Antibacterial, Antioxidant Potency, and Chemical Composition of Essential Oils from Dried Powdered Leaves and Flowers of Hypericum revolutum subsp. keniense (Schweinf.)

Hypericum revolutum subsp. keniense is a plant mainly used to treat diarrhoea, rheumatism, nervous disorders, and wounds in African traditional medicine. The objective of the current work was to establish antibacterial, antioxidant potency, and chemical composition of essential oil from the leaves and flowers of Hypericum revolutum subsp. keniense. The oils were isolated by steam distillation. Antibacterial activity against methicillin-resistant Staphylococcus aureus, Staphylococcus aureus (ATCC 12393), Escherichia coli (ATCC 25922), Acinetobacter baumannii (ATTC 19606), Salmonella enteritidis (NCTC12023), Salmonella typhimurium (ATCC 14028), Pseudomonas aeruginosa (ATCC 15442), and Haemophilus influenzae (ATCC 49766) was carried out by agar disk diffusion and microtiter broth dilution methods. Antioxidant activities of the essential oils were examined by different methods, DPPH, FRAP, and H2O2 assays. Chemical characterization was performed using gas chromatography interfaced with mass spectrometry, Fourier-transform infra-red (FTIR) spectroscopy, and quantification of phenolics and flavonoids by Folin–Ciocalteu reagent and aluminium nitrate, respectively. The oils showed potential antibacterial activity with mean zone of inhibition ranging from 20.67 ± 0.33 to 32.00 ± 1.00 mm at 100% oil concentration against the tested bacteria. Furthermore, the minimum inhibitory concentrations (MICs) in all the tested microorganisms were in the range from 250 to 15.6250 μg/ml. The essential oils derived from the leaves revealed varied antioxidant activity levels with the different methods of assay. The IC50 of values obtained from the three methods, DPPH, FRAP, and H2O2 were ˃1000 μg/ml, 0.31 μg/ml, and 12.33 μg/ml, respectively. Caryophyllene (22.1%) and 2, 3, 4-trimethylhexane were the major chemical components of the essential oils derived from the leaves and flowers, respectively. FTIR spectroscopy of the essential oils from the leaves and flowers showed similarity at peaks for hydroxyl, unsaturated olefinic, and amine functional groups. Further findings indicated that the total phenolic and flavonoid contents essential oils derived from leaves were 130.4 6 ± 10.5 mg GAE/g dry weight and 0.911 ± 0.04 mg CE/g dry weight, respectively. It was therefore concluded that essential oils from the leaves and flowers of H. revolutum subsp. keniense have compounds that have antibacterial and antioxidant potency.


Introduction
Essential oils are mainly used as antimicrobial agents [1]. Also, they are used as insecticides, insect repellents, antimalaria, antidepressant, anticancer, antimutagenic, anti-infammatory, analgesic, antipyretics, antioxidant, anticonvulsants, conjunctivitis, tuberculosis, stress, bronchitis, anxiety, neurological disorders, ulcers, liver conditions, and Parkinson's disease [2][3][4]. Te diverse biological activities of essential oils are attributed to the complex nature of chemical composition [2]. Te main components of essential oils are monoterpenes and sesquiterpenes, and they exert antibacterial efects by structural and functional damages of the bacterial cell wall [5]. Te known antimicrobial activities of essential oils are based on the ability to destroy the cell wall and cell membranes, and they act by causing destabilization of bacterial membranes, damaging the membrane proteins, depleting proton motive force, and releasing cell contents [6]. Essential oils are accumulated mainly by plants as secondary metabolites though a few of them are found in animals and microorganisms [7]. Essential oils are relatively distributed in the plant kingdom in about 60 plant families that comprise close to 2000 species. Te major plant families that yield commercially signifcant essential oils are found mostly in dicotyledonous plants [8]. However, some monocotyledonous and gymnospermatophyta type of plants also produce the essential oils found in the markets [8,9]. Some essential oils have also been reported in pteridophytes, bryophytes, algae, and fungi too [7].
Plant species of the genus Hypericum have a wide scope of secondary metabolites, which are responsible for their many biological activities [10]. Scores of research studies have demonstrated that essential oils have chemical compounds with varied biological and pharmacological properties. In the Hypericum genus, Hypericum perforatum is the most popular and best studied of all the Hypericum species across the world and has been found largely useful in mild to moderate depression, neuroprotection, antineuralgic, and antiviral properties [11]. Also, biological activities such as antibacterial, antioxidant, analgesic, anti-infammatory, hypotensive, and spasmolytic properties have been reported for Hypericum perforatum [12]. Te other Hypericum species have demonstrated antioxidant, antimicrobial, antiinfammatory, cytotoxic, antimalaria, analgesic, wound, and burn healing properties [12,13], and out of the various biological activity studies of the essential oils from Hypericum species, antimicrobial activities are the most reported [13,14].
Te species Hypericum revolutum subsp. keniense is used in traditional medicine to treat skin disorders, nervous disorders, gastrointestinal disorders, and rheumatism [15,16]. Limited works on biological, pharmacological, and phytochemical studies of Hypericum revolutum subsp. keniense have been reported for the organic and aqueous extracts, that is, antimicrobial, brine shrimp lethality, and oral acute toxicity and preliminary phytochemical studies that revealed the presence of saponins, cardiac glycosides, favonoids, tannins, coumarins, carotenoids, and volatile oils [17] and isolation of betulinic acid and coumarins [17,18]. To the best of our knowledge, there are no reports on the biological or pharmacological activities of the essential oils from Hypericum revolutum subsp. keniense. Te objective of the current study was to establish the antibacterial and antioxidant potency and characterize the composition of the essential oil components from this plant. Te fndings from this study imperatively provide scientifc data to validate the ethnomedicinal claims of using the plant extracts for the management of gastrointestinal, skin, and musculoskeletal disorders.

