Chemical Constituents and Biological Activities of Essential Oils of Hydnora africana Thumb Used to Treat Associated Infections and Diseases in South Africa

Abstract

Hydnora africana (HA) Thumb is a member of the genus Hydnora. The roots are used in the treatment of infectious diseases in South Africa folk medicine. Though the root extracts are used to treat various human diseases including inflamed throat, there is a dearth of scientific data on the biological activities of essential oil isolated from this plant in the literature. Therefore, the present study was conducted to determine the chemical components and certain biological activities of the essential oil using standard bioassay methods. The plant essential oil exhibited a moderate free radical scavenging activity that was dependent on the radical species. Similarly, the essential oil was active against the growth of all thirteen opportunistic bacteria apart from Salmonella typhimurium, Pseudomonas aeruginosa and Proteus vulgaris. The essential oil was also active against Aspergillus niger among all the nine fungi selected. In addition, the species is typified by substantial amounts of classes of compounds including; carboxylic acids (30.68%), terpenes (10.70%), alkyl aldehydes (4.86%) and esters (0.82%), identified as antioxidant and antimicrobial agents. The essential oil of H. africana could be said to have pharmacological properties, and these agents in the essential oil of H. africana could justify the folklore usage of this plant in the treatment of infections and related diseases.

1. Introduction

Medicinal plants have contributed immensely to health care in Africa. These plants are easily accessible and cheap sources of therapeutic agents [1]. Essential oils from medicinal plants contain certain constituents that play a pharmacological role in the prevention and treatment of chronic diseases such as cancer, cardiovascular disease, diarrhoea diseases and infections [2].

Infection is the invasion of the body tissues by disease-causing agents like bacteria, virus and fungi. The host tissue’s reaction to these organisms as well as the toxins produced by the organisms is attributed to inflammation in patients with HIV or AIDS, cancer, solid organ transplantation, immune deficiencies, chronic inflammatory gastrointestinal, liver, respiratory, urinary tract and other infections [3]. The bacteria associated with common infections like wounds, gastrointestinal and urinary tract infections include Klebsiella pneumonia, Streptococcus pyogenes and Serratia mercescens [4,5]. Essential oils are known to soothe infections by acting as antimicrobial agents, or by reducing and neutralising reactive oxygen species (ROS) generated during the pathophysiology of disease infections [6].

Medicinal plants play important roles as a source of antimicrobial and antioxidant. One of these plants is the genus Hydnora africana (HA) Thumb (Hydronaceae (Piperales)) (Figure 1A,B). The plant is also known as Jackal food, Ubnklunga (Xhosa) or Umavumbuka (Zulu) [7]. It comprises 15 species in South Africa. This plant, similar to about ten species of parasitic flowering plant, lives a greater part of its life cycle underground and is unable to create chlorophyll or perform photosynthesis. In traditional medicine, the root of Hydnora species, tuber, fruits, leaves and fruit pulp (such as potato) is used for the treatment of infectious diseases such as dysentery, diarrhoea, amenorrhoea, bladder and kidney complaints. Other ailments include swollen glands and inflamed throat [7]. Although progress has been made in validating the chemical components and biological activities of the root parts of the plant, the chemical composition, antioxidant and antimicrobial properties of HA essential oil remain obscure.

Therefore, this study aimed at identifying the chemical constituents of H. africana essential oil using gas chromatography interfaced with mass spectrometry (GC-MS). We also aimed to assess its antioxidant, antibacterial and antifungal potential, and to relate our findings to its possible use in folk medicine.

2. Materials and Methods

2.1. Plant Collection and Preparation

The roots of H. Africana as shown in Figure 1B were collected in December 2013 at the Ntselamanzi area location in Nkonkobe Municipality, Eastern Cape Province, South Africa. This lies at the latitude and longitude as described by Masika and Afolayan [8]. The samples were collected and the specimen of the voucher (Win 2014/1) was kept in the Giffen’s herbarium, University of Fort Hare, Alice, South Africa. The plant materials were washed using tap water and then stored in the refrigerator at 4 °C until it was time for essential oil extraction.

2.2. Extraction of the Essential Oil

The essential oil was isolated from H. Africana roots by hydro-distillation following the procedure from European Pharmacopoeia [9]. Here, 250 g of the dry roots were subjected to hydro-distillation for 3 h in an all-glass Clevenger (model and manufacturer name). Through the supply of heat to the heating mantle (50 °C), the essential oil was extracted with 4 litres of water in 3 h until no more essential oil was coming out. The average percentage yield was 0.3%. Appropriate 10% DMSO vehicles in between 80 were utilized to dissolve the essential oil for more in vitro bioassay activities.

