Antibacterial Effect of Hagenia Abyssinica (Bruce) JF Gmel Against on Selected Pathogens - Juniper Publishers

 Biotechnology & Microbiology - Juniper Publishers

Abstract

Hagenia abyssinica (Bruce) JF Gmel is a species of flowering plant and crucial for a remedy for intestinal parasites that appeared on humans. Globally medicinal plants used as alternative drugs acquired from plants naturally. This study was conducted to investigate the antimicrobial activities of different solvent extracts of Hagenia abyssinica (Bruce) JF Gmel flowers and leaves against 3 selected pathogenic microorganisms. At the same time to conserve it. Field surveys were incorporated to assess owns herbal medicinal values. The Experimental study design was performed/implemented, Microbiology laboratory of AASTU. Agar well diffusion method was used to In vitro bioassay. It was detected by measuring the zone of inhibition (excluding the diameter of wells) that appeared after overnight incubated at 370C. Ethanol and chloroform extracts showed significant inhibitory activity against Escherichia coli, Shigella flexneri, and Staphylococcus aureus. Ethanol flower extract was the highest impact on all test organisms (5mm). Ethanol leaf extract was the smallest effect against Shigella flexneri (2mm). Other studies already undertaken in Ethiopia and Brazil revealed that different medicinal plants have unique antimicrobial capacity or efficiency. It was conserved because it has implications for example to extraordinary research purpose and to save nature. Medicinal plants are fruitful to treat human and animal ailments. This study showed that different solvent extracts have strengths and weaknesses regarding inhibiting the growth of gram-negative species. Hagenia abyssinica (Bruce) JF Gmel were screened for an antimicrobial agent. The finding could reflect the areal part has a bioactive constitute. Determination of minimum inhibitory concentrations and full screening of phytochemicals were mandatory for extra advantages.

Keywords: Antibacterial; Crude extracts; Flower; Leaf; In vitro

Introduction

Hagenia abyssinica (Bruce) JF Gmel is a species of flowering plant and a native to the high-elevation Afromontane regions of central and eastern Africa. It is a multipurpose dioecious tree used as consumption, prevention and treatment of disease particularly a remedy for intestinal parasites particularly against Cestodes-Tapeworm in humans. Besides being a source of medicine, it has been utilized for various other purposes such as construction, furniture, fuel wood, and soil fertility management [1-4]. Almost all regions of the world, peoples are depending on traditional knowledge and medicinal plants to meet some of their primary health care needs. Medicinal plants have active biochemical compounds that function to the inhibit growth of pathogenic organisms [5,10]. Traditional medicinal plants are responsible for the treatment and preventive practice since prehistoric times. Local researchers focus on the identification and documentation of traditional herbal plants, but its potentials are scientifically uncharacterized [4,9]. Former studies do not provide sufficiently detailed information on the antibacterial profile of Hagenia abyssinica (Bruce) JF Gmel This paper may address the untouched features of scientific communities. Nowadays, Antimicrobial resistance has become a global concern. Drug scientists provide special attention to innovative plant-based antimicrobials instead of synthetic substances to combat pathogenic diseases. Natural products are considered as effective sources of novel compounds and termed as a core competitive advantage. There is a need to develop safe, highly effective and broad-spectrum antimicrobial agents that possess potent biological activities with a novel mode of action [11-21]. The number of diseases that infect humans not only increasing throughout the world but also the frequency of life-threatening infections caused by pathogenic organisms is also increasing worldwide. In recent years, antimicrobial characteristics of herbal plants are announced from various zones of continents. Normally, it is predicted that extracts exhibited target sites other than those used by antibiotics will be effective against multiple drug-resistant microbial pathogens [10].

The spread of drug-resistant microorganisms is a big threat to successful therapy of microbial diseases especially in children [15]. E.coli and Shigella are developing increasing antibiotic resistance. Usually taken antibiotics also cause diarrheal and antibiotic-associated diarrhea is the most common adverse effect of treatment with general antibiotics. Increased prevalence of resistant bacteria still now a scientific and political issue, together with absence and unfair cost of new generation drugs has escalated acute and chronic infection. Surprisingly, medicinal plants are used for the treatment of medical and clinical pathogenic bacteria. Infectious diseases of bacterial origins (Salmonella Spp., and Shigella Spp.) represent the major cause of morbidity and mortality in developing countries [22-26]. However, many Ethiopian medicinal plants are still waiting for scientific validation of their anti-microbial potentials [2]. Then searching for a new antibacterial agent is from medicinal plants is even more urgent in Ethiopia. The first aim of the study was to evaluate the in vitro antibacterial assay of the crude extracts of Hagenia abyssinica (Bruce) J.F. Gmel against Escherichia coli, Shigella flexneri and Staphylococcus aureus. Finally, this typical genetic resource was conserved via the ex-situ approach.

Materials and Methods

Study area

A preliminary field survey was carried out around Kotebe Metropolitan University All laboratory activities were conducted in Microbiology Laboratory, Addis Ababa Science and Technology University (AASTU) from April 1 to May 20, 2015 (Figure1). Addis Ababa is the Federal Capital of Ethiopia and a Chartered City, having three layers of Government City Government at the top, 10 Sub City Administrations in the Middle, and 99 Kebele Administrations at the bottom. It is located between 8055’ and 9005’ North Latitude and between 38040’ and 38050’ East Longitude. The total land area is covering 54,000 hectares. The date of establishment was November 1887 by Emperor Menelik II and Empress Taitu. The estimated population is More than 3 million. Its average elevation is 2,500 meters above sea level, and hence has a fairly favorable climate and moderate weather conditions. Addis Ababa is the capital city of Ethiopia, the seat of the African Union (AU) and the United Nations Economic Commissions for Africa (UNECA).

