The Effect of Botanical Tinctures and Essential Oils on the Growth and Morphogenesis of Candida albicans

Objective: Candida albicans is an opportunistic and polymorphic fungal pathogen that affects mucosal membranes and squamous epithelia as well as being part of the normal human flora. Historically, there have been many botanical-based remedies used to treat fungal conditions, including C. albicans. This study examined the efficacy of both botanical tinctures and essential oils on the growth and morphological differentiation of C. albicans. Methods: The in vitro growth and differentiation of C. albicans was monitored following treatment with ethanol-based tinctures and essential oils prepared from several commonly used botanicals. Results: Results demonstrated that all ethanol-based botanical tinctures tested did not inhibit the growth of C. albicans, but several tinctures, including Marsdenia condurango, Juglans nigra (Black walnut), Anemopsis californica (Yerba mansa) and Piper cubeba (Cubeb berry), significantly reduced the morphological differentiation of the yeast into the invasive hyphae form. Alternatively, several botanical essential oils, including those from Thymus vulgaris (Thyme), Rosmarinus officinalis (Rosemary) and Cymbopogon citratus (Lemon grass) had a dramatic effect on inhibiting the growth of C. albicans. Conclusions: These results suggest that botanical tinctures commonly used in the treatment of C. albicans infections may act by blocking the differentiation of the yeast into a more virulent hyphal form while not affecting the growth rate. In comparison, therapeutic essential oils may target both the differentiation and growth rate of C. albicans. The results support that different active constituents are present in botanical tinctures as compared to oils thereby contributing to our understanding of how these botanicals may be effective therapeutics in the treatment of C. albicans infections.


Introduction
Candida albicans is a commensal organism in the human cutaneous and mucosal flora, but can become a major human fungal pathogen given the proper conditions allowing for hyphal differentiation, biofilm formation, and overgrowth. Some of the most common areas for candidiasis include the oral and gastrointestinal tract, vaginal canal and topically on the skin [1]. In patients experiencing vulvovaginal candidiasis, common signs include burning, itching and redness. This condition affects up to 75% of women at least once in their lifetime [2]. Skin infections are common in burn victims and neonates due to their depressed or underdeveloped immune system [2]. In the AIDS patient population, there is an increased likelihood of developing oral and esophageal candidiasis, due to decreased immune function [2]. In the general population, Candida overgrowth in the gastrointestinal tract is exacerbated by high-sugar diets or medications including steroids and antibiotics. In these cases, people will present with digestive discomfort, gas or bloating. More seriously, C. albicans  [3]. C. albicans has become the fourthleading cause of hospital-acquired blood infections in the world and mortality rates for the more severe infections remain high at about 50% [1,[4][5][6].
During growth, C. albicans may undergo a reversible morphological transition from single-celled yeast; round budding spores (also known as blastospores), to filamentous pseudohyphae and finally, multicellular, elongated tubal cells attached end-to-end known as true hyphae [1,2,6]. This transition, which is induced upon exposure of C. albicans to a number of host conditions, is required for increased virulence. The most common host conditions required for differentiation include the presence of serum, the environmental temperature, and pH levels [2]. In studying mucosal infections, the hyphal form of C. albicans has been found embedded and overgrown in tissue leading to the conclusion that in its multicellular form is when C. albicans is at its most invasive and infectious stage [7].
Currently, there are a large variety of anti-Candida treatments dependent on the anatomical location of the yeast overgrowth.
Pharmacological oral agents such as fluconazole and amphotericin B have been used in cases of internal or invasive Candida overgrowth [8]. Topical agents such as nystatin and clotrimazole have been used to treat cutaneous candidiasis as well as certain oral candida overgrowths. Miconazole and tioconazole are more commonly used topically in the vaginal canal for treatment of vaginal yeast infections [3,8]. Due to Candida being a eukaryotic pathogen, many of the antifungal agents available interact negatively with human cells. These anti-fungal treatments have many adverse effects including hepatotoxicity as the most common and concerning [2]. Invasive and systemic forms of candidiasis are more difficult to treat using these medications because of their high side effect profile, particularly in patients who are already immunocompromised [3].
In this study, we examined the efficacy of botanical tinctures and essential oils to inhibit both the growth and morphological transformation of C. albicans.

