The essential oils of Grewia Lasiocarpa E. Mey. Ex Harv.: chemical composition, in vitro biological activity and cytotoxic effect on Hela cells

Abstract

The chemical composition and biological activity of the essential oil extracted from the fresh leaves and stem bark of Grewia lasiocarpa was determined for the first time in this study. The essential oils were extracted by hydrodistillation and identified by GC–MS and FTIR. The antibacterial, antioxidant activity and total phenolic content of essential oils were determined. The major compounds identified were phytol (22.6%); α-farnesene (8.62%); n-hexadecanoic acid (7.24%); farnesol (4.61%) in the leaves, and 2-methylheptadecane (7.24%); heptacosane (7.60%); heptadecane, 2,6,10,14-tetramethyl (7.30%). The presence of aromatic, alkanes and phenolic compounds were revealed by FTIR analysis. The in silico oral prediction shows that some of the components are orally safe. The essential oil from the leaves showed cytotoxic activity at 1mg/mL(IC50 =555.70 μg/mL) against HeLa cells. The oils exhibited no significant antioxidant activity (IC50 >1 000 μg/mL) with <100 mg/g GAE of total phenol. The essential oils showed different degrees of activities against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 314588) at 10 μg/mL, 5 μg/mL and 2.5 μg/mL. These results might provide a future reference basis for further exploration of more of its medicinal application.

Key words
Grewia lasiocarpa; essential oil; antibacterial; antioxidant; Cytotoxicity; HeLa cells

INTRODUCTION

Oil is the carrier of the ‘essence’ of plants thus, plant volatiles are referred to as ‘essential oils’ this description was given by Haagen-Smit (1949) (Raut & Karuppayil 2014). Although these essential oils, which are volatile and lipophilic, are commonly obtained from plants, they are also present in bryophytes, e.g., Liverworts (Figueiredo et al. 2008).

The plant family Malvaceae is known to have a significantly high composition of secondary metabolites rich in volatile compounds and essential oils (Baser 1995). The presence of these metabolites supports the economic significance of plants belonging to this family, e.g., food okra- Abelmoschus esculentus, fibre cotton -genus Gossypium) etc. (Zuzarte & Salgueiro 2015, Flachs 2016). Grewia lasiocarpa E. Mey. ex Harv. (Malvaceae) is an indigenous South African plant that is not yet well documented in the literature; however, there are recent reports (Akwu et al. 2019a, b, c, 2020a, b, 2021). Globally, numerous plant genera, including the genus Grewia (family Malvaceae sensu lato) are being exploited for the phytocompounds they possess based on their ethnomedicinal properties (Morton 1987, Robertshawe 2011, Rehman et al. 2013, Ayurveda 2015). Reports on the presence of essential oils/volatile oils in some Grewia species have been given viz. in the stem bark of Grewia venusta FRES (Nep et al. 2013) and the leaf, stem bark and roots of G. mollis (Rehman et al. 2013). The essential oil of the seed bark of G. asiatica Linn. (G. subinaequalis DC) contains β-amyrin, lupeol and betulin, while the essential oil of the flower contains glycosides pelargonidin, cyanidin, and delphinine (Oliver-Bever 1986), 1,2-epoxy [5.6%], 1-(2, cyano2-ethyl butyl)3-isopropyl urea [5.9%] and hexadecanoic acid [6.3%] (Langford et al. 2010). The essential oil present in the flower of G. bicolor (syn G. salicifolia) constitutes a sedative, farnesol, which is also peculiar to other plants in the Tiliaceae family to which Grewia was previously taxonomically grouped (Burt 2004, Kubmarawa et al. 2007, Pathak 2017). Essential oils are good sources of bioactive compounds, and they possess several biological activities (Shagal et al. 2012), e.g. antioxidant, antimicrobial, cytotoxic, antiviral etc. (Shaaban et al. 2012, Hassan et al. 2016), resulting in an application upsurge in biomedicine (Ullah et al. 2012). Owing to their complexity, characterisation, and identification of essential oils or volatile organic compounds are done using advanced chromatographic and spectroscopic techniques such as gas chromatography and mass spectrometry (GC-MS). Fourier Transform - Infra-Red Spectroscopy (FTIR) is a technique is employed for the characterisation and identification of functional groups. The substance to be analysed could be in any form, namely liquid, solid, solution, fibre, gas, film or with a surface coating (using the attenuated total reflectance (ATR) technique) (Coates 2000, Stuart 2005). As the use of computational technology (in silico methods), to predict the biological activity of substances that are found in humans and the environment, is gaining grounds (Maree et al. 2014, Erukainure et al. 2018). Hence, it is crucial to evaluate the pharmacognistic potentials, oral toxicity, and characterization of these substances using in silico methods.

