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Wednesday 11 March 2020

Pokok Anti Virus : Gelam


Melaleuca alternifolia (Tea Tree) Oil: a Review of Antimicrobial and Other Medicinal Properties




ANTIMICROBIAL ACTIVITY IN VITRO

Of all of the properties claimed for TTO, its antimicrobial activity has received the most attention. The earliest reported use of the M. alternifolia plant that presumably exploited this property was the traditional use by the Bundjalung Aborigines of northern New South Wales. Crushed leaves of “tea trees” were inhaled to treat coughs and colds or were sprinkled on wounds, after which a poultice was applied (). In addition, tea tree leaves were soaked to make an infusion to treat sore throats or skin ailments (). The oral history of Australian Aborigines also tells of healing lakes, which were lagoons into which M. alternifolia leaves had fallen and decayed over time (). Use of the oil itself, as opposed to the unextracted plant material, did not become common practice until Penfold published the first reports of its antimicrobial activity in a series of papers in the 1920s and 1930s. In evaluating the antimicrobial activity of M. alternifolia oil and other oils, he made comparisons with the disinfectant carbolic acid or phenol, the gold standard of the day, in a test known as the Rideal-Walker (RW) coefficient. The activity of TTO was compared directly with that of phenol and rated as 11 times more active (). The RW coefficients of several TTO components were also reported, including 3.5 for cineole and 8 for cymene (), 13 for linalool (), and 13.5 for terpinen-4-ol and 16 for terpineol (). As a result, TTO was promoted as a therapeutic agent (-). These publications, as well as several others (), describe a range of medicinal uses for TTO. However, in terms of the evidence they provide for the medicinal properties of TTO, they are of limited value, since by the standards of today the data they provide would be considered mostly anecdotal.
In contrast, contemporary data clearly show that the broad-spectrum activity of TTO includes antibacterial, antifungal, antiviral, and antiprotozoal activities. Not all of the activity has been characterized well in vitro, and in the few cases where clinical work has been done, data are promising but thus far inadequate.
Evaluation of the antimicrobial activity of TTO has been impeded by its physical properties; TTO and its components are only sparingly soluble in water (Table (Table2),2), and this limits their miscibility in test media. Different strategies have been used to counteract this problem, with the addition of surfactants to broth and agar test media being used most widely (). Dispersion of TTO in liquid media usually results in a turbid suspension that makes determination of end points in susceptibility tests difficult. Occasionally dyes have been used as visual indicators of the MIC, with mixed success ().

Antibacterial Activity

The few reports of the antibacterial activity of TTO appearing in the literature from the 1940s to the 1980s () have been reviewed elsewhere previously (). From the early 1990s onwards, many reports describing the antimicrobial activity of TTO appeared in the scientific literature. Although there was still a degree of discrepancy between the methods used in the different studies, the MICs reported were often relatively similar. A broad range of bacteria have now been tested for their susceptibilities to TTO, and some of the published susceptibility data are summarized in Table Table3.3. While most bacteria are susceptible to TTO at concentrations of 1.0% or less, MICs in excess of 2% have been reported for organisms such as commensal skin staphylococci and micrococci, Enterococcus faecalis, and Pseudomonas aeruginosa (). TTO is for the most part bactericidal in nature, although it may be bacteriostatic at lower concentrations.
The activity of TTO against antibiotic-resistant bacteria has attracted considerable interest, with methicillin-resistant Staphylococcus aureus (MRSA) receiving the most attention thus far. Since the potential to use TTO against MRSA was first hypothesized (), several groups have evaluated the activity of TTO against MRSA, beginning with Carson et al. (), who examined 64 MRSA isolates from Australia and the United Kingdom, including 33 mupirocin-resistant isolates. The MICs and minimal bactericidal concentrations (MBCs) for the Australian isolates were 0.25% and 0.5%, respectively, while those for the United Kingdom isolates were 0.312% and 0.625%, respectively. Subsequent reports on the susceptibility of MRSA to TTO have similarly not shown great differences compared to antibiotic-sensitive organisms ().
For the most part, antibacterial activity has been determined using agar or broth dilution methods. However, activity has also been demonstrated using time-kill assays (), suspension tests (), and “ex vivo”-excised human skin (). In addition, vaporized TTO can inhibit bacteria, including Mycobacterium avium ATCC 4676 (), Escherichia coliHaemophilus influenzaeStreptococcus pyogenes, and Streptococcus pneumoniae (). There are anecdotal reports of aerosolized TTO reducing hospital-acquired infections (L. Bowden, Abstr. Infect. Control Nurses Assoc. Annu. Infect. Control Conf., p. 23, 2001) but no scientific data.

Mechanism of antibacterial action.

