Phytochemical Analysis, Antifungal, and Antioxidant Properties of Two Herbs (Tristemma mauritianum and Crassocephalum bougheyanum) and One Tree (Lavigeria macrocarpa) Species

Phytochemicals present in medicinal plants (herbs, shrubs, and trees) are endowed with high antimicrobial and antioxidant properties. The aim of this work was to study the chemical composition, antioxidant, and antifungal activities of Tristemma mauritianum, Crassocephalum bougheyanum, and Lavigeria macrocarpa. Chemical composition of the plant extracts was determined using standard methods. The antioxidant activities were performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), nitric oxide (NO), and hydroxyl (OH) scavenging assays. The antifungal activity of plant extracts and their combinations with antifungals was evaluated against eleven Candida spp. using the broth microdilution method by determining the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). The quantitative chemical analysis of the extracts of T. mauritianum, L. macrocarpa, and C. bougheyanum showed that they contain phenols, tannins, and flavonoids that vary according to the plant species and extracts. All the plant extracts presented promising antifungal (MIC = 64–2048 µg/mL) and antioxidant activities. The extract of T. mauritianum displayed the highest antifungal (MIC = 64–256 µg/mL) and antioxidant (IC50 = 19.052 ± 1.11 μg/mL) activities which can be explained by its high phenolic content. Interestingly, extracts of T. mauritianum, L. macrocarpa, and C. bougheyanum displayed synergistic effects (fractional inhibitory concentration index, FICI ≤ 0.5) with ketoconazole against clinical resistant isolates. The results of the present study demonstrate promising antifungal and antioxidant activities of the tested plants that are associated to their phenol, tannin, and flavonoid contents. Hence, extracts of T. mauritianum and L. macrocarpa could be deeply investigated as antifungal alone and in combination with conventional antifungal drugs to treat infections caused by Candida spp.


Introduction
Infectious diseases appear as a major cause of mortality and morbidity in humans. Amongst them, invasive fungal infections have dramatically increased mainly in immunocompromised individuals [1]. Candida species that are the most common isolated in clinical fungal invasive infection are C. albicans, C. tropicalis, C. parapsilosis, and C. glabrata [2]. Despite current antifungal therapies, Candida infections show unpleasant high mortality rates. For example, the previous study indicated that mortality attributable to invasive Candida infections was about 19-24% among hospitalized patients [3]. Limited antifungal arsenal, diverse side efects, and drug-resistant strains appear as the main factors that contribute to that scenario [4]. In recent years, the emergence of clinically resistant strains is a major cause of failure in the treatment of invasive fungal infections. Te use of prolonged or repeated treatment with antifungals, such as fuconazole, is responsible for this scenario [5]. Terefore, the search for new antifungals is necessary and natural sources deserve attention because of the perception that they cause minimal side efects and have a long history of use in folk medicine for the treatment of fungal infections and oxidative stress conditions [6,7]. Low levels of reactive oxygen species (ROS) or free radicals are essential for cells to carry out normal biochemical functions such as cell signalling and apoptosis of defective cells [8]. Terefore, excessive generation of free radicals damages biological components that lead to aging and several chronic diseases such as cancer and cardiovascular diseases in humans [9]. Although an endogenous system of antioxidant is present in our body to get rid of excessive free radicals, exogenous antioxidants are recommended [10]. Chemically synthesized antioxidant compounds such as butylated hydroxytoluene have been questioned due to reports of their carcinogenicity [11]. Terefore, alternative antioxidants that have minimal side efects are highly needed. Te bioactive constituents extracted from the root and aboveground biomass of medicinal plants contain secondary metabolites (also known as phytochemicals), which represent a diverse group of natural products including alkaloids, phenols, favonoids, terpenoids, steroids, saponins, tannins, quinones, coumarins, and glycosides [12]. Phenolic compounds (including favonoids and tannins) are the most abundant phytochemicals in plant kingdom that serve as a supply source of health-benefcial properties such as antimicrobial and antioxidant activities in the human diet [12,13].
Crassocephalum bougheyanum (Compositae) is a fowering herb from subtropical or tropical dry forest and is commonly found in Cameroon [14]. In Cameroon, Congo, Gabon, and Nigeria, it is used as medicines and vegetables [15]. Essential oils obtained from C. bougheyanum had shown to contain α-phellandrene, pcymene, pinenes, myrcene, limonene, and (E)-β-ocimene, which are all monoterpene hydrocarbons [16]. Lavigeria macrocarpa (Icacinaceae) is a shrub having an extensive liana of 24 m long cauliforous, with a large underground tuber and rusty stellate pubescent and ecliptic leaves [17]. L. macrocarpa is mostly found in Cameroon forest area. It is used to treat rheumatism, poisoning, snakebites, malaria fever, and other feverish conditions [18,19]. Tristemma mauritianum (Melastomataceae) is a plant with a height of 1.25 m, and it is mostly found in marshy and moist places such as Senegal, West Cameroon, Equatorial Guinea, Congo Brazzaville, and upper Shari [20]. T. mauritianum is also used to treat oral and digestive candidiasis in children, paralysis, epilepsy, convulsions, and spasm [21]. Stem and leaf decoction of T. mauritianum is used to treat diarrhea, dysentery, and skin infections [22,23]. Previous research has shown its antisalmonella and antioxidant properties [24]. Te GS/MS analysis has shown that T. mauritianum contains 2,4-di-tert-butylphenol, 2-((octyloxy) carbonyl) benzoic acid, and sitosterol [25]. Te aim of this work was to study the chemical composition, antioxidant, and anticandidal activities of T. mauritianum J. F. (Gmel), C. bougheyanum C. D. Adams, and L. macrocarpa (Oliv.) Pierre.