To the best of our knowledge, there are no published reports on the chemical composition, cytotoxicity, total phenolic content, antioxidant and antibacterial activity of the essential oils of the Grewia lasiocarpa. This present study aimed to extract, identify the chemical composition, evaluate the antioxidant, total phenolic content and biological activity of the essential oils of G. lasiocarpa (leaves and stem bark).

MATERIALS AND METHODS

Collection of plant material

Fresh, healthy plant organs (leaves and stem bark) of G. lasiocarpa were collected from the Umdoni Trust Park area of KwaZulu-Natal’s southern countryside, South Africa. The plant material were taxonomically identified and authenticated by Dr. Syd Ramdhani, curator of the School of Life Sciences, University of KwaZulu-Natal, and a voucher specimen was deposited at the Herbarium with herbarium number (Nneka 002).

Extraction of essential oils

The chopped fresh leaves and stem bark (730 g) each were separately subjected to successive hydrodistillation at normal pressure in a standard Clevenger-type apparatus for 4 h each, in the ratio 1:4 (v/w) of plant material to distilled water. The extracted essential oils were dried over anhydrous sodium sulphate (Merck, Darmstadt, Germany) and measured. The essential oils were stored in screw-capped glass vials in the dark at 4°C until analysis.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis of essential oils

The GC-MS analysis was performed on a GCMS-QP2010 Plus Shimadzu instrument and fitted with capillary chromatographic column of 30 m x 0.25 mm ID x 0.25/0.24 µm film thickness of diphenyl dimethyl polysiloxane (5% diphenyl and 95% dimethyl polysiloxane) model Rtx® –5 ms (RESTREK). For the analytical conditions, the oven temperature was programmed as follows: the initial temperature at 50°C, for 1.5 min, then increased to 200°C at a rate of 4°C min^-1, and then increased up to 300°C at the rate of 10°C min-¹ held for 7 min. The injector and interface temperatures were 240 and 220°C, respectively. The carrier gas was helium, adjusted to a column velocity flow of 1.2 mL/min and 2 µL of essential oil diluted (1/100) in n-hexane (≥99%, GC grade, Sigma-Aldrich) and filtered using a 0.22 µm filter, which was injected into the “splitless” mode system. The mass detector scan mode range was 40 to 500 atomic mass units (amu) or 40-500 m/z., with a total running time of 59 min.

Identification of compounds

The identification of individual compounds was based on the comparison of the retention indices (Kovats indices-IK) and the obtained mass spectral fragmentation patterns with those of known compounds reported and stored in the database of the National Institute of Standards and Technology, Washington, DC, USA (NIST) and literature (Davies 1993, Adams 2007, Babushok et al. 2011).

Fourier transform infra-red analysis

The essential oils were dissolved in 10% DMSO, and the diluents were subjected to Fourier transform infrared (FTIR) spectroscopy (mid-IR spectra) on a Perkin-Elmer FTIR using spectrum software version 6.1. The measurements were carried out at 25 - 27°C, and the spectrum recorded from 4000-400 cm^-1 with a spectral resolution of 4 cm^-1. The peak frequencies were compared to the reference literature (Coates 2000, Stuart 2005) to determine the functional groups.

Biological activities of the essential oil

In vitro cytotoxicity/MTT assay

HeLa cells were maintained in EMEM supplemented with 10% (v/v) gamma-irradiated FBS and 1% antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin) at 37 °C and 5% CO₂, in a HEPA Class 100 Steri-Cult CO₂ incubator (Thermo-Electron Corporation, Waltham, Massachusetts, USA).