Research design

A cross-sectional based and in vitro experimental design was adopted to conduct the study. Hagenia abyssinica (Bruce) JF Gmel collected and conserved for sustainable utilization [4 ,17].

Sample collection

The samples were upper plant parts. Representative informants were asked to share their background, knowledge, and experience about Hagenia abyssinica (Bruce) JF Gmel formally. Effective communication occurred on it used part, utilization, means of application, mode of preparation, dosage and extra purposes seen in their life. Respondents were Semi-structured interviewed during their operation time to know the status of the species, drivers, barriers, management efforts and conservational activities taken place in the study area but excluded here.

Sample size and sampling technique

Twelve Hagenia abyssinica (Bruce) JF Gmel a species were selected for treatment of ailments caused by bacterial agents and also for planting. Purposive sampling was implemented with twenty key informants, such as elderly and traditional healers. They already had traditional and indigenous knowledge about it [4].

Plant material

Plant specimens were gathered from the Yeka sub-city around the Koteb site. In the study area, the vernacular name was termed as Kosso in the Amharic language. Hagenia abyssinica (Bruce) JF Gmel specimens were collected, pressed, submitted, identified and confirmed by a taxonomist and herbal experts. General authentications were carried out morphologically in National Herbarium, Addis Ababa University [4]. To ensure the desired plant species from the side of informants, Morphological identification key or attributes had inclusive (Figure 2).

Preparation of crude extracts

The raw plant parts (flower and leaf) were collected from their natural habitat in plastic bags and washed with distilled water to remove unnecessary particles like debris and dust. It was dried under shade at room temperature for about a week. The airdried plant materials were ground using a grinder or commercial blender to obtain a homogenous powder. The powders of each plant sample were weighed using the precision standard electronic balance. 1 gram of flower sample was immersed in each of three conical flasks containing 100ml distilled water, 95% ethanol, and chloroform solvent. The same procedure used for leaf also. Soaked samples were stayed for 72 hours with shaking with an orbital shaker (Gemmy Industrial Corporation, VRN-480) at 100 rpm and an occasional manual stirring with a glass rod. After 3 days, the macerates of each plant were filtered in separate flasks by using standard filter paper Whatman number 1. The residues after the filtration process were discarded while the filtrate parts were introduced to a vacuum rotary evaporator (GM- 0.3111, 30 letter /ml ultimate vacuum 50 Mbar) machine for extraction with at 25- 35 °C temperature adjustment. In addition to this extract, solvents were allowed to evaporate by using dry oven (35 °c for forty-five minutes). The dry weight of the extracts was used to determine the concentration in mg/ml. The remains dried extract was stored in a sterile container at +4 °C refrigerator for further application [2, 7,11].

Bacterial species

The three gram-negative bacteria (E.coli, S. aures and S. flexneri strains) known to cause foodborne bacterial infection. These test microorganisms used in the present study were obtained from the Microbial culture collection, New Delhi, India. Then it kept at -80 °C until use. The Bacterial strains were reactivated by subculturing on nutrient broth (HI media) at 37 °C for 24 hr. The Purity and viability of the organisms were checked by culturing nutrient agar (HI media) at 37 °C overnight in the presence of oxygen. It became maintained on nutrient agar slant at 4 °C for further activity. The selection criteria of bacterial were based on their contingency pattern of multi-drug resistance, cytotoxicity, and accessibility.

Antibacterial Assay

Muller Hinton agar was used for the antimicrobial tests whereas Nutrient agar (HIMEDIA Laboratories) was also used for routine stock cultures and sub-culturing. The medium was prepared plus autoclaved (121 °C and pressure of 15 pounds/ square inch /for 15 minutes) then cooled at 45-50 °C. Antibacterial activity of potential plant extract against bacterial pathogens by agar well diffusion assay technique (Figure 3). Fresh plate culture of 0.5 Mc Farland turbidity standard which corresponds to approximately 1.5 × 108 CFU/ml bacterial suspensions were uniformly spread on the solidified Mueller-Hinton (HIMEDIA Laboratories) plate via sterile swab to form lawn cultures. Then, allowed to dry for about five up to ten minutes. The use of a sterile Cork borer, holes of 9 millimeters in diameter was made in the seeded agar. With the help of micropipette, 50 microliters of each of the extracts (from the concentration 100 milligrams/ml or equivalent to 1 gram/ml stock solution) were introduced into the respective wells aseptically. Antibiotic was purchased from an authorized drug store in Addis. A working solution (50𝜇g/ml) of amoxicillin was prepared from a stock solution as a positive control. The experiment was performed in triplicate. The media were kept in the biosafety cabinet for 1 hour for proper diffusion. The plates were incubated at 37 °C for 24 hours aerobically. The final results were obtained by a measured zone of inhibition with a caliper [7, 11,17].

Data analysis

The outcome of antibacterial tests was examined, recorded and analyzed by applying Microsoft spreadsheet 2010 and finally taken the averages (Table 1). The ultimate inhibition zone was interpreted in a simple table.

Discussion

The present in vitro experimental study was reviled the antibacterial effectiveness of Hagenia abyssinica (Bruce) JF Gmel extracts against the target human pathogens. It could serve for future benefits and fill the concrete gap in the area of pharmacology. The ultimate evaluation outcome showed that ethanolic flower extract was more active than other water and chloroform extracts against tested organisms. It became verified by inhibiting and treating those selected bacteria strains. This study outcome helps to decide and declare the presence of bioactive compounds or phytochemicals abundantly. Previously reported papers indicated that medicinal plants have unique and pure biochemical constitutes like Alkaloids Glycosides, Saponins, Tannins, Flavonoids, and other unknowns. Those are lead to effective antibacterial activity against clinical isolates of Shigella and Salmonella [26,27].