2.3. Chemicals and Reagents Used

The chemicals used consist of 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), butylated hydroxyl toluene (BHT), vanillin, potassium persulphate, rutin, sodium nitroprusside (Na₂[Fe(CN)₅NO]2H₂O), glacial acetic acid (CH₃COOH), sulfanilic acid, gallic acid, tannic acid, ferric chloride (FeCl₂), aluminum chloride (AlCl₃), potassium ferricyanide [K₃Fe(CN)₆], trichloroacetic acid (TCA), Folin-Ciocalteu reagent, sodium carbonate (Na₂CO₃), hydrochloric acid (HCL), sodium dihydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), Mueller-Hinton agar (MHA), dimethyl sulfoxide (DMSO), ascorbic acid, ciprofloxacin and amoxicillin, 2-thiobarbituric acid (TBA), potassium acetate (CH₃CO₂K), Mueller-Hinton dextrose broth (MDB), diclofenac sodium and sodium chloride (NaCl), These chemicals were bought from Merck and Sigma-Aldrich, Gauteng, South Africa. Every chemical utilised in this study was of analytical grade.

2.4. Antioxidant Assay

The antioxidative capacities of the essential oil of H. africana were assessed in terms of DPPH, nitric oxide, hydrogen peroxide and ABTS inhibitory assays.

2.4.1. 1,1-diphenyl-2-picrylhydrazyl DPPH Radical Scavenging Activity Assay

The method of Liyana-Pathiranan and Shahidi [10] was employed to determine DPPH free radical scavenging activity. A solution of 0.135 mM 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in methanol was briefly prepared. Briefly, 1.0 mL of this solution was mixed with 1.0 mL of the HA oil and standard drugs (BHT and gallic acid) (0.025 to 0.5 mg/mL). The reaction mixture was then vortexed and put in the dark at room temperature for a period of 30 min. The absorbance of the mixture was measured using a spectrophotometer at 517 nm. All tests and analysis were run in triplicates. The plant extract’s scavenging ability was then calculated using the following equation:

DPPH Scavenging activity (%) = [(Abs control − Abs sample)/(Abs control)] × 100

where Abs control is the absorbance of DPPH + methanol and Abs sample is the absorbance of DPPH radical + sample (sample or standard).

2.4.2. 2,2′-Aazino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) Radical Scavenging Assay

The method described by Oyedemi et al. [11] was adopted for the determination of ABTS scavenging activity. The stock solution of 7 mM ABTS solution and 2.4 mM potassium persulfate solution was prepared. The working solution was prepared by mixing the two stock solutions in equal proportions and then keeping it in the dark room for 12 h. The solution was then diluted by mixing 1 mL ABTS⁺ solution with 60 mL of methanol to get an absorbance of 0.708 ± 0.001 units at 734 nm with the use of the spectrophotometer. The plant’s essential oil (1 mL) and the controls were allowed to react with 1 mL of the ABTS⁺ solution and the absorbance was taken at 734 nm after 7 min using a spectrophotometer. The ABTS⁺ scavenging capacity of the essential oil was then compared with that of the standards. The percentage inhibition was then calculated as follows:

Inhibition % = [(A blank − A sample)/A blank] × 100

(1)

where A blank is the absorbance of ABTS radical + methanol used as control and the A sample is the absorbance of ABTS radical + sample extract/standard.

All the tests were carried out in triplicate. The activity was expressed as 50% inhibitory concentration (IC₅₀). The lower the IC₅₀ value, the higher the antioxidant activity.

2.4.3. Nitric Oxide Scavenging Activity Assay

The nitric oxide radical scavenging activity was determined by the modified method described by Oyedemi et al. [11]. A volume of 2 mL of 10 mM of sodium nitroprusside prepared in 0.5 mM phosphate buffer saline (pH 7.4) mixed with 0.5 mL of plant extracts and standards, separately, to make different concentrations from 0.025 to 0.5 mg/mL. The mixture was incubated at 25 °C for a period of 150 min after which 0.5 mL of incubated solution was mixed with 0.5 mL of Griess reagent (1.0 mL of sulfanilic acid reagent (0.33% prepared in 20% glacial acetic) acid at room temperature for 5 min with 1 mL of naphthylenediamine dichloride (0.1% w/v). After incubating the mixture was for 30 min and taking absorbance at 540 nm, the amount of nitric oxide radicals inhibited by the essential oil was calculated by the use of the equation below:

NO radical scavenging activity (%) = [(Abs control − Abs sample)/(Abs control)] × 100

where Abs control is the absorbance of NO radical + methanol and the Abs sample is the absorbance of NO radical + extract or standard.