Botanical tincture and essential oil preparation
Plant material was obtained from reputable sources with documentation of authenticity. All plant material was subsequently verified by qualified botanical specialists using herbal pharmacopoeia monographs and reference keys. A voucher specimen of all plant material was deposited in a repository.
For tincture preparation, the dried botanicals were ground to a fine powder, resuspended in extraction solution and incubated for 3 days at room temperature. The extract was centrifuged at 3000 × g for 10 min to remove cell debris and the extraction solution filtered through a 0.

Microscopy differentiation analysis
At 24 hours post-treatment, light microscopy was done to evaluate the morphological state of the C. albicans. Each broth culture was stained with Lactophenol Cotton Blue and visualized by light microscopy. A minimum of 100 cells were counted and the percent of single cells verses hyphal/pseudohyphal cells determined.

Results
In this study, several botanical tinctures and oils were utilized based on their historical and/or suggested therapeutic efficacy in the treatment of C. albicans infections. Upon testing tinctures for growth inhibitory effects on C. albicans, little or no effect was observed. Figure 1  Since morphological transformation of C. albicans to hyphal form is a key virulence factor, the herbal tinctures were tested for their ability to inhibit this transformation process.   Table 2 The source and preparation method for each essential oil.
represents the percent hyphae formation of C. albicans cultures treated with herbal tinctures for 24 hours. As shown, when C. albicans cultures were grown at 37°C with the addition of serum, 80-100% of the culture formed hyphae, whereas when grown at 30°C without serum, typically only 10% of the culture formed hyphae with 90% as single celled form. Upon the addition of Marsdenia condurango tincture at 10 μl/ml at 37°C with serum, decreased hyphae formation was observed to about 15% of the population and further decreased to 5% of the population at 30 μl/ml. This was the strongest acting tincture upon hyphae inhibition with Piper cubeba, Juglans nigra, Anemopsis californica and Cinnamomum zeylanicum also being significantly potent with only 10-25% hyphae growth at 30 μl/ml. At 10 μl/ml these botanicals provided between 50-75% hyphal growth.
Visually, Figure 3A shows microscopic images of the C. albicans grown at 0 μl/ml, 10  Herbal Medicine: Open Access P. cubeba at 30 μl/ml, pseudohyphae were still prevalent, but as shorter structures. These results agree with Figure 1 where the growth rate of C. albicans was not affected by the herbs, but rather the morphological state of the cells was affected.
The effect of these tinctures on C. albicans differentiation can also be observed at a gross level with the broth cultures. Figure  3B shows M. condurango treatment of C. albicans cultures at 0 μl/ml, 10 μl/ml and 30 μl/ml at 37°C plus serum and 30°C without serum. At 30°C without serum, the broth cultures were turbid at every herb concentration due to the C. albicans growing in a single-celled form. When grown at 37°C with serum and no herb, the culture appeared flocculent due to large, hyphae formation ( Figure 3B). This then decreased to a less flocculent and more turbid appearance at 10 μl herb/ml and a completely turbid appearance (similar to 30°C without serum) at 30 μl herb/ml.
For other herbs tested, intermediate acting herbs showed a significant effect on the morphological growth of C. albicans only at the higher doses of 30 μl/ml and were able to inhibit hyphal growth to 40-60% of the population (Figure 2). These herbs included Chilopsis linearis, Alpinia officinarum and Carum carvi. More minor effects on growth, with 75-80% of the culture still forming hyphae was seen with Olea europea, Thymus vulgaris and Cymbopogon citratus at the highest (30 μl/ml) concentrations of each tincture.