Collection of Plant Materials.
Te fresh leaves and fowers of Hypericum revolutum subsp. keniense were collected from Kieni village, Gakoe sublocation in Kiambu County. Te plant was authenticated by a taxonomist at the East Africa Herbarium of the National Museums of Kenya. A voucher specimen number GS001-2020 was prepared and deposited at the Department of Botany in the East Africa Herbarium. Te collected leaves and fowers were air dried at room temperature and pressure for two weeks. Te dried plant materials were ground into course powder using a blender.

Extraction of Essential Oil from Leaves and Flowers of
H. revolutum subsp. keniense. Te leaves and fower powders of H. revolutum subsp. keniense (500 g) were extracted using Clevenger-like apparatus for 2 hours according to the British pharmacopoeia steam distillation method [19]. Te essential oils that were extracted were collected and dried over anhydrous sodium sulphate; thereafter, the oils were weighed and stored in airtight sealed vials at 4°C until antibacterial and GC-MS analysis of the essential oils was conducted.  [22] with minor modifcations as described in 2016 by Hudzicki [23]. Each bacteria inoculum of approximate 1.5 × 10 8 CFU/ mL was diluted in a ratio of 1 : 100 using sterile normal saline to obtain a colony suspension of 1 × 10 6 CFU/mL in an inoculum tube. Ten, a sterile swab was dipped into an inoculum tube and the swab was rotated against the side of the tube (above the fuid level) using frm pressure to remove excess fuid and swap dripping wet. Tereafter, the dried surface of a MH agar plate was inoculated by streaking the swab three times over the entire agar surface. Te plate was rotated approximately 60 degrees each time to ensure an even distribution of the inoculum. Sterile disks (6 mm in diameter) were impregnated with 10 μl of (100%, 50%, 25%, and 12.5% v/v) of the H. revolutum subsp. keniense leaf and fower essential oils. Te impregnated disks were placed on the surface of the agar, using sterilized forceps at a distance of more than 24 mm apart. Experiments for each sample were conducted in triplicates and 10 μl of ciprofoxacin (0.5 mg/ml) was used as positive controls while 10 μl of dimethylsulfoxide (0.1%) was used as a negative control. Te inoculated plates containing the essential oil impregnated disks were incubated for 24 hours at 37°C and respective zones of inhibition were measured in mm using the digital zone of inhibition measuring caliper.

