prawn diluted in 10mL of solvent plus 1mL of Croton zambesicuswater extract.
In the second phase, the dilution ratio in 100mg/mL were: A: 1g of Moringaoleifera(diethylether extracts) was dissolved in 10mL solvent containing 2.5mL of DMSO and 7.5mL of water. B: 1g of Moringaoleifera(water extracts) was dissolved in 10mL solvent containing 2.5mL of 499 DMSO and 7.5mL of water. C: 0.25g of Croton zambesicus(diethylether extract) was dissolved in 2.5mL of solvent containing 0.6mL of DMSO and 1.9 mL of water. D: 0.25 of Croton zambesicus(water extracts) was dissolved in 2.5mL of solvent containing 0.6mL of DMSO and 1.9mL of water. One (1%) percent, that 0.1mL of Chitosan from crayfish was added to the 10mL prepared extract dissolved with DMSO to synergize it.

Results:-
This study shows the antimicrobial potential and synergistic properties of Croton zambesicus, Moringaoleiferaand Chitosan. Table 1 shows the results of antimicrobial assay studying synergistic reaction of water extracts of Croton zambesicus, Moringaoleiferaand Chitosan synthesized from both crayfish and prawn. Chitosan extracted from crayfish and the synergized extracts of Chitosan sources with Croton zambesicuswere effective against organisms like Candida albicans,Vibrio cholerae,Enterobacter cloacaeandSalmonella typhiwith 30mm, 13mm and 12mm zones of inhibition respectively. In Table 2, water and diethylether extracts of Croton zambesicus, synergized with one (1%) Chitosan extracted from crayfish were only effective against Bacillus cereus and Eschericha coli. Interpretive categories of zone diameter in the nearest whole mm Staphylococcus aureus    The in-vitro anti-microbial analysis for some extracts showed bacteriocidaland bacteriostatic effect which is also an inhibitory antimicrobial effect as also reported by James et al., 2019 thereby confirming the efficacy levels of medicinal plants and its susceptibility. Although, a plant might exhibit bacteriostatic effect, it might still be effective against microorganism, therefore other factors such as exploring different solvents and other micro-organism before conclusion is important.

501
In a bid to get the active compounds from the extracts for better comparism, the solvent diethylether was used for extraction as seen in table 2 above. The solvent extracts (diethylether) of Croton zambesicuswas susceptible against Bacillus cetus and Eschericha coli in a wide range of consideration, while the water extracts of Croton zambesicuswas susceptible to the same organism at an intermediary range. As seen in studies carried out by Goy et al., 2016 andTayelet al., 2010, the use of more amount of extracts in terms of the ratio and solvent have a huge impact on its microbial activities especially for chitosan. Therefore, it is worth noting that the potency of the Chitosan extract may be more accurately evaluated by increasing the concentration, as the zone of inhibition might be influenced by solubility and diffusion rate of the phytocompounds which might be the reason for no reaction with some of the microorganism seen in table 1 and 2. This factors taken into consideration will be useful for subsequent research on Chitosan, its commercialization and drug design.

Conclusion:-
It can be deduced that Chitosan in general can be used as a potential constituent in the quest for developing drugs such as immune boosters against prevailing disease conditions. This deduction is hinged on the fact that Chitosan was sensitive to a wide range of microorganism as shown in this research. Futhermore, the synergism of Chitosan with other ethnomedicinal plants such as Croton zambesicus, Moringaoleiferaand others are potential breakthroughs to new drug design that are cost effective which enhances a wide range of reachability to the community, Nigeria and the world at large.

Recommendation:-
This research is reproducible and translational. Exploring other solvents and ethnomedicinal plants in synergy with Chitosan for therapeutic purposes underscores the importance of further research. Furthermore, it is necessary to carry out in vivo studies to determine the toxicity of active constituents, their side effects, circulating levels, immunomodulatory effects, pharmacokinetic properties and diffusion in different body sites, for exploitation as an immune booster.