The mechanism of action of TTO against bacteria has now been partly elucidated. Prior to the availability of data, assumptions about its mechanism of action were made on the basis of its hydrocarbon structure and attendant lipophilicity. Since hydrocarbons partition preferentially into biological membranes and disrupt their vital functions (), TTO and its components were also presumed to behave in this manner. This premise is further supported by data showing that TTO permeabilizes model liposomal systems (). In previous work with hydrocarbons not found in TTO () and with terpenes found at low concentrations in TTO (), lysis and the loss of membrane integrity and function manifested by the leakage of ions and the inhibition of respiration were demonstrated. Treatment of S. aureus with TTO resulted in the leakage of potassium ions () and 260-nm-light-absorbing materials () and inhibited respiration (). Treatment with TTO also sensitized S. aureus cells to sodium chloride () and produced morphological changes apparent under electron microscopy (). However, no significant lysis of whole cells was observed spectrophotometrically () or by electron microscopy (). Furthermore, no cytoplasmic membrane damage could be detected using the lactate dehydrogenase release assay (), and only modest uptake of propidium iodide was observed () after treatment with TTO.
In E. coli, detrimental effects on potassium homeostasis (), glucose-dependent respiration (), morphology (), and ability to exclude propidium iodide () have been observed. A modest loss of 280-nm-light-absorbing material has also been reported (). In contrast to the absence of whole-cell lysis seen in S. aureus treated with TTO, lysis occurs in E. coli treated with TTO (), and this effect is exacerbated by cotreatment with EDTA (C. Carson, unpublished data). All of these effects confirm that TTO compromises the structural and functional integrity of bacterial membranes.
The loss of viability, inhibition of glucose-dependent respiration, and induction of lysis seen after TTO treatment all occur to a greater degree with organisms in the exponential rather than the stationary phase of growth (; S. D. Cox, J. L. Markham, C. M. Mann, S. G. Wyllie, J. E. Gustafson, and J. R. Warmington, Abstr. 28th Int. Symp. Essential Oils, p. 201-213, 1997). The increased vulnerability of actively growing cells was also apparent in the greater degree of morphological changes seen in these cells by electron microscopy (S. D. Cox et al. Abstr. 28th Int. Symp. Essential Oils, p. 201-213). The differences in susceptibility of bacteria in different phases of growth suggest that targets other than the cell membrane may be involved.
When the effects of terpinen-4-ol, α-terpineol, and 1,8-cineole on S. aureus were examined, none was found to induce autolysis but all were found to cause the leakage of 260-nm-light-absorbing material and to render cells susceptible to sodium chloride (). Interestingly, the greatest effects were seen with 1,8-cineole, a component often considered to have marginal antimicrobial activity. This raises the possibility that while cineole may not be one of the primary antimicrobial components, it may permeabilize bacterial membranes and facilitate the entry of other, more active components. Little work on the effects of TTO components on cell morphology has been reported. Electron microscopy of terpinen-4-ol-treated S. aureus cells () revealed lesions similar to those seen after TTO treatment (), including mesosome-like structures.
Mechanism of action studies analogous to those described above have not been conducted with P. aeruginosa. Instead, research has concentrated on how this organism is able to tolerate higher concentrations of TTO and/or components. These studies have indicated that tolerance is associated with the outer membrane by showing that when P. aeruginosa cells are pretreated with the outer membrane permeabilizer polymyxin B nonapeptide or EDTA, cells become more susceptible to the bactericidal effects of TTO, terpinen-4-ol, or γ-terpinene ().
In summary, the loss of intracellular material, inability to maintain homeostasis, and inhibition of respiration after treatment with TTO and/or components are consistent with a mechanism of action involving the loss of membrane integrity and function.



        Antifungal Activity

Comprehensive investigations of the susceptibility of fungi to TTO have only recently been completed. Prior to this, data were somewhat piecemeal and fragmentary. Early data were also largely limited to Candida albicans, which was a commonly chosen model test organism. Data now show that a range of yeasts, dermatophytes, and other filamentous fungi are susceptible to TTO () (Table (Table4).4). Although test methods differ, MICs generally range between 0.03 and 0.5%, and fungicidal concentrations generally range from 0.12 to 2%. The notable exception is Aspergillus niger, with minimal fungicidal concentrations (MFCs) of as high as 8% reported for this organism (). However, these assays were performed with fungal conidia, which are known to be relatively impervious to chemical agents. Subsequent assays have shown that germinated conidia are significantly more susceptible to TTO than nongerminated conidia (), suggesting that the intact conidial wall confers considerable protection. TTO vapors have also been demonstrated to inhibit fungal growth () and affect sporulation ().


Mechanism of antifungal action.