Broth Microdilution Assay.
Minimum inhibitory concentration (MIC) of H. revolutum subsp. keniense essential oils was evaluated by the broth microdilution assay according to the procedures described in 2017 by Ohikhena et al. [24] and modifed in 2020 by Mogana et al. [25]. First, Mueller Hinton broth (100 μl) was put into the wells from the second to the eleventh column from row B to G. Second, hundred microliters of H. revolutum subsp. keniense essential oils extracts at concentration 500 μg/mL and the standard drug (ciprofoxacin at concentration 0.8 μg/mL) were added into row B of column 2-9 in quadriplicate using multichannel pipette. Hundred microliters were removed from the starting concentrations (columns 2-9 in row B) and transferred to the next row and properly mixed with the 100 μl broth that was already in the wells. Two-fold serial dilution from row C to G was performed by transferring 100 μl from row C to row D systematically, followed by mixing gently using microchannel pipette. Te procedure was repeated up for row (E), (F), and (G). Te last 100 μl that was removed from row G was discarded. Te 10 th and 11 th columns were treated as a negative control which served as growth control (drugfree) columns. Hundred µl of the diluent (1% DMSO in MH broth) were added into 100 μl in row B of column of the 10 th and 11 th columns. Two-fold serial dilution was performed from row B to row G of the 10 th and 11 th columns. Finally, the resultant volume in all the test wells with the extracts, the standard drugs, and the negative controls was 100 μl. A volume of 5 μl of the 1 × 10 6 CFU/ml bacteria inoculum was transferred into all wells except for the wells in the row G which served as a blank control (microorganism and drugfree).
Te microtiter plates were incubated at 37°C for 18 hours after which, 60 μl of Alamar blue resazurin was added to all the wells and then incubated for 20 minutes after which the colour change from blue/purple to pink was observed to indicate minimum inhibitory concentration. Te viable cells produce oxidoreductases which convert blue resazurin to resorufn (pink) therefore indicating microbial growth and used to determine minimum inhibitory concentration [25,26].

2.5.3.
Determination of Minimum Bactericidal Concentration. Minimum bactericidal concentrations (MBCs) were determined according to the procedure described by Man et al. [27] with slight modifcations. Tree microliters of Mueller Hinton broth were taken from the last three wells of each row that indicated no bacterial growth and was subcultured in the petri dish plates containing 20 ml fresh Mueller Hinton agar (MHA) and were incubated for 24 h at 37°C. Te lowest concentration without visible microbial biomass growth under optical microscope was defned as MBC, indicating the death of 99.5% of the original inoculum.

Disposal of Microbiological
Waste. Te used petri dishes, 96-micro-titre well plated, and bacteria inoculum were autoclaved before disposing them into the trash.

Evidence-Based Complementary and Alternative Medicine 3
Test concentrations of Hypericum revolutum subsp. keniense leaf essential oil extract and standard (L-ascorbic acid) were prepared at concentrations of 1000 μg/ml, 100 μg/ml, 10 μg/ ml, 1 μg/ml, 0.1 μg/ml, and 0.01 μg/ml in methanol. DPPH (0.1 mM) was prepared in methanol and 2.4 ml were added to 1.6 ml of the diferent concentrations of the extract and the standard L-ascorbic acid. Te control for the current study consisted of a mixture of 2.4 ml of (0.1 mM) 2, 2diphenyl-2-picrylhydrazyl (DDPH) solution in methanol and 1.6 ml of methanol. Te test samples and the standard were prepared in triplicate test tubes which were then kept in the dark at room temperature for 15 minutes to allow reaction to take place. Absorbances were measured at a wavelength of 517 nm against methanol as the blank using the UV spectrophotometer. Te test was conducted in triplicates and the percentage radical scavenging activity was calculated using the formula as follows: % means the percentage, RSA means the radical scavenging activity, and abs. is the absorbance. Te antioxidant potency was expressed as a value of concentration of Hypericum revolutum subsp. keniense leaf essential oil that was responsible for 50% inhibition of DPPH radicals (IC 50 ). IC 50 values for the oil and the standard were calculated from the relationship curve of RSA versus concentrations of the respective sample and standard curves. Te antioxidant activity levels were classifed based on the IC 50 values, very strong (IC 50 of less than 50 μg/ml), strong (IC 50 of between 50 and 100 μg/ml), medium (IC 50 of between 100 and 150 μg/ml), weak (IC 50 of between 150 and 200 μg/ml), and very weak (IC 50 of more than 200 μg/ ml) [29].

Ferric Reducing Antioxidant Power (FRAP) Assay.
Te reducing power of H. revolutum subsp. keniense essential oil moisture was evaluated by protocol of Moriasi et al. [28] with minor modifcations. Te essential oil and L-ascorbic acid diferent concentrations (0.01, 0.1, 1, 10, 100, and 1000 μg/ml) were prepared in methanol. Exactly one-milliliter of the extract or the standard at diferent concentrations was added into 2.5 ml of 200 mM phosphate bufer (pH 6.6) and 2.5 ml of 30 mM potassium ferricyanide. Te resulting mixture was incubated for 20 min at 50°C. Tereafter, 2.5 ml of 600 mM trichloroacetic acid (TCA) was added into the cooled reaction mixture and then centrifuged at 3000 rpm for 15 minutes. A volume of 2.5 ml of the supernatant was mixed with an equal volume of distilled water. Finally, 0.5 ml of 600 mM ferric chloride was added to the mixture. Te absorbance of both the standard (ascorbic acid) and the extracts at diferent concentrations were measured at a wavelength of 700 nm against the blank using a spectrophotometer (Lab tech model double beam UV-Vis). Te concentrations of the oil and standard that yielded an absorbance value of 0.5 were determined from the graph of absorbance at 700 nm against extract concentrations and considered as the median efective concentration (EC 50 ). Increase in the absorbance indicated high antioxidant activity. In each case, the experiment was performed in triplicate.

