molecules

Article
Antioxidant and Antimicrobial Evaluations of Moringa oleifera
Lam Leaves Extract and Isolated Compounds
Mmabatho Kgongoane Segwatibe 1 , Sekelwa Cosa 2 and Kokoette Bassey 1, *

1 Department of Pharmaceutical Sciences, School of Pharmacy, Sefako Makgatho Health Sciences University,
Molotlegi Street, Ga-Rankuwa, Pretoria 0204, South Africa
2 Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Private Bag X20, Hatfield,
Pretoria 0028, South Africa
* Correspondence: edward.bassey@smu.ac.za

Abstract: Moringa oleifera, native to India, grows in tropical and subtropical regions around the world
and has valuable pharmacological properties such as anti-asthmatic, anti-diabetic, anti-inflammatory,
anti-infertility, anti-cancer, anti-microbial, antioxidant, and many more. The purpose of this study
was to assess the free radical scavenging ability of two extracts and two pure compounds of M. oleifera
Lam (hexane, ethanol, compound E3, and compound Ra) against reactive oxygen species, as well as
their reducing power and antimicrobial activities. Bioautography antioxidant assay, 2,2-diphenyl-1-
picrylhydrazyl (DPPH), hydrogen peroxide (H2 O2 ) free radical scavenging, and iron (iii) (Fe3+ to
Fe2+ ) chloride reducing power assays were used to assess the extracts’ qualitative and quantitative
free radical scavenging activities. Furthermore, the extract and the compounds were tested against
both Gram-positive and Gram-negative bacterial strains suspended in Mueller–Hinton Broth. The
extracts and pure compounds showed noteworthy antioxidant potential, with positive compound
bands in the Rf range of 0.05–0.89. DPPH), H2 O2 , and Fe3+ to Fe2+ reduction assays revealed that
ethanol extract has a high antioxidant potential, followed by compound E3, compound Ra, and finally
hexane extract. Using regression analysis, the half maximal inhibitory concentration (IC50 ) values
for test and control samples were calculated. Compound Ra and ethanol exhibited high antioxidant
activity at concentrations as low as ≈0.28 mg/mL in comparison with n-hexane extract, compound
Citation: Segwatibe, M.K.; Cosa, S.; E3, ascorbic acid, and butylated hydroxytoluene standards. The radical scavenging activity of almost
Bassey, K. Antioxidant and all M. oleifera plant extracts against DPPH was observed at 0.28 mg/mL; however, the highest activity
Antimicrobial Evaluations of Moringa was observed at the same concentration for ascorbic acid and butylated hydroxytoluene (BHT) with
oleifera Lam Leaves Extract and
a low IC50 value of 0.08 mg/mL and compound Ra and ethanol with a low IC50 of 0.4 mg/mL,
Isolated Compounds. Molecules 2023,
respectively. The extracts and pure compounds of M. oleifera have little to no antibacterial potential.
28, 899. https://doi.org/
M. oleifera extracts contain antioxidant agents efficient to alleviate degenerative conditions such as
10.3390/molecules28020899
cancer and cardiovascular disease but have little activity against infectious diseases.
Academic Editor: Francesco
Cacciola Keywords: antimicrobial; antioxidant; bioautography
Received: 12 December 2022
Revised: 3 January 2023
Accepted: 5 January 2023
Published: 16 January 2023 1. Introduction
Natural products derived from medicinal plants, whether as pure compounds or
standardized extracts, offer countless prospects for new drug discovery. This is due to the
unparalleled availability of chemical diversity in them. According to the World Health
Copyright: © 2023 by the authors.
Organization (WHO), medicinal plants serve the primary healthcare needs of more than
Licensee MDPI, Basel, Switzerland.
80% of the world’s population [1–4]. In particular, countries such as China, India, Japan,
This article is an open access article
Sri Lanka, Thailand, and Korea stand to gain the most from these plants [5–7]. Plant-based
distributed under the terms and
medicines are well-known for their reliability, accessibility, and affordability. These plants’
conditions of the Creative Commons
medical usefulness comes from their bioactive phytochemical components, which have
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
defined physiological effects on the human body. Some of the most significant bioactive
4.0/).
plant secondary metabolites are alkaloids, flavonoids, tannins, terpenoids, saponins, and

Molecules 2023, 28, 899. https://doi.org/10.3390/molecules28020899 https://www.mdpi.com/journal/molecules

