In Vitro Investigation of the Antioxidant and Cytotoxic Potential of Tabernaemontana ventricosa
Hochst. ex A. DC. Leaf, Stem, and Latex Extracts
Abstract: Tabernaemontana ventricosa (Apocynaceae) a latex-bearing plant is used in traditional
medicine for its therapeutic benefits in reducing fever and hypertension and wound healing.
Due to limited information on the plant’s pharmacological activities, this study aimed to
investigate the antioxidant potential of the leaf, stem, and latex extracts of T. ventricosa,
using the Folin-Ciocalteu (total phenolics), aluminum chloride colorimetric (total flavonoids),
2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays.
The cytotoxic activity was evaluated in the human HEK293 (embryonic kidney), HeLa (cervical
carcinoma), and MCF-7 (breast adenocarcinoma) cell lines using the MTT assay. The latex
extracts possessed the highest total phenolic content (115.36 ± 2.89 mg GAE/g), followed by
the stem hexane extracts (21.33 ± 0.42 mg GAE/g), the chloroform leaf (7.89 ± 0.87 mg
GAE/g), and the chloroform stem (4.69 ± 0.21 mg GAE/g) extracts. The flavonoid content was
substantially high ranging from 946.92 ± 6.29 mg QE/g in the stem hexane, 768.96 ± 5.43 mg
QE/g in the latex, 693.24 ± 4.12 mg QE/g in the stem chloroform, and 662.20 ± 1.00 mg QE/g
in the leaf hexane extracts. The DPPH assays showed the highest percentage of inhibition at
240 µg/mL, for the stem hexane (70.10%), stem methanol (65.24%), and stem chloroform
(60.26%) extracts, with their respective IC50 values of 19.26 µg/mL (stem hexane), 6.19
µg/mL (stem methanol), and 22.56 µg/mL (stem chloroform). The FRAP assays displayed
minimal inhibition ranging from 4.73% to 14.40%, except for the latex extracts which
displayed moderate inhibition at 15 µg/mL (21.82%) and substantial inhibition at 240 µg/mL
(98.48%). The HeLa and MCF-7 cell lines were the most sensitive to the extracts, with the
hexane, chloroform, and methanol leaf and stem, and latex extracts significantly affecting the
percentage cell survival. Overall, the various parts of T. ventricosa exhibited strong
antioxidant activity correlating to its cytotoxicity. Further studies should focus on the
isolation of specific antioxidant compounds that could be investigated for their anticancer
potentia
1. Introduction Cancer is a composite terminal disease whereby irregular cells rapidly divide
and often attack several tissues or migrate into various other locations [1]. There are three
significant and common causes of cancer, which include improper diet [2] genetic character
[3], and ecological factors [4] such as ultraviolet (UV)-light, infectious agents, and pollution
[4]. Thehasty incline in the critical contributors to this disease has directly increased cancer
rates over the past decade [5–7]. According to the global cancer statistics (2018),
approximately 18.1 million people are likely to be diagnosed with cancer, and an additional
10 million cancer-related deaths were anticipated in 2020 [5,8–10]. Global cancer statistics
have revealed that lung cancer is the foremost cause of cancer mortality (18.4%), closely
followed by breast cancer (11.6%), stomach cancer (8.2%), liver cancer (8.2%), prostate
cancer (7.1%), and colorectal cancer (6.1%) [10]. According to Made et al. [11], nearly 38,000
(8%) of the total deaths in South Africa in 2014 were associated with cancer. Regardless of
the widespread usage of chemotherapy, radiation therapy, and immunotherapy, these
treatments introduce significant consequences ranging from multidrug resistance (MDR),
exhaustive side effects, and the generation of reactive oxygen species (ROS), which
ultimately induce detrimental effects to DNA (deoxyribonucleic acid) and cellular signaling
pathways [12–14]. Recently, the discovery of novel compounds from medicinal plant extracts
has been considered for the treatment of cancer [7,15,16]. These compounds are often
preferred over conventional techniques since natural compounds display minimal side effects
and act as a modulator of MDR [17–19]. Approximately 60% of anticancer agents are often
generated from plant-based compounds [20–23]. The most common plant-derived
anticancer products include vinca alkaloids, combretastatin, taxanes, camptothecin, and
epipodophyllotoxin [24–27]. Additionally, phenolics, flavonoids, and volatile essential oils are
often utilized in cancer research due to their rich source of antioxidants [28–30]. These
compounds are exploited to combat ROS, resulting in the reduction of oxidative stress,
improvement of immune function, and an increase in longevity [31–33]. According to Kam et