MTT assay

The cells were trypsinized and seeded into 48-well plates at a seeding density of approximately 3 x 10⁴ cells/well and incubated at 37°C for 24 h to allow for attachment. The medium was then replaced with fresh medium, and the essential oils of the leaves and stem bark (1 mg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL) was added to the wells and allowed to incubate for 48 h at 37°C. A negative control containing only cells was set as 100% cell survival/viability. After the 48-h incubation period, the spent medium was removed, and 200 μL fresh medium and 20 μL MTT reagent (5 mg/mL in PBS) was added to each well.

The cells were incubated at 37°C for an additional 4 h, after which the MTT-medium solution was removed, and 200 μL DMSO was added to solubilize the formazan crystals. Absorbance was read at 540 nm using a Mindray MR-96A microplate reader (Vacutec, Hamburg, Germany). All assays were performed in triplicate.

Antibacterial assay (Disc diffusion method)

Test-bacterial strains

Six clinical isolated strains Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC BAA-1683), Klebsiella pneumoniae (ATCC 314588) and Salmonella typhimurium (ATCC 14026), provided by Dr. Chunderika Mocktar, School of Pharmacy and Pharmacology, University of KwaZulu-Natal, maintained in 75% glycerol at -80°C, were used to assess the antibacterial activity of the essential oils. 100 µL of each bacterial strain stock were added to 5 mL of nutrient broth (Merck), and the cultures were grown overnight in a test-tube shaker for 24 h at 36 ± 1°C; after which the bacterial cultures (inoculum) were further diluted with sterile nutrient broth to an OD of 0.08-0.1 at 625 nm using a UV–vis spectrophotometer (Agilent Cary 60 Spectrophotometer, USA) to yield a final concentration of approximately 1 × 10⁸ – 1 × 10⁹ bacteria cells /mL and then swabbed onto the Müller-Hinton agar (MHA) plates.

Sample preparation

The essential oils of the leaves and stem bark were dissolved in 10% DMSO to a concentration of 1 mg/mL, from which final concentrations of 10, 5 and 2.5 µg/mL were prepared. 10 µL of each of the different sample concentrations was impregnated on a sterile Whatmann 1 filter paper disc (6 mm in diameter) and kept dry in sterile Petri dishes. For the positive control, the filter papers were soaked in 10 µg/mL of Gentamicin (Gram-negative) and Streptomycin (Gram-positive) to get a concentration of 10 µg/disc. For the negative control, the discs were soaked in 10% DMSO.

Disc diffusion assay

MHA (Biolab, South Africa) was prepared, poured into sterile Petri dishes and allowed to set and dry at room temperature. The standardized bacteria broth solutions were then swabbed onto the prepared MHA plates and allowed to air dry for 15 min. The impregnated discs were then placed on the prepared agar’s surface and incubated at 36°C for 18 h. The diameter of the zone of inhibition was measured in mm inclusive of the disk diameter. The assay was done in triplicate.

Total phenolic content and Antioxidant assay

Preparation of stock solution

Stock solutions (1 mg/mL in methanol) of the leaves and stem bark essential oil was prepared, from which 15, 30, 60, 120 and 240 µg/mL were prepared for the in vitro studies.

Estimation of total phenolic contents

The Folin–Ciocalteu reagent assay was used in determining the total phenolic content of each essential oil (Liu & Yao 2007). To 30 µL of each oil (240 μg/mL), 150 µL of 10% diluted Folin-ciocalteau reagent, and 120 μL of 0.7 M Na₂CO₃ were added, and the mixtures were incubated for 30 min at room temperature. Absorbance was measured at 765 nm, and results expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight using the formula (equation 2.1).

$$C_{tp}=C\ast V/m$$

where C_tp = Total phenolic content (mg/g) in GAE (gallic acid) equivalent

C=Concentration of gallic acid obtained from calibration curve in mg/mL

V=volume of extract in mL

m=mass of extract in gram

In vitro antioxidant assays

DPPH scavenging activity

The 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined using the method of Braca et al. (2002). To 500 µL of each oil, 50 µL of 0.3 Mm of DPPH in methanol was added and the microplate was incubated in the dark at room temperature for 30 min. Ascorbic acid was used as the standard. The absorbance was then read at 517 nm against blank without sample or standard. The IC₅₀ was derived from the inhibition curves by plotting the percentage inhibition against the concentration logarithmic scale. The compound’s scavenging ability was calculated using equation 2.2:

$$\text{DPPH Scavenging activity (\%)}=\left[\frac{({\text{Abs}}_{\text{control}}-{\text{Abs}}_{\text{sample}})}{{\text{Abs}}_{\text{control}}}\right]\ast 100$$

where Abs _control is the absorbance of DPPH and methanol

Abs _sample is the absorbance of DPPH radical + sample (compound or standard)

