Antibacterial Efficiency of Croton Bonplandianum Plant Extract Treated Cotton Fabric

Croton bonplandianum ( C . bonplandianum ) is a monoecious exotic weed belonging to the euphorbiaceae family, is grown unwantedly at a large scale in the cultivation land of eastern India, along with the main crop. In this present work, sap from the C . bonplandianum plant is extracted through aqueous extraction method, which is further applied on a cotton fabric through pad-dry-cure method. The fabric obtained immediately after the treatment and from different stages of washing are then evaluated for their anti-bacterial activity against both the gram +ve and gram –ve pathogenic strains. It is observed that C bonplandianum sap and treated cotton fabric exhibit very promising results against both the gram +ve and gram –ve bacteria and it retains its properties even after five washes.


Introduction
Under the shed of protective textiles, antibacterial textile has its own importance and it becomes more significant whenever a pandemic situation (like ongoing Covid 19 situation) arises. In its inherent state, a textile material is susceptible to the microbial attack but with suitable chemical treatment it can resist the same. Degeneration of fiber, foul order, change in color, unwanted stains etc. are the symbols of microbial attack, mainly a symbol of bacterial attack on textiles [1][2][3]. Bacteria are unicellular organisms which grow rapidly under a suitable warm and humid conditions and some of them are deadly to the human races [4]. Hence, an antibacterial finishing on textile is very essential to protect its wearer from the infectious bacteria attack. Moreover, antibacterial fabric also helps to control secondary bacterial infection or co-infection during viral pandemics [5]. Lots of synthesized antibacterial agents are available in the market. A textile fabric treated with one of these synthetic chemicals exhibits excellent antibacterial behavior. However, these synthetic antibacterial agents are toxic and harmful to the environment [6]. Evaluation of an eco-friendly, bio-based antibacterial agent can meet the aforesaid contradictory demand of antibacterial agents for textiles. There are already some studies on bio-based antibacterial finishing on textiles has been carried out, among those sericin, chitosan, neem extract etc. shows very promising results. However, researchers are still going on with an aim of further improvement.
Croton bonplandianum (C. bonplandianum) a member from Euphorbiaceae family, is an exotic weed commonly known as three-leaved caper [7]. It is an annual herb having a length around 60cm long while its leaves are varying between 3cm and 5cm. A photographic image of a Croton bonplandianum plant is shown in Figure 1. C. bonplandianum is an unwanted plant grows in sandy clay soil along the roadside, irrigation canal banks, in plantations, in agricultural fields and on waste ground [8][9][10]. This plant is known for its meditational value for long time. The leafs of C. bonplandianum is antiseptic and are used for the treatment of skin diseases, cuts and wounds. They are also used for controlling high blood pressure and to cure fever caused infection in the glands. The latex of this plant has wound healing activity and the fresh juice of the leaves is used against headache [7,11]. Looking at the high meditational value of this plant, the present study aims to evaluate the antibacterial properties of C. bonplandianum plant extract when applied on cotton fabric. In this regard, fresh stem sap and aqueous leaf extract of C. bonplandianum are applied on the cotton fabrics through pad-dry-cure method. The treated fabrics are washed number of times to understand the durability of the treatment. Finally, the treated fabric and the fabric from different washing stages are evaluated for their antibacterial properties and test results are reported here.

Cytotoxicity of C. bonplandianum Plant extract
Potential toxicity of a plant extracts on living human cells determine its cytotoxicity when ingested, inhaled or being absorbed by skin. Cytotoxicity of a plant extract depends on its Phyto constituents, extracting solvent and on cell lines used to assess cytotoxicity [12,13]. Phyto constituents of any plant extract is the determining factor for its antibacterial activity. Extracting solvents ranging in polarity used to extract different plant parts and ultimately contributes to the wide variety of cytotoxicity results [13]. Researchers have prepared leaf extract of C. bonplandianum plant in different medium such as aqueous, methanol [14], acetone [11], hexane and ethyl acetate. It is observed that the aqueous extract of C. bonplandianum leaf is less cytotoxic compared to that of methanol, acetone, hexane and ethyl acetate extracts. Hence, to study the antibacterial activity of C. bonplandianum plant extract on cotton fabric, fresh stem sap and aqueous leaf extracts are used in this study. The aqueous extract of C. bonplandianum plant leaf contains carbohydrates, amino acids, proteins, tannins, saponins, flavonoids alkaloids, anthrocyanins, β-cyanins, quinones, glycosides, terpenoids, phenols, coumarins, steroids and resins [15,16].

Materials
Apparently healthy C. bonplandianum plants are collected personally by the researchers from the roadside of Bhubaneswar, Odisha, India. After collection, the plants are washed thoroughly for multiple times, initially using tap water followed by using deionized water in the lab. These cleaned C. bonplandianum plants are further used for the study purpose. A cotton fabric having a GSM of 95, 27 ends per cm and 24 picks per cm, warp and weft yarn count 32 s and 31 s Ne, respectively is used in the present study. The cotton fabric is purchase from the local fabric market of Bhubaneswar, India. Before the antibacterial treatment, the fabric is desized, scoured and bleached in the lab respectively. Bleached fabric is then treated with 5ml per liter citric acid which acts as a crosslinking agent between cotton fabric and C. bonplandianum extract [17]. Staphylococcus aureus (S. aureus), a gram +ve and Escherichia coli (E. coli) a gram -ve bacteria is used to check antibacterial activity of C. bonplandianum stem sap and leaf extract applied on cotton. The pure culture of abovementioned pathogenic strains is obtained from Orissa University of Agriculture & Technology Laboratory, Bhubaneswar.