Hydrogen Peroxide Radical Scavenging Capacity Assay.
Te method of Arika et al. [30] with minor modifcations was adopted. Te H. revolutum subsp. keniense essential oil and L-ascorbic acid were prepared to yield diferent concentrations in methanol. Hydrogen peroxide solution (40 mM) was prepared using a phosphate bufer (pH 7.4). One-milliliter of diferent concentrations of the essential oil/standard (1000-0.01 μg/ml) was added to 0.6 ml of hydrogen peroxide solution and incubated for 10 min at room temperature. Te absorbance of the solutions was measured at 230 nm against a blank consisting of phosphate bufer (pH 7.4) only. All the tests were performed in triplicates, and the scavenging activity of hydrogen peroxide was calculated using the formula stipulated by Legas Muhammed et al. [31].
where A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of the samples or standards.

Gas Chromatography-Mass Spectrometry (GC-MS)
Analysis. Chemical composition of the essential oil isolated from H. revolutum subsp. keniense leaves and fowers were analyzed by gas chromatography-mass spectrometry SHIMADZU 2010 plus (GC-MS). Te GC-MS analyses were performed in three independent repetitions. A 7890A gas chromatography interfaced with mass spectrometer triple quad system ftted with 7693 auto-sampler, Agilent Technologies, and installed with Mass Hunter Workstation Software Version B.05.00 build 5.0.519.0, service pack I © Agilent technologies 2011, for qualitative analysis. A capillary column DB-5, 30 m × 0.25 mm ID, and column flm thickness of 0.25 μm composed of 100% dimethylpolysiloxane was used. Helium of 99.9999% purity was used as a carrier gas at a fow rate of 1 ml/min. A sample of essential oil was introduced to the injector at a volume of 1 μl and the split ratio of 10 : 1. Te inlet temperature was maintained at 250°C. Te oven temperature was programmed at 110°C for 4 min and then increased to 240°C and fnally to 280°C at a rate of 20°C for 5 minutes. Te total run time for the extract was 40 minutes. Te MS transfer line was maintained at a temperature of 250°C, and the source temperature was maintained at 280°C. Te mass fragments were analyzed using electron impact ionization at 70 eV and a scan interval of 0.5 seconds, and the fragments were read from 45 to 450 Da, and the obtained data were evaluated using the total ion count (TIC). H. revolutum subsp. keniense essential oils from the leaves and fowers were mixed with spectroscopic grade potassium bromide (KBr) in the ration of 1 : 10 by a mortar and pestle. Te resultant fne powder mixture was compressed using a hand press (5 × 10 6 Pa) in an evacuated die to produce a clear transparent disc of 13 mm diameter and 1 mm thick. Te disc was placed in a sample handler and was scanned twenty times at the frequency regions of between 4000 and 450 cm −1 and the spectra produced were recorded.

Evaluation of Total Phenolic Content (TPC).
Te total phenolic content of Hypericum revolutum subsp. keniense leaf essential oil extract was determined based on Folin-Ciocalteu reagent assay, as described by Saeed et al. [33] with slight modifcations. Diferent concentrations range from 150 to 4.6875 μg/ml of gallic acid (0.3 ml) were prepared, and they were mixed with 1.5 ml of Folin-Ciocalteu's phenol reagent (10%) by swirling using test tubes. Hypericum revolutum subsp. keniense leaf essential oil extracts (0.3 ml) were put in a separated test tube and mixed with 1.5 ml Folin-Ciocalteu's phenol reagent (10%) by swirling using test tubes too. Tereafter, 1.5 ml of 7.5% Na 2 CO 3 solution were made in deionized water and were mixed with 0.3 ml of diferent concentrations of gallic acid and Hypericum revolutum subsp. keniense leaf essential oil extracts. Te mixtures were incubated for 2 minutes in the dark at room temperature, after which the absorbance was read at 760 nm. Te TPC was determined from the extrapolation of gallic acid standard calibration curve. Te estimation of the phenolic compounds was carried out in triplicate. Te TPC was expressed as milligrams of gallic acid equivalents (GAEs) per g of the sample in dry weight. Results were calculated and expressed as a microgram of gallic acid equivalents (GAEs) per gram extract using the formula described by Siddiqui et al. [34].
where C in mgGAE per gram dry weight of the sample, c is the concentration of gallic acid (μg/ml) extrapolated from the curve equation, v is the volume in which the sample was diluted (ml), and m is the mass of the extract weighed in grams.

