Evaluation of Anti-Candida albicans Activities of Herbal Preparations Sold at the Kumasi Central Market in the Ashanti Region of Ghana

Candida albicans (C. albicans) is predominantly the leading cause of candidiasis among women with urogenital candidiasis. Since most people in resource-limited countries depend on herbal medicine for their primary care needs, many herbal drugs are sold to manage various infectious diseases. This study, therefore, evaluated the anti-C. albicans activities of five selected herbal preparations indicated for treating candidiasis sold at the Kumasi Central Market in the Ashanti Region of Ghana. The market was divided into five clusters, and one herbal preparation was randomly selected from each cluster. Using the Kirby Bauer disc diffusion antimicrobial susceptibility test, the herbal preparations were tested against clinically isolated C. albicans. Fluconazole, a standard antifungal drug, was included in the evaluation as a positive control. The experiments were performed on three different days and each in triplicates. Among the five selected herbal preparations, only one was effective against C. albicans with a mean inhibition zone of 19.1 mm. This effective herbal drug was prepared from Centella asiatica sap, Turnera microphylla leaves, and Vitex agnus-castus leaves. The results suggest that not all the herbal preparations selected were effective against C. albicans. Hence, we recommend that the authorities continually check the effectiveness of the herbal preparations on the market.


Background
Candidiasis is a fungal disease caused by Candida spp., mostly Candida albicans (C. albicans) accounting for about 60% of the reported cases [1]. Other non-albicans, including Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida vaginitis, and Candida auris, have also been identifed to cause candidiasis [2,3]. Candidiasis spans from mucocutaneous mycoses such as urogenital and oral candidiasis to life-threatening invasive forms such as candidaemia and hepatosplenic candidiasis [4]. In Ghana, most studies have focused on urogenital candidiasis, with a prevalence of 21-25.5% [5,6]. As a result, diferent herbal preparations are indicated for treating urogenital candidiasis on the market.
Plants have been used in traditional herbal medicine for years [7,8]. In some parts of the world, plants and herbs are still the primary sources of remedies used in treating diseases [9]. For example, Centella asiatica is used in managing varied conditions including, but not limited to, diseases of the female urogenital tract, fever, psoriasis, and wounds [10]. Also, in low-and middle-income countries (LMICs), about 80% of people depend on medicinal plants for their primary healthcare needs [11]. Moreover, these medicinal plants are easily accessible in these countries [12]. Although some herbal preparations on the market are efective, not much can be said about others because there is limited postmarket surveillance. Also, some people are into preparing herbal drugs because of the proceeds and not the efectiveness of their preparations [13].
Herbal preparations on the market are generally safe but can cause severe adverse risks among consumers [14]. Also, the efectiveness of these herbal preparations cannot always be guaranteed [15,16]. In Ghana, eforts are being made by the Food and Drugs Authority to assess the safety and effcacy of herbal preparations before they are introduced into the market. However, some herbal preparations still get to the market without safety and efcacy evaluation. Also, there is limited postmarket safety and efcacy surveillance on herbal preparations, thereby subjecting consumers to inefective treatment of their disease conditions. A study in Kumasi in the Ashanti Region of Ghana using a qualitative semistructured interview reported that some herbal preparations on the market are inefective, according to responders [16]. Also, there are limited studies on validating antimicrobial herbal preparations on the markets in Ghana.
Over the years, herbal preparations have been used in managing yeast and bacterial infections due to their efectiveness and low cost [17]. A previous study has shown that two medicinal plants, Lawsonia inermis and Portulaca oleracea have signifcant anti-Candida albicans (anti-C. albicans) activities [18]. Also, several plant extracts have been reported to have anti-C. albicans activities, including Allium sativum, Cinnamomum verum, and Origanum vulgare [19][20][21][22][23]. Moreover, extracts from Alchornea cordifolia (A. cordifolia), Spathodea campanulata (S. campanulata), and Afzelia africana (A. africana) have been reported to inhibit the growth of C. albicans [24][25][26]. Tese studies assessed individual medicinal plants without the combinations of the plant extracts. However, one fascinating thing about herbal medicine products is that most herbal formulations on the market are polyherbal. Also, there are limited studies on the postmarket efcacy of most polyherbal formulations. Hence, this study was imperative to evaluate the anti-C. albicans activities of herbal preparations indicated for treating candidiasis sold at the Kumasi Central Market in the Ashanti region of Ghana. Tus, this study targets a reallife concern of evaluating herbal preparations on the market for managing candidiasis.

Selection of Herbal Preparations. Te Kumasi Central
Market was divided into fve clusters, and one herbal preparation with an indication for treating candidiasis was randomly selected from each cluster. Te herbal preparations were given coded names due to ethical considerations (A, B, C, D, and E).