Crude ethanolic extracts were used for the evaluation of biological activities because ethanol can extract most of the polar and moderately polar constituents. Based on past findings, in the current demonstration ethanol has been selected as a solvent to extract active compounds from the plant products [25]. In the present study, high anti-bacterial activities were exhibited from ethanol flower extract on E.coli (5 mm). And the minimum inhibition zone was observed on leaf extract in S. flexneri strains (2 mm). However, the activities were weak, and the zones of inhibition were generally much smaller than those made by the standard. It might be vital to reducing the negative consequences of antibiotics when the target plant is optimized. This result consisted of another research in Myanmar. E. coli appeared to be most strongly affected by 95% ethanol extract of Terminalia chebula Retz., with a 17 mm diameter zone of inhibition, while the standard, chloramphenicol, had a diameter of 24 mm [18].

Chloroform extracts of Hagenia abyssinica (Bruce) JF Gmel showed that limited. The methanolic extracts of A. indica showed maximum inhibition of both Salmonella (70%) and Shigella (80%) isolates at a concentration of 2 mg/ml which is almost comparable with tetracycline. but methanolic and chloroform extracts up to a concentration of 1 mg/ml were not able to inhibit bacterial growth appreciably suggesting the probable variations between the organic solvents used that result in different extract profiles in terms of quality and quantity [26]. During the assessment, water extract does not show a zone of inhibition against those microorganisms. As Semeneh et al.,[24] stated that the antimicrobial activities of ethanol methanol chloroform and water extracts of Moringa stenopetala Leaves against Escherichia coli pathogens was 8mm, 12mm, 11.5mm, and 8mm respectively through disc diffusion method. According to Alemtshay [2] ethanol extract of Guizotia schimperi Sch. Bip. ex Walp. showed higher antibacterial activity for the test S. aureus ATCC 33591, ATCC 33592, SA3 and SA5 strains (128–256 μg/ml) than oxacillin (512μg/ml). Petroleum ether extract of seed of Nigella sativa exhibited activity against both the laboratory isolated and certified strain of Bacillus cereus. Zone of inhibition indicated that 44 ± 0.31 mm and 40 ± 2.33 mm respectively as mentioned by Ketema [13].

Enterococcus faecalis was reported as the etiology of endodontic failure. The methanolic extracts of Eucalyptus galbie and Myrtus communis L. had antibacterial activity against E. faecalis. The diameter of the inhibition zone was near 9.6 mm and 7.6 mm respectively [17]. Ethanolic extract of Origanum vulgare demonstrated lower cell viability than the aqueous extract and has significant antiviral activity against some viruses of veterinary importance Equine Arteritis Virus (EAV) and both aqueous and ethanolic extracts have antiviral action against Canine Distemper Virus (CDV) [5]. Methanolic extract of Rhizome coptidis inhibits the early viral entry steps of Hepatitis C Virus infection, which deserves further evaluation for use as an anti-HCV agent [27]. During the evaluation study, tested bacteria was influenced by the type of extraction solvent and other laboratory condition. Ultimately could have caused variation in the antimicrobial activity results. For example, Staphylococcus aureus growth in the presence of plant extract had inhibited already depends on the amount of dose, time of incubation and concentration of plant extracts [22]. Bioassay of 50% ethanol Ginkgo biloba leaf extracts had an 18.25 mm inhibition zone against Pseudomonas putida in vivo [8]. Antibiotics had associated with adverse impact on the host including hypersensitivity, and allergic reactions. Probably this paper might explain the use of these plants by the indigenous people against many infections for generations. Hagenia abyssinica (Bruce) JF Gmel traditionally used for antihelminthic activity especially Tape warm. It could be a pivotal role in antimicrobial discovery. In recent years many studies are focusing on natural products for the screening of new and potential antimicrobial agents [6]. Purified active antimicrobial compounds from Persicaria pensylvanica may serve as an alternative natural product to treat staphylococcal infections in humans and animals [22].

Synergistic interactions of phytochemicals and antibacterial drugs were advanced approaches to reduce drug resistance and toxicity. Phytochemical synergists are functional as inhibitors of active site modification, enzymes that degrade/modify drugs, and permeability enhancers/efflux pumps [19]. The formulation of the synergistic combination was accepted in the world. When C. zeylanicum extract was used in combination with azithromycin, it showed strong synergistic activity against P. gingivalis and T. denticola. A combination of C. zeylanicum with metronidazole and tetracycline showed synergistic outcomes against A. actinomycetemcomitans and P. gingivalis, respectively [25]. Rubus chingii extract combined with fluconazole showed significant synergy against fluconazole resistant Candida albicans [3].

The action of the aqueous and Ethanolic extract of Hagenia abyssinica (Bruce) JF Gmel was particularly interesting to tested organisms, as the microbes were almost partially inactivated in the presence of the extract when compared with the control. The mechanism of action of ethanolic extracts against Eshercial and Shiglla spp was not fully determined. It might be sourced from the antagonistic approach of extract leads to inhibiting biological potentiality.