Evaluation of the Total Flavonoid Content (TFC).
Total favonoid content (TFC) of Hypericum revolutum subsp. keniense leaf essential oil extract was determined by aluminium nitrate colorimetric method described by Cosmulescu et al. [35] with some modifcations. In brief, 0.125 ml of 1% Hypericum revolutum subsp. keniense leaf essential oil extract in methanol and diferent concentrations of catechin (0.625 to 20 μg/ml) were prepared using test tubes in triplicates. Five percent of 0.075 ml sodium nitrate (NaNO 3 ) was added to the test tubes containing the test sample and the diferent concentrations of the standard (catechin). Tereafter, the mixture was incubated for six minutes at room temperature. Tereafter, the mixtures in the test tubes were incubated for six minutes at room temperature, then followed with the addition of0.15 ml of 10% aluminium nitrate (AlNO3) to each preparation. 0.75 ml of 4 % sodium hydroxide was added to the mixtures, and fnally, the volume of each mixture in the test tube was made up to 2.5 ml using distilled water. Te absorbance of the reaction mixtures was measured at 510 nm using UV spectrophotometer. Te blank was setup by following the same procedure; however, the plant extract was replaced with an equal volume of methanol. All the determinations were carried out in triplicate. Te total favonoid content was expressed as a milligram of catechin equivalents (CEs) per gram dry weight of the extract, respectively, using the formula described by Siddiqui et al. [34].
where C in mgCE per gram dry weight of sample, c is the concentration of cathecin in μg/ml that was extrapolated from the curve equation, v is the volume in which the sample was diluted (ml), and m is the mass of the extract weighed in grams.

Statistical Analyses.
Antimicrobial and antioxidant results were presented as the mean ± standard error of the mean. Te one-way ANOVA with Turkey's posthoc test was performed for the comparison between the means. Te statistical analyses were performed using Minitab statistical software version 20 (Minitab, LLC., USA). Te data were considered signifcant when the P value was <0.05.

Extraction Yield.
Te dried leaves and fowers had a percentage essential oil yield of 7.5 ± 07% and 3.8 ± 03%, respectively. Te essential oil mixture obtained from the leaves was yellow in colour with sweet foral aroma while the one from the fower was brown with sweet fruity aroma.

Antibacterial Activity of H. revolutum subsp. keniense
Essential Oils. In this study, the fower and leaf derived essential oils of H. revolutum subsp. keniense produced signifcantly larger growth inhibition zones against the tested bacteria at 100% concentration than at lower concentrations (P < 0.05; Table 1). Tere were no signifcant statistical diferences in growth inhibition of the tested bacteria observed between plates incubated with 12.5% v/v and 25% v/v and 25% v/v and 50% v/v of the fower and leaf oils of H. revolutum subsp. keniense (P > 0.05; Table 1). Notably, the inhibition zones produced by the leaf and fower essential oils against methicillin-resistant S. aureus and H. infuenzae and the fower oil against A. baumanii and S. aureus were not signifcantly diferent from those produced by the standard antibiotic (ciprofoxacin 0.05% w/v) (P > 0.05; Table 1). Besides, the inhibition zones produced by the two types of essential oils at each concentration against all the test bacteria, except methicillin-resistant S. aureus, were not signifcantly diferent (P > 0.05; Table 1). On the methicillin-resistant S. aureus, the leaf derived essential oil of H. revolutum subsp. keniense showed a statistically signifcant larger inhibition zone than the fower derived essential oil (P < 0.05; Table 1); however, the inhibition zones produced by the two oils at all the other concentrations are not signifcantly (P > 0.05) diferent as shown in Table 1.
A summary of the lowest concentrations of the Hypericum leaf and fower essential oils which prevented visible growth (MIC) of the tested bacteria is tabulated ( Table 2). Te lowest MIC values that were revealed in this study were 15.625 (μl/ml); this was demonstrated by the essential oils from the leaves of H. revolutum subsp. keniense against methicillin-resistant Staphylococcus aureus, Salmonella enteritidis, and Escherichia coli. Also, the lowest concentration of the oils that was bactericidal (MBC) was 15.625 (μl/ml) for methicillin-resistant Staphylococcus aureus. Te MBC: MIC ratio was either one (1) or two (2) throughout the experiment.