2.4.4. Hydrogen Peroxide (H₂O₂) Scavenging Activity Assay

The H₂O₂ inhibition activities of the extracts were assessed by the method of Oyedemi et al. [11]. A solution of hydrogen peroxide (4 mM) was prepared in phosphate buffer (0.1 M; pH 7.4) and incubated for 10 min after which 1 mL of the essential oil (0.025 to 0.5 mg/mL) was added to a 0.6 mL of hydrogen peroxide solution (4 mM). After 10 min of incubation, the absorbance of the reacting mixture was measured spectrophotometrically at 230 nm, against a blank solution containing phosphate buffer solution without hydrogen peroxide. BHT and Gallic acid (0.025 to 0.5 mg/mL) were used as positive control. Below is the formula used to calculate percentage scavenging of hydrogen peroxide of samples:

H₂O₂ inhibition capacity (%) = [1 − (Absorbance of sample/Absorbance of blank)] × 100

(2)

2.5. Antimicrobial Activity

2.5.1. Microorganisms and Media

The bacteria and fungi employed in this study were selected mainly on the basis of their significance as opportunistic pathogens of humans with diarrheal diseases. The bacterial strains include Shigella flexneri KZN, Proteus vulgaris, Klebsiella pneumonia ATCC 4352, Staphylococcus aureus, Enterococcus faecalis ATCC 29212, Streptococcus pyogenes, Pseudomonas aeruginosa ATCC 19582 and Serratia mercescens ATCC 9986. The fungi strains of the American Type Culture Collection (ATCC) include Triphophytonrubrum ATCC 28188, Trichophyton tonsurans ATCC 28942, Trichophyton mucoides ATCC 201382, Microsporum canis ATCC 36299, Microsporum gypseum ATCC 24102, Aspergillus niger ATCC 16888, Aspergillus fumigatus ATTC 204305, Penicillium chrysogenum ATCC 10106 and Penicillium aurantiogriesum ATCC 16025. The bacteria test organisms were obtained from the Department of Biochemistry and Microbiology, University of Fort Hare, South Africa and fungi test organisms were purchased from Davies Diagnostics (Pty) Ltd., Gauteng, South Africa, respectively.

The Mueller–Hinton dextrose agar (MHA), Mueller-Hinton dextrose broth (MHB), potato dextrose agar (PDA) and Sabouraud dextrose broth (SDB) were prepared according to the instruction from the manufacturer. The nutrient agar was suspended in demineralized water, heated and stirred until it completely dissolved and then sterilised by autoclaving at 121 °C for 15 min. The fungi and bacteria were maintained at 4 °C on MHA and PDA. Scraped cell mass in 0.85% salt solution was diluted and adjusted to 0.5 McFarland standards and authenticated by spectrophotometric reading at 580 nm assays [12] to prepare the inoculums for the assays.

Finally, the cell suspensions were diluted 1:100 in nutrient broth to present an estimated inoculum of 10⁴ CFU·mL⁻¹ in comparison with McFarland standard for use in the assays [12].

2.5.2. Antibacterial and Antifungal Susceptibility Test

The agar well diffusion technique as described by Prabuseenivasan et al. [2] was modified and used to test for the antibacterial and antifungal activity. Briefly, 100 μL of 0.5 McFarland solutions of bacterial or fungal strain cultures in 0.85% sterile distilled water (SDW) was placed over the surface of an agar plate and spread with a sterile inoculation loop. In each agar plate, three wells were cut with a cooled, flamed cork borer of 5 mm diameter, while a sterile needle was used to remove the agar plugs. A 50 μL of the amoxicillin (0.0125 mg/mL) or nystatin (0.03 mg/mL) were added to the first well for antibacterial and antifungal susceptibility test respectively to serve as a positive control. In the second well, 50 μL of the corresponding extract solvent were added while in the third, 50 μL of the acetone extract/aqueous extract/essential oil (50 mg/mL) were added. The essential oil had been prepared by dissolving in 10% DMSO with Tween 80 (0.5% v/v for easy diffusion) and sterilised by filtration through a 0.45 μm membrane filter [2]. Preliminary studies confirmed that the carrier vehicle did not inhibit the growth of bacteria. Each test was done in triplicate. The culture plates were incubated at 37 °C, and the results were examined after 24 h and 72 h for antibacterial and antifungal susceptibility test, respectively. The clear zone around each of the wells was measured in mm, showing the plant fractions’ activity against the tested organisms.

2.5.3. Minimum Inhibitory Concentration (MIC) Assay

The broth microdilution method using 96 well microtiter plates was used to verify the minimum inhibitory concentration (MIC) of the plant extracts which indicated antibacterial or antifungal activity [12]. After adding 120 μL of SDW into every well of the first (A) and the last (H) rows and into every well of the last column, 120 μL of Nutrient broth (NB) was put in all the wells of the second row (B) after which a 150 μL of NB was put in the wells left in the first column and 100 μL in the remaining wells from the second column towards the right. There was then the addition of fifty microliters of the essential oil (20 mg/mL) into the third well of the first column as well as the addition of 50 μL of the positive (amoxicillin or nystatin) and negative control (SDW) independently into the wells left in the first column. A serial dilution was carried out in two folds. The contents in all the wells of the first column (beginning from the third row) were mixed and 100 μL was transferred into the second well of the same row. This process was replicated up to the 11th well after which the 100 μL was discarded. Consequently, the dilution of the fractions of the plant and the control in the wells brought about a range of concentration from 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.019, 0.0098 and 0.005 mg/mL.