al. [34], several species belonging to the genus Tabernaemontana contain indole alkaloids,
which may suppress the growth and development of tumor cells. Tabernaemontana
ventricosa Hochst. ex A. DC. (Forest toad tree) is a latex-bearing plant belonging to the
Apocynaceae and a genus consisting of medium-sized flowering trees within
Tabernaemontana [35]. This species emits a pungent sweet scent from tubular salvershaped
whitish-yellow flowers [36]. Approximately 100 species of the Tabernaemontana genus are
primarily distributed in tropical and subtropical regions of the world, including Africa, Asia,
and America [17,36]. The Tabernaemontana species T. ventricosa and T. elegans are reported
to occur in South Africa [1,36,37]. Previous phytochemical analysis on T. ventricosa indicated
the presence of major alkaloidal components and large quantities of triterpenes [38]. Parts of
T. ventricosa are reportedly used in traditional medicine systems in KwaZulu-Natal to palliate
fever, reduce blood pressure, treat wounds, and heal sore eyes [36,39]. Insufficient research
is available on T. ventricosa, although a few studies have investigated the biological activity
of certain chemical compounds. Van Beek et al. [40] reported on the alkaloidal compounds
conopharyngine and akuammincine, which showed healing and opioid activity, respectively.
Considering the scarcity of information available on the pharmacological activities of T.
ventricosa and the crucial discoveries of curative agents from innovative plant-based
products, the current investigation aimed to determine the antioxidant activities of the
hexane, chloroform, methanol leaf and stem, and latex extracts of T. ventricosa using various
protocols such as the Folin-Ciocalteu (total phenolics), aluminum chloride colorimetric (total
flavonoids), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power
(FRAP) assays. The cytotoxic activity of all extracts was also evaluated in the human cell lines;
HEK293 (human embryonic kidney cells), HeLa (cervical carcinoma), and MCF-7 (breast
adenocarcinoma) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) assay. 2. Materials and Methods Leaves, stems, and latex exudate from fully grown T.
ventricosa plants (Figure 1) of wild origin were collected from the University of KwaZulu-
Natal (Westville campus), South Africa (29◦49003.300 S 30◦56032.700 E). A combination of
emergent, young, and mature leaves and stems were collected for analysis. The plant
material and exudate were taxonomically identified, and a voucher specimen (18222) was
deposited at the Ward herbarium, School ofLife Sciences, University of KwaZulu-Natal. The
plant material was inspected for any signs of microbial and fungal contamination. Thereafter
the leaves and stems were separately air-dried for three months at 23 ◦C and ground at high
speed into a fine powder using a grinder (Mellerware, Model: 29105, Durban, South Africa).
The powdered material was kept in an airtight consolTM glass jar, out of direct sunlight, at 23
◦C, until further use. The latex exudate was aseptically collected by careful incisions in the
soft stems of the plant with a sharp blade. The latex was diluted in distilled water (1:1; v/v),
and the sample was centrifuged at 5000 rpm/ref for 10 min using an Eppendorf centrifuge
(Model: 5415R, Marshall Scientific, Hampton, NH, USA). The pellet was removed, and the
supernatant was stored at −8 ◦C until further use.
Figure 1. Tabernaemontana ventricosa captured at the University of KwaZulu-Natal (UKZN)-
Westville Campus, Durban, South Africa. Co-ordinates: 29◦49003.3” S 30◦56032.7” E. (A)
Dichotomously branched tree, (B) punctured leaf showing white latex exudate, (C) white
salver-shaped flowers, and (D) obliquely ellipsoid follicle fruit joined at the base. 2.1. Solvent
Extraction Powdered leaf (10 g) and stem (10 g) samples were placed separately into round
bottom flasks and dissolved in 100 mL of hexane. The flasks containing the solvent mixture
were attached to a Soxhlet apparatus, heated at 60 ◦C for four sessions (3 h each), and
filtered after each session using Whatman No. 1 filter paper. The extraction process was
repeated using chloroform and methanol solvents, separately. The resulting leaf and stem
extracts for each solvent were stored individually in sterilized consolTM glass jars, in a dark
room. 2.2. Evaporation and Concentration Following extractions, the leaf and stem extracts,
and latex supernatants were entirely air-dried in a well-ventilated dark room (~23 ◦C) for
approximately 30 days. The dried
