ANTIBACTERIAL EFFICACY OF SPECIES ON

PATHOGENIC BACTERIA 1 2,* Sonam S.K and Archana Tiwari

1School of Sciences, Noida International University, Uttar Pradesh, (India)

2Associate Professsor, School of Sciences, Noida International University, Uttar Pradesh, (India)

ABSTRACT The antibacterial activity of Aloe species such as Aloe barbadensis, , Aloe juvenna and Aloe maculata var. pulchra (Gasteria pulchra) leaf extracts were tested against Bacillus cereus. Agar well diffusion method was used to determine the inhibitory effect of leaf extracts of on Bacillus cereus. The leaf extracts of all the aloes showed varied levels of antimicrobial activity against the tested pathogen. The leaf extract produced inhibitory zone ranging from 10.3 mm to 22.3 mm against Bacillus cereus. Maximum zone of inhibition was given by Aloe rupestris (22.3 ± 1.52 mm) than Aloe maculata var. pulchra (Gasteria pulchra) (21 ± 2 mm), Aloe juvenna (13.3 ± 1.57 mm) and Aloe barbadensis (10.3 ± 0.57 mm). So it can be concluded that the leaf extracts of these aloes can be used against Bacillus cereus infection.

Keywords: Aloe barbadensis, Aloe Juvenna Aloe Maculata var. Pulchra (Gasteria Pulchra), Aloe Rupestris, anti-Bacterial.

I. INTRODUCTION

Medicinal play an important role in health care. Both infectious and non-infectious diseases have been cured by the action of the medicinal plants (Saba Irshad et.al., [2011]). Public health is facing many problems due to the microbial resistance to antibiotics. Medicinal plants are a rich source of antimicrobial agents and normally produce bioactive secondary metabolites and many of them exhibit activity, hence can be used as antimicrobial drugs (Muhammad, [1997]; Mahesh et.al., [2008]). Some antibiotic agents fail to bring an end to microbial infections due to super resistant strains. For this reason new researches are performed to invent new antimicrobial agents, either by the design and synthesis of new agents or through the search of natural sources (Cock, [2007]). Aloes are the large genus of plants containing over 500 species of flowering plants belonging to family from sub-Saharan Africa, Madagascar and parts of Arabia. Some species have been adopted as medicinal plants since ancient times and others are locally used in folk medicine. Aloe vera (Linn), also known as Aloe barbadensis of family Liliaceae is widely distributed in Asia, Africa and other tropical areas (Asma Bashir et.al., [2011]). It is a stemless or very short-stemmed spreading by offsets and root sprouts growing to 80- 100 cm tall in length. The leaves are lanceolate, thick and fleshy, green to grey-green, with a serrated margin

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(Surjushe et.al., [2008]). The are produced on a spike up to 90 cm tall, each pendulous, with a yellow tubular corolla 2-3 cm long (Sampath Kumar et.al., [2010]). The active compound includes aloesin, aloin, aloe-emodin, aloemannan, acemannan, aloeride, naftoquinones, methylchromones, flavonoids, saponin, sterols, amino acids and vitamins. The levels of these compounds are highly variable according to species and strain, as well as growth conditions in Aloe plants (Anderson, [1996]; Reynolds et.al., [1999]; Vogler and Ernst, [1999]; Ndhlala et.al., [2009]; Pandey and Mishra, [2010]). The leaf consists of inner gel and outer rind. The chemical composition of leaf pulp includes anthraquinones, anthrones, chromones, carbohydrates, enzymes, essential and non- essential amino acids, lipids, inorganic compounds, vitamins, proteins, and miscellaneous organic compounds (Shelton [1991], Vogler and Ernst [1999], Hamman, [2008]). It is reported that A. vera possesses antifungal, antiviral, antibacterial and acaricidal activity against skin infections such as acne, herpes and scabies (Mantle et.al., [2001]; Hart et.al., [1990]). Aloe rupestris is a fast growing mostly single stemmed tree aloe with very showy flowers. Leaves are deep green, unspotted and bordered by red teeth and form a spiralled rosette at the stem apex. 7-O-methylaloesin was isolated as a natural product from the leaf exudate of Aloerupestris (Daniel Bisrat, [2000]). Aloe juvenna is a tiger tooth aloe with multibranching stems. It is very attractive and forms smallish spikey rosettes from base and becomes redden on exposure to heavy sunlight. Aloe maculata var. pulchra (Gasteria pulchra) is very decorative, tall and succulent with long sword finger-like marbled foliage. The aim of this study is to evaluate the antibacterial efficacy of these Aloe species against pathogenic bacteria Bacillus cereus.

