Introduction

Candida albicans is among the most common human yeast that adheres and colonizes epithelial tissues of  the mucosal membranes including the oral, gastrointestinal tract, bladder and genitalia especially vagina (Rodrigues et al., 2019).  Candida infection was raised dramatically in terms of  severity mainly due to the increase of  disorder incidences – such as cancer and AIDS-, chemotherapy, etc. (WHO, 2014 and Darvishi et al. 2015). The incidence of  fungal infections by Candida spp. is increasing in both the community and hospital environments. C. albicans causes over 50% of  oral infections, while as high as > 90% of  vaginal candidiasis (Irish et al. 2006; Bahadoran et al., 2010 and Cortegiani et al., 2018). Vaginal candidiasis is common in Saudi Arabia in which 70.2% is yeast vaginitis caused by C. albicans (Al-Aali 2013). Overgrowth of  yeast can result from pregnancy, high-dose oral estrogen use, contraceptives tablets, over usage of  broad-spectrum antibiotics, uncontrolled diabetes, corticosteroids and obesity (Darvishi et al. 2015 and  Yokoyama et al., 2019).  The increase of  drug-resisting C. albicans encouraged the researchers to find alternative treatment.

Over thousands of  years, the old Egyptian, Greek and Muslims have used honey as a folkloric medicine for many diseases (Hegazi, 2012). Honey has biological activity as enhance immune response (Hegazi et al., 2015 and 2017a), potential antibacterial activity (Hegazi et al.,2014a,  2014b and 2017b), antitumor and antioxidant  ((Hegazi et al.,2014c) and antioxidant (Hörmet-Öz et al. 2009). However, the differences between honey types are due to maturation of  the honey itself inside the honey bees and the derivation from different kinds of  flower sources (Michalkiewicz et al. 2008). Moreover, seasonal climatic, geographic origin, harvesting time, storage and processing conditions all play significant roles of  honey bioactive effects (Michalkiewicz et al. 2008). In Arab cultures, honey is extremely used for both nutrition and therapy, however, there is a limited number of  research investigations on the antimicrobial activity of  honey published in Arabian region (Hegazi 2011 and Hegazi et al.,2017b).

Other alternative suggestion for candida treatment is the rose oil. Rosa species members of  the Rosacea family are important decorative plants with pink flowers and have been referred to as the “King of  Flowers” and the “Sambal of  Love” (Mahboubi 2016). Rosa damascena is one of  the most important Rosa species whose products are rose concrete, rose water, rose oil, rose absolute, and dried petals (Abdel-Hameed et al. 2013). These products have long been used in cosmetics, perfumes, medicinal purposes and food industries (Mahboubi 2016). Rose essential oil in addition to its perfuming effects was reported to possess a wide range of  biochemical activities in folkloric medicine such as hypnotic, analgesic, antispasmodic, anticonvulsant, anti-inflammatory, anti-oxidants and anti-microbial activities (Ulusoy et al. 2009). Taifi rose (Ward Taifi, e.g., Rosa damascena trigintipetala Dieck) is one of  the most important commercially propagated rose in Taif, Saudi Arabia. Taifi rose essential oil is very expensive and is known for its good perfumery applications and the use in cosmetic products (Hagag et al. 2014).

Up to our knowledge, no research on the influence of  Taifi rose oil alone or in conjunction with honey as anti-microbial agent was reported. In this study, the anti-candidia effects of  different kinds of  Saudi honey in addition to Taifi rose essential oil was investigated individually and in combination.

Materials and Methods

Microorganism and culture conditions

One local C. albicans isolate used in this study was kindly provided by the Microbiology Unit, East Jeddah Hospital, Jeddah, Saudi Arabia. The C. albicans isolate was previously identified by VITEK microbial identification system. The isolate was originally cultured on Sabouraud dextrorse agar (SDA) media (Oxoid, UK) and incubated at 37°C for 48-72 h. One single colony was subcultured in three replicates on Nutrient Broth (NB) (Himedia). Rate of  candida growth was evaluated in suspension cultures by spectrophotometer at OD 600 and diluted to 1.0 OD by nutrient broth media (NB).

Preparation of  honey and Taifi rose’s essential oil samples

Four kinds of  monof oral honey from different flower source and/or geographic origin, were used. Three of  them (Markh, Qatad and Sider) were obtained from a commercial Al Nahl Al Gawal Aprifarm (AlGuthami Foundation, Jeddah, Saudi Arabia), while the fourth is Manuka honey (New Zealand). All honey types were stored in sterile bottles in the dark at 4^oC temperature until used. Four different concentrations (50, 80 and 95%) of  honey were used in addition to the control (nutrient broth media without honey). Dilutions were prepared just before use. Essential oil extracted from Taifi rose (Rosa damascena trigintipetala) was kindly provided by farms of  Taif rose. Two different concentrations of  the Taifi rose oil (1 and 2%) were used in addition to the control (nutrient broth media without oil).