Confrmation of Clinically Isolated Candida albicans.
Clinically isolated C. albicans were obtained from the Microbiology Laboratory of the Komfo Anokye Teaching Hospital in Kumasi, Ghana. Te C. albicans were inoculated on the sabouraud dextrose agar (SDA) and incubated for 24 hours at 37°C. Te isolates were confrmed using the Integral System Yeasts Plus (Lioflchem, Italy), a system for identifying clinically important yeasts, by following the manufacturer's instructions [27]. Also, the confrmation was supported by performing a germ tube test on the C. albicans isolates [28].

Anti-Candida albicans Susceptibility
Testing. Using the Kirby Bauer disc difusion antimicrobial susceptibility test, the herbal preparations were tested against the clinically isolated C. albicans. A sterile flter paper was cut into about 6 mm and impregnated with the herbal preparations by allowing the paper to absorb the preparations until it could not absorb any more and allowed to dry as previously described [17]. Mueller Hinton agar plates were inoculated with 0.5 McFarland C. albicans suspension and dried for 5 minutes. Each herbal preparation was assigned a plate with fuconazole as a positive control in the experiment. Te impregnated discs were placed on the plates and incubated for 24 hours at 37°C. A flter paper impregnated with sterile water was included as a negative control. Te zone of inhibition was assessed as previously described [17]. Te experiments were performed on three separate occasions and each in triplicates.

Data
Analysis. Data were analysed using the Statistical Package for Social Sciences Statistical Software (version 20.0, IBM Corporation, USA). Te data were expressed in means and standard deviations. Where appropriate, the diferences in the means of inhibition zones were assessed using a student's t-test or two-way ANOVA (analysis of variance). Te statistical signifcance was accepted in all comparisons at a p value of less than 0.05.

Constituents of the Selected Herbal Preparations.
From the fve randomly selected herbal preparations at the Kumasi Central Market in the Ashanti Region of Ghana, A. cordifolia was the most common plant constituent. Also, the herbal preparations were constituted using at least two medicinal plants (Table 1).

Anti-C. albicans Activities of the Selected Herbal
Preparations. Among the fve selected herbal preparations indicated for treating candidiasis or yeast infection sold at the market, only the herbal preparation C inhibited the growth of C. albicans (Figure 1(c), plate C).
Intriguingly, the impregnated discs used for the herbal preparation C did not afect the zone of inhibition (p � 0.6553). Also, the diferent days on which the tests were performed did not afect the zone of inhibition (p � 0.1254), as shown in Table 2.

