Preliminary Investigation Associated with Antibacterial Potency of Both Juice Extract and an Isolate from Aloe vera Gel

Objective: The aim of this study was to investigate the antibacterial activity of both the juice extract and an isolate of Aloe vera leaves of gel from Ghana on clinical isolate bacteria found to prolong wound infections. Methods: Using sharp knife, the rind (outer cover) of the leaf and the layer immediately below the rind were removed to obtain the internal gel matrix portion (fillet). The isolate was precipitated from propylene glycol of the Aloe juice extract and was used to inhibit Escherichia coli and Staphylococcus aureus . Agar diffusion bioassay was used for inhibitory test of both the whole aloe juice and the isolate against E. coli and S. aureus. Results: Our findings exhibited that there was no significant difference in activity of both the isolate and the whole juice on E. coli at all concentrations used. S. aureus was resistant to both the isolate and the whole juice at all concentrations used, compared with the positive control, chloramphenicol which showed an intermediary zone of inhibition of 13.5 mm at a concentration of 10 g/L. A one-way analysis of variance (ANOVA) at (P ≤ 0.05) of the diameter zone of inhibition values compared with the activity of Chloramphenicol (positive control) indicated significant inhibitory activity by both the isolate and the whole juice extract against E. coli thus suggesting their efficacy in treating E. coli infections within the concentration used in this inhibitory studies. Conclusion: Both the A. vera juice extract and the isolate showed high activity against E. coli. These seem to justify the widespread use of the plant in the treatment of gastrointestinal diseases and its application in many pharmaceutical products.


INTRODUCTION
There is a growing demand for medicinal plant and plants products as an option to orthodox medicines principally in developing countries. The potential of higher plants as sources for new drugs is still chiefly uncharted. Medicinal plants are valuable antimicrobial agents and source of many potent and powerful drugs [1]. Over the ages right up to these modern era, the A. vera plant has been used to treat a broad catalog of diseases as well as its application in a host of cosmetic purposes. The inner gel has been used to sooth flesh wounds such as burns, cuts, scrapes and stings; it has also been used for headaches, gum sores, hemorrhoids, skin disorders, acne, dandruff and even baldness [2]. Aloe's role on healing process and its ability to penetrate target areas and stimulate cell regeneration has also been scientifically documented along with a multitude of other properties that buttress A. vera's role as a healer, a restorer and a protector [3]. Research work by Tanka et al. [4] in Phytotherapy Research has found A. vera to contain two anti-diabetic active compounds and therefore have shown the plant's ability to stabilize blood sugar in diabetics.
Scientific research has shown that the polysaccharides within A. vera have strong immune boosting and cancerous fighting capabilities [5,6].
The polysaccharides are believed to exhibit potent macrophage activators and produce increase volume of nitric acid, a compound that has tumor fighting potential [7]. One major polysaccharide, acemannan, a phytonutrient found in A. vera has been established to improve wound healing [8], it has also proven to exhibit antiviral activity, inhibiting reproduction of herpes [9,10], measles and the HIV virus In vitro [11]. Acemannan, which is the active ingredient in A. vera plant has been found not only important in sealing wounds but also keeping out bacteria agents. In this research we obtained the crude extract from the leaves of the A. Vera plant and further isolated a crude product from the extract. We then investigated the effects of both the isolate and the juice extract on clinically isolated bacteria, E. coli and S. aureus.

Source of Test Bacteria
Clinical isolates of E. coli and S. aureus were obtained from the Medical Microbiology Laboratory of the Bolgatanga Regional Hospital Ghana. Pure cultures of each of the bacterial isolates were obtained by sub-culturing the isolates on Chocolate agar (Oxoid, Basingstoke, Hampshire, England)

Preparation of Aloe vera Juice Extract
The leaves, 8.8 kg of A. vera were cut at the base without breaking or damaging any part of the leaves using a sharp knife . The leaves were washed thoroughly under running water to remove surface dirt and traces of any contaminants. The lower end portions of the leaves were cut using sharp knife in other to drain the yellow sap in the leaves and the remaining portions of the leaves were cut into segments and placed uprightly in an aqueous solution to drain all remaining yellow sap for about thirty minutes. Using sharp knife, the rind (outer cover) of the leaf and the layer immediately below the rind were removed to obtain the internal gel matrix portion (fillet). The fillets were washed under running water to remove any yellow sap and rinds that might have adhered to the fillet. The internal gel matrixes were drained for approximately an hour by placing the fillets on a mesh to obtain a gel matrix strip. The drained gel matrix strips was weighed and recorded as 600 g .

Isolation of the Crude Product (′isolate′) from the A. vera Juice
The gel matrix strips, 500 g were transferred into a blender and rotated at a high speed for two minutes. The blended fillets were then cooled in a refrigerator to allow the foam formed to settle and the cooled fillets were transferred into a Pyrex measuring cylinder and the volume was found to be 500 ml. The pH of the blended fillets was adjusted to 3.1 by addition of 5 ml 30% hydrochloric acid. Ethanol 70% (v/v) was then added gently to the measured volume in the ratio 3:1 ethanol/fillets mixture while stirring. This was done to precipitate most of the polysaccharides in the mixture. The flocculent residue floating on the ethanol layer was removed using a stirrer. The mixture was allowed to settle for four hours at room temperature and the ethanol layer was decanted gently without disturbing the precipitate at the bottom of the mixture. The precipitate was then transferred into a test tube and centrifuged at 5000 rpm for two minutes. The supernatant was decanted and the precipitate transferred into a VWR brand test tube for use in the next experiment.
Propylene glycol, 80%, 20 mL, was added to the precipitate recovered from the ethanol mixture and the mixture was vigorously shaken and then centrifuged at 5000 rpm for two minutes. The thick smooth gel forming the supernatant, 15 mL, was collected and diluted with 28 mL distilled water to obtain a 30% (v/v) propylene glycol mixture. The solution was kept at -30ºC for 24 hours and then mounted on a freeze-dryer overnight to afford a thick gel of 1.5 g.