Subsequently, there was the inoculation of 20 μL of 0.5 McFarland bacterial or fungal suspensions into the wells apart from the ones which had SDW. The test organism’s growth was measured through the determination of the absorbance at 620 nm with an automatic ELISA microplate reader (Synergy Mx, BioTek^®, Winooski, VT, USA) prior to, and following incubation. The plates were incubated at 37 °C for 24 h and 72 h for bacteria and fungi, respectively. The MIC 50 was defined and recorded as the concentration of the test antibacterial and antifungal agent that revealed 50% inhibition of bacteria and fungi growth, respectively.

2.6. Chemical Composition Evaluation

2.6.1. Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analyses were performed on Agilent 5977A MSD and 7890B GC System, Chemetrix (Pty) Ltd., Midrand, South Africa; Agilent Technologies, Deutschland, Germany with a Zebron-5MS column (ZB-5MS 30 m × 0.25 mm × 0.25 um) (5%-phenylmethylpolysiloxane). The column and temperature conditions employed include GC grade helium at a flow rate of 2 mL/min and splitless 1 mL injection was also employed. The injector, source and oven temperatures were set at 280 °C, 280 °C and 70 °C, respectively. The ramp settings were; 15 °C/min to 120 °C, then 10 °C/min to 180 °C, then 20 °C/min to 270 °C and held for 3 min.

2.6.2. Identification of Components

The identification of the chemical components of the essential oil was influenced by their GC retention times, percentage composition (area %) and retention indices. The interpretation and identification of their mass spectra were confirmed by mass spectral incorporated library. The identification was further confirmed by search using the National Institute of Standards and Technology (NIST) database (NIST/EPA/NIH mass spectral library 2014) with those of published data [13]. Empirical searches were conducted using the PubChem Project (https://pubchem.ncbi.nlm.nih.gov/) and Drug Bank (www.drugbank.ca/) to identify the known pharmacological properties associated with these compounds.

2.6.3. Statistical Analysis

All experiments were carried out in triplicates and the results were expressed as Mean ± SD. Where appropriate, the data were subjected to one-way analysis of variance (ANOVA) employing the Minitab program (version 12 for windows). p < 0.05 were considered significant.

3. Results and Discussion

3.1. Antioxidant Activities

In this study, the DPPH, ABTS, nitric oxide and hydrogen peroxide scavenging activities have been used as in vitro models to evaluate the antioxidant capacity of the essential oil of Hydnora africana, in comparison with the standards BHT and Gallic acid. Figure 2 illustrates the DPPH radical scavenging activity of the essential oil of Hydnora africana compared with BHT and Gallic acid. We observed weak DPPH inhibitory activities of the essential oil of Hydnora africana, compared with the reference compounds used. Previous studies have indicated that essential oils from different sources possess varying capacities to scavenge DPPH radicals, probably due to the differences in their composition. Factors like stereo-selectivity of the radicals or the solubility of the oil in the different testing systems have been reported to affect the capacity of essential oils in quenching different radicals [14]. Our result was similar to that of Negue et al. [15] who also found weak DPPH radical scavenging activity using the essential oil of P. brazzeana (RSA = 14% for 1 g/L, SC50 = 1.5 mg/mL and SC50 = 3.23 mg/mL). In contrast, Ndoye et al. [16] showed that the essential oil of Allium sativum was a more effective DPPH radical scavenger (SC50 = 7.67 mg/mL). It was observed that increasing the concentration of the oil or standards was not accompanied by any significant increases in their DPPH radical scavenging activities. It appears that the lowest concentrations tested (0.025 mg/mL) was sufficient, in each case, to produce the maximum antioxidant capacity of the standards. It is possible, therefore, that much higher doses of the oil than those tested in this study may produce higher DPPH radical scavenging activities. The IC₅₀ values could not be determined precisely for both standards as they were lesser than the lowest concentration tested (0.025 mg/mL). Similarly, IC₅₀ for the essential oil was greater than the highest concentration tested (0.5 mg/mL) and could not be precisely determined. The results observed in this study indicated that the Hydnora africana essential oil hasa low effectiveness in donating a hydrogen proton to the lone pair electron for the radical.

