Assessment of herbal drugs for promising anti-Candida activity

Plants were collected from different locations within the city of Sharjah, U.A.E. on April, 2016 as indicated in Table 1. The plants were taxonomically identified by Dr. Ali El-Keblawy at the Department of Applied Biology, University of Sharjah and voucher specimens were deposited at the University of Sharjah Herbarium on April 2016. The fresh aerial parts of the plants were cut into small sections and ground to very fine paste/powder. The paste/powder was extracted either with ethyl acetate or 95% ethanol three times followed by filtration. The organic solvent extracts were combined separately and evaporated using rotary evaporator at 50 °C till dryness. The residual extract either used directly or left at room (~25 °C) or ~4 °C temperatures for 4 months. The residual extracts were dissolved in sterile water prior to antimicrobial testing and in PBS washing buffer prior to toxicity testing.

The antimicrobial activity of each plant extract was studied against C. albicans (SC5314) and bacteria strains, namely: S. aureus, P. aeruginosa, E. coli, and the multidrug resistant (MDR) A. baumannii and Klebsiella pneumoniae. All bacterial strains are clinical isolates from patients who were seen at Harbor-UCLA Medical Center, Torrance, CA, U.S.A. The antimicrobial activities of all plant extracts were tested either by disc diffusion assay, in liquid media and by measuring the minimum inhibitory concentration (MIC).

Each plant extract was divided into three portions; one left at room temperature (~ 25 °C) for 4 months, another one was refrigerated at ~4 °C for 4 months and a last one used directly once the extraction was done. This was followed by disc diffusion assay of each treatment and the diameter of zone of inhibition (in mm) was read at 24 h.

The cytotoxic assay of the plants extracts was measured as the amount of hemoglobin released by the lysis of human erythrocytes [41, 42]. Briefly, fresh whole blood from healthy individual was collected into heparinized vacutainer from Harbor-UCLA Hospital and 1 mL whole blood was immediately centrifuged at 500 g for 10 min using benchtop centrifuge (Eppendorf 5804R Refrigerated Benchtop). The erythrocytes were washed three times with DPBS supplemented with 1 mg/mL bovine serum albumin (BSA) and then re-suspended to 3 × 10⁷ cells/ mL in DPBS. Washed cells (3 × 10⁶ cells per well) were incubated with the total plant extracts dissolved in the washing buffer at different concentrations (ranging from 3.6 to 100 μg/mL) in round-bottomed 96-well plates in a final volume of 200 μL. Washing buffer and 0.1–1% Triton X-100 were used as negative and positive controls, respectively. The plate was incubated at 37 °C for 30 min, followed by 30 min incubation on ice, and the intact cells were precipitated by centrifugation at 500 g for 10 min at 4 °C and the supernatants (125 μL) were transferred to a flat-bottom 96-well plate to measure hemoglobin release by absorbance at 405 nm using a microplate reader. The absorbance values for each sample were subtracted from the absorbance value obtained for washing buffer-treated cells and the hemolytic activity (%) was calculated. The experiment was conducted in triplicate and the data was analyzed using two-way analysis of variance (ANOVA).

The 50% cytotoxic concentration (CC₅₀) values were calculated as the concentration of plant extract caused 50% hemolysis compared to 100% hemolysis of erythrocytes treated with 1% triton X-100. And selective activities of the extracts were calculated according to the following formula “Selectivity index (SI) = (CC₅₀ in mg/mL)/ (MIC in mg/mL)” [43].

The data was collected and graphed using Microsoft Excel^®. Data was then exported to Graph Pad 5.0 for Windows (GraphPad Software, La Jolla, CA, USA) for statistical analysis. The effects of plant extracts on C. albicans inoculated onto solid agar media, liquid broth and during biofilm formation was analyzed using one-way analysis of variance (ANOVA) using Dunnett’s Multiple Comparison Test. P value <0.05 was considered as significant.

In this study, the potentiality of seven medicinal plants including Avicennia marina, Fagonia indica, Lawsania inermis, Portulaca oleracea, Salvadora persica, Ziziphus spina- Christi and Asphodelus tenuifolius were compared for their activities against C. albicans. The effect of both ethyl acetate and alcoholic (95% ethanol) plants extracts of the aforementioned medicinal plants were tested against wild type C. albicans (SC5314) on LB-agar media using disc diffusion assay. Paper discs saturated with plant extracts at 25, 50, and 100 μg/mL were applied on LB solid media streaked with C. albicans and incubated at 37 °C for 72 h. A. tenuifolius, S. persica, L. inermis and P. oleracea alcoholic extracts inhibited growth of C. albicans after 24 h of incubation (Table 2); However only L. inermis and P. oleracea alcoholic extracts showed significant (P < 0.05) growth inhibition activity up to 72 h (Table 2).

Candida infections are complicated by many factors including nutritional conditions, planktonic versus biofilm modes of growth, and the adaptability of the pathogen [44]. All factors together should be considered to provide an effective inhibition of the microbe; so sequential experiments were conducted in order to decide a promising lead extract out of tested plant extracts.