Plant-derived extracts and essential compounds had demonstrated antibacterial activity via interfering in particular with the protein synthesis, enzymes inhibition, production of cell wall complexes, formation of disulfide bridges and intercalation with the cell wall and/or DNA [26]. It was known that antimicrobial action can vary to various microorganisms and even in different stages of infection; the action may be extracellular, in the adsorption to the cells, intracellular, during the replication or also in the stage of the liberation of the viral, and bacterial and fungal particles [12, 23]. Microbial diseases are a challenge for human health not only clinical severity but also financial losses. The application of antimicrobial drugs for prevention and treatment had characterized by several restrictions such as reduced action spectrum, limited therapeutic utility, microbial resistance, high costs, and limited availability. The emergence of Multidrug-Resistant (MDR) pathogens due to the irrational use of these agents, an increasing number of immune-compromised individuals, aging, transplant complications, and stress. The resistance mechanism is target site/receptor modification mediated via mutation in the target site which leads to a decline in the antimicrobial efficacy of drugs. Typical examples of this kind of resistance are structural alteration in the Penicillin- Binding Proteins (PBP) and mutations in DNA gyrase and RNA polymerase, which render several drugs inactive [14]. All study informants were publicized creating awareness and communitybased management and plantation programs urgently needed and give more emphasizes. Then, Ex-situ conservation was adopted for the implication of many years, genetic stability, distribution, commercialization, and other related work activities by authors. According to Marma [16], conservation of biodiversity considered as a strategy for improving human health and well-being. It helps to mitigate climate change, generating income, accessing and benefit-sharing. In addition to this, create new dimensions of sustainable agriculture and forestry (bio-innovations) also [1]. In Ethiopia, herbal practitioners actively engaged in crude preparations. Biruktayet [4] suggested that Hagenia abyssinica (Bruce) JF Gmel. were efficiently utilized and overexploited by rural communities of Bale, Kofele, and Debark, but not conserved even in agroforestry ways and urgently announced should be taken action.

Conclusion

Ethiopia has a long history of using medicinal plants for the treatment of various diseases. Hagenia abyssinica (Bruce) JF Gmel flower, ethanol extract showed that more effective than the leaf extract against S. flexneri which indicates 5mm and 2mm respectively. To generate socio-economic returns and to save the genetic resource conservation always implemented. All study key representatives had participated and give full informed consent about Ethno-medicine and its implications, but not included in this paper. The outcome presented in the above section was useful to the proposed idea inside of clinical researches. To end, this paper could serve as a preliminary source to motivate others for future exploration.

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Antibacterial and Cytotoxicity Studies of Barringtonia Asiatica - Juniper Publishers

 Anatomy Physiology & Biochemistry - Juniper Publishers

Abstract

Objective: The hexane leaf extract of Barringtonia asiatica has biological activity, however, the study was carried out with an objective to ascertain its effects on Escherichia coli (ATCC©25922), Salmonella typhi (ATCC©14028), Staphylococcus aureus (ATCC©25923) and Kliebsielia Pheumonia and to evaluate the cytotoxicity of the leaves extract using brine shrimp lethality assay.

Methods:Barringtonia asiatica extract was evaluated for its antibacterial activity. Antibacterial activity assessment was performed by Disc diffusion assay the leaves of the plant were extracted with n-hexane, dichloromethane, ethyl acetate, chloroform and methanol and then vaporized to give respective extracts. Antimicrobial activity against Escherichia coli, salmonella typhi, Staphylococcus aureus and Klebsielia pneumonia, was determined. The optical density of the broth using UV mini spectrophotometer and zone of inhibition by the crude extract were determined.

Results: The results showed that of n-hexane extracts of varying concentration the 500ppm and 1000ppm displayed more activity with 4.00 ± 0.10, 4.30 ± 0.10b, 3.70 ± 0.10, 4.07 ± 0.12mm and 4.67 ± 0.12a, 4.35 ± 0.07a, 4.05 ± 0.07a, 4.55 ± 0.07mm respectively on all the pathogen subjected to the studies displayed where aSignificantly (p<0.05) higher compared to different extract at the same concentration b Significantly (p<0.05) lower compared to the control, than others at 25-1000 ppm per disc of the extracts concentration tested. However, the result of the cytotoxicity showed that Barringtonia asiatica Leaf extract were toxic on brine shrimp larvae with LC₅₀ value of 208.091 when compared with the control 7.455 thus having toxicity when referred to the fact that LC₅₀ value of less than 1000μg/mL is toxic while LC₅₀ value of greater than 1000μg/mL is non-toxic.

Conclusion: The present results showed the potential of the medicinal plant used by traditional herbal medical practitioners as natural antimicrobial agents, thus can be further used to determine the bioactive products that may provide as leads in the development of new drugs.

Keywords: Barringtonia asiatica; Extract; Cytotoxicity; Antibacterial

Introduction

Plants are important sources of medicinal products, they are recognized for their ability to produce a rich source of secondary metabolites and humans have long before now used many species to treat various kind of disease and ailment [1]. Barringtonia asiatica is a species of Barringtonia native to mangrove habitats on the tropical, it is a common plant in the Malaysian Mangroves and wetlands such as the Kuching wetlands Sarawak and Bako National Park,

It is also found in tropical Africa, Nigeria and Madagascar. Its large pinkish-white, pompon flowers give off a sickly-sweet smell to attract bats and moths which pollinate the flowers at night. It is grown along streets for decorative and shade purposes in some parts of Sarawakian houses and it’s also known as Box Fruit due to the distinct box-shaped of the fruit, it is a medium-sized tree growing to 7-25 m tall. [2,3].

The leaves are narrow obovate, 20–40 cm in length and 10–20 cm in width matured foliage colour is green, smooth glossy shiny leathery thick simple and evergreen. It is used as sausage food among the native of sarawakian in the kampong as well as a medicinal plant, inhabitants of several West African countries, Nigeria and the Polynesian Islands use liquid from the crushed bark of Barringtonia asiatica to treat chest pains and heart troubles. The same plant is used in Papua New Guinea to treat stomach-aches, the top leaves from this tree are squeezed into water and the liquid taken orally [4]. The plant when mature the bark texture is smooth and woody with the root type fibrous and tap root.

In cytotoxicity study, the brine shrimp cytotoxicity assay is considered as a convenient method for preliminary assessment of toxicity, testing. However, limited studies have reported bioactivities of Barringtonia asiatica and the antimicrobial activity

Thus, this in-vivo lethality assay is the simplest zoological organism (brine shrimp) which can be used as a convenient monitor for screening and fractionation in the discovery and monitoring of bioactive natural product, it is a general assay and capable of detecting various bioactivity present in crude extracts of medicinal plants and has been used as an indicator for general toxicity and as a guide for the detection of antitumor and pesticidal compounds. Since its introduction by Meyer et al. [5].