The ABTS radical scavenging activity of H. africana essential oil is illustrated in Figure 3. The ABTS radical scavenging activity of the essential oil was also significantly lower (p > 0.05) when compared to those of BHT and Gallic acid at all the concentrations investigated (Figure 3). The % inhibition of ABTS by the essential oil was found to be concentration- dependent. The IC₅₀ value for the ABTS radical scavenging activity of the essential oil was 0.55 mg/mL, while those of the standards could not be determined precisely as they were lower than the lowest concentration tested. This result here is similar to the work by Mamadalievaa et al. [17] who found that three Uzbek Scutellaria species also exhibited weak ABTS radical scavenging activity.

The nitric oxide radical scavenging activities of the essential oil and the reference compounds are presented in Figure 4. At all the concentrations tested, the essential oil produced a significantly higher (p < 0.05) nitric oxide radical scavenging activity, when compared with both BHT and Gallic acid. However, the capacities of all the tested compounds to scavenge nitric oxide were largely concentration-dependent. Nitric oxide, in itself, can alter cell structure and function by modifying different signalling pathways. Its toxicity usually results when it combines with the superoxide anion to form peroxynitrite, which is a very strong oxidant [18]. The ability of the essential oil of H. africana to effectively scavenge nitric oxide radicals in this study suggests it could be useful against oxidative conditions involving this radical.

The scavenging activities of the essential oil and the standards against hydrogen peroxide are depicted in Figure 5. For both the oil and the standards, the amount of hydrogen peroxide remaining in the reaction mixtures, decreased as their concentrations were increased. There was no significant difference observed between the oil and BHT at nearly all the concentrations (p < 0.05) except at 0.05 mg/mL where BHT showed a significantly higher activity than the oil (p > 0.05). At all the concentrations tested (0.025 to 0.5 mg/mL), the essential oil showed lesser hydrogen peroxide antioxidant activity as compared to the standards (BHT and gallic acid), only able to reduce hydrogen peroxide by less than 20%, while gallic acid had the highest activity, reducing the H₂O₂ by over 80%.

3.2. Antimicrobial Analysis

The essential oil was tested against thirteen opportunistic bacteria. The results showed that the plant was active against the growth of all organisms except Salmonella typhimurium, Pseudomonas aeruginosa and Proteus vulgaris. The essential oil of H. africana showed activity against growth of some pathogenic bacteria that cause infections associated with diarrhoeal diseases (

Table 1. Inhibition zone diameters caused by the essential oil (50 mg/mL) in the tested opportunistic bacteria.

Organism	Essential Oil	Positive Control (Amoxicillin)
Salmonella typhimurium −	NA	36 ± 1.1 a
Enterococcus faecalis −	17 ± 0.2 a	34 ± 1.2 b
Escherichia coli −	15 ± 1.3 a	35 ± 0.2 b
Pseudomonas aeruginosa −	NA	NA
Bacillus cereus +	10 ± 1.6 a	32 ± 1.2 b
Shigella sonnei −	15 ± 0.3 a	35 ± 2.2 b
Streptococcus pyogens +	22 ± 0.2 a	35 ± 2.1 b
Bacillus subtilis +	22 ± 0.2 a	40 ± 0.2 b
Shigella flexneri −	19 ± 0.2 a	40 ± 1.1 b
Klebpneumoniae −	23 ± 2.1 a	33 ± 2.2 b
Staphylococcus aureus +	25 ± 3.1 a	37 ± 0.2 b
Proteus vulgaris −	NA	38 ± 1.1 a
Serretiamercenscens −	25 ± 3.1 a	35 ± 2.2 b

The bacteria are denoted thus by: St (Salmonella typhimurium), Ef (Enterococcus faecalis), Ec (Escherichia coli), Pa (Pseudomonas aeruginosa), Bc (Bacillus cereus), Ss (Shigella sonnei), Sp (Streptococcus pyogens), Bs (Bacillus subtilis), Sf (Shigella flexneri), Kp (Kleb pneumoniae), Sa (Staphylococcus aureus), Pv (Proteus vulgaris) and Sm (Serratia marcescens). Data expressed as means ± SD; n = 3; values along a row with different subscripts are significantly different (p ≤ 0.05).

and

Table 2. Minimum inhibitory concentrations (MIC) of the H. africana essential oils against the tested opportunistic bacteria.

Solvent	Inhibition Zone Diameter (mm)
	St	Ef	Ec	Pa	Bc	Ss	Sp	Bs	Sf	Kp	Sa	Pv	Sm
Essential oil	NA	2.5 a	>5 a	NA	>5 a	>5 a	0.02 a	0.02 a	0.02 a	2.5 a	0.02 a	NA	0.02 a
Positive control	0.01	0.01 b	0.01 b	NA	0.01 b	0.01 b	<0.01 b	0.01 b	0.01 b	<0.01 b	0.01 b	0.01 b	0.01 b

The bacteria are denoted thus by: St (Salmonella typhimurium), Ef (Enterococcus faecalis), Ec (Escherichia coli), Pa (Pseudomonas aeruginosa), Bc (Bacillus cereus), Ss (Shigella sonnei), Sp (Streptococcus pyogens), Bs (Bacillus subtilis), Sf (Shigella flexneri), Kp (Kleb pneumoniae), Sa (Staphylococcus aureus), Pv (Proteus vulgaris) and Sm (Serratia marcescens). Data expressed as means ± SD; n = 3; values with the different superscript in the same column are significantly different (p ≤ 0.05). NA indicates not active.