In Silico oral toxicity prediction of the identified compounds

The oral toxicity classes of the major compounds were reported as LD₅₀ values (mg/kg) and categorized in accordance with the globally harmonized system of classification of labelling of chemicals (GHS) on ProTox (http://tox.charite.de/protox_II/) (Drwal et al. 2014).

Statistical analysis

All experiments were carried out in triplicate, and the data derived were subjected to statistical analysis using SPSS 25 for Windows, IBM Corporation, New York, USA. Tukey’s-honest significant difference multiple range post hoc test. The values expressed as means ± standard deviation and significant difference established at p<0.05.

RESULTS AND DISCUSSION

Extraction of essential oils

The essential oil of the leaves and stem bark had a pungent aromatic smell. The leaves’ essential oil was liquid at room temperature with a yield of 0.008% (w/w) greenish liquid, and that of the stem bark was 0.003% (w/w) viscous colourless liquid.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis: Chemical composition of the identified constituents of G. lasiocarpa essential oils

The GC-MS analysis revealed nineteen compounds were present in the leaves, representing (90.20%), out of which twelve were identified (Table I), and in the stem bark, twenty compounds were eluted representing (92.34%), out of which ten were identified (Table II). The major compounds in the essential oil of the leaves were phytol (22.6%); α-farnesene (8.62%); n-hexadecanoic acid (7.62%); farnesol (4.61%), and 2-methylheptadecane (7.24%); heptacosane (7.60%); heptadecane, 2,6,10,14-tetramethyl (7.30%) in the stem bark. The compound farnesol was present in the leaves and stem bark but with variations in area percentage. In a relatively large number of plant essential oils that have been investigated, the low-molecular-weight aliphatic compounds, terpenes/terpenoids and aromatic are often present in a large percentage (Pichersky et al. 2006). This general observation agrees with the essential oil composition of the leaves (Table I) but not of the stem bark (Table II). The compounds hexadecanoic acid, tetratricontane, farnesol and their derivatives might be part of the chemotaxonomic essential oil tool for this genus since there are reports of these compounds in other Grewia spp. (Chen et al. 2008, Langford et al. 2010).

Table I
Chemical composition of Grewia lasiocarpa leaves essential oil.

S/N	RT	Compounds	RIcalc .	RILit	Identification Method	Peak area (%)
1	12.92	trans-2,7-dimethyl-3,6-octadien-2-ol	1100	-	MS	2.16
2	23.87	α-Farnesene	1500	150119	MS, RI	8.62
3	26.48	Farnesol	1600	-	MS	4.61
4	40.35	n-Hexadecanoic acid	1800	-	MS	7.24
5	41.93	Phytol	2100	2116.419	MS, RI	22.60
6	42.52	Heptadecane,2,6,10,15-tetramethyl	2100	-	MS	1.71
7	42.61	Hexacosane	2600	260010	MS	2.17
8	42.98	Tetracosane	ND	-	MS	1.56
9	52.37	Octacosane	2800	280010	MS, RI	2.12
10	53.95	Squalene	3000	29141	MS	0.91
11	55.05	Hexatriacontane	3600	36001	MS; RI	2.41
12	57.07	Tetratetracontane	4400	-	MS	0.92

• RI^calc. =Calculated retention indices, RI_Lit.=Retention indices from literature.

Table II
Chemical composition of Grewia lasiocarpa stem bark essential oil.