The aim of this research is study the hexane leaf extract of Barringtonia asiatica has biological activity and to ascertain its effects on Escherichia coli (ATCC©25922), Salmonella typhi (ATCC©14028), Staphylococcus aureus (ATCC©25923) and Klebsiella Pneumonia as well as to evaluate the cytotoxicity of the leaves extract using brine shrimp lethality assay.

Materials and Methods

All chemicals used in this investigation were of analytical grade and were obtained from SIGMA. Standard antibacterial agent (30μg) tetracycline, antimicrobial susceptibility test discs and Nutrient agar (CM0003) were obtained from Oxoid Ltd, Wade Road, Basingstoke, Hants, RG2 8PW, UK.

Preparation of agar plates

Preparation of agar plates was performed based on method described by Pundir and Jain [6].

Preparation of bacteria broth

All the selected bacteria were used to evaluate the antibacterial activities of the crude extracts of Barringtonia asiatica; Escherichia coli (ATCC©25922), Salmonella typhi, (ATCC©14028), Staphylococcus aureus (ATCC©25923) and Kliebselia pneumonia, (ATCC©19155). They were all obtained from the stock culture provided by Virology Laboratory, Universiti Malaysia Sarawak, the nutrient broth was prepared according to manufacturer’s instruction, with 2.6 g of the dried broth dissolved in 200 mL distilled water followed by sterilization in autoclave at 121°C.

The bacterial was sub-cultured in a 10 mL of broth, each in universal glass bottle for 16 hours inside an incubator equipped with shaker at 37°C [7]. After 16 hours incubation, turbidity (optical density/OD) of the bacterial broth was measured by using UV mini spectrophotometer (model 1240 of Shimadzu brand), comparable to that of nutrient broth standard tube for further use [8]. Measurement was performed at wavelength 575 nm and the bacterial broth was ready to be used when its turbidity was between OD 0.6 to 0.9. Nutrient broth was used to adjust the turbidity until the desired value was obtained.

Plate Inoculation

Inoculation of the bacteria was carried out in a biohazard cabinet and the procedure was based on method described by Pundir & Jain [6]. Approximately 1 mL of the ready bacterial broth were transferred into mini centrifuge tubes. A sterile cotton swap was dipped into the mini centrifuge tube containing bacteria broth and streaked over entire of the agar plate surface, performed in 4 different directions. The agar plate was then left for 5-10 minutes before applying the test samples.

The disc used was 6 mm diameter. A volume of 10 μL of the test samples of concentration 25, 50, 100, 250, 500, 1000 ppm were each pupated onto the discs and placed onto the agar plate by using sterile forceps and gently pressed to ensure contact. Next to be placed on the agar plate was the disc pupated with methanol as negative control, followed by 30 μg of tetracycline as standard antibacterial agent (positive control). The plates were left at room temperature for 10 minutes to allow the diffusion of the test samples and the standards into the agar. Each crude extract was tested in triplicate for each bacterium used. The plate samples were then incubated at 37°C for 24 hours before the inhibition zone around every sample disc being examined. The inhibition zone was measured in diameter to indicate the presence of antibacterial activity for each sample, as compared to the positive control.

Brine Shrimp (Artemia salina) Lethality Test

Toxicity test against brine shrimp (Artemia salina) developed by [5] was used in this study. The brine shrimp hatch, 1.5 g of Artemia salina cysts (Sanders Great Salt Lake, Brine Shrimp Company U. S. A.) was aerated in 1 L capacity glass container containing filtered seawater (collected from Damai beach in Kuching-Sarawak).

Air pump was fitted to the water to ensure complete aeration of the cysts after 48 hrs. of incubation at room temperature between 27-29°C under continuous illumination of fluorescence lamp, newly hatched free-swimming nauplii were harvested from the bottom of the glass container. The freshly hatched nauplii were used for the bioassay.

Exactly 5mg of sample was dissolved in 5 mL methanol, and the mixture was sonicated to ensure homogeneity of the extract. six different volumes of 500, 100, 50, 25, 10 and 1μL each from the stock solution were transferred into NUNC multidisc in triplicate. Solvent could evaporate under a running fume hood for overnight and followed by the addition of 0.2 mL DMSO and 4.8 mL seawater to give final concentration of 500, 100, 50, 25, 10 and 1 μg/mL, respectively.

Ten brine shrimp nauplii were transferred into each concentration in NUNC multidisc, and was observed every 6 hours for 24 hours. The amount of dead nauplii were calculated. Thymol was used as positive control, whereas 0.2 mL DMSO and 4.8 mL seawater was used as negative control. The data was analyzed to determine the concentration of the samples that kill 50% of brine shrimp at 24 hours or known as LC₅₀.

Statistical analysis

The results were expressed as means ± Standard deviation (SD) of three parallel measurements with one-way ANOVA. The LC₅₀ values for toxicity assay was calculated and determined by performing Profit analysis in IBM SPSS Statistic software of version 21.

Result and Discussion

Result

In the antibacterial and cytotoxicity studies, the hexane extract of Barringtonia asiatica exhibited the presence of antibacterial bioactive component (Table 1). The antibacterial activity of the extract was in concentration dependent manner. Activity was gradually increased with the concentration, from low concentration level to higher concentration level. The hexane extract exhibited dose dependent inhibition of bactericidal in comparison to the control.

Determination Values are Mean ± SD for five

aSignificantly (p< 0.05) higher compared to different concentration on same organism in each row bSignificantly (p< 0.05) higher compared to at the same organism at different concentration in each column.