Agar Diffusion Bioassay
The modified agar well diffusion method was employed. Colonies of a pure culture of each test organism was suspended in 100 mL of sterile peptone water to get a turbidity of about 2 McFarland Standard. The suspended test organism, 1 mL was surface-spread on Mueller Hinton Agar (OXOID) until complete diffusion. Plates were left to stand until complete diffusion of the formulations into the medium. Plates were incubated in inverted positions at 37°C for 48 hours after which they were observed for inhibitory activity depicted by zones of inhibition around the wells. Inhibition zone diameters were measured using a ruler and recorded in millimeters (mm). The experiments were repeated three times to check for reproducibility.

Statistical Analysis
Means and standard mean errors were calculated for the zones of inhibition measured for the three sets of experiments in each case. These means were statistically scrutinized using the one -way ANOVA to determine if they were significantly different at P ≤ 0.05.

Extraction
The juice, a jelly-like residue was extracted from 8.8 kg of A. vera. An isolate was precipitated from 500 mL of the aloe juice extract to obtain a light yellowish residue. The precipitate was dissolved in propylene glycol and freeze dried to obtain a thick gel of 1.5 g yield. The gel was characterized by applying simple physical properties such as pH, solubility and alkaline test. The pH of the gel was found to be 3.1. The gel formed less viscous homogenous solution in propylene glycol and alkali test indicated a nonviscous gel, an indicative of the cleavage of an oacetyl bond in a polysaccharide, which suggested the presence of acemmannan.

Inhibitory Studies of the Isolate and
Juice Extract on E. coli E. coli were resistant at lower concentrations of 2 g/L and 4 g/L of the A vera juice. The bacteria became susceptible to the juice extract when the concentrations were increased from 6, 8 and 10 g/L with corresponding zone of inhibition, 17.5, 19.0, and 23.0 mm respectively (Fig. 1). This means that a study increase in concentration increases the activity of the A. vera juice with no significant toxicity.

DISCUSSION
Wound healing is affected by innumerable wound healing factors and the deficiency of any of these factors can result in the retardation of the healing process. Several mechanisms have been proposed for the wound healing effects of acemannan, which include keeping the wound moist, increase epithelial cell migration, more rapid maturation of collagen and reduction in inflammation [12]. In the wound healing process, In this current research, we intended to extract pure beta-(1,4)-linked acetylated mannan (acemannan) from the aloe juice and physicochemically and spectroscopically characterize using 1 H and 13 C NMR, IR spectroscopy, GC/MS, after HPLC purification to homogeneity. We further aimed at comparing the antibacterial activity of the pure mannan to that of the crude juice extract on wound infecting microbes. Since there was not enough physicochemical characterization to elucidate the structure of the isolated compound, we termed it an "isolate". Even though the solubility of the isolate in propylene glycol, the pH and alkali test gave positive indication for the presence of acemannan it was inconclusive without other vital spectroscopic characterization. Alkali treatment of the isolate displayed less jelly-like forming property, indicating the disruption of an o-acetyl bond and resulting in decreasing entirely its viscosity. The de-acetylated product was not soluble in water due to perceptibly increased stronger hydrogen bonding. Moreover, a 0.2% (w/v) of the isolate gave a pH 6.31±0.33 which was approximately similar to the value determined by a scientific research group [13].
The zones of inhibition of both A. vera juice and the isolate on E. coli showed linear correlation. The activities were dose dependent with increasing zones of inhibition with a corresponding increase in concentration. Although the activity of the isolate indicated a higher potency against E. coli, it was not statistically significant compared with that of the juice extract. The probability of accepting or rejecting the null hypothesis (pr) was greater than the α-value at 95% confidence interval, that is, (pr > 0.05). This meant that the difference in their activity was statistically insignificant. The pvalue for the extracts together with chloramphenicol on E. coli was also greater than the α-value at a 95% confidence interval, that is, (pr > 0.05). Similarly the p-value for the extracts on S. aureus was also greater than the α-value. This statistics showed that the efficacy of the aloe isolate was independent on other compounds present in the juice.

CONCLUSION
The comparatively high activities shown by both the A. vera juice extract and the isolate against E. coli seem to justify the widespread use of the plant in the treatment of gastrointestinal diseases and its application in many pharmaceutical products. The high activity exhibited also seems to favour aloe's ability to penetrate target areas and stimulate cell regeneration. Isolation, purification, characterization and structure elucidation of the isolate could lead to the development of novel drugs from A. Vera and this is the future work in our laboratory.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

The
Medical Microbiology (Bacteriology) Laboratory of the Bolgatanga Regional Hospital, for the clinical isolates of S. aureus and E. coli. Head of Pharmacology and Phytochemistry Department, Centre for Scientific Research into Plant Medicine (CSRPM), Akwapim-Mampong.