Antibacterial effect of Dimethyl sulfoxide extract of Aloe vera ( Aloe barbadensis ) leaf gel against Staphylococcus aureus , Pseudomonas aeruginosa , Escherichia coli and Klebsiella pneumoniae

An exploratory study based on laboratory experiment was carried out to determine the antibacterial effect of Dimethyl sulfoxide (DMSO) extract of Aloe vera leaf gel (DAE) against standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae in the Department of Pharmacology & Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. DMSO extract was used in five different concentrations (100, 200, 300, 400 and 500 μg/ml). Dose dependent inhibitory effect was seen against the test organisms using disc diffusion method. Zone of inhibition (ZOI) were 8 mm, 13 mm, 15 mm, 16 mm and 21 mm against S. aureus; 0 mm, 8 mm, 13 mm, 15 mm and 18 mm against P. aeruginosa; 8 mm, 11 mm, 13 mm, 16 mm and 20 mm against E. coli; 0 mm, 9 mm, 12 mm, 14 mm and 18 mm against K. pneumoniae at 100, 200, 300, 400 and 500 μg/ml respectively. The minimum inhibitory concentration (MIC) was assessed by broth dilution technique. The MICs of DAE for S. aureus, P. aeruginosa, E. coli and K. pneumoniae were 300 μg/ml, 400 μg/ml, 400 μg/ml and 450 μg/ml respectively. From the study it was observed that DMSO extract of Aloe vera leaf gel possesses antibacterial effect against the test pathogens. The findings highlight the need for further extensive study to detect and isolate the biologically active ingredients present in the Aloe vera leaves which are responsible for antibacterial effect. Hopefully, that would lead to the discovery of new and more potent antimicrobial agents originated from Aloe vera.


Introduction
Microorganisms such as bacteria, fungi and virus have the ability to cause life-threatening infections in human beings all over the world. Diseases due to the pathogenic bacteria represent a critical problem to human health and safety. Antimicrobial resistance is a serious threat to mankind because many of the infection causing bacteria have become multidrug resistant. The resistance developed by the bacteria to various drugs restricts the choice of antibiotics for therapy. It is of utmost importance to search for new infection fighting strategies. 1 For centuries, medicinal plants have been used in the traditional system of medicine in the curing of various types of diseases. Different species of medicinal plants have been analyzed for biologically active ingredients known to have pharmacological properties and many of the studied plants have shown antimicrobial property. 2 The use of plant extracts, with known antimicrobial properties, can be of great significance in the treatment of various microbial infections. 3 Aloe vera (Aloe barbadensis miller) is a plant, which belongs to the Liliaceae family and is mostly succulent having a whorl of elongated, pointed leaves. The name is derived from the Arabic word 'Alloeh' which means 'bitter'. It refers to the bitter taste of the liquid contained in the leaves. Modern clinical use of Aloe vera began in the 1920s. Since then, numerous researches have been conducted regarding its significant therapeutic potentials when used both topically and parenterally. 4 Aloe vera is a stem less or sometimes very shortstemmed succulent plant growing up to 60-100 cm tall. The green leaves are thick and fleshy with some varieties showing white flecks on the upper and lower stem surfaces. 5 When the green skin of a leaf is removed, a clear mucilaginous substance is seen that contains fibers, water and also the ingredient to retain the water in the leaf. The gel of Aloe vera is contained in the leaves. The gel contains 99.3% of water, the remaining 0.7% is made up of solids with carbohydrates constituting for a large part of it. 3 Aloe vera has been described as an antibacterial agent. Aloe vera gel has anthraquinones as an active compound, which is structural analogue of tetracycline. The anthraquinones act like tetracycline which inhibits bacterial protein synthesis by blocking the ribosomal A site (where the aminoacylated tRNA enters). Therefore, the bacteria cannot grow in the media containing Aloe vera extract. Polysaccharides of Aloe vera gel have been attributed direct antibacterial activity through the stimulation of phagocytic leucocytes to destroy bacteria. 6 Several studies have indicated the effectiveness of Aloe vera gel against both gram positive and gram negative bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus pyogenes, Bacillus subtilis etc. 3,4,5 Therefore, the aim of the present study is to evaluate the antibacterial effects of Ethanolic extract of Aloe vera leaf gel. This study may be helpful to emphasize the importance of natural products to control antibiotic resistant bacteria, which have been a major threat to human health.

Materials and methods
This laboratory based exploratory study was carried out in the Department of Pharmacology & Therapeutics in collaboration with the Department of Microbiology, Mymensingh Medical College, Mymensingh, during the period from July 2017 to June 2018.

Materials required
• Aloe vera leaves were bought from local market of Mymensingh. •

Extraction of aloe gel:
Mature, healthy and fresh leaves of Aloe vera were bought from local market of Mymensingh city. The leaves were washed out in running tap water for 5 minutes and rinsed with distilled water (D/W). After that they were dissected longitudinally and the colourless parenchymatous tissue (aloe gel) was scraped out without the fibres using a sterile knife.