Preparation of Extracts.
Te plant materials were washed thoroughly under running water, air-dried under room temperature, and crushed to powder using mixer-grinder. Te air-dried and powdered material from each plant was soaked separately in methanol (1/4 w/v) for 48 h at room temperature with shaking fve times per day. Te mixture was fltered through a Whatman flter paper No. 1, and the fltrate was concentrated by evaporation at 65°C using a rotatory evaporator (Buchi R-200) to obtain the crude extract that was dried in an oven at 40°C. Te extract was fnally kept at +4°C until further use.

Phytochemical Analysis of the Plant Extracts
2.3.1. Qualitative Phytochemical Screening. Standard methods described by Harbone [26] were used to perform the qualitative phytochemical screening of plant extracts. Te various plant extracts were screened for the presence of triterpenes, steroids, phenols, saponins, tannins, favonoids, anthraquinones, and alkaloids.

Determination of Total Phenolic Content (TPC).
Te total phenolic content (TPC) was determined as described by Ramde-Tiendrebeogo et al. [27]. Te reaction mixture in this test consisted of 20 µL of extracts (2 mg/mL), 100 µL of the Folin-Ciocalteu reagent (diluted 10 times in water), and 80 µL of a sodium carbonate solution 20%. Te mixture was stirred and incubated in a water bath at 20°C for 30 min, and then the absorbance was measured with a spectrophotometer (Biobase Bk-D590 Double Beam Scanning UV/Vis) at 765 nm. Te extracts were replaced with distilled water for control tubes. A calibration curve was plotted using gallic acid (concentrations ranged from 0.015 to 2 mg/mL). Results were expressed as milligram of gallic acid equivalent per gram of extract (mg·GAE/g).

Determination of the Total Flavonoid Content (TFC).
Te total favonoid content (TFC) of the extracts was determined using the aluminium chloride colorimetric method [28]. A volume of 100 µL of extracts (2 mg/mL) was mixed with 50 µL of aluminium chloride (1.2%), and then 50 µL of potassium acetate (120 mM) was added. Te mixture was incubated for 30 min at room temperature, and the absorbance was measured with a spectrophotometer (Biobase Bk-D590 Double Beam Scanning UV/Vis) at 415 nm. Te extracts were replaced with distilled water for control tubes. TFC was calculated using the quercetin calibration curve (concentrations ranged from 0.015 to 2 mg/mL), and results were expressed as milligram quercetin equivalent per gram of extract (mg·QE/g).

Determination of the Total Tannin Content (TTC).
Te total tannin content (TTC) of the extract was determined using the Folin-Ciocalteu method as previously described [29]. Te reaction mixture consisted of 100 µL of extracts (2 mg/mL), 500 µL of the Folin-Ciocalteu reagent (diluted 10 times in distilled water), 1000 µL of sodium carbonate solution at 35%, and 8.4 mL of distilled water. Te mixture was stirred and incubated at room temperature for 30 minutes, and then the absorbance was measured in a spectrophotometer (Biobase Bk-D590 Double Beam Scanning UV/Vis) at 700 nm. Te extracts were replaced with distilled water for control tubes. A calibration curve was plotted using tannic acid (concentrations ranged from 100 to 500 µg/mL). Te results were expressed in milligram equivalent of tannic acid per gram of extract (mg·TAE/g).