Studies investigating the mechanism(s) of antifungal action have focused almost exclusively on C. albicans. Similar to results found for bacteria, TTO also alters the permeability of C. albicans cells. The treatment of C. albicans with 0.25% TTO resulted in the uptake of propidium iodide after 30 min (), and after 6 h significant staining with methylene blue and loss of 260-nm-light-absorbing materials had occurred (). TTO also alters the permeability of Candida glabrata (). Further research demonstrating that the membrane fluidity of C. albicans cells treated with 0.25% TTO is significantly increased confirms that the oil substantially alters the membrane properties of C. albicans ().
TTO also inhibits respiration in C. albicans in a dose-dependent manner (). Respiration was inhibited by approximately 95% after treatment with 1.0% TTO and by approximately 40% after treatment with 0.25% TTO. The respiration rate of Fusarium solani is inhibited by 50% at a concentration of 0.023% TTO (). TTO also inhibits glucose-induced medium acidification by C. albicansC. glabrata, and Saccharomyces cerevisiae (). Medium acidification occurs largely by the expulsion of protons by the plasma membrane ATPase, which is fuelled by ATP derived from the mitochondria. The inhibition of this function suggests that the plasma and/or mitochondrial membranes have been adversely affected. These results are consistent with a proposed mechanism of antifungal action whereby TTO causes changes or damage to the functioning of fungal membranes. This proposed mechanism is further supported by work showing that the terpene eugenol inhibits mitochondrial respiration and energy production ().
Additional studies have shown that when cells of C. albicans are treated with diethylstilbestrol to inhibit the plasma membrane ATPase, they then have a much greater susceptibility to TTO than do control cells (), suggesting that the plasma membrane ATPase has a role in protecting cells against the destabilizing or lethal effects of TTO.
TTO inhibits the formation of germ tubes, or mycelial conversion, in C. albicans (). Two studies have shown that germ tube formation was completely inhibited in the presence of 0.25 and 0.125% TTO, and it was further observed that treatment with 0.125% TTO resulted in a trend of blastospores changing from single or singly budding morphologies to multiply budding morphologies over the 4-h test period (). These cells were actively growing but were not forming germ tubes, implying that morphogenesis is specifically inhibited, rather than all growth being inhibited. Interestingly, the inhibition of germ tube formation was shown to be reversible, since cells were able to form germ tubes after the removal of the TTO (). However, there was a delay in germ tube formation, indicating that TTO causes a postantifungal effect.

 

Antiviral Activity

The antiviral activity of TTO was first shown using tobacco mosaic virus and tobacco plants (). In field trials with Nicotiniana glutinosa, plants were sprayed with 100, 250, or 500 ppm TTO or control solutions and were then experimentally infected with tobacco mosaic virus. After 10 days, there were significantly fewer lesions per square centimeter of leaf in plants treated with TTO than in controls (). Next, Schnitzler et al. () examined the activity of TTO and eucalyptus oil against herpes simplex virus (HSV). The effects of TTO were investigated by incubating viruses with various concentrations of TTO and then using these treated viruses to infect cell monolayers. After 4 days, the numbers of plaques formed by TTO-treated virus and untreated control virus were determined and compared. The concentration of TTO inhibiting 50% of plaque formation was 0.0009% for HSV type 1 (HSV-1) and 0.0008% for HSV-2, relative to controls. These studies also showed that at the higher concentration of 0.003%, TTO reduced HSV-1 titers by 98.2% and HSV-2 titers by 93.0%. In addition, by applying TTO at different stages in the virus replicative cycle, TTO was shown to have the greatest effect on free virus (prior to infection of cells), although when TTO was applied during the adsorption period, a slight reduction in plaque formation was also seen (). Another study evaluated the activities of 12 essential oils, including TTO, for activity against HSV-1 in Vero cells (). Again, TTO was found to exert most of its antiviral activity on free virus, with 1% oil inhibiting plaque formation completely and 0.1% TTO reducing plaque formation by approximately 10%. Pretreatment of the Vero cells prior to virus addition or posttreatment with 0.1% TTO after viral absorption did not significantly alter plaque formation.
Some activity against bacteriophages has also been reported, with exposure to 50% TTO at 4°C for 24 h reducing the number of SA and T7 plaques formed on lawns of S. aureus and E. coli, respectively ().
The results of these studies indicate that TTO may act against enveloped and nonenveloped viruses, although the range of viruses tested to date is very limited.


Antiprotozoal Activity

Two publications show that TTO has antiprotozoal activity. TTO caused a 50% reduction in growth (compared to controls) of the protozoa Leishmania major and Trypanosoma brucei at concentrations of 403 mg/ml and 0.5 mg/ml, respectively (). Further investigation showed that terpinen-4-ol contributed significantly to this activity. In another study, TTO at 300 mg/ml killed all cells of Trichomonas vaginalis (). There is also anecdotal in vivo evidence that TTO may be effective in treating Trichomonas vaginalis infections ().




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