II. MATERIALS AND METHOD

Four different Aloe specimens such as Aloe barbadensis, Aloe rupestris, Aloe juvenna and Aloe maculata var. pulchra (Gasteria pulchra) were collected from different plant nurseries of Ghaziabad (Uttar Pradesh) and Alwar (Rajasthan) in India. Fresh leaves were dissected and washed properly with cold running tap water to remove the dust particles. Again it was rinsed in hot water to sterilize them and allowed to dry.

III. PREPARATION OF PLANT EXTRACT

Aloe leaves were harvested from different Aloe species. The base and the tip of the leaves were removed. The spikes were cleaned off by using a sterilized sharp knife. Again it was rinsed in running tap water so as to remove the yellow exudate that oozes out. The leaves so collected were lacerated into small pieces using sterilized knife and blended in an electric grinder to obtain the whole leaf extract. These extracts were collected in separate vials and stored at 4° C in refrigerator.

IV. TEST ORGANISM

Slant culture of Bacillus cereus was subcultured to inoculum using 0.85 NaCl2.

V. EVALUATION OF ANTI-BACTERIAL 5.1 Activity Antibacterial activity of Bacillus cereus was done in the plant extracts of Aloe barbadensis, Aloe rupestris, Aloe juvenna and Aloe maculata var. pulchra (Gasteria pulchra) by agar well diffusion method in triplicates. Nutrient

144 | P a g e agar was prepared and poured in petriplates. 100µL of the inoculum was spreaded on the nutrient agar. A sterile cork borer has been used for ensuring wells at the center of the agar plates. 50µL of plant extracts of different Aloe species were loaded in corresponding wells. Plates inoculated with the test organism and plant extract were incubated at 37°C for 24 hrs. The results so obtained from triplicates is processed by statistical means of Mean ± Standard deviation (SD) using the computer programme Microsoft Excel.

VI. RESULTS AND DISCUSSION

The anti-bacterial activity of Aloe barbadensis, Aloe rupestris, Aloe juvenna and Aloe maculata var. pulchra (Gasteria pulchra) leaf extracts were evaluated against Bacillus cereus by measuring the diameters of the zones of inhibition. It was found that all the Aloe species were effective against Bacillus cereus. The measurements of zone of inhibition varied in each species. Maximum zone of inhibition was seen in Aloe rupestris leaf extract than other Aloe species. The zone of inhibition is 22.3 ± 1.52 mm (Fig No. 2) whereas Aloe barbadensis possessed 10.3 ± 0.57 mm (Fig No.1) and Aloe maculata var. pulchra (Gasteria pulchra) showed 21 ± 2 mm zone of inhibition (Fig No.4) and least inhibition zone of inhibition was shown by Aloe juvenna i.e, 13.3 ± 1.57 mm (Fig No.3) (TABLE 1). Antibacterial activity tended to vary with Aloe species. Aloe juvenna was the least susceptible against Bacillus cereus. Among the four species tested Aloe rupestris participated effectively in inhibiting Bacillus cereus (Fig 5).

6.1 Figures and Tables Table 1: Measurements of Zone of Inhibition in Mean and Standard Deviation (mm) by Aloe Species. ALOE SPECIES ANTIBACTERIAL SENSITIVITY AGAINST BACILLUS CEREUS

Aloe barbadensis 10.3 ± 0.57

Aloe rupestris 22.3 ± 1.52

Aloe juvenna 13.3 ± 1.57

Aloe maculatavar. pulchra 21.0 ± 2.0

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Fig No.1: Antimicrobial Activity of Aloe Barbadensis Leaf Extract (11mm Zone of Inhibition) in Bacillus Cereus.

Fig No.2: Antimicrobial Activity of Aloe Rupestris Leaf Extract (23mm Zone of Inhibition) in Bacillus Cereus.