Molecules 2023, 28, 899 2 of 22

phenolic compounds [8–10]. The biological function of antioxidants in medicinal plants

is crucial. This is because oxidative stress, which is brought on by excessive free radi-
cals, is linked to several degenerative conditions, such as cancer, ischemic heart disease,
atherosclerosis, diabetes mellitus, and neurological illnesses. Since free radicals play a
crucial physiological function in metabolic processes, it is necessary to maintain a homeo-
static equilibrium between their presence and antioxidants [11,12]. One of the major public
health concerns worldwide is infectious diseases caused by pathogens such as Staphylococ-
cus aureus, Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, and Streptococcus
pyogenes [13,14]. The increased antibiotic resistance of pathogens has resulted in escalating
mortality and morbidity rates [15]. The World Health Organization (WHO) estimates that
each year infections caused by multidrug resistant (MDR) bacteria cause approximately
700,000 deaths globally, affecting people of all ages, including 200,000 newborns [16]. Thus,
one possible treatment for these MDR bacteria is the use of natural products such as medici-
nal plants utilized by traditional healers in the management of diseases caused by microbial
pathogens. Many foods, including fruits and vegetables, contain antioxidants. Plants and
animals maintain sophisticated systems of many types of antioxidants, such as glutathione,
vitamin C, vitamin A, and vitamin E, as well as enzymes such as catalase, superoxide
dismutase, and different peroxides. Traditional herbal treatments and dietary foods were
ancient peoples’ principal sources of antioxidants and antimicrobials, which protected them
from free radical damage and antibiotic resistance [17]. Gram-positive bacteria (GPB) are
non-spore producing, facultative anaerobic bacteria that can affect the respiratory tract,
skin, and soft tissues [18]. Streptococcus pyogenes and Staphylococcus aureus cause prevalent
infections such as furuncles, pneumonia, phlebitis, meningitis, urinary tract infections,
pharyngitis, localized skin infections, rheumatic fever, rheumatic heart disease, and strep-
tococcal toxic shock syndrome [19]. The Gram-negative bacteria of Neisseria gonorrhoeae,
Pseudomonas aeruginosa, and Escherichia coli are non-spore forming bacteria that are known
to cause erythematous exudate of pharynx, lung infections, and urinary tract infections,
respectively [20–22]. This high burden of infectious diseases, along with the poor health sys-
tems found in most African nations, heightens the danger of antimicrobial resistance (AMR)
spreading and its repercussions [23]. The insufficiency of effective treatments, pathogen
resistance to antibiotics, and oxidative stress brought on by free radicals in the human body
system have all led to the search for novel therapeutic alternatives from plants. A possible
source of effective and safe antioxidant and antimicrobial natural products is the medicinal
plants used by traditional healers and claimed to have potential for the management of
these conditions. This is because the plants or other natural products’ derived antioxidants
are rich in phenolics, flavonoids, phenolic acids, lignans, and stilbenes secondary metabo-
lites. These compounds often serve as the plants’ defense mechanism against adverse
effects including ultra violet radiation, temperature, and mechanical damage. In addition,
by reacting directly with the oxidation products of fatty acids, phenolic compounds can
prevent adverse changes from occurring in living organisms. Phenolic compounds also
exhibit antimicrobial activity, causing the inhibition of microbial growth by interfering with
the transport of nutrients that are important to their function [24]. One such medicinal
plant used by traditional healers is Moringa oleifera Lam. It is a perennial softwood tree
with low-quality timber and a straight, long trunk (10–12 m in height). Moringa oleifera,
popularly called drumstick tree or horseradish tree, is widely researched for its plethora of
pharmacological properties that are affiliated to the many bioactive compounds identified
and in come cases isolated from the plant. Many studies have been carried out to inves-
tigate the medical significance of the bioactive compounds, possessing several biological
activities such as antimicrobial, anti-inflammatory, and antioxidant. Even though nearly all
the plant parts are useful to mankind and livestock, the leaves have found application as
food and nutritional supplements, which are formulated and marketed in different dosage
forms. However, the biological activities and the isolation of compounds from Moringa
oleifera of the South African ecotype are poorly documented in the literature. As a result,
Molecules 2023, 28, 899 3 of 22

the antioxidant and antimicrobial properties of hexane extract, ethanol extract, compound
E3, and compound Ra are reported in this study.

2. Results
2.1. Dry Mass and Percentage Yield of the Extracts
The mass and the percentage yield of the dried hexane and ethanol extracts were
recorded as 14.99 g (2.91%) and 8.59 g (1.72%), respectively, from 500 g plant powder.