Extraction of C. bonplandianum sap and its Application on Cotton Fabric
Fresh leaves and green soft stems are plucked manually from the cleaned C. bonplandianum plant, which are further used for the next process. During plucking white coloured latex or stem sap comes out from the damaged part of the plant. This sap is collected into sterile plastic containers and stored at 4ºC prior to its further use. On the other hand, the plucked leaves and stems are then grinded using a grinder after adding 100ml of distilled water to 500gms of collected leaves. The grinded paste is then filtered using a clean muslin cloth and then again through a Whatman filter paper. The filtrated is then kept in an air oven and is converted to powder form. Two different solutions of C. bonplandianum sap is prepared. In first case, 5ml of stem sap extract is added in 20ml of distilled water to prepared 200μl/ml s stem sap solution while in second cases 1gm of leaf extract powder is added in 20ml distilled water to prepare 50mg/ml aqueous leaf extract solution. Both the solutions are then applied on preprocessed cotton fabric through Pad-Dry-Cure method separately. Padding was done using a laboratory padding mangle with 100% wet pickup to maintain

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the add on of 200μl of stem sap and 50mgs of leaf extract per gram of fabric, respectively. Padded fabrics are then dried and cured at 140ºC for 180 seconds.

Washing of the treated fabric
Effectiveness after washing is one of the major criteria for any kind of finishing applied on textiles. If the durability of the antibacterial finish does not last for a good number of washes, then the antibacterial finish applied on the garment will be of no use. Hence the washing durability of the treated samples are very important. Both the fabric sample i.e. fabric treated with stem sap solution and fabric treated with aqueous leaf extract were washed in launder-o-meter for one, three and five wash cycles using 5grams per litre non-ionic detergent with M:L ratio 1:10. Finally, the antibacterial properties of all samples are evaluated.

Evaluation for bacterial growth
100μl of each type of the pathogenic strains contained in test tubes are added to each of the two Petri plates and spread all over by the means of the cotton swabs. Then by means of sterilized forceps, the treated fabric specimens are placed in each Petri plate. On first and second petri plates base fabric (F B ), fabric treated with citric acid (F CA ), fabric treated with stem sap solution (F SS ) and fabric treated with aqueous leaf extract (F LE ) were kept with different pathogenic strain. Similarly, in third and fourth plates, washed fabric samples are kept with different pathogenic strain. The petri plates are kept in an incubator at 37°C for about 24hours. After stipulated time, the antibacterial activity of the treated cotton fabric is evaluated by the agar diffusion method and the developed 'zone of inhibition' is measured in mm.

Results and Discussion
Zone of inhibition during antibacterial test of base fabric, fabric treated with citric acid, samples treated with stem sap solution and aqueous leaf extract solution, on different pathogenic strains by standard agar diffusion test method are reported in Table 1. The test results obtained from the study are revealed that C. bonplandianum stem sap and aqueous leaf extract possesses significant antibacterial property against S. aureus and E. coli bacteria. Fabric treated with stem sap solution showed a zone of inhibition 17±1.3 and 16.5±1.5mm against S. aureus and E. coli bacteria respectively, whereas aqueous leaf extract showed 15.5±1.2 and 15±1.3 mm respectively as shown in Figure 2. High inhibition rate against S. aureus and E. coli bacteria due to the presence of diversity of compounds including Phenols, flavonoids, alkaloids, tannins and triterpenoids in C. bonplandianum stem sap and leaf and extracts [18]. According to Mabhiza et al. [19], Alkaloids inhibits bacterial growth at a concentration of 1.67mg/ mL, comparable effects to ampicillin, a standard antibiotic. According to Xie et al. [20], flavonoids have excellent antimicrobial activity against a wide spectrum of microorganisms and could be an alternative for tackling the so-called "antibiotic resistance crisis". According to Ketema et al. [17], phenolic compound present in the plant extract showed excellent anti-bacterial activity against the gram-positive bacteria S. aureus as well as the gram-negative bacteria E. coli. Table 2 showed the zone of inhibition of treated fabric samples after washing. Even after 5 wash cycles, antibacterial activity of the C. bonplandianum stem sap and aqueous leaf extract treated fabric found promising. It is mainly due to the pretreatment of cotton fabric with citric acid which enhances the washing durability of the finished fabric.

Conclusion
The results of the present study indicate that the stem sap and aqueous leaf extract of C. bonplandianum possesses promising antibacterial activity against S. aureus and E. coli bacteria.
The effectiveness of the treatment on cotton fabric withstand even after 5 washes, which open up the avenue to apply the C. bonplandianum stem sap and aqueous leaf extract on textile fabrics to get antibacterial apparel products.