Antioxidant Assays
2.4.1. DPPH Radical Scavenging Assay. Te antiradical activity of each plant extract was evaluated using the protocol described previously [30]. Briefy, a volume of 900 μL of DPPH methanol solution (20 mg/L) was mixed with 100 μL of test sample. Te samples were prepared in methanol and tested at concentration range of 12.5 to 200 μg/mL. Te mixture was incubated in a dark room at room temperature for 30 minutes and the absorbance was read in spectrophotometer (Biobase Bk-D590 Double Beam Scanning UV/ Vis) at 517 nm. L-ascorbic acid was used as a standard antioxidant (12.5 to 200 µg/mL). Te experiments were carried out in triplicate for each concentration. Te optical densities obtained were converted to percentage inhibition, and the percentages of DPPH°scavenged (%RSa) by test samples were calculated as %RSa = [(A 0 − A 1 )/A 0 ] × 100, where A 0 is the absorbance of the DPPH alone and A 0 is the absorbance of the mixture. Te half-maximal inhibitory concentration (IC 50 ) values were estimated from the %RSa versus log of concentration plots using a nonlinear regression algorithm.

Ferric Reducing Assay.
Te reducing power of plant extracts was determined by applying the method described previously [31]. Briefy, 1 mL of each plant extract at diferent concentrations (200, 100, 50, 25, and 12.5 µg/mL) was mixed with 2.5 mL of a 0.2 M phosphate bufer solution (pH 6.6) and 2.5 mL of 1% potassium ferricyanide (K 3 Fe(CN) 6 ). Te resulting solution was incubated in a water bath at 50°C for 20 min. Ten, 2.5 mL of 10% trichloroacetic acid was added to stop the reaction, and the tubes were centrifuged at 300 rpm for 10 min. An aliquot (2.5 mL) of the supernatant was mixed with 2.5 mL distilled water and 0.5 mL of 0.1% FeCl 3 methanol solution. Te absorbance was read at 700 nm using a spectrophotometer (Biobase Bk-D590 Double Beam Scanning UV/Vis). Vitamin C was used as a standard antioxidant (12.5 to 200 µg/mL).

Hydroxyl Radical Scavenging Assay.
Te hydroxyl scavenging activity of the extracts was determined using Fenton reaction as previously described [32]. Briefy, 60 µL of FeCl 3 was mixed with 90 µL of 1,10-phenanthrolin (1 mM). Ten, 2.4 mL of phosphate bufer (0.2 M, pH 7.4) and 150 µL of H 2 O 2 (0.17 M) was added. A volume of 1.5 mL of extract at concentrations ranging from 12.5 to 200 µg/mL was introduced in the mixture. Te reacting mixture was incubated for 5 min at room temperature. After incubation, the absorbance was read at 560 nm in a spectrophotometer (Biobase Bk-D590 Double Beam Scanning UV/Vis). Vitamin C was used a as standard antioxidant (12.5 to 200 µg/ mL). Te percentage of hydroxyl radical scavenging activity (%HRSA) is calculated by the following formula: where A 0 is the absorbance of the control and A 1 is the absorbance of the mixture.

Nitric Oxide Scavenging Assay.
Nitric oxide scavenging activity of the extracts was carried out as previously described [33] with some modifcations. In a quartz cuvette, to 0.75 mL of sodium nitroprusside(10 mM) in phosphate bufer, 0.5 mL of extract/standard (vitamin C) (concentrations varying from 12.5 to 200 µg/mL) was added. Te resulting mixture was incubated at room temperature for 60 min. Ten, 1.2 mL of Griess reagent (10% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-napthyl) ethylenediamine dihydrochloride in distilled water) was added. Te fnal concentration varied between 12.5 and 200 µg/mL. After 5 min of incubation in a dark room at room temperature, the absorbance of chromophore formed was read using a spectrophotometer (Biobase Bk-D590 Double Beam Scanning UV/Vis) at 540 nm. Te control tubes contained methanol rather than extracts. Te radical scavenging percentage (NRS) of the various extract was calculated as follows: NRS (%) = [(A 0 − A 1 )/A 0 ] × 100, where A 0 is the absorbance of the control and A 1 is the absorbance of the extract/standard.