S/N	RT	Compounds	RI calc .	RILit	Identification Method	Peak area (%)
1	29.27	Tetradecane, 4-methyl	1500	-	MS	4.36
2	33.99	Tetradecane, 4-ethyl	1600	-	MS	5.89
3	34.78	Pentadecane, 2-methyl	1600	-	MS	3.93
4	39.09	2-Methylheptadecane	1800	-	MS	7.24
5	39.71	Hexadecane, 5-butyl	2000	-	MS	3.18
6	53.92	Farnesol	-	-	MS	1.72
7	55.30	Heneicosane	2100	-	MS	4.58
8	55.89	Heptadecane,2,6,10,14-tetramethyl	2100	-	MS	7.30
9	56.02	Tricosane, 2-methyl	2300	-	MS	3.62
10	57.05	Heptacosane	2700	-	MS	7.60

• RI^calc. =Calculated retention indices, RI_Lit. =Retention indices from literature.

In vitro Cytotoxicity/MTT assay

The major constituents of essential oils determine the biological properties (Bakkali et al. 2008). The significant cytotoxic activity observed at 1 mg/mL of the essential oils from the leaves (Figure 1) may be attributed to the high percentage of phytol present in the leaves (Table I) (Kumar et al. 2010, Silva et al. 2014), which is in agreement with the report of Tisserand & Balacs (1999), which states that phytol is grouped among the uncommon constituents of essential oils and most often when they occur they are in high percentage. The other compounds present in the essential oil of the leaves are tetratricontane (Dandekar et al. 2015), squalene (Murakoshi et al. 1992, Rao et al. 1998, Smith et al. 1998, Reddy & Jose 2013), n- hexadecanoic acid (Ji et al. 2005), tetradecane, 4-methyl (Begum et al. 2016) in the stem bark and farnesol (Asakawa et al. 2013) present in both, have cytotoxic properties.

Figure 1
In vitro cytotoxicity activity of the leaves and stem bark of Grewia lasiocarpa against HeLa cells. a -c letters above the bars for a given concentration are significantly different from each other (Tukey’s honest significant difference multiple range post hoc test p<0.05 IBM SPSS version 25). GL_LEO=Grewia lasiocarpa leaves essential oil, GL_SBEO=Grewia lasiocarpa stem bark essential oil.

Antibacterial assay

Quite a number of essential oils exhibit remarkable antibacterial activity due to their ability to bind to and fragment the lipids in the cell membranes and mitochondria, resulting in the cytosol’s release and its contents (Burt 2004). In addition, essential oils exhibit more inhibitory activity against Gram-positive bacteria than Gram-negative bacteria owing to the cellular morphology of the cell membrane of the former (Burt 2004). On the contrary, the oils from G. lasiocarpa leaves and stem bark showed no activity against the Gram-positive bacteria Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC BAA-1683) and Salmonella typhimurium (ATCC 14026), but against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 314588) with a range of dose-dependent antibacterial activity (Table III). The antibacterial activity of the oils from the leaves and stem bark might be due to the presence of phytol (Kumar et al. 2010, Ghaneian et al. 2015), n-hexadecanoic acid (Kumar et al. 2010), hexacosane (Rukaiyat et al. 2015) in the leaves and heneicosane (Uma & Parvathavarthini 2010), farnesol (Asakawa et al. 2013) present in the leaves and stem bark. However, the antibacterial activity of the essential oils from the leaves and stem bark may be a synergistic effect of these reported active compounds or some other compounds in the oil.

Table III
Antibacterial activity of the essential oils from the leaves and stem bark of Grewia lasiocarpa.

Zone of inhibition [mm] (Mean ± SD)
Bacteria Strain	GL_LEO				GL_SBEO
	10 µg/mL	5 µg/mL	2.5 µg/mL	Positive control	10 µg/mL	5 µg/mL	2.5 µg/mL	Positive control
Pa	7.25±1.06	6.50±0.00	R	9.30±0.56	7.80±0.29	8.30±1.53	6.50±0.00	10.00±1.73
Ec	8.67±.58	7.50±0.71	6.25±0.00	11.00±3.00	8.50±0.71	6.75±0.35	6.50±0.00	10.33±2.08
Kp	6.50±0.00	7.23±1.54	6.20±0.00	10.00±2.00	7.50±1.41	6.75±0.35	6.50±0.00	9.83±1.04

• R= Resistance, Pa= Pseudomonas aeruginosa (ATCC 27853), Ec=Escherichia coli (ATCC 25922), Kp=Klebsiella pneumoniae (ATCC 314588), (n = 3), Positive control (Gentamicin [10 µg/disc]). Methicillin-resistant Staphylococcus aureus [MRSA] (ATCC BAA-1683), Staphylococcus aureus (ATCC 25923) and Salmonella typhimurium (ATCC 14026) were observed to be resistant to both essential oils.