The extract showed 4.00 ± 0.10mm, 4.30 ± 0.10mm, 3.70 ± 0.10mm, 4.07 ± 0.12 mm and 4.67 ± 0.12mm, 4.35 ± 0.07mm, 4.05 ± 0.07mm, 4.55 ± 0.07mm inhibition of activity at the doses of 500 and 1000 ppm, respectively while tetracycline showed 5.50±0.91mm, 5.68±0.59mm, 5.83±0.29mm, 6.73±0.77 inhibition of bacteria. While, at various concentration of the extract (1, 10, 25, 50, 100 and 500ppm) the average death of Artemia salina of hexane crude extract of the Leaf caused the death rate to increase with increase in concentration, given rise to LC₅₀ 208.091 μg/mL when compared to the test control thymol with LC₅₀ 7.455μg/mL. The result is mean+SD. N = 30 (Table 2).

Discussion

The hexane extract of Barringtonia asiatica various concentration gave an impressive inhibition against the pathogen (Salmonella typhi, E. coli, Staphylococcus aureus, Klebsielia Pneumonia) with a diameter of inhibition within the range of 2.35 ± 0.07mm and 4.67 ± 0.12mm for 25pmm-1000ppm. However, the crude extract showed a greater antibacterial activity against Salmonella typhi with inhibition zone of 4.67 ± 0.12mm at concentrations of 1000ppm, when compared to positive standard of tetracycline as well as other concentration with the inhibition zone.

The inhibition of Escherichia coli (E. coli) by the crude extract at various concentration is within the diameter range of 2.60 ± 0.10 to 4.35 ± 0.07mm for 25-1000ppm. Most of the crude extract inhibition gave an increase in the inhibition with increase in concentration, this was followed by Staphylococcus aureus and Klebsielia Pneumonia as shown in the (Table 1). Barringtonia asiatica at 1000ppm is significant and active on all the pathogen, with aSignificantly (p< 0.05) higher compared to different concentration in each rows and bSignificantly (p< 0.05) higher compared to different extract at the same concentration in each column.

However, the result of the cytotoxicity showed that Barringtonia asiatica Leaf extract were toxic on brine shrimp larvae with LC₅₀ value of 208.091 when compared with the control thymol at LC₅₀ 7.455 thus having toxicity when referred to the fact that LC₅₀ value of less than 1000μg/mL is toxic while LC₅₀ value of greater than 1000μg/mL is non-toxic.

Conclusion

The hexane extract of the leaves of Barringtonia asiatica indicated varied levels of antibacterial activities. The concentration of the plant extracts at various concentration level exhibited a high cytotoxicity activity on Brine shrimps. Thus, the plant is said to have a reasonable potential as antimicrobial compounds against microorganisms especially in the case of Salmonella typhi, E. coli, Klebsielia Pneumonia and lastly Staphylococcus aureus with increase in concentration. However, the plant extracts can be used as a novel drug against the development of resistance strains and in the treatment of infectious diseases caused by resistance bacteria.

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Antibacterial Finishing of Cotton Textiles with Extract of Citrus Fruit Peels - Juniper Publishers

Fashion Technology & Textile Engineering - Juniper Publishers

Abstract

The objective of this study is to explore, evaluate and compare the antibacterial activity of a cotton fabric treated with essential oils extracted from green, orange & black (a mixture of both green and orange) lemon peel (Citrus limon). The Citrus limon peel is rich in nutrient such as flavonoid and essential oil that can be used for antimicrobial activity. The finishing agent, lemon peel extract was extracted by treatment with methanol using steam distillation techniques. The antimicrobial activities were evaluated against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria by zone of inhibition measurement. Cotton finished with green lemon peel extract showed strong antimicrobial activities against Staphylococcus aureus (24-30mm) and Escherichia coli (22-26mm) bacteria as compared to orange and black lemon. Black (50% green and 50% orange) lemon peel extract showed better antimicrobial activities against Staphylococcus aureus (18-26mm) and Escherichia coli (18-25mm) bacteria than the orange lemon peel. In addition to this, the durability of the natural finishing agent on cotton was measured before or after washing and has got the same effect. From this study, it was revealed that citrus lemon have better, durable antibacterial potential with the green lemon peel extract having a more efficient effect compared to others.

Keywords: Antibacterial; Lemon peel; Essential oil; Zone of inhibition; Cotton fabric

Introduction

The antimicrobial finishing process imparts the ability, to textile substrate, to inhibit the growth (-static) or reproduction of at least some types of microorganisms or to kill (-cidal) at least some types of microorganisms” [1-3]. Therefore, an antimicrobial finish should be capable to kill the microbes by breaching the cell wall or alter cell membrane permeability, obstructing the synthesis of proteins of microbes, blocking enzyme production necessary for microbes’ food [1]. The major use of the antimicrobial was in the medical and the pharmaceutical industry. However, newer applications are possible. The textile fibers are these days increasingly treated with antimicrobial reagents. The other examples include the applications in food packaging and food storage, and medical, surgery and hygienic products etc [4-6]. With the improvement of life standards, the demand of hygienic products is increasing for biocidal finishes in textiles (sports-wears, undergarment, bedlinen) and water filtration. The antibacterial finish treatment has become vital area of medical, surgical and healthcare activities due potential pathogenic microorganisms present in hospital environment and cause cross-infection diseases [7-11]. The types of micro-organisms include different kinds of organisms such as virus, bacteria, unicellular plants and animals, certain algae and fungi [12]. Classification in bacteria family is “gram- positive, gram-negative, spore bearing or non-spore bearing type”. Some of the bacteria are of pathogenic nature that may cause infections to human [13].