Physiochemical analysis of  honey

The physiochemical analysis of  honey samples (Hegazi et al., 2018). The physicochemical properties in terms of  glucose, fructose, and sucrose contents and parameters of  identity and quality of  local honeys in terms of  moisture content and acidity. The Codex Alimentarius Committee (SASO, 1990) and Hegazi et al., 2018 ) permitted a maximum value in commercialized honey.

Estimation of  anti-candida activity

A freshly grown C. albicans suspension was cultured in Falcon tubes (50 ml) with different honey (50, 80 and 95%) or Taifi rose oil (1 and 2%) dilutions in nutrient broth (Himedia) in three replicates. Based on the results, an extra culture of  C. albicans was prepared to include the lowest concentration of  a selected local honey that showed the highest anti-candida activity mixed with Taifi rose oil at the low concentration (1%). All tubes were incubated at 37^oC for 48 and 72 h incubation periods with continuous shaking at 150 rpm. For control, microbe-free media was used. Each broth culture (100 µl) was serially diluted by sterile water to the dilution factors 10², 10³ or 10⁴. Then, cultures were spread into SDA agar plates and incubated at 37°C for 72 h. The colony forming unit (CFU) was measured by open CFU 3.9.0 sof tware (Geissmann 2013).

Determination of  total phenols and flavonoids in Markh honey

For the determination of  free phenols and flavonoids, the Markh honey sample was diluted 10 times with (80%) ethanol solution. For determination of  total phenols and flavonoids, the honey samples were further diluted 10 times by 2M HCl and heated at 95°C for 30 min to liberate the ether binding phenols and flavonoids. The total phenols were determined by Lowry method (Lowry et al. 1951) , while the total flavonoids were determined in both extracts by the aluminum chloride method (Akbay et al. 2003; Barker 2019)

The phenolic compounds and flavonoids were detected in hydrolysate solution of  honey by mass spectrometry using Xivo TQD mass unit: Waters, after electrospray ionization (ESI) using daughter scan mode and negative ion detection [M-H]. The capillary and cone volts were 2.7 KV and 30 V, respectively, while the desolvation gas flow was 600 L/h. The MW and daughter ions for the different compounds were detected. Gallic acid has MW of  170, while daughter ions of  150.6, 125 and 97. Caffeic acid has MW of  180, while daughter ions of  161, 107.1 and 88.7. Vanillic acid has MW of  182, while daughter ions of  59.2, 67.1, 78.8, 93.0, 106.4 and 120.8. Quercetin has MW o f302, while daughter ions of  273, 178.9 and 151.

GC-MS analysis of  volatile oils and alkaloids

The analysis was performed using Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG–5MS (30 m x 0.25 mm x 0.25 µm film thickness). The column oven temperature was initially held at 50°C, then increased by 5°C/min to 230°C and held for 2 min, then increased again to the final temperature of  290°C by 30°C/min and held for 2 min. The injector and MS transfer line temperatures were kept at 250 and 260°C, respectively. Helium was used as a carrier gas at a constant flow rate of  1 ml/min. The solvent delay was 3 min and diluted samples of  1 µl were injected automatically using Autosampler AS1300 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of  m/z 40–1000 in full scan mode. The ion source temperature was set at 200°C. The components were identified by comparison of  their retention times and mass spectra with those of  WILEY 09 and NIST 11 mass spectral database.

Results

General description of  the different honey types used in the present study is shown in Figure 1. It included the physicochemical properties in terms of  glucose, fructose, and sucrose contents and parameters of  identity and quality of  local honeys in terms of  moisture content and acidity. The results indicated that contents of  glucose and fructose are almost the same in the three used local honey. Sucrose content was much less in the three types of  honey especially Qatad. Moisture content is almost similar for the three types of  honey averaging ~15%. Acidity was much higher in Markh (40 meq/kg) as compared with the other two types of  honey (Figure 1).