Tinctures which proved to have no effect on hyphae growth were Artemisia absinthium, Arcostaphylos uva-ursi, Nepeta cataria, Allium sativum, Cuminum cyminum, Arctium lappa, Tabebuia avellanedae and Origanum vulgare (data not shown). In addition, vehicle control samples treated with 60% ethanol also showed no effect on hyphae growth or differentiation (data not shown).
Since essential oils are also historically used in treating C. albicans infections, the effect of various essential oils on the growth and differentiation of C. albicans was also examined. Figure 4 shows that certain essential oils were able to inhibit the replication of C. albicans at both 30°C without serum and 37°C with serum. Although Thymus vulgaris and Cymbopogon citratus as tinctures showed no effect on the replication rate of C. albicans, the essential oils inhibited growth approximately 1000fold at 1 µl/ml and over 10,000-fold at doses of 3 µl/ml or higher. This appeared to be a fungicidal effect since levels of viable cells decreased over 2-logs below the initial concentration. For other essential oils, including those from Rosmarinus officinalis, Melaleuca alternifolia, Mentha balsamea, Organum vulgare, Lavandula officinalis, and Cymbopogon martini, a more dose dependent inhibition in growth was observed (Figure 4). These herbs generally inhibited growth approximately 10-fold at 1 µl/ ml, 100-1,000-fold at 3 µl/ml, and greater than 10,000-fold at 10 µl/ml and higher. Not all essential oils tested inhibited C. albicans growth. Palmarosa oil showed no activity on the growth of the C. albicans even at the higher concentrations tested (Figure 4). Similar non-inhibitory effects were observed with essential oils from Copaifera officinalis, Oenothera biennis and Cannabis sativa (data not shown).
Since the herbal tinctures had an effect on the morphological differentiation of C. albicans, similar assays were done using the essential oils. As shown in Figure 5, Lavandula officinalis, Rosmarinus officinalis, Mentha balsamea and Melaleuca alternifolia oils were able to inhibit hyphal formation at concentrations below that which inhibited replication. For example, L. officinalis reduced hyphal formation to 5-15% at 10 and 1 µl/ml, respectively, but did not significantly affect the replication rate of C. albicans until 30 µl/ml. For Thymus vulgaris, Cymbopogon citratus and Origanum vulgare oils there was no significant effect on hyphal formation at doses that did not lead to growth inhibition (Figure 5). These results may suggest that herbs like R. officinalus, may have separate constituents which affect both growth and morphological differentiation of C. albicans, whereas herbs like T. vulgaris, only a constituent which affects only the growth rate. Cymbopogon martini essential oil showed no inhibition of hyphal differentiation at even the highest concentration of the essential oil ( Figure 5). Other essential oils tested with no effect on hyphal growth include Copaifera officinalis, Oenothera biennis and Cannabis sativa (data not shown).
The previous results demonstrated that several tinctures and oils could inhibit the differentiation of C. albicans from a singlecelled yeast to a hyphal form. In order to test if these herbs could also reverse differentiation from a hyphal form to a yeast form, pre-existing hyphal C. albicans was maintained under growth conditions of 37°C with serum and subsequently treated with various herbs and examined for morphological changes. These studies had the goal of indirectly assessing if an already  Figure 6A and 6B, with M. condurango, the initial culture started out with approximately 90% hyphae population. At 24 hours with no treatment, the hyphal growth remained unchanged. At 10 μl tincture/ml, a significant reduction to pseudohyphae and single cells was observed at 24 hours post treatment. At 30 μl tincture/ml, hyphae formation was decreased to 95% single-celled yeast following 24 hours post-treatment. Figure 6B shows similar results for the other tinctures and oils tested with Piper cubeba tincture, Melaleuca alternifolia oil, Rosmarinus officinalis oil and Lavandula officinalis oil all significantly reversing hyphal growth to a single-celled yeast form.