Fourier transform infrared spectroscopy (FTIR) analysis

To further confirm that the inhibitory effect was due to the essential oil present in the diluent (10% DMSO), FTIR analysis was done. Hence, the functional groups present in the EOs were identified. As typical of aromatic compounds, two sets of bands around 1600 cm^-1 and 1500 cm^-1 were observed (Figure 2) (Coates 2000, Stuart 2005). According to Morar et al. (2017), the relatively low number of peaks in the FTIR spectra may be attributed to the low concentrations of most of the essential oil components, and the spectra regions between 640-1840 cm^-1 and 2770-3070 cm^-1 ranges are considered as regions with molecular structural information. However, the broad bands 3400 and 2500 cm^-1 were observed because of moisture or O-H stretching (Figure 2). The presence of characteristic functional groups such as phenols (1646.74 cm^-1 and 1320.38 cm^-1), alkanes (1407.26 cm^-1 and 1437.85 cm^-1) and aromatic compounds (1646.74 cm^-1 and 1635.30 cm^-1) was revealed by the FTIR analysis in the essential oils of the leaves and stem bark respectively (Figure 2). The leaves have more cytotoxic compounds than the stem bark, as portrayed by their IC₅₀ values (555.70 and >1000 µg/mL), respectively (Table IV).

Figure 2
Overlay of the FTIR spectra of 10%DMSO, essential oils of Grewia lasiocarpa leaves and stem bark dissolved in 10%DMSO.

Table IV
Biological activities: IC₅₀ values of the essential oils from the leaves and stem bark of Grewia lasiocarpa.

Sample	Cytotoxicity (µg/mL)	DPPH (mg/mL)
GL_LEO	555.70±25.29	1.04±3.32b
GL_SBEO	>1000	39.87±1.87c
AA	-	0.008±17.85a

• The IC₅₀ of the cytotoxicity and DPPH assay of the essential oil is the concentration that can result in 50% decrease of cell growth and the concentration needed to scavenge 50% of the DPPH free radicals, respectively. Data are presented as mean ± SD, n=3, of triplicate determinations. ^a-c Different superscript letters within the column for a given parameter are significantly different from each other different Tukey’s honest significant difference multiple range post hoc test p<0.05 IBM SPSS version 25). GL_LEO=Grewia lasiocarpa leaves essential oil, GL_SBEO=Grewia lasiocarpa stem bark essential oil, AA=Ascorbic acid.

Total phenolic contents

There are several assays for evaluating the antioxidant activity of lipophilic substances like essential oils; however, scientists prefer to use the DPPH technique because of its simplicity and sensitivity (Miguel 2010). The % inhibition capacity for scavenging DPPH free radicals was observed to be dose-dependent, and a comparison between the leaves and the stem bark % inhibition, the former showed a slightly higher inhibition percentage in all concentration although not significantly different (Figure 3). This observation may be due to the antioxidant properties of terpenoids present in the leaves (Kumar et al. 2010), since the antioxidant activity of essential oils is dependent on the bioactive compounds percentage occurrence (Reddy 2001), most especially owing to the high percentage of the diterpene - phytol (Ruberto & Baratta 2000, Lanfer-Marquez et al. 2005, Santos et al. 2013) and other antioxidants e.g. tetratricontane (Dandekar et al. 2015). However, the low IC₅₀ value of the essential oils from the leaves (IC₅₀=1.04 mg/mL) and stem bark (IC₅₀=39.87 mg/mL) when compared with ascorbic acid (IC₅₀=0.008 mg/mL) (Table IV) this might be due to the inactive concentrations of the phenolic compounds present in the leaves and stem bark.

Figure 3
In vitro antioxidant activity of the leaves and stem bark of Grewia lasiocarpa.

The total phenolic content of the leaves and stem bark (Table V) might have also influenced the % inhibition observed (Figure 3). However, the IC₅₀ values are significantly different; this implies that any substance’s antioxidant capacity is influenced by the quality and quantity of its constituents, i.e. the chemical composition and amount of individual compounds.