). The antibacterial activity could also be attributed to the presence of some chemical constituents in the essential oil fraction of H. africana. For example, Terpinen-4-ol, both in aqueous solutions and vapour form, acts as an antimicrobial agent against bacteria of Legionella genus [19]. β-cymene possesses antibacterial and antifungal activities [20]. Napthalene has antibacterial and antifungal properties against various human pathogens [21]. Diphenyl ether possesses bactericidal activity against gram-positive and gram negative bacteria [22]. (−)-Spathulenol, an oxygenated sesquiterpene, has antifungal, antibacterial, antiviral and cytotoxic activities [23].The zones of inhibition varied from 10 to 25 mm (Table 1). The HA essential oil showed a significant growth inhibitory effect against the ten organisms (p < 0.05) having IC₅₀ values ranging from 5 to 0.005 mg/mL (Table 2). The lowest MIC was observed with Enterococcus faecalis (p < 0.05) (Table 2). Of the selected and tested nine fungal isolates, the plant species inhibited only the growth of Aspergillus niger whose MIC activity was significantly similar to the control drug (p < 0.05) (

Table 3. Inhibition zone diameters caused by the essential oil (50 mg/mL) in the tested opportunistic fungi.

Organism	Essential Oil	Positive Control (Nystatin)
Aspergillus fumigates	NA	30 ± 1.1
Aspergillus niger	20 ± 1.2 a	20 ± 1.2 a
Microsporium gypsum	NA	25 ± 0.2
Triphophytonrubrum	NA	25 ± 0.2
Triphophytontonsurans	NA	30 ± 1.2
Triphophytonmucoides	NA	25 ± 2.2
Penicillium aurantrognereum	NA	30 ± 2.1
Penicillium chrysogenum	NA	25 ± 0.2
Microsporum canis	NA	35 ± 1.1

The fungi are denoted thus by: Af (Aspergillus fumigatus), An (Aspergillus niger), Mg (Microsporium gypsum), Tr (Triphophyton rubrum), Tt (Triphophyton tonsurans), Tm (Triphophyton mucoides), Pa (Penicillium aurantrognereum), Pc (Penicillium chrysogenum) and Mc (Microsporum canis). Data expressed as means ± SD; n = 3; values along a row with different subscripts are significantly different (p ≤ 0.05). NA indicates not active.

and

Table 4. Minimum inhibitory concentrations (MIC) of the H. africana essential oils against the tested opportunistic fungi.

Solvent	Inhibition Zone Diameter (mm)
	Af	An	Mg	Tr	Tt	Tm	Pa	Pc	Mc
Essential oil	NA	2.5 a	NA	NA	NA	NA	NA	NA	NA
Positive control	0.01	2.5 a	0.02	2.5	<0.01	0.02	0.01	0.02	0.01

The fungi are denoted thus by: Af (Aspergillus fumigatus), An (Aspergillus niger), Mg (Microsporium gypsum), Tr (Triphophyton rubrum), Tt (Triphophyton tonsurans), Tm (Triphophyton mucoides), Pa (Penicillium aurantrognereum), Pc (Penicillium chrysogenum) and Mc (Microsporum canis). Data expressed as means ± SD; n = 3; values along a row with differentsubscripts are significantly different (p ≤ 0.05). NA indicates not active.

).

Although compounds in the oil are known to have antimicrobial activity independently, they could be acting independently or in combination to potentiate the plants’ potentials [24]. Therefore, it is crucial to isolate and elucidate the bioactive compounds and determine their pharmacological properties. This should facilitate the identification of novel antimicrobial agents.

GC-MS analysis of the essential oil fraction revealed fraction revealed the presence of 67 compounds in different chemical classes including terpenoids, aldehydes, ketones, fatty acid esters and carboxylic acids (

Table 5. Chemical composition of the essential oils of the root part of H. africana.

