aloe emodin, aloe vera, anthraquinone, antibacterial, antifungal, miller
I Cock. Antimicrobial Activity of Aloe barbadensis Miller Leaf Gel Components. The Internet Journal of Microbiology. 2007 Volume 4 Number 2.
Methanolic extracts of
Financial support of this work was provided by the School of Biomolecular and Physical Sciences, Griffith University.
Bacterial resistance to antibiotics is increasingly becoming a concern to public health. Currently used antibiotic agents are failing to bring an end to many bacterial infections due to super resistant strains. For this reason the search is ongoing for new antimicrobial agents, either by the design and synthesis of new agents or through the search of natural sources for as yet undiscovered antimicrobial agents. Herbal medications in particular have seen a revival of interest due to a perception that there is a lower incidence of adverse reactions to plant preparations compared to synthetic pharmaceuticals. Coupled with the reduced costs of plant preparations, this makes the search for natural therapeutics an attractive option.
Despite the therapeutic possibilities of this plant, there have been limited reports on the antimicrobial effects of isolated Aloe vera components. Ferro et al. (2003) have shown that Aloe vera leaf gel can inhibit the growth of the two Gram-positive bacteria
Materials and Methods
Aloe emodin (Sigma, purity >95%) was prepared freshly before use by dissolving in distilled water to give a concentration of 500 µg ml -1 . Aloe vera juice was obtained from Aloe Wellness Pty Ltd, Australia and was stored at 4 o C until use. Aloe vera juice was used undiluted in the antimicrobial assay.
Fresh clean whole
Preparation of Crude Extracts
1 g of lyophilised
HPLC Separation of Extract Components
The extract was analysed and further fractionated by RP-HPLC using a Shimadzu HPLC system. The system consisted of twin LC-10AT pumps, a DGU-12A degasser, a SIL-10AD automatic injector using a 20 µl injector loop and a SPD-M10A diode array detector, all under the control of a SCL-10A system controller. All solvents were of HPLC grade and were obtained from Lab-Scan.
HPLC separations were performed using a Waters Spherisorb® C18 column (5 cm × 4.6 mm). 20 µl samples of Aloe gel extract were injected and chromatographed using a gradient from 20 % methanol to 60 % methanol as follows: 2 minutes isocratically at 20 % methanol followed by an 8 minute gradient to 40 % methanol. This was followed by isocratic elution at 40 % methanol for a further 5 minutes. The methanol was increased to 60 % over a further 10 minutes. The column was washed with 100 % methanol before reequilibrating to 20 % methanol for further chromatograms. Samples from multiple chromatograms (30 repeats) were collected and pooled. These samples were dried by rotary evaporation in an Eppendorf concentrator 5301 and were resuspended in 1 ml distilled water and stored at 4 o C for further analysis. For comparison, 20 µl of aloe emodin (500 µg ml -1 ) was chromatographed using the same HPLC gradient.
Electrospray Ionisation (ESI) Mass Spectroscopy
Electrospray ionisation (ESI) mass spectroscopy was performed using a single quadrupole VG platform 2 mass spectrometer equipped with an electrospray (ES) ionisation source at an electrospray energy of 3.5 kV. Mass spectra were recorded in both the positive mode (cone voltage = 30 V) and negative mode (cone voltage = 50 V). Samples (100 µg ml -1 ) were introduced into the spectrometer at a flow rate of 1 µl min -1 in 80 % acetonitrile.
All microbial strains were obtained from Tarita Morais, Griffith University. Stock cultures of
Evaluation of Antimicrobial Activity
Antimicrobial activity of Aloe vera juice, extract and of the HPLC purified compounds was determined using a modified Kirby-Bauer (Bauer et al. 1966) disc diffusion method. Briefly, 100 µl of the test bacteria/fungi were grown in 10 ml of fresh media until they reached a count of approximately 10 8 cells ml -1 for bacteria, or 10 5 cells ml -1 for fungi. 100 µl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained.