Antioxidant Activity of H. revolutum subsp. keniense Leaf
Essential Oil Mixture. Tree in vitro antioxidant activity assay methods were used to demonstrate the ability of the Hypericum essential oil from H. revolutum subsp. keniense to scavenge for free radicals. Te methods were based on spectrophotometry, namely DPPH, fE (III) complex, and H2O2 assays.. Te percentage radical scavenging activity of the essential oil obtained from H. revolutum subsp. keniense in the current study revealed IC 50 values of the oil as more than 1000 µg/ml for DPPH assay, 0.31 μg/ml for FRAP assay, and 12.33 μg/ml in the case of hydrogen peroxide assay.

Antioxidant Activity by DPPH Assay.
Te three methods of assay had variant IC 50 values, and this is consistent with literature that explains that diferent methods utilize diferent  [36]. On the basis of the description performed in 2020 by Minsas et al. [29], the DPPH assay revealed very weak antioxidant activity (IC 50 > 200 μg/ml) when compared to that of L-ascorbic acid which was very string (IC 50 < 50 μg/ ml). Te very high IC 50 values in the DPPH method of assay are ascribed to the inadequacy of the method to the lipophobic systems and given that the essential oil in this case provides for the nonpolar system [37,38].

Ferric-Reducing Antioxidant Power of the Leaf Essential
Oil of H. revolutum. Te FRAP method a nonfree radical involvement method focusses on the monitoring of the process of reducing of ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ). Te concentration of the Hypericum essential oil under this study was 0-1000 μg/ml, and the calculated ferric reducing antioxidant power that converted half of the Fe 3+ -Fe 2+ known as IC 50 was 0.31 μg/ml. As indicated in the results in Table 2, the absorbances of the reaction mixtures containing the leaf oil of H. revolutum subsp. keniense at concentrations of 0.32 and 1.6 as well as 8.0 and 40.0 (μg/ml) were not signifcantly diferent (P > 0.05); however, these absorbances were signifcantly lower than those recorded at concentrations between 200.0 and 1000.0 μg/ml (P < 0.05). Furthermore, the standard (L-ascorbic acid) showed a signifcant concentration-dependent increase in the measured absorbances (P < 0.05; Table 3). Notably, the diferences between the absorbances of the H. revolutum subsp. keniense leaf oil and L-ascorbic acid recorded at concentrations of 0.32, 200, and 1000 μg/ml were insignifcant (P > 0.05); however, at other concentrations, the absorbances recorded by L-ascorbic acid were signifcantly higher than those recorded by the studied oil (P < 0.05; Table 3).

Hydrogen Peroxide Scavenging Activity.
Te percentage hydrogen peroxide scavenging activity of the H. revolutum subsp. keniense leaf oil increased signifcantly (P < 0.05) in a concentration-dependent manner, as shown in Table 4. However, the percentage of hydrogen peroxide scavenging activity of L-ascorbic acid recorded between concentrations of 40.0 and 200.0 μg/ml, 1.6 and 8.0 μg/ml, and 0.32 and 1.6 μg/ml, respectively, were not signifcantly diferent (P > 0.05) whereas that recorded at a concentration of 1000.0 was signifcantly higher than that recorded at all the other concentrations (P < 0.05; Table 4). Notably, the leaf oil of H. revolutum subsp. keniense had a signifcantly higher percentage of hydrogen peroxide scavenging activity than Lascorbic acid at all concentrations (P < 0.05; Table 4). Te ability of H. revolutum subsp. keniense leaf essential oil to scavenge for hydrogen peroxide (H 2 O 2 ) was studied. Te IC 50 value was calculated to be 12.33 μg/ml, which indicated that such concentration was required to quench half of the available H 2 O 2 molecules, therefore ofering protection from the tissue damage [32,39].

Fourier Transform Infrared Spectra of H. revolutum subsp. keniense Essential Oils.
Te Fourier transform infrared spectra that were obtained from the current study revealed eighteen and seventeen peak values that were interpreted to represent diferent functional groups. FTIR spectral analysis was used to identify the functional groups of the secondary metabolites that are associated with medicinal values. Te region above 1200 cm −1 revealed eleven and nine spectral peaks due to the vibration of individual bonds or functional groups of the compounds that were in the essential oils that were obtained from Hypericum revolutum subsp. keniense leaves and fowers, respectively ( Table 6 and Figures 1 and 2). On the other hand, six spectral peaks were observed in the region below 1200 cm −1 , which is known as the "fngerprint region" that indicates bands due to the vibrations of the complete bioactive molecule. Te fngerprint region was notable with many infrared bands.
Te bands represent diferent vibrations, including those of O-H, -CH (CH 2 ), C�O, C�C-C, C-H, and C-N single bond stretches.