A gram-positive bacterium contains peptidoglycan and teichoic acid, peptidoglycan comprises of 90% of cell walls and made of amino acid and sugar. One example of gram-positive bacteria is Staphylococcus aureus (S.aureus) that is in form of pair, short chain or graphic like cluster. Its size range is 0.5μm to 1.0μm and grows in temperature range of 35 to 40°C. S aureus is major cause of cross infection in hospital environment and 19% of total surgical infection. It’s also responsible for boils and also cause scaled skin infections. Gram-negative bacteria are firm to reduce as compared to gram positive bacteria for the reason that of extra cell walls. An example of gram-negative bacteria is Escherichia coli (E. coli); its shape is similar to a bacillus and dwell in intestine of human. E. coli can be proliferated during eating and/or usage of raw food stuff [13]. Antimicrobials control the growth of microorganisms and their negative effects of odour, staining and deterioration. Antimicrobial finishing prevents or inhibits the growth of microorganisms or microbes. The enormous majority of antimicrobials work by leaching or moving from the surface on which they are applied [14-16]. Most modern antimicrobial finished textiles are based on synthetic products, and current consumer demands must be correlated with obtaining environmental friendly final products. Conventional antimicrobial finishing includes treatment with quaternary ammonium compounds, triclosan, N-halamines, poly biguanides, nanoparticles of noble metals (nanosilver treatment) and metal oxides [17,18] but also treatment with titanium oxide doped with various elements for photocatalytic and antimicrobial induced properties.

New trend in antimicrobial finishing promotes plant-based dyes [19] over synthetic ones that can also act as antimicrobial agents [20]. Plant extracts provided an attractive source of eco- friendly antimicrobial finish. The natural cure using plant extracts is increasingly receiving interest in the development of antimicrobial textiles. Plant extracts can be used as finishing agents during textile processes or can be encapsulated for inducing controlled release properties (acacia based capsule wall filled with herbal extracts [21]. Current researches regarding use of various plants extracts for the treatment of antimicrobial finished fabrics include functionalization of 100% cotton bed linen fabric with neem (Azadirachta indica) and Mexican daisy extracts [22], fabrics treated with turmeric rhizomes extract (Curcuma longa) pomegranate fruit rinds extract (Punica granatum), aloe vera extract [23], tea oil, eucalyptus oil, tulsi leaves extract, with high antimicrobial efficiency [24] against a series of fungi and Gram-positive and Gram-negative bacteria [25]. Beside extracts treatment, bioactive functionalisation of textile fibers includes compounds such as phenolic and polyphenols, alkaloids, lectins, poypeptide, polyacetylene, terpenoids etc [26]. Although the antimicrobial properties of various plants extracts has been thoroughly researched, the antimicrobial active functionalization of textile materials using plants extracts still require intensive documentation.

Lemon (Citrus sinensis) peel is an agro-horticultural waste produced in huge quantities from various fruit processing industries. It is normally discarded and dumped in the environment that can create environmental concerns [27,28]. Citrus limon belongs to Rutaceae family; its common name is lemon and this originated from South East Asia, probably in India or Southern China. Lemon is a pale yellow, elliptical or globe shaped berry fruit. Citrus fruit, in general contain sugar, polysaccharide, organic-acid, lipids, carotenoids, vitamins, minerals, flavonoids, bitter lemonoids and volatile compounds. Lemon is a good source of potassium, calcium & vitamin C. Limon or lime juice have been reported to exhibit antimicrobial activity against Vibrio cholera [29, 30]. Citrus by-products, if utilized fully, could be major sources of phenolic compounds. The peels, in particular, are an abundant source of natural flavonoids, and contain higher amount of phenolics compared to the edible portions. It has been reported that the contents of total phenolics in peels of lemons, oranges, and grapefruit were 15% higher than those in the peeled fruits [27,31]. Flavonoids in citrus are a major class of secondary metabolites. The peel contains the highest amount of flavonoids than other parts and those flavonoids present in citrus fruits belong to six peculiar classes according to their structure. They are: flavones; flavanones; flavonols; is of lavones; anthocyanidins and flavanols [31].

Recent research suggests that citrus fruits possess another health benefit phytochemicals called limonoids, highly oxygenated triterpenoid. Citrus limonoids appear in large amounts in citrus juice and citrus tissues as water soluble limonoid glucosides or in seeds as water insoluble limonoid aglycones. The limonoid aglycones are responsible for the development of delayed bitterness in citrus and are converted to the non-bitter limonoid glucosides during fruit maturation [27]. The objective of the present work is imparting antimicrobial finish on cotton by using natural fruit peel extract to fabric and to reduce the effect of microorganism on human body and a fabric.

Materials and Methods

Materials and Chemicals

Lemon fruits (Citrus sinensis) were obtained from local Ethiopian market. The lemon peels were collected immediately after the fruit was peeled. Full bleached plain cotton fabric with 24 ends per inch, 18 picks per inch and having a GSM of 142g/ m2 supplied by Kombolcha Textile Share Company was used. All chemicals used were of analytical-reagent grade and obtained from Chemical Engineering Laboratory (KIOT, Ethiopia).

Experimental Procedure

In this study the following experimental procedure was followed to finishes cotton with ant-bacterial by treatment with essentials oils of lemon peels (Figure 1).

Preparation of lemon peel

The lemon peels were obtained from the local juice vendors in Kombolcha (Ethiopia). The fruits collected from local market must be fresh and not affected bacteria before then it will change the properties of microbes during application and testing. The peels were sorted, cleaned and washed in sterile distilled water, air dried and peeled off further; the peels were dried in sun, packed in envelops for drying in hot air oven at 65℃ for 3 days and used as raw material for the extraction of antimicrobial compounds.

Methods of Extraction of Essential Oil

Essential oils were obtained by steam distillation from lemon peels. Steam distillation was preferred to direct extraction by heating, in order to avoid loss and denaturation of constituent chemicals. The cotton fabric was treated by impregnation with essential oils of orange and green colored lemon peel separately. In this study three different essential oils was extracted from orange, green and black (mixture of orange and green) lemon peel (Table 1).