The extract and fractionated components were tested using 6 mm sterilised filter paper discs. Discs were impregnated with 10 µl of the test sample, allowed to dry and placed onto inoculated plates. The plates were allowed to stand at 4 o C for 2 hours before incubation with the test microbial agents. Plates inoculated with
Results and Discussion
The antimicrobial activity of Aloe vera juice was investigated by agar disc diffusion against a panel of bacteria, fungi and yeast (Table 1). Aloe vera juice showed antibacterial activity against only the Gram-negative bacteria
The components of lyophilised
Aloe vera juice and gel are known to contain the anthraquinone aloe emodin which has previously been shown to have antimicrobial activity (Wu et al., 2006). For this reason, the antimicrobial activity of pure aloe emodin was also examined in the current study. Pure aloe emodin possessed similar antimicrobial activities as the juice, being able to inhibit the growth of both
To further study the antimicrobial growth inhibitory activity of the individual components of the Aloe vera leaf gel, the extract was further fractionated by reverse phase HPLC. 20 µl samples of the leaf gel extract were injected onto the column and fractions were collected and pooled. In total, 30 chromatograms were run and their fractions were pooled for further analysis. Figure 1(a) shows a typical HPLC profile. All 14 of the HPLC separated fractions were tested for antimicrobial activity. Only fractions 1, 2, 5, 8 and 9 showed any antimicrobial activity (Table 1).
Fraction 1 had the broadest antibacterial activity of any of the fractions, inhibiting the growth of the majority of the Gram-negative rod bacteria tested (
Electrospray ionisation (ESI) mass spectroscopy of fraction 1 showed complicated patterns that indicated the presence of multiple molecular species, thus a definitive structural characterisation was not possible. However, when dry, this fraction was a crystalline solid with an orange red colour and a slightly tacky consistency. A methanolic solution absorbed UV light between 250 and 290 nm with a maxima at 265 nm in the UV region. For comparison, anthraquinones are characterised by their orange red colour and their absorbances in the UVB range. Hirata and Suga (1977) have listed the UV absorbance peaks for aloe emodin as 221, 253, 266 and 289 nm. Furthermore, pure aloe emodin had a similar elution volume to fraction 1 when chromatographed under identical conditions (Figure 1b). Thus it is likely that whilst by no means pure, fraction 1 contains aloe emodin. This fraction has a broader antimicrobial specificity than pure aloe emodin, indicating other antimicrobial compounds may also be present in fraction 1. More work is necessary to fully characterise the molecular composition of this fraction.
The other fractions tested were much more selective in their antimicrobial activities, being capable of only inhibiting the growth of specific Gram-negative rod bacteria. Fraction 2 inhibited the growth of
Fraction 5 only inhibited
Fraction 8 had antibacterial activity against
Previous studies have demonstrated the antimicrobial activity of other chromones from diverse plant species such as
Fraction 9 only inhibited
The fungi tested in the present study were shown to have limited susceptibility to Aloe vera gel and extracted fractions.
The findings of this study have established the susceptibilities of a broad range of bacteria to fractions isolated from Aloe vera inner leaf gel. Gram-negative rod bacteria were found to be particularly susceptible to Aloe vera gel components. Of the bacterial classes tested, only the Gram-positive cocci bacteria were resistant to the Aloe vera components.
It is evident that fractions derived from Aloe vera inner gel have potential as antimicrobial agents, especially against Gram-negative bacteria and against resistant strains of
Financial support for this work was provided by the School of Biomolecular and Physical Sciences, Griffith University, Australia.
Dr Ian Cock, Biomolecular and Physical Sciences, Logan Campus, Griffith University,University Drive, Meadowbrook, Queensland 4131, Australia Tel.: +61 7 33821378; fax: +61 7 33821034. E-mail address: I.Cock@griffith.edu.au