Figure 1: Physiochemical properties of Examined honey samples Click here to view figure

As shown in Table 1and Figure 2, all four honey types completely inhibited the C. albicans growth at both concentrations 95% and 80% after either incubation period, e.g., 48 or 72 h. On the other hands, Markh and Manuka honey continued to completely inhibit C. albicans growth at 50% concentration, while Qatad and Sider honey gave counted growth at both incubation periods of  48 and 72 h (Figure 2). Much heavier growth was shown for the latter two types of  honey as compared with Markh and Manuka honey at 50% concentration. Overall, Sider seemed to have the least anti-candida effects, while Manuka had the highest effects at both incubation periods as compared to the other types of  honey. The data of  Taifi rose oil indicated that 2% had completely inhibited C. albicans growth at the incubation periods of  48 and 72 h, while 1% concentration caused a declined C. albicans growth (Table 2). Based on the above-mentioned results, Markh was selected for further analysis in order to detect the accumulative effects of  mixing honey at either 30 or 50% with rose oil at 1% for either incubation period of  48 or 72 h, respectively. The results indicated that the two mixed honey/oil treatments were capable to completely inhibit C. albicans growth (Table 2).

Table 1: Influence of  different kinds of  honey dilutions (at 95, 80, 50 and 30%) on in vitro growth of  C. albicans.

Table 2: Influence of  Markh honey and combinations with Taifi rose oil (at 1 and 2%) on in vitro growth of  C. albicans.

The analysis of  phenols showed the presence of  0.1 % free phenols and 0.96% of  total phenols (free and ether bind phenols). The mass spectrometry analysis for detection of  phenolic compounds and flavonoids showed the presence of  caffeic acid, gallic acid and quercetin (Figure 3). The mass spectrum of  gallic acid clearly showed the [M-H] ion at m/z 169 and all daughter ions of  150.6, 125 and 97 were detected (Figure 2a). The mass spectrum of  caffeic acid clearly showed the [M-H] ion at m/z 178.9 and all daughter ions 161, 107.1 and 88.7 were detected (Figure 2b). The mass spectrum of  quercetin showed the [M-H] ion at m/z 301.1 and all daughter ions of  273, 178.9 and 151were detected (Figure 2c).

Figure 2: C. albicans growth at 103 CFU for 48 h incubation on Saboraud Dextrouse agar medium with 50% concentration of  honey. (a) = Manuka honey, (b) = Markh honey, (c) = Qatad honey, (d) = Sider honey.   Click here to view figure

Figure 3: The mass spectra of  gallic acid (a), caffeic acid (b) and quercetin (c) showing the [M-H] ion of  gallic at m/z 169, caffeic at m/z 168.9 and quercetin at m/z 301.1 in addition to the daughter ions for each compound. Click here to view figure

Discussion

The mechanism of  the anti-candida effect of  honey is not completely understood, however, several honey characteristics have been proposed. They include acidity and osmolarity due to high sugar content up to a certain threshold (Hegazi et al. 2017; Zam et al. 2018 and Hegazi et al., 2018 ). In this study, Markh honey had the highest free acidity as compared with the other two kinds of  local honey (Table 1), thus, had the highest anti-candida effect (Table 2). Free acidity is an important parameter related to the deterioration of  honey. Then, we speculate that Markh is potent to more storage periods. Free acidity is characterized by the presence of  organic acids in equilibrium with several other elements such as lactone, internal esters and some inorganic ions such as phosphates, sulfates and chlorides (Moreira et al. 2010 and Hegazi et al. 2017and Hegazi et al., 2018 ). The free acidity in Markh honey was 40. The Codex Alimentarius Committee on Sugars stated that values higher than 50 may be indicative of  fermentation of  sugars into organic acids especially in monof oral honeys (Fett et al. 2015). Therefore, many organic acids have influence on honeys’ free acidity (SOS, 1999, Alves et al. 2013; Tornuk et al. 2013; Hegazi et al. 2018).

The antifungal activity of  honey also relies on several other factors such as activity of  glucose oxidase to retard ripening of  nectar, hence, reduce the level of  hydrogen peroxide (AL-Waili et al. 2013; Hegazi 2011; Hegazi et al. 2017; Zam et al. 2018). Moreover, geographic origin, harvest season and flower source contribute to antibacterial mechanism of  honey  Hegazi et al. 2017; Zam et al. 2018). In the current study, four different kinds of  honey from different flower source in different geographic origins were studied. Markh and Qatad are monof loural types of  honey from Leptadenia pyrotechnica and Astragalus spinosus flowers, respectively, growing in Tihama, Madinah Almonawarah region, Saudi Arabia. They have ambergris and light ambergris colours and are grown at spring and autumn seasons, respectively. Sider is also a monof loural honey from Ziziphus nimmularia flowers growing in Fakhrah region in Saudi Arabia. It has an ambergris colour and is grown at autumn season. The fourth kind of  honey is Manuka New Zealand honey, from the flowers of  native Manuka tree. Manuka has a light ambergris colour.

Prior results indicated that the flower source of  Markh honey (Leptadenia pyrotechnica) has components such as phytols, cardenoids, flavonoids, alkaloids, steroidal glycosides and terpenes (Ghaneian et al. 2015; Verma et al. 2014). Phytol (3,7,11,15-tetramethylhexadec-2-EN-1-OL) is an important member of  branched chain unsaturated terpene, and is a product of  chlorophyll metabolism in plants. Interestingly, phytol can inhibit microbes (Ghaneian et al. 2015). Manuka honey has been also shown to contain high levels of  methylglyoxal (MGO) produced by the non-enzymatic conversion of  dihydroxyacetone that presents at high concentrations in the nectar of  Leptospermum scoparium flowers. Reports indicate that neutralization of  MGO in Manuka honey abolished the antimicrobial activity of  the honey against Staphylococcus aureus, but did not abolish the antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa (Kwakman et al. 2011; McLoone et al. 2016; Matzen et al. 2018). The authors concluded that MGO is not fully responsible for Manuka honey’s non-peroxide antimicrobial activity and that other components, possibly polyphenols, may be responsible.

In contrary to our results, Hegazi and others indicated that Qatad honey has the highest antibacterial activity on Klebsiella pneumoniae as compared with other Saudi honey (Hegazi et al. 2017). However, the latter study was concentrated on the anti-bacterial, rather than anti-candida, influence. Al-Waili et al. (2013) indicated that the minimum inhibitory concentration to inhibit candida growth is 70%. They also indicated that the rate of  growth after 72 h was similar to that after 24 h when candida was cultured on the same honey concentration. Other reports indicated that honey at 80% could completely inhibit the candida growth when incubated for 2-6 hour (Khosravi-Darani et al. 2013). Our results recommend the further use of  either 30 or 50% in treating vaginitis. Higher concentration might be sticky enough to prevent penetration of  the honey within the tissues, thus, retards the anti-candida effect.

The detected phenolic compounds are among those of  the aerial part of  Leptadenia pyrotechnica plant (Khasawneh et al. 2011). The quercetin is a flavonoid exists in the form of  quercetin-3-β-D-glucoside in the plant (Moustafa et al.2009). This compound is well known as an antioxidant and antimicrobial agent. Quercetin also showed antifungal activity against Cryptococcus spp. (Oliveira et al. 2016). It was the most active flavonoid tested against dermatophytes (Bitencourt et al. 2014). The alcoholic extract of  Sebastiania commersoniana exhibited significant antifungal activity against the dermatophytes Microsporum gypseum, Trichophyton mentagrophytes and Trichophyton rubrum. The active components in this extract include quercetin and gallic acid that are among flavonoid and phenolic compounds (Hnatyszyn et al. 2007). Among the tested phenolic compounds against Candida sp., gallic acid showed the highest MIC value (Alves et al. 2014). The GC/MS analysis of  volatile oils and alkaloids showed absence of  any volatile oil compounds, while the alkaloid 5-Heptyl-2-pyrrolidinone was detected at retention time 6.2 min. Pyrrolidin derivatives have been extensively studied as antifungal compounds (Bharose and Gajera 2018; Moradi et al. 2013). Interestingly, previous investigations of  the chemical composition of  Manuka honey also showed the presence of  gallic acid, caffeic acid, and quercetin (Alvarez-Suarez et al. 2014).. Therefore, the antimicrobial activity of  Manuka honey can also be attributed to the presence of  these compounds.

In the present study, the possible accumulative impact of  Taifi rose oil mixed with Markh honey on the candida growth, rather than the separate impact of  either treatment (1% rose oil or 30% Markh honey), was suggested. Prior reports showed that honey and ginger extract had a more significant inhibition of  Candida albicans growth (Khosravi-Darani et al. 2013). Darvishi et al. (2015) also indicated that honey mixed by yogurt is more effective on treating vaginal candidiasis than clotrimazole vaginal cream. The present study its kind to provide a substantial in vitro investigation of  the anti-candida effects of  Markh honey and Taifi rose oil, either separately and mixed. Further research is required to determine the anti-candida activity of  the mixture when treating vaginal candidiasis in the human body. Successful results might suggest the use of  the new mixture as a therapeutic drug for patients with vaginal candidiasis.

Conclusion

From the current results, it is concluded that a substantial in vitro investigation of  the anti-candida effects of  Markh honey and Taifi rose oil, either separately or mixed, indicates the possible use as complementary anti-candida agent.

Acknowledgments

The authors are grateful for the financial support by the Al Guthami Foundation, Saudi Arabia.

Conflicts Interests

The authors declare no competing interests.

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