Antioxidant Activity
3.3.1. DPPH Radical Scavenging Activity. Te capacity of the extract to scavenge the DPPH radical was determined and expressed in IC 50 . It was observed that all the extracts possess a concentration-dependent antiradical activity (Figure 1 and Table 3). Apart from the root extract of L. macrocarpa, all the extracts inhibit signifcantly (p < 0.05) the DPPH radical. Te extract of C. bougheyanum presented the highest percentage of DPPH radical scavenging activity mainly at a concentration of 200 µg/mL (94.69 ± 2.98%) followed in decreasing order by those of T. mauritianum (93.42 ± 7.85%) and L. macrocarpa leaves (74.83 ± 4.05%) (Figure 1). Based on the calculated IC 50 values, extracts showed medium-to-strong DPPH scavenging activity ranging from 19.05 to 259.21 μg/mL. Te extract of T. mauritianum showed the most potent antioxidant activity (IC 50 � 19.052 ± 1.11 μg/mL) followed by that of C. bougheyanum (IC 50 � 23.70 ± 1.18 μg/mL). Vitamin C used as standard antioxidant was globally more potent than the extracts at all the tested concentrations, with IC 50 of 8.13 ± 1.78 μg/mL (Table 3).

Ferric Reducing Antioxidant Power (FRAP) of Extracts.
Te reducing power of iron was determined by the transformation of Fe 3+ into Fe 2+ in the presence of extracts and expressed as any increase in absorbance at 700 nm. Te results showed that the extract of L. macrocarpa leaves presented the best reducing power at all tested concentration compared to other extracts. Vitamin C used as standard drug presented the highest ferric reducing activity than that of the plant extracts at concentration of 200 µg/mL (Figure 2).

Hydroxyl Radical Scavenging Activity.
Te hydroxyl radical scavenging activities of the selected plant extracts are presented in Table 4. It was observed that the extract of C. bougheyanum showed the highest inhibition percentage (94.03%) at a concentration of 200 µg/mL than that of other extracts, not signifcantly diferent (p ≥ 0.05) from that of the reference antioxidant, vitamin C which  61.82 ± 0.14 e 6.57 ± 0.14 a 9.08 ± 0.62 b TPC: total phenolic content; TFC: total favonoid content; TTC: total tannin content.

Combination Efects of Ketoconazole/Fluconazole with the Plant Extracts.
Te results of the interaction study between known antifungal drugs (ketoconazole and fuconazole) and the tested plant extracts at their subinhibitory concentrations against yeast species are presented in Tables 7 and 8. Globally, we found that all the extracts showed synergistic efect with ketoconazole/fuconazole against at least two yeast species. Synergy ( FIC ≤ 0.5) was observed for the combinations of ketoconazole with the extracts of L. macrocarpa, T. mauritianum (leaves), and C. bougheyanum against fve (50%), four (40%), and three (30%) of the ten drug-resistant yeasts tested, respectively. Moreover, C. bougheyanum displayed 4 cases (40%) of additive efects (0.5 < ΣFIC ≤ 1) in combination with ketoconazole (Table 7). Interactions between the tested extracts and fuconazole were mainly indiferent efects (1 < ΣFIC ≤ 2) whereas some synergistic and additive interactions were noted for these combinations. For example, synergistic efects were observed against 3 (30%) and 2 (20%) out of 10 drug-resistant yeasts tested, with the combinations