Phenolic Contents of H. revolutum subsp. keniense Leaf
Essential Oil Extract. Te phenolic content of the Hypericum oil isolated from the leaves of H. revolutum subsp. keniense was measured using the Folin-Ciocalteu reagent. Te results were derived from a calibration curve (Y � 0.009x + 0.1158, R 2 � 0.9975) of gallic acid (0-150 μg/ ml) and expressed in gallic acid equivalents (GAE) per gram dry extract weight (Figure 3). Te total phenolic content in the essential oils derived from the leaves was calculated and found to be 130.4 6 ± 10.5 mg GAE/g dry weight.

Flavonoid Content of H. revolutum subsp. keniense Leaf
Essential Oil Extract. Te quantitative determination of favonoids in the essential oils derived from the leaves of total leaves of H. revolutum subsp. keniense was carried out by aluminium nitrate colorimetric method. Te results were derived from the calibration curve (Y � 0.0208x + 0.0149, R 2 � 0.952) of catechin (0.625-20 μg/ml) ( Figure 4) and expressed as mg catechin equivalent (CE) per gram of essential oils dry weight. Te calculations revealed that the total favonoid content essential oil from the dried leaves of H. revolutum subsp. keniense was 0.911 ± 0.04 mg CE/g dry weight.

Discussion
Te current study provides a report on the antibacterial, antioxidant, and chemical composition of H. revolutum subsp. keniense essential oil mixture obtained from leaves and fowers for the frst time. However, the essential oils from other Hypericum species have been reported to have activity against a range of bacteria [40,41]. Te antibacterial activity of essential oils extracts from H. revolutum subsp. keniense leaves and fowers was interpreted according to Alves et al. [42] as a very high antibacterial activity at 100% undiluted oil. Te essential oils demonstrated broad spectrum antibacterial activity against both gram-positive and gram-negative bacteria. Te MIC values of both leaves and fowers essential oils extracts less than 8000 μL/ml and can be interpreted according to Voukeng et al. [43] as signifcant antibacterial activity. Te MBC/MIC ratio was found to less than 4 (<4) for the H. revolutum subspecies keniense leaf and fowers oils extracts against all the assayed bacteria in this study. Terefore, it was interpreted to mean that both the oils had bactericidal efects [25]. More so, the antioxidant activities of the Hypericum oil from H. keniense subsp. keniense in the current study are consistent with the literature of the essential oils from other Hypericum species, more especially the exceptionally and highly studied Hypericum perforatum [44,45]. A considerable number of chemical constituents in the essential oils detected in this study were consistent with the ones found in other Hypericum species in literature though it was noted that quantities of the composition were diferent Values are presented as x ± SEM for three replicates. Means with diferent superscript lower-case alphabets within the same column are signifcantly diferent (P < 0.05; one-way ANOVA with Turkey's posthoc test), whereas means with diferent subscript uppercase alphabets within the same row are signifcantly diferent (P < 0.05; independent student's t-test statistic). HRLEOs: H. revolutum leaf essential oils; L-ASA: L-ascorbic acid. Values are presented as x ± SEM for three replicates. Means with diferent superscript lower-case alphabets within the same column are signifcantly diferent (P < 0.05; one-way ANOVA with Turkey's posthoc test), whereas means with diferent subscript uppercase alphabets within the same row are signifcantly diferent (P < 0.05; independent student's t-test statistic). HRLEOs: H. revolutum leaf essential oils; L-ASA: L-ascorbic acid. 8 Evidence-Based Complementary and Alternative Medicine    Evidence-Based Complementary and Alternative Medicine from those of other Hypericum species. Some of the major compounds that were identifed in the essential oils derived from Hypericum leaves and fowers have been reported previously by Crockett and Del Monte et al. [40,46], and caryophyllene, caryophyllene oxide, ocimene, pipene, spathulenol, myrcene, amorphene, selinene, and undecane were reported in the current study of the essential oils H. revolutum susp. keniense. However, germacrene D and limonene which are commonly identifed in the essential oils of other Hypericum species were not identifed. Te chemical content and percentage variations in the components of essential oils and the variations that were observed in the Hypericum oil in the current study are attributed to environmental factors, plant parts, harvesting time, drying procedures, and storage conditions as described by Bergonzi et al. [47]. Te antibacterial and antioxidant activities of some of the major compounds that we found in the leaves and fowers of H. revolutum subsp. keniense have been reported. For