Inoculation of bacterial cultures on nutrient medium:
H Pure cultures were maintained on nutrient agar plates regularly.
H Inoculum from the primary culture plate was prepared by touching the top of the colony of the test organism with a sterile wire loop.
H The loop was streaked all over the surface of the medium three times, rotating the plate through an angle of 60°C after each application. Finally, the loop was passed round the edge of the agar surface.
H The cultures were kept in incubator for 24 hours at 37°C and stored at 4°C.

Preparation of Bacterial Suspension:
For each of the test organisms the separate inoculums were prepared in the following ways. 3-5 similar colonies from 18-24 hours old nutrient agar plates were picked up with sterile wire loop and mixed with sterile normal saline. The turbidity was adjusted with 0.5 McFarland turbidity standards by adding saline or colony to the suspension as required. The turbidity of 0.5 McFarland standards corresponds to 1.5×108 organisms/ ml.

Preparation of DMSO stock solution:
1 gm of DMSO extract powder was dissolved in 10 ml of distilled water (D/W) to get a concentration of 0.1 gm i.e. 100 mg/ml which was labeled as stock solution-1. Again from the above solution 1 ml was taken and dissolved in 99 ml distilled water to get a concentration of 1mg/ml which was labeled as stock solution-2. From the above stock solution-2, different concentrations such as 100, 200, 300,400 and 500 µg/ml were prepared.

Calculation:
1 ml of stock solution-2 was mixed with 9 ml of D/W. So, the total amount is 10 ml.
This 10 ml solution contains 1000 µg extract (as described before).
So, the final concentration is 100 µg/ml.
Similar procedure was applied to prepare working solutions having concentrations of 200, 300, 400 and 500 µg/ml. This calculation is given below in tabulated form: blank discs and left for five minutes. Then the plates were incubated at 37°C for 24 hours. After that zone of inhibition for respective organisms were measured in mm by using ruler.

Determination of the Minimum Inhibitory Concentration (MIC):
The minimum inhibitory concentration (MIC) is the concentration giving the least inhibitory activity and below which there is no further inhibition. The MIC of the DMSO extract was obtained by dissolving various volume of DAE stock solution-2 (1 mg/ml or 1000 µg/ml) in various volume of nutrient broth.

Preparation of different concentrations of working EAE solution:
Set-I: Working solution was made by mixing 9 ml of DAE stock solution-2 with 1 ml of Nutrient broth (N/B). So the total amount became 10 ml. As stated previously, 1 ml of DAE stock solution-2 contains 1 mg extract.
The final concentration is 900 µg/ml.
Three types of controls were also used.
Control-I was made with 10 ml of DAE stock solution-2 (inoculated with bacterial suspension) in test tubes.

Control-2 was made with 10 ml of nutrient broth medium (inoculated with bacterial suspension) in test tubes.
Control-3 was made with 10 ml of nutrient broth medium (not inoculated with bacterial suspension) in test tubes (Table 02).

Examination of growth of test organisms in different concentrations of DAE:
After 18 to 24 hours of incubation at 37 °C, the growth of test organisms in different preparations of DAE were examined and compared against that of controls by matching their turbidity. The clear preparations were considered as "No growth" of bacteria and turbid ones, as "Growth of bacteria". The MIC was reported as lowest concentration of DAE required to prevent the visible growth of test organisms.

Results
A. Results of Antibacterial sensitivity test by disc diffusion method: Figure 01 shows

B. Results of Minimum Inhibitory Concentration (MIC) by broth dilution technique:
As stated before, the minimum inhibitory concentration (MIC) is the concentration giving the least inhibitory activity and below which there is no further inhibition. Table 03 shows that the initial MICs for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae are 300, 400, 400 and 500 µg/ml respectively.
. After performing the initial experiment, a second experiment was done to find out more precise MICs for the test organisms. Now, measured amounts of DMSO extract, Nutrient broth and organisms are taken in different test tubes to make required concentration of DAE. The calculation is given below in tabulated form. Antimicrobial activity of Aloe vera gel investigated against multi-drug resistant (MDR) Pseudomonas aeruginosa isolated from patients with burn wound infections. 9 Aloe vera gel was extracted using DMSO. MIC of Aloe vera was measured by broth dilution technique. The results obtained from antibacterial activities of Aloe vera gel showed that 89.4% isolates were inhibited by Aloe vera gel extract at the MIC � 400 µg/ml. The MIC values of Aloe vera gel against the remaining 10.6% isolates were 800 µg/ml. In present study MIC against Pseudomonas aeruginosa was 400 µg/ml which is compatible for majority of the organisms used in the aforementioned research.

Conclusion
From the study it is clearly evident that DMSO extract of Aloe vera leaf gel has a dose dependent inhibitory effect against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Relatively higher concentrations of the extracts showed higher degrees of inhibition against the test organisms. Further studies are required to detect and isolate the biologically active ingredients present in the Aloe vera leaves which are responsible for antibacterial effect. The practice of using medicinal plants like Aloe vera as supplementary or alternative medicine in developing countries will reduce not only the clinical burden of drug resistance development but also the side effects and cost of the treatment as compared to synthetic compounds.