#	RT	Name	Area (%)	Class of Compound	Molecular Formula	Molecular Weight
1	3.585	2-Heptanone	1.07	Ketone	C7H14O	114
2	3.674	Heptanal	0.97	Aldehyde	C7H14O	114
3	3.714	2H-Isoindole-1,3(1H,3H)-dione, 5-amino-2-(2-methoxyphenyl)-	0.52	n/a	n/a	n/a
4	4.215	Benzaldehyde	0.45	Aldehyde	C7H6O	106
5	4.351	2-Hepten-2-one, 6-methyl-	0.21	n/a	n/a	n/a
6	4.393	Beta-Myrcene	0.64	Monoterpene	C10H16	136
7	4.487	Octanal	0.27	Aldehyde	C8H16O	128
8	4.675	Benzene, 1-methoxy-3-methyl	0.47	n/a	C8H10O	122
9	4.725	p-Cymene	0.58	Alkylbenzene	C10H14	134
10	4.765	o-Limonene	3.05	Monoterpene	‎C10H16	136
11	5.008	Octyl chloroformate	0.37	n/a	C9H17ClO2	193
12	5.199	8-Nonen-2-one	1.31	Ketone	C9H16O	140
13	5.250	2-Nonanol	0.28	Alcohol	C9H20O	144
14	5.308	Nonanal	0.84	Aldehyde	C9H18O	142
15	5.527	2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, trans-	0.18	n/a	C10H18O	154
16	5.617	Cyclopentane, 1,3-bis (methylene)-	6.38	n/a	C7H10	94
17	5.756	Bicyclo [2.2.1] heptan-2-one, 1,7,7-trimethyl-, (1S)-	0.42	Monoterpenoid	C10H16O	152
18	5.808	Nona-3,5-dien-2-one	0.47	Ketone	C9H14O	138
19	5.916	Cyclohexanol, 5-methyl-2-(1-methylethyl)-, (1. alpha, 2. beta, 5. alpha.)-(.+/−.)-(Menthacamphor)	0.30	n/a	C10H20O	156
20	5.976	Terpinen-4-ol	0.22	Monoterpenoid	C10H18O	154
21	6.012	Sorbic acid vinyl ester	0.63	n/a	C8H10O2
22	6.089	Naphthalene	0.81	n/a	C10H8	128
23	6.184	2(3H)-Naphthalenone, 4,4a,5,6,7,8-hexahydro-	0.28	n/a	C10H14O	150
24	6.452	Pulegone	0.16	Monterpenoid	C10H16O	152
25	6.520	2-Decanal, (E) −	0.17	Aldehyde
26	6.588	3,5-Dimethoxytoluene	0.23	n/a	C9H12O2	152
27	6.696	Phenol, 4-ethyl-2-methoxy-	0.50	n/a	C9H12O2	152
28	6.735	Phenol, p-tert-butyl	0.38	n/a	C10H14O	150
29	6.927	2,4-Decadienal, (E,E) −	0.17	Aldehyde	C10H16O	152
30	6.959	Benzene, 4-ethyl-1,2-dimethoxy-	0.26	n/a	C10H14O2	166
31	7.243	Cyclotetradecane	0.24	Cycloalkane	C14H28	196
32	7.302	6-Bromomethyl-5-methyl-bicyclo[3.1.0]hexan-2-one	0.51	n/a	n/a	n/a
33	7.362	Butanoic acid, butyl ester	0.20	Fatty acid ester	C8H16O2	144
34	7.610	Diphenyl ether	0.77	n/a	C12H10O	170
35	7.833	5,9-Undecadien-2-one, 6,10-dimethyl-,(Z)-(Nerylacetone)	0.37	n/a	C13H22O	194
36	8.008	2-Undecanone, 6,10-dimethyl-	0.18	n/a	C13H26O	198
37	8.142	Methyl (Z)-dec-2-en-4,6-diynoate	0.24	n/a	C11H12O2	176
38	8.244	Beta-Bisabolene	1.49	Sesquiterpene	C15H24	204
39	8.682	1,7-Dimethyl-4-oxa-tricyclo[5.2.1. 0(2,6)]decane-3,5,8-trione	0.52	n/a	n/a	n/a
40	8.787	(−)-Spathulenol	0.87	Sesquiterpenoid	C15H24O	220
41	8.848	Lanceol, cis	0.68	Sesquiterpenoid	C15H24O	220
42	8.898	3-Pyridinecarboxylic acid, 1,6-dihydro-6-oxo-	2.29	Carboxylic acid	C6H5NO3	139
43	8.965	Alloaromadendrene	0.43	Sesquiterpene	C15H24	204
44	9.054	Longifolene	0.23	Sesquiterpene	C15H24	204
45	9.098	3-Cyclohexene-1-acetaldehyde, alpha.,4-dimethyl-(Carvomenthenal)	1.99	Aldehyde	C10H16O	152
46	9.225	3-Methyl-2-butenoic acid, 2-methyloct-5-yn-4-yl ester	1.12	Fatty acid ester	C14H22O2	222
47	9.283	Bicyclo[3.1.1]heptan-3-one, 2-(but-3-enyl)-6,6-dimethyl-	0.45	n/a	n/a	n/a
48	9.325	Triisopropylphosphite	0.96		C9H21O3P	208
49	9.360	2-Cyclohexen-1-one, 3,6-dimethyl-6-(1-methylethyl)-	0.75	n/a	n/a	n/a
50	9.472	3-Methylbut-2-enoic acid, 3,5-dime	2.23	n/a	n/a	n/a
51	9.567	s-Indacene, 1,2,3,5,6,7-hexahydro-1,1,4,8-tetramethyl-	0.27	n/a	n/a	n/a
52	9.714	4-[3-(4-Nitrophenoxy)propyl]-1H-imidazole	3.55	n/a	n/a	n/a
53	9.818	Ethyltetramethylcyclopentadiene	2.84	n/a	n/a	n/a
54	9.902	Dodecanoic acid, 10-undecen-1-yl ester	0.37	n/a	n/a	n/a
55	9.971	Neoisolongifolene, 8,9-epoxy-	2.86	n/a	n/a	n/a
56	10.048	3-Metthoxybenzyl alcohol	1.02	n/a	n/a	n/a
57	10.268	1,2-Benzenedicarboxylic acid, butyl octyl ester	0.50	Fatty acid ester	C20H30O4	335
58	10.305	4,4′-Bis (tetrahydrothiopyran)	1.02	n/a	n/a	n/a
59	10.365	3-Fluorobenzoic acid, 3,5-dimethyl cyclo hexyl ester	0.34	n/a	n/a	n/a
60	10.501	5,9,13-Pentadecatrien-2-one, 6,10,14-trimethyl-	0.30	n/a	C18H30O	262
61	10.571	Hexanoic acid, 2-tetradecyl ester	0.49	n/a	n/a	n/a
62	10.745	Dibutyl phthalate	2.78	n/a	C16H22O4	278
63	10.877	Cis-9-hexadecenoic acid	4.09	Carboxylic acid	C16H30O2	254
64	11.355	1,5,9-Decatriene, 2,3,5,8-tetramethyl-	10.52	n/a	C14H24	192
65	11.396	n-Hexadecanoic acid	24.30	Carboxylic acid	C16H32O2	256
66	11.421	7-Oxabicyclo[4.1.0]heptane, 1,5-dimethyl-	3.18	n/a	n/a	n/a
67	12.218	Phenanthrene, 7-ethenyl-1,2,3,4,4a,4b,5,6,7,8,8a,9-dodecahydro-1,1,4b,7-tetramethyl-, [4aS-(4a.alpha., 4b.beta.,7.alpha.,8a.alpha.)]-	2.51	Diterpene	C20H32	273
Total			100.05