Discussion
Historically, many botanical treatments have been used as therapeutic agents for fungal infections. In this study, botanical tinctures and essential oils were tested individually for their efficacy towards both inhibiting the growth and preventing differentiation of C. albicans. Juglans nigra has been used to treat oral infections and has been shown to have inhibitory compounds towards C. albicans [9,10]. Piper cubeba has reported constituents with bacteriostatic, fungistatic and fungicidal properties that have been found useful historically in treating oral pathogens [11][12][13][14]. Anemopsis californica has reported very strong antifungal properties making it a potentially effective topical treatment for fungal infections [15]. Some herbs, such as Marsdenia condurango are commonly used by practitioners without laboratory-based studies or evaluations. Similarly, essential oils have a long history of therapeutic use for fungal infections. Thymus vulgaris, as an essential oil, has been suggested to inhibit the growth of C. albicans intracellularly [16][17][18]; Rosmarinus officinalis was demonstrated to inhibit germ tube formation as an essential oil [19,20]; and Cymbopogon citratus has been suggested to inhibite mycelial formation as well as yeast bud formation [21,22].
The botanical tinctures used in this study were prepared as ethanolbased herbal extractions. Unexpectedly, based on historical use, no herbal tincture was able to inhibit the growth rate of C. albicans. However, several tinctures inhibited the morphological differentiation of C. albicans to a hyphal form. These results may suggest that therapeutic herbal tinctures may act by inhibiting the progression of single-celled yeast into the more invasive hyphae form. The strongest herbs with this mechanism of action included Marsdenia condurango, Piper cubeba, Juglans nigra, Anemopsis californica and Cinnamomum zeylanicum. C. albicans is typically a serious problem only when it differentiates into a hyphal form, therefore, it may be therapeutically valuable to limit differentiation of C. albicans instead of completely inhibiting growth and maintaining the yeast form as part of the stable flora of the microbiome in the human body.
There have been very limited previous studies done on herbs and their constituents that effectively act on inhibiting hyphal growth. Gymnemic acid is found in the herb Gymnema sylvestre and has been traditionally used in the treatment of diabetes. Similar to the herbs tested in this study, gymnemic acid did not have an effect on the growth of C. albicans, but did inhibit differentiation of the single-celled yeast to hyphae. In this case, the effective constituent was isolated and tested in hyphaeinducing conditions, which supports the future possibility of isolating novel compounds from various botanicals with which effect hyphal growth and virulence of C. albicans [5].
To further study the original goal of the experiment, which was to evaluate the fungicidal effects of herbs, essential oils were also evaluated. Essential oils are the steam-extracted, volatile aroma compounds of plants. They typically have different constituents and a higher concentration of active constituents requiring a small dose for a therapeutic effect. In this study, several essential oilbased botanicals were able to completely block hyphae formation as well as inhibiting the replication of C. albicans. Thymus vulgaris and Cymbopogon citratus were the most effective essential oils, although they had no fungicidal effect as botanical tinctures. These two herbs as tinctures only had an effect on inhibiting differentiation of C. albicans. This suggests that the same herb extracted into either oil or tincture may contain different active constituents.
Since C. albicans in patients with severe infections is commonly differentiated into the hyphal form, several tinctures and oils were also tested for their ability to reverse the hyphal form to a single-celled yeast form. M. condurango was the most effective tincture against preventing differentiation and it, along with the other active tinctures and oils were shown to be highly effective in the reversal trial. These results support the possibility for these tinctures and oils to be used in active C. albicans infections.
Tinctures are appropriate to treat both oral and topical infections, whereas essential oils are more suitable to treat topical infections due to their highly concentrated and caustic nature. Most of the botanical tinctures and oils used in this study have a low side-effect profile when taken internally or applied topically, respectively, when compared to conventional anti-fungal treatments [3]. There is a potential for both the tinctures and the oils to be combined in a synergistic blend that could be more efficacious in the overall treatment of a C. albicans overgrowth by providing different mechanisms of action.
Botanicals offer a therapeutic effect on many aspects of a fungal or microbial imbalance. Future research can explore the potential risk of antifungal resistance to these herbs, which may support the use of combined botanical preparations as opposed to a singleherb therapy and provide support for which herbs may work synergistically in order to best control C. albicans overgrowth in the human body.

Financial Support
Support for this project was provided by internal funding from the Southwest College of Naturopathic Medicine.  Herbal Medicine: Open Access