Discussion
In Ghana, most herbal medicine practitioners combine various plant parts or species to make herbal mixtures. Tis combination is due to these medicinal plants' additive or synergistic efects. Over-the-counter herbal preparations are popular, with an estimated 12% of the world population accessing herbal medicine. Also, in resource-limited countries, over 80% rely on medicinal plants for their primary healthcare needs, resulting in an estimated world market of 50 billion USD in the commercial herbal medicine industry, with an annual growth of 6.5% [11,29]. Although herbal supplements are popular worldwide, most herbal preparations on the market have unreliable efcacy and low quality. Hence, clinicians and scientists are usually concerned about the safety, efectiveness, and consistency of these herbal mixtures (reviewed in [30]).
Among the fve herbal preparations selected for the study from the market, only one of them inhibited the growth of C. albicans with an appreciable zone of inhibition (19.1 mm). Tis efective herbal mixture was prepared from Centella asiatica (C. asiatica), Turnera microphylla (T. microphylla) and Vitex agnus-castus (V. agnus-castus). Hence, the anti-C. albicans activity of this herbal preparation could be an additive or synergistic efect between the active ingredients of the medicinal plants because C. asiatica and V. agnus-castus have been reported to exhibit anti-C. albicans activities [31,32]. However, there is limited data on the anti-C. albicans activity of T. microphylla; hence, further studies are required to assess this plant to know its contributing efect in this efective herbal preparation for managing candidiasis.
Extracts from C. asiatica have been used in ethnomedicine for managing lupus, eczema, psoriasis, wounds, and diseases of the female urogenital tract [10,33]. Also, 100 g of fresh C. asiatica delivers 13.8 mg of vitamin C (reviewed in [34]). Vitamin C activates the body's immune system by stimulating leukocyte activities [35]. Hence, these medicinal and nutritional benefts of C. asiatica support the anti-C. albicans activity of the herbal preparation. Ethnomedically, V. agnus-castus extracts are used to manage menopausal problems and menstrual disorders. Also, this medicinal plant has other pharmacological efects, including antifungal, antibacterial, and anti-infammatory efects (reviewed in [36]). Tese reported pharmacological efects support the inclusion of V. agnus-castus in the herbal preparation with efective anti-C. albicans activity.
Tere are several mechanisms through which agents exhibit their antifungal efects. For example, fuconazole, like the other azoles, involves disruption of the conversion of lanosterol to ergosterol and subsequent disruption of fungal membranes. Also, nystatin binds to sterols in the plasma membranes of fungi leading to fungal cell death. C. asiatica contains pentacyclic triterpenes including, but not limited to, asiaticoside, madecassic acid, and asiatic acid [37]. Asiatic acid has various therapeutic properties, including antifungal activity against C. albicans. Drug efux overexpression is one of the most common mechanisms of drug resistance in C. albicans (reviewed in [38]). Interestingly, asiatic acid has been shown to inhibit efux pump activity and   19.0 ± 0.1 19.0 ± 0.1 19.2 ± 0.1 SD, standard deviation; C1-C3, triplicate discs impregnated with herbal preparation C. Te statistical diference was assessed using a two-way ANOVA. 1 Te impregnated discs used did not afect the zone of inhibition (p � 0.6553), and 2 the diferent days did not afect the zone of inhibition (p � 0.1254).
Evidence-Based Complementary and Alternative Medicine 3 morphological transformation in C. albicans [39]. Hence, it could be concluded that the inhibition of efux pump activity and morphological transformation by asiatic acid could be one of the molecular mechanisms via which the efective herbal preparation inhibits C. albicans growth. Moreover, V. agnus-castus contains essential oils, which have been reported to inhibit the growth of C. albicans similar to amphotericin [40]. Farnesyl pyrophosphate synthase is a vital enzyme in Candida spp. required for ergosterol biosynthesis, the main component in the cell membrane [41][42][43]. Hence, targeting this enzyme is suicidal for Candida spp., and essential oils from V. agnus-castus (caryophyllene and verticiol) have good binding afnities to this enzyme. Caryophyllene binds to the enzyme's active site via hydrophobic interactions, whereas verticiol forms a hydrogen bond with the Asp156 in the enzyme's active site, justifying the anticandidal activity of V. agnus-castus [40]. Also, the binding activities of caryophyllene and verticiol could be another molecular mechanism via which the effective herbal preparation inhibits C. albicans growth. Te active herbal preparation against C. albicans exhibited an inhibition zone of 19.1 mm. Compared to the standard drug, fuconazole, there was a statistical diference in the inhibition zone, with fuconazole having a higher zone of inhibition (19.5 mm). Even though there is no standard interpretation for the zone of inhibition for the active herbal preparation, it could be accepted to be efective for treating candidiasis since it has a zone of inhibition of ≥19 mm. However, a previous study that assessed the antifungal activity of Flos rosae chinensis on C. albicans found that this medicinal plant was more efective than fuconazole in inhibiting the growth of C. albicans with an inhibition zone of 22 mm compared to fuconazole (18 mm) [44]. Although this current study reported an antifungal activity of a polyherbal, it still supports the efectiveness of herbal medicines for managing infectious diseases, including candidiasis.
Plant constituents of some of the selected herbal preparations, such as A. cordifolia, S. campanulata, and A. africana, have been reported to inhibit the growth of C. albicans [24][25][26]. However, the polyherbal mixture containing these plant extracts could not inhibit the growth of C. albicans. Tis low efcacy could be attributed to inconsistent processing methods used by manufacturers and diferent therapeutic potentials in herbal preparations resulting from the age, harvesting time, geographical location, and postharvest handling of the plants. Moreover, the combination of these plants could hamper their antimicrobial activities via antagonism [45].

Conclusions and Recommendations
Only one of the fve selected herbal preparations indicated for treating candidiasis was efective against the growth of C. albicans. Te efective herbal preparation was prepared from C. asiatica, T. microphylla, and V. agnus-castus. Previous studies have shown that active agents from C. asiatica and V. agnus-castus inhibit C. albicans growth by inhibiting efux pump and farnesyl pyrophosphate synthase activities [39,40], supporting the anti-C. albicans activity of the efective herbal preparation. Tis study has revealed that not all herbal preparations on the market are efective. Hence, authorities should continually check the efectiveness of herbal preparations on the market. Further, persons should use herbal preparations prescribed by authorised herbal medicine practitioners. We also recommend similar studies in diferent markets in Ghana using larger sample sizes.

Limitations of the Study
Te study was performed using a small sample size due to fnancial constraints, which could account for the low effective rate of the herbal preparations. Nonetheless, our fndings could serve as the baseline data for further studies on commercially processed herbal preparations.

Data Availability
Te data supporting the current study are given in the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
SAD conceptualized and supervised the work. GK and MB conducted the experiments and analysed the data. SAD wrote the manuscript. All authors have read and approved the fnal version of the manuscript. Evidence-Based Complementary and Alternative Medicine 5