example, caryophyllene and α-pinene are indicated to have antibacterial properties in literature [48][49][50][51]. D-camphene is also known for its antioxidant activities [48]. Te interpretation of FTIR spectra with sixteen bands provide information of varied classes of chemical components of the essential oils. Te main classes being alcohols, phenolics, carbonyl ketones, amides, and aldehydes. Te band at 3749.4 cm −1 for the leave essential oil, indicated the nonbonded O-H stretch of the hydroxyl group, and the broad band between 3315.5 and 3160.2 cm −1 and 3417 and -3255.6 cm −1 for the leaf and fower essential oils, respectively, depicts the O-H of the hydroxyl group of alcohols. Both the leaf and fower essential oils FTIR spectra revealed the bands between 2923 cm −1 and 2862.2 cm −1 , which are characteristic of asymmetric and symmetric stretch vibrations of C-H groups. Also, the band at 2731.8 represented the C-H group of aldehydes [52]. C-H group of the essential oils was confrmed by the presence of the spectra band at 1450.4 cm −1 -987.5 cm −1 by Burman et al. [53]. Te bands at 1635.5 cm −1 and 1643.2 cm −1 in the FTIR spectra for the leaf and fower essential oils, respectively. Tese were the characteristic of the presence of C=C that absorbs between 1680-1620 cm −1 [54]. Te presence of C=C band on the FTIR spectra was associated to the presence compounds with olefnic unsaturated group. In this case, an example is farnesene [55]. Te bands at 1558.4 cm −1 and 1566.1 cm −1 of the leaf and fower essential oil spectra, respectively, represent the presence of stretch vibrations that were indicative of compounds containing carboxylate or carboxylic acid salt that is proposed to originate from 1-octyl trifuoroacetate [56]. Te two peaks FTIR spectrum of the H. revolutum subsp. eniense essential oils that appear at ∼1450.4-∼1380.9 cm −1 originate possibly from caryophyllene due to the presence of isopropyl and gemdimethyl groups which give rise to a split umbrella mode [57,58].
Te plant extracts that contain phenolic and favonoid molecules are known to have antibacterial and antioxidant properties [59]. Te antibacterial potency of the phenolic and favonoid compounds is attributed to their ability to destroy the membrane of bacteria, inhibit the bacterial virulence factors such as enzymes and toxins, and suppress bacterial bioflm formation [60]. On the other hand, these molecules donate hydrogen to neutralize and deactivate the free radicals and have therefore gained popularity for preventing diseases caused by oxidative stress over the synthetic antioxidants [61,62]. Te available literature indicates that some groups of compounds in the essential oils of plants are classifed as terpenes, straight-chain compounds not containing any side chain, phenylpropanoidsand miscellaneous group having varied structures not included in frst three groups (sulfuror nitrogen-containing compounds). Compounds from these groups have antibacterial and antioxidant potency plant extracts that have phenolic and favonoid molecules, inhibiting bacterial growth as well as preventing cellular damage to the action of free radicles [33,59,63,64]. Te extracts and essential oils from Hypericum species have demonstrated compounds with antibacterial and antioxidant properties [41,44,45,65]. However, there is no study that has been reported over the essential oils revived from the leaves or fowers of H. revolutum subsp. keniense.

Conclusions and Recommendation
Based on the results from the current study, it was concluded that the essential oils obtained from the leaves and fowers of Hypericum revolutum subsp. keniense had signifcant antibacterial activity against methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Acinetobacter baumannii, Salmonella enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli, and Haemophilus infuenzae. Te fndings for antibacterial activity are consistent with the traditional uses of H. revolutum subsp.  Evidence-Based Complementary and Alternative Medicine kenience in folklore medicine in the treatment of diarrhoea, burns, and wounds. Based on the FRAP and H 2 O 2 methods of the antioxidant assay, the essential of the leaves have compounds with antioxidant properties. It was presumed that the antibacterial antioxidant potency was due to the compounds such as caryophyllene, α-pinene, and Dcamphene. It was recommended that research on antioxidant activities and quantifcation of phenolics and favonoids be carried out using the fower derived essential oils. Further eforts are needed to assess the wound healing properties and toxicological profles of the H. revolutum subsp. keniense essential oils.

Data Availability
Te statistical data used to support the fndings of this study are included within the article.

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