Fig No.3: Antimicrobial Activity of Aloe Juvenna Leaf Extract (14mm Zone of Inhibition) in Bacillus Cereus. 146 | P a g e

Fig No.4: Antimicrobial Activity of Aloe Maculata Var. Pulchra (Gasteria Pulchra) Leaf Extract (22 mm Zone of Inhibition) in Bacillus Cereus. TABLE 2: Antibacterial activity of leaf extracts of A.barbadensis, A.rupestris, A.juvenna and Gasteria pulchra against Bacillus cereus expressed in terms of mean. Experiments were performed in triplicates with ± standard deviation.

The leaf extract used contains almost all the active constituents such as anthraquinones, saponins etc. which have antibacterial property. Studies have proved that the leaf extracts includes whole leaf components such as anthraquinones, (Garcia-Sosa et.al., [2006]; Dabai et.al., [2007]) dihhydroxyanthraquinones (Wu et.al., [2006]), and saponins (Reynolds et.al.,[1999]; Urch, [1999] which have direct antibacterial properties. This fact supports our study. An investigation was carried out in aqueous and methanolic extracts of the roots of Aloe vera against Bacillus cereus and methanolic extract was found to more effective than aqueous extracts (Krishnakumar et.al., [2012]; Karpagam and Aruna, [2011]). Maximum antibacterial activity of Bacillus cereus was seen in methanol, ethanol and acetone gel extracts of Aloe barbadensis (Rubina et.al., [2009]). Martinez et al. [1996] observed no antimicrobial activity in the aqueous extract of A. vera leaves. Aloe vera gel showed no activity in dimethyl sulphoxide solution against Bacillus cereus (Thu, [2013]).

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Most of the studies have been carried out in the gel extracts with organic solvents of Aloe barbadensis. It was also found that the gel and Aloe vera juice were effective against Gram positive bacterias such as Staphylococcus aureus (Jothi, [2009]), Mycobacterium smegmatis, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus and Bacillus sphericus (Alemdar and Agaoglu [2009]), Bacillus subtilis (Pratibha et.al., [2011]), Streptococcus progenies, Streptococcus pyrogenes (Nwankwo et.al., [2014]); Gram negative bacterias such as E.coli, Shigella flexneri (Ferro et. al., [2003]), Pseudomonas aeruginosa, Klebsiella pneumonniae, Salmonella typhimurium, (Alemdar and Agaoglu, [2009]), Proteus sp.(Joshua et.al., [2010]) and fungus such as A. flavus, A. glaucus, Candida albicans, C. tropicalis, Trichophyton mentagrophytes and T. rubrum (Sitara et.al., [2011] ; Manafi, [2012] ; ArunKumar et.al., [2009]), Penicillium sps (Bandaru Narasinga Rao et.al., [2012]). Reports are there on the fact that the Staphylococcus aureus isolated from the wound infections is susceptible to both Aloe vera gel and ethnolic leaf extracts while Pseudomonas auruginosa was susceptible to Aloe vera ethnolic leaf extracts only (Okoko and Nwanade, [2010]). Methanolic extracts of Aloe barbadensis Miller inner leaf gel were fractionated by RP-HPLC and were tested for inhibitory activity and found that it was capable of inhibiting the growth of a nystatin resistant strain of the fungus Aspergillus niger (Cock I, [2007]). Kawai et.al., (1998) studied activity of Aloe arborescens Miller var. natalensis. Ali et al. [1999] found that the extracts of fresh leaves of A. barbadensis and A. arborescens have anti-fungal potential against Aspergillus niger, Cladosporium herbarum and Fusarium moniliforme. Aloe vera extract have been shown to inhibit the growth of fungi that cause tinea (Sumbul et.al., [2004]). Information regarding the antimicrobial activity of Aloe rupestris, Aloe juvenna and Aloe maculata var. pulchra (Gasteria pulchra) is lacking. In this present study it revealed that these aloes were more effective in inhibiting the growth of Bacillus cereus.

VII. CONCLUSION

All the four species such as Aloe barbadensis, Aloe rupestris, Aloe juvenna and Aloe maculata var pulchra (Gasteria pulchra) were effective against Bacillus cereus. There is only a slight difference among the antibacterial activity among the species. This study justifies that the leaf extracts of aloe species can be used as antimicrobial agents against Bacillus infection.

VIII. ACKNOWLEDGEMENT

This study was supported by School of Sciences laboratories of Noida International University. The authors also wishes to thank Vandana and Emraan Husain for technical assistance and for providing bacterial strain.

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