Table V
Total phenolic contents of the essential oils from the leaves and stem bark of Grewia lasiocarpa.

Sample	Total phenols (mg/g GAE)
GL_LEO	86.29±0.25a
GL_SBEO	78.22±0.25a

• Data presented as mean ± SD of triplicate ^a Superscript letter within each column for the given parameter is not different from each other Tukey’s honest significant difference multiple range post hoc test p<0.05 IBM SPSS version 25). GL_LEO=Grewia lasiocarpa leaves essential oil, GL_SBEO=Grewia lasiocarpa stem bark essential oil.

In Silico prediction of oral lethal dose toxicity class of the identified compounds

The advantages of applying the in silico method are virtual (no live animal required), quick and reliable. The oral lethal dose toxicity classes are Class I: fatal if swallowed (LD₅₀ ≤ 5), Class II: fatal if swallowed (5 < LD₅₀ ≤ 50), Class III: toxic if swallowed (50 < LD₅₀ ≤ 300), Class IV: harmful if swallowed (300 < LD₅₀ ≤ 2000), Class V: may be harmful if swallowed (2000 < LD₅₀ ≤ 5000), Class VI: non-toxic (LD₅₀ > 5000) (Drwal et al. 2014), based on this it is unsafe to orally administer some of the compounds (Table VI and VII); thus the internal use of EOs without a thorough analysis of its chemical composition is not advised. Some essential oils are not safe for consumption (Baser 1995). However, they are generally regarded as safe (GRAS), but it is claimed that they do not cause an adverse detrimental effect when administered orally in considerable low concentrations (hydrosols) (Baser 1995). Phytol is used in aromatherapy; the in silico oral prediction of its non-toxicity (Table VI) further supports its safe use in aromatherapy.

Table VI
In Silico oral toxicity prediction of the Identified compounds in the leaf of Grewia lasiocarpa.

Compounds	Predicted LD50 (mg/Kg)	Toxicity class	Prediction accuracy (%)
trans-2,7-dimethyl-3,6-octadien-2-ol	3500	5	67.38
α- Farnesene	5000	5	68.07
Farnesol	5000	5	100
Hexadecanoic acid	130	3	100
Phytol	5000	5	100
heptadecane,2,6,10,15-tetramethyl	ND	ND	ND
Hexacosane	154	3	100
Tetracosane	154	3	100
Octacosane	154	3	100
Squalene	5000	5	100
Hexatriacontane	154	3	100
Tetratetracontane	154	3	100

• ND=Not determined.

Table VII
In Silico oral toxicity prediction of the identified compounds in the stem bark of Grewia lasiocarpa.

Compounds	Predicted LD50 (mg/Kg)	Toxicity class	Prediction accuracy (%)
Tetradecane, 4-methyl	154	3	100
Tetradecane, 4-ethyl	154	3	100
Pentadecane, 2-methyl	154	3	100
2-Methylheptadecane	ND	ND	ND
Hexadecane, 5-butyl	154	3	100
Heptadecane,2,6,10,15-tetramethyl	ND	ND	ND
Hexadecane, 5-butyl	154	3	100
Heneicosane	154	3	100
Heptadecane,2,6,10,14-tetramethyl	ND	ND	ND
Tricosane, 2-methyl	154	3	100

• ND=Not determined.

CONCLUSIONS

This study presented the first data for the leaves’ essential oil constituents and the stem bark of Grewia lasiocarpa through hydrodistillation, GC-MS, and FTIR analyses. Grewia lasiocarpa has significant antibacterial and cytotoxic potential based on the results obtained; isolation of the individual compounds for further bio-assays might be promising for safe drug development. Therefore, more studies should be conducted on the essential oils from the genus Grewia to establish more bioactive components.

ACKNOWLEDGMENTS

The authors would like to acknowledge the Organisation for Women in Science for the Developing World (OWSD), and Swedish International Development Cooperation Agency (SIDA) for their intellectual and material contribution (financial assistance). We acknowledge the consumables supply by the National Research Foundation (NRF). We are grateful to Dr. Chunderika Mocktar for the provision of the clinical bacteria strains.

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Publication Dates

• Publication in this collection
  28 May 2021
• Date of issue
  2021

History

• Received
  24 Apr 2019
• Accepted
  2 July 2019