2.2. Structural Characterization and Elucidation of Compound E1

2.2.1. HPLC-PDA and UPLC-MS of Compound E1
The HPLC chromatogram of E1 revealed a single peak at Rt = 2.9 min for the com-
pound, while high-resolution UPLC-MS analysis of E1 resulted in a single peak at Rt of
11.48 with m/z ratio of 383.2020 (M+1). In addition, the mass fragments of E1 obtained
from the high-resultion UPLC-MS/MS at 383, 259, 183, and 129 conform with its struc-
tural major fragments at 259 (M-C18 H27 O), 183 (M-C18 H21 O2 ), and 129 (M-C7 H13 O2 ) (See
Supplementary Materials).

2.2.2. One- and Two-Dimension NMR Analysis of E1

The carbon-13 NMR analysis of E1, conducted after filtering out the noise due to the
experimental background, resulted in 25 clear carbon signals. These signals appeared at δ
173, 147, 139, 124, 123, 119, 114, 60, 50, 49, 39, 37, 34 33, 32, 31, 30, 29.6, 29.5, 28, 27, 24, 22,
19, and 14 ppm. As for the H-1 (proton) NMR of E1, the integration of the proton signals
afforded a total of 35 hydrogens as potentially being an integral part of E1. The multiplicity
and the coupling constant of the protons as well as the C-13 assignments are summarized
in Table 1.

Table 1. Proton and carbon-13 signals and the multiplicity of E1.

Position C-δ (ppm) H-δ (ppm), J (Hz)

1 147 Cq
2 114 7.14 (H, d, J = 8.4)
3 119 7.54 (H, dd, 11.1, 3.1)
4 124 7.37 (H, d, J = 14.0)
5 123 Cq
6 34 2.29 (2H, m)
7 24 1.69 (2H, dt, J = 11.4, 7.4)
8 39 1.30 (H, m)
9 28 2.02 (H, m)
10 139 Cq
11 27 2.09 (2H, dd, J = 11.3, 4.2)
12 31 2.36 (2H, m)
13 50 3.51 (H,
14 29.6 1.30 (H, m)
15 30 2.10 (2H, t, J = 5.7)
16 49 Cq
17 37 1.32 (2H, m)
18 32 1.44 (H, dd, J = 12.1, 2.9)
19 173 Cq
20 33 2.36 (2H, dd, J = 11.3, 3.2)
21 22 0.89 (2H, m)
22 29.5 1.30 (2H, m)
23 60 4.17 (2H, dd, J = 11.5, 3.0)
24 19 0.89 (3H, s)
25 14 0.90 (3H, s)
Cq = quarternary carbons.
 23 60 4.17 (2H, dd, J = 11.5, 3.0)
24 19 0.89 (3H, s)
Molecules 2023, 28, 899
25 14 0.90 (3H, s) 4 of 22
Cq = quarternary carbons.

The distortionless enhancement by polarization transfer (DEPT) of E1 was useful in

The distortionless
confirming the saturated enhancement
carbons that by werepolarization
present intransfer
E2. It was (DEPT) of E1 from
deduced was useful
the
in confirming the saturated carbons that were present in E2. It
experiment that indeed E1 consisted of two methyl (‐CH3) groups as positive signals, was deduced from the
ten
methylene (‐CH2) groups as negative signals, and a further 5 methylene (‐CH) groups asten
experiment that indeed E1 consisted of two methyl (-CH 3 ) groups as positive signals,
methylene
positive (-CHThe
signals. 2 ) groups
number as of
negative signals,
quaternary and a further
carbons, five in the 5 methylene (-CH)
case of E1, wasgroups
not
as positive signals. The number of quaternary carbons, five in
revealed in this experiment. The HSQC signals of E1 positively confirmed the number the case of E1, wasofnot
protonated, and by extension the quaternary carbons, inherent in the structure of E1. Theof
revealed in this experiment. The HSQC signals of E1 positively confirmed the number
protonated, and by extension the quaternary carbons, inherent in the structure of E1. The
experiment produced the observation that E1 comprised 20 protonated and 5 quaternary
experiment produced the observation that E1 comprised 20 protonated and 5 quaternary
carbons in agreement with the DEPT results. Following the NMR interpretation of the
carbons in agreement with the DEPT results. Following the NMR interpretation of the
other experiments, especially the C‐13, H‐1, DEPTH, and HSQC NMR experiments of E1,
other experiments, especially the C-13, H-1, DEPTH, and HSQC NMR experiments of E1,
it was evident that E1 was made up of different moieties. These different units include a
it was evident that E1 was made up of different moieties. These different units include
saturated cyclic or aryl unit at between 14 and 50 ppm, an O‐C unit at 60 ppm, an –OH‐
a saturated cyclic or aryl unit at between 14 and 50 ppm, an O-C unit at 60 ppm, an
substituted phenyl unit, as well as a carbonyl carbon (C=O) at δ 147 and 173 ppm,
–OH-substituted phenyl unit, as well as a carbonyl carbon (C=O) at δ 147 and 173 ppm,
respectively. To determine how the different units linked up to form the structure of E1,
respectively. To determine how the different units linked up to form the structure of E1,
an HMBC experiment was performed. From the HMBC long‐J couplings that translate to
an HMBC experiment was performed. From the HMBC long-J couplings that translate to
how the respective units in E1 are connected, the diagnostic HMBC correlations from the
how the respective units in E1 are connected, the diagnostic HMBC correlations from the
experiment were constructed and are depicted in Figure 1A. Based on the assigned HMBC
experiment were constructed and are depicted in Figure 1A. Based on the assigned HMBC
correlation of
correlation ofE1,E1,
itsits
skeletal structure
skeletal structurewas
wasconstructed
constructedand
and is isdisplayed
displayedininFigure
Figure1B. 1B.