Since Candida shows medium-dependent expression of hyphae specific genes with prominent expression in liquid media compared to solid media [45, 46], all plants extracts under study were evaluated for their ability to inhibit C. albicans in liquid LB media. Similar to disc diffusion assay, plant extracts at 25, 50 and 100 μg/mL were added to LB broth media inoculated with C. albicans and incubated for 72 h at 37 °C. A. tenuifolius and S. persica as well as L. inermis and P. oleracea alcoholic extracts significantly (P < 0.05) inhibited growth of C. albicans to 24 and 72 h post-incubation, respectively (Table 3). The other plant extracts including A. marina, F. indica and Z. spina- Christi increased the growth of C. albicans at lower concentrations, similar to some plant extracts such as green tea leaf extract [47] and cabbage leaf extract [48] that can selectively inhibit and stimulate different microbial growth. All ethyl acetate extracts showed no activity either on solid or liquid media (data not shown).

An important feature of C. albicans growth is its ability to switch between yeast and hyphae forms [49]. The hyphae form is importantly required for disease progression by invading host cells and causing tissue damage [50, 51], and for formation of biofilm [52]. Because both L. inermis and P. oleracea showed significant inhibitory effect on C. albicans in solid and liquid media, they were tested against biofilm formation. Both alcoholic plant extracts showed significant (P value < 0.05) inhibitory effect on C. albicans biofilm formation (Fig. 1a) within the range of MIC (Table 4). The MIC of both L. inermis and P. oleracea were measured to be 10 μg/mL (Table 4) compared to 1 μg/mL ketoconazole (Sigma) as control. And the minimum fungicidal concentrations (MFC) of both L. inermis and P. oleracea was ≤25 μg/mL.

Another complication with in vivo Candida infection is its frequent ability to form mixed infections with bacterial species including Pseudomonas aeruginosa usually found in combination in biofilm formation and recovered from patient with lung infection [53], Staphylococcus aureus and Escherichia coli in inflamed palatal mucosa, enterococci and Klebsiella in labial lesion and other infections that can induce life-threatening septicemia [54]. Both L. inermis and P. oleracea alcoholic extracts showed consistent broad spectrum antibacterial activity to all tested microorganisms including E. coli, S. aureus, A. baumanii, K. pneumoniae and P. aeruginosa compared to other aforementioned plant extracts (Fig. 1b). Other plant extracts including A. marina, F. indica, S. persica, Z. spina, and A. tenuifolius showed modest species-specific antibacterial activities (Fig. 1b).

The relative susceptibility of C. albicans to antibiotics is dependent on the age of culture because the culture environment is rapidly changing and the cell populations becomes more physiologically heterogeneous [55] and hence, more resistant with age [56‐58]. So it is beneficial to test the effect of both plant extracts on C. albicans culture in its stationary phase of growth [59]. A C. albicans culture was developed by growing LB broth inoculated with C. albicans for 24 h prior to treating separately with L. inermis or P. oleracea alcoholic extracts. Both extracts caused aggregation and precipitation of the C. albicans culture (Fig. 1c). Inoculation of plant extract treated-cultures into fresh antibiotic-free LB broth followed by incubation for 24 h at 37 °C showed >90% inhibition in growth compared to control LB broth that received the same volume of untreated C. albicans culture (Fig. 1c). The results indicated that C. albicans cultures showed high sensitivity to both plant extracts even at increased growth rate and the effect of the two plant extracts are cidal.

Usually antimicrobials are under suspicion of diminishing activities either because of admixture and dispensing to be stored at home or shelf storage before use [60]. The stability during shelf half-life storage of both L. inermis and P. oleracea alcoholic extracts were tested by storing both plant extracts for 4 months at room temperature (~25 °C) followed by incubation with aforementioned microbes. The results showed that both extracts possess activities similar to those used fresh or stored at 4 °C (data not shown). The results indicated that both plant extracts are stable at wide range of temperatures making them adequate for long storage, and different handling environment. These stability features make both extracts desirable for further development as potential antifungal agents [61].

The adverse drug effects associated with the use of antimicrobials can be of a major concern especially with antifungal agents due to the eukaryotic nature of the organism being targeted. Therefore, it is important to test the toxicity of plant extracts prior to application as antimicrobials. Among the cytotoxicity tests is hemolytic activity assay of human erythrocytes [62]. A cytotoxicity assay was conducted by testing different plant extracts at different concentrations and by using fresh human erythrocytes. The results showed that all plant extracts under study except A. marina and F. indica are safe and not toxic at a wide range of growth inhibitory concentrations (3–30 μg/mL) (Fig. 2). Both CC50 (cytotoxicity) and selective activity of the plant extracts were measured (Table 5). Our data show that both L. inermis and P. oleracea exhibit high selective antimicrobial activities. The relatively high selectivity indices of both L. inermis and P. oleracea indicate that both extracts are likely useful in managing infections due to C. albicans and other bacterial infections in humans [43]. The total activity of both L. inermis and P. oleracea plants were also calculated as 1.7 and 2.1 mL/g, respectively indicative of higher potency of both plants against C. albicans. And the results from this research indicate that both L. inermis and P. oleracea plants could be promising antimicrobials once they promoted for in vivo and clinical studies.