n/a = compound details not found in the NIST database. Please note the names in red are synonyms for the ones in black. They could be used instead. The name, nature, retention time (RT), molecular weight, molecular formula and percentage composition (%) of the identified compounds in essential oil.

). The details of certain compounds could, however, not be confirmed in the NIST database. Among identified compounds, carboxylic acids, dominated by n-Hexadecanoic acid and cis-9-hexadecanoic acid, constituted the largest group of compounds (30.68% of total oil composition). The higher content of carboxylic acids in the essential oil, compared to other groups of compounds, is an unusual finding, as most other essential oils reportedly contain terpenes and other hydrocarbons. The results of this study, however, bear a similarity to those observed for the composition of essential oils from Tetrapleura tetraptera Udourioh and Etkudoh, [25], who also reported a high content of carboxylic acids in the oil.

Terpenoids identified in the oil made up 10.70% of the total oil composition. They included Beta-Myrcene, o-limonene, Terpien-4-ol, pulegone, Beta-bisabolene, (−)-spathulenol, cis-lanceol, alloaromadendrene, Longifolene, Phenanthrene, 7-ethenyl-1,2,3,4,4a,4b,5,6,7,8,8a,9-dodecahydro-1,1,4b,7-tetramethyl-, [4aS-(4a.alpha.,4b.beta.,7.alpha.,8a.alpha.)]- and Bicyclo [2.2.1] heptan-2-one,1,7,7-trimethyl-, (1S)-Bicyclo. Aldehydes (4.86%); Ketones (2.85%) and fatty acid esters (1.82%) were also among compounds whose identity was confirmed from the library. The low percentage of terpenoids and relatively high content of carboxylic acids in the oils extracted from Hydnora africana may suggest that the oil may not be considered as a true essential oil [25]. This may be the reason for the weak antioxidant effects shown by the oil against DPPH, ABTS and nitric oxide. However, the potential applications of the oil in the cosmetic industries, due to the presence of some terpenoids may be very useful.

4. Conclusions

In this study, we conclude that the essential oil of H. africana possess antioxidant and antibacterial activities and could be attributed to the presence of bioactive compounds observed in the study. However, the oils exhibited poor antifungal activity which could be due to the weak effect of its component in eradicating opportunistic fungi. These results have partially justified the folklore usage of this plant in the treatment of infections and some diarrhoeal diseases.