Extraction Mechanism of Essential Oil from Lemon Peel

A green and orange colored lemon was used in this study to extract essential oil for coating of cotton fabric in order to impart antibacterial finish to cotton. Steam distillation techniques were used for extraction of the essential oils from lemon. The essential oils were separated from water using density separator methods. The extraction process for the green lemon and orange lemon was presented in (Figures 2&3) respectively.

Optimization of Application Conditions

Pad-dry-cure technique was used in this study to apply the extracted essential oils onto cotton fabric. Optimization of the pad-dry-cure process was done by varying squeezing pressure, drying temperature, drying time, curing temperature and curing time during the application of the essential oils on cotton. For finishing of the extracted natural antimicrobial agent on cotton optimization of application conditions were tried at 60, 70, 80, 90 and 100℃ of drying temperature; 3, 5, 7, 9 and 11 minutes of drying time; 120, 130, 140 150 and 160℃ of curing temperature; 1, 2, 3, 4 and 5 seconds of curing time. This optimization process was done separately for green, orange and black lemon peel types. Throughout this study, all the samples and results presented are at the optimized applications conditions after a number of trials.

Evaluation of Antimicrobial Activities

The antimicrobial activity of the samples was tested against E.coli as gram negative bacteria and S.aureus as gram-positive bacteria. The antibacterial activity was tested by means of liquid (turbidity evaluation) and solid medium (agar diffusion) antibacterial using nutrient broth medium [32,33]. Antibacterial finishes on cotton fabric was assessed according to AATCC 100- 2004 standard test method. Test specimens were cut in 4.8 +0.1 cm diameter using a steel die. 100μl working culture inoculated test specimens, individually in sterile Petri plates. After inoculation, specimen were placed screw cap jar contained 100ml neutralizing agent. The toxicity of neutralizing agent against tested organisms was reexamined and no toxicity was determined. Jars were shaked vigorously for one minute, serial dilutions were made. From each of three suitable dilutions, 0.1ml liquid was drawn and transferred to TSA [34]. The number of survivors was determined after 48 hour incubation at 37°C by counting the colonies as colony forming units per millimeter (CFU/ml) using a colony counter device (Acolyte Super colony Counter, Symbiosis). Furthermore, additional jars were prepared to provide information about the bactericidal activity of treatment over contact period (60 minutes).

Durability is tested by two ways, by washing and by zone of inhibition. The durability of antimicrobial activity by washing is one of the major concerns of textile researchers and users because textiles are subjected to frequent laundering. The antimicrobial activity of the finished samples was evaluated after being subjected to several wash cycles by ISO: 6330.2012E. In this investigation 5 times washing cycles was used for durability test throughout the whole observation. The content of lemon (Citrus Limon) peel in antimicrobial textile before or after washing was measured and has got the same effect [35]. This is the reason that the antimicrobial textile treated with lemon (Citrus Limon) peel had good antimicrobial ability after 5 times washing cycles and also implied that even only small quantity of essential oil extracted from lemon peel existed on fabrics would offer a good inhibition to E coli and S aureus.

Results and Discussion

In this research work, the antibacterial activities of orange and green colored lemon peel extract were determined and compared by finishing cotton fabric. The result shows the antimicrobial activity on cotton fabric after 5 washing cycles. The gram positive and gram-negative bacteria were taken to observe their effectiveness and since they behave differently for citrus lemon finishes.

Extraction of the Essential Oil

The same procedure and parameters were used to extract the essential oils from the green and orange colored lemon. 10 grams of each plant materials were dissolved separately in 100ml of distilled water with 10ml of methanol and heated at 100℃ for 75 minutes. In this steam distillation technique of finishing agent extraction the essential oil was separate from water using density separator.

Optimization of the Application Conditions

Coating of cotton with natural finishing agent obtained from lemon peel extract were carried out at different application conditions in order to determine the maximum inhibition zone and minimum scorching of the fabric. More effective antibacterial activities were obtained at the optimal parameters in the pad-drycuring process and the results were presented in Table 2.

Antibacterial Activity of Lemon (Citrus Limon) Peel Extract

The lemon (Citrus Limon) peel extract were determined for their antibacterial activity against E. coli and S.aureus using muller hinton agar (MHA) method (Figure 4). Diameter of inhibition zone of lemon (Citrus Limon) peel extract was shown in Table 3. From Table 3 and Figure 5, it was observed that the antibacterial activities of green lemon peel extract finished cotton showed higher effectiveness in both gram-negative (E.coli) and gram-positive (S.aureus) than orange and black lemon. (Figure 5&6) indicate the maximum and minimum zone of inhibitions against both types of bacteria respectively. Cotton sample which was finished with green lemon peel is more effective under E.coli types of bacteria by 22-26 mm zone of inhibition and under S.aureus bacteria by 24-30mm zone of inhibition. This result showed that maximum zone of inhibition against both types of bacteria was observed by finishing of cotton with green lemon peel extract. However, the antimicrobial activity of black (mixture of green and orange) lemon peel extract finished cotton sample showed better effect than the orange lemon peel extract treated cotton in both under E. coli types of bacteria by18-25mm and under S.aureus bacteria by 18-26mm zone of inhibition. The black sample which compose a mixture of 50% green and 50% orange lemon peel was more effective than 100% orange lemon because of the combinational effect.

Conclusion

In this study, the antibacterial activities of green, orange and black colored lemon was discussed and compared. The result shows that the coating of cotton using lemon peel extracts have good antimicrobial activity against Escherichia coli and Staphylococcus aureus bacteria. Green lemon peels extract finished cotton showed strong antimicrobial activity against these bacterial. Finishing of cotton using natural plant products waste like lemon peel has high potential to exhibit antimicrobial activity. 

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