Figure
Figure 1. 1.
HMBCHMBC correlations
correlations that
that linked
linked thethe various
various moieties
moieties (A)(A)
to to form
form thethe proposed
proposed skeletal
skeletal
structure (B) of
structure (B) of E1. E1.

At this juncture, the proposed structure of E1 (Figure 2) and the name for E1 was
elucidated as 10-hydroxy-1,3-dimethylchrysen-3-yl)-5-hydroxypentan-1-one with m/z ratio
of 344.2664 calculated from C25 H36 O3 . To verify the authenticity of the proposed structure,
the calculated mass of E1 was compare to the high-resolution mass obtained from UPLC
analysis of E1. As far as all the reported parameters are concerned, one can say that the
proposed structure of E1 was true. This is because the calculated mass of E1 based on
 At this juncture, the proposed structure of E1 (Figure 2) and the name for E1 was
elucidated as 10‐hydroxy‐1,3‐dimethylchrysen‐3‐yl)‐5‐hydroxypentan‐1‐one with m/z
ratio of 344.2664 calculated from C25H36O3. To verify the authenticity of the proposed
Molecules 2023, 28, 899 5 of 22
structure, the calculated mass of E1 was compare to the high‐resolution mass obtained
from UPLC analysis of E1. As far as all the reported parameters are concerned, one can
say that the proposed structure of E1 was true. This is because the calculated mass of E1
the proposed
based structure was
on the proposed in agreement
structure with the UPLC-MS
was in agreement mass-to-charge
with the UPLC‐MS ratio. In
mass‐to‐charge
addition, the mass fragments
ratio. In addition, the mass of E1 obtained
fragments from
of E1 the high-resultion
obtained UPLC-MS/MSUPLC‐
from the high‐resultion at 383,
259,
MS/MS at 383, 259, 183, and 129 conformed with its structural major fragments at 259 183
183, and 129 conformed with its structural major fragments at 259 (M-C 18 H27 O), (M‐
(M-C
C18H27 H21183
18O), O2 ),(M‐C
and 18129
H21(M-C 7 H13129
O2), and O2 ).(M‐C7H13O2).

Figure 2.
Figure 2. Structure
Structure of
of E1
E1 (10-hydroxy-1,3-dimethylchrysen-3-yl)-5-hydroxypentan-1-one).
(10‐hydroxy‐1,3‐dimethylchrysen‐3‐yl)‐5‐hydroxypentan‐1‐one).

2.3.
2.3. Structural
Structural Characterization
Characterization and
and Elucidation
Elucidation of
of Compound
Compound E3
E3
2.3.1. HPLC-PDA and UPLC-MS of Compound E3
2.3.1. HPLC‐PDA and UPLC‐MS of Compound E3
The HPLC results of compound E3 afforded a single peak that resolved at a retention
The HPLC results of compound E3 afforded a single peak that resolved at a retention
time (Rt) of 2.5 min in a total run time of 15 min. The purity of the peak was determined as
time (Rt) of 2.5 min in a total run time of 15 min. The purity of the peak was determined
97% and is considered good for a compound that has been isolated from a natural product.
as 97% and is considered good for a compound that has been isolated from a natural
Similar to the analysis of E