Discussion
Phytomedicines have become increasingly popular for their potential use in curing many kinds of ailments with higher therapeutic value, lower toxicity, and fewer side efects when compared to allopathic medicines [36]. In the current study, the quantitative chemical analysis of the plant extracts was carried out with the aim of determining the content of secondary metabolites, which could explain their antioxidant and antifungal activities. In fact, the biological activity of medicinal plants is correlated with the presence and level of one or more classes of bioactive secondary metabolites [37,38]. Te results of this work indicate that all the studied plant extracts contain steroids and phenols such as favonoids and tannins. In comparison to our results, bioguided fractionation of the MeOH extract of T. mauritianum aerial parts led to the identifcation of luteolin-3′-O-β-Dglucuronopyranosyl butyl ester, quercetin-3-O-β-Dglucuronopyranosyl butyl ester, arjunolic acid-28-β-Dglucopyranosyl ester (Arjunglucoside II), asiatic acid-28β-D-glucopyranosyl ester (Quadranoside IV), β-sitosterol, oleanolic acid, ellagic acid, casuarinine, luteolin, pterocaryanin C, quercetin-3-O-β-D-glucopyranoside, and 6hydroxyapigenin-7-O-β-D-glucopyranoside [39]. Other study has found that T. mauritianum extract possesses signifcant quantities of phenols and favonoids [25]. However, this is a pioneer study performing the phytochemical composition of C. bougheyanum and L. macrocarpa extracts.
As multiple mechanisms are involved in the initiation of the oxidative stress, a single method is not sufcient to conclude about the antioxidant property of a sample [40]. Hence, in this work, the antioxidant activity of the plant extracts was confrmed by four tests (DPPH, NO, OH, and FRAP) even at low concentrations. Indeed, the DPPH radical scavenging assay is based on the ability of the stable free radical 2,2-diphenyl-1picrylhydrazyl to react with hydrogen donors including phenolic acids, favonoids, and tannins [41,42]. In the physiology condition, the interaction between ferric ion and superoxide anion induced the formation of hydroxyl radical, which induced the oxidation of DNA, lipid peroxidation, oxidation of proteins, and the activation of kinases [43]. Tus, the reduction of ferric ion can prevent those damages. Te presence of reductants (antioxidants) in the tested plant extracts can cause the reduction of Fe 3+ /ferrocyanide complex to ferrous form [44]. Hydroxyl radical is the major active oxygen species that causes lipid oxidation and important biological damage reacting with polypeptides, saccharides, nucleotides, and organic acids [45]. Te role of the free radical (NO) in infammatory processes is well known [46]. Total phenols, favonoids, and tannins can be linked to the antioxidant properties of the tested plants by acting as reducing agents, hydrogen donors, and singlet oxygen quenchers. Other groups of compounds that possess antioxidant activity, such as alkaloids, can contribute to this antioxidant potency. Indeed, it is well documented that synergies between various chemicals must be taken into consideration when predicting their biological activities [47,48]. An extract is considered as having signifcant antioxidant potential when IC 50 < 20 µg/mL, moderate when 20 ≤ IC 50 ≤ 75 µg/mL, and weak when IC 50 > 75 µg/ mL [49]. Based on that, extracts of T. mauritianum (IC 50 = 19.052 ± 1.11 μg/mL) and C. bougheyanum (IC 50 = 23.70 ± 1.18 μg/mL) have signifcant antioxidant activity. Tese results are highly supported by the quantities of TPC and TFC present in these extracts.
Te extracts of L. macrocarpa (leaves) and C. bougheyanum (aerial parts) displayed no MFC values against all the tested yeast species on which MIC values were determined, indicating that these extracts have fungistatic efect (MFC/MIC > 4) [7]. However, the extract of T. mauritianum (aerial parts) showed fungicidal activity (MFC/MIC ≤ 4) against C. dubliniensis 5 : 152 and C. albicans ATCC 10231 while that of the roots of L. macrocarpa showed fungicidal efect (MFC/MIC ≤ 4) against C. albicans ATCC 10231 and C. albicans 7Ca.  Advances in Pharmacological and Pharmaceutical Sciences  10 Advances in Pharmacological and Pharmaceutical Sciences With the increased incidence of drug-resistant fungi, synergistic combinations have been explored between conventional drugs and natural bioactive substances resulting in a new direction in antifungal drug discovery and antifungal therapy. Hence, research on the use of combinations of antifungals with natural substances to overcome fungal resistance has attracted considerable attention [1,2]. In this study, the extract of L. macrocarpa displayed the most relevant synergistic efect (fractional inhibitory concentration index, FICI ≤ 0.5) with ketoconazole against 50% clinical resistant isolates. Tis result suggests that the combinations of the extract of L. macrocarpa with antifungal drugs could be an alternative to treat invasive fungal infections involving drug-resistantCandida spp. Te current fndings also indicated that the tested extracts and mainly that of T. mauritianum have promising antifungal activity, which might be attributed to the presence of phenols, especially favonoid and tannin contents. Overall, the results of the current study are in agreement with those of Ngoudjou et al. [25] who demonstrated that T. mauritianum extracts had signifcant antioxidant and antibacterial activities. To our knowledge, this is the frst study showing anticandidal activity of T. mauritianum. Additionally, this is a pioneer study demonstrating the antioxidant and antifungal activities of L. macrocarpa and C. bougheyanum and the synergistic efect between these plant species and ketoconazole.

Conclusion
Te results of the present study demonstrate the antifungal and antioxidant activities of the tested plants that could be attributed to their phenolic contents. Hence, extracts of T. mauritianum and L. macrocarpa could be deeply investigated as antifungal alone and in combination with conventional antifungal drugs to treat infections caused by Candida spp. [54]. Minimal inhibitory concentration TCA: Trichloroacetic acid TFC:

Abbreviations
Total favonoid contents TPC: Total polyphenolic contents.

Data Availability
Te datasets generated and analysed during the current study are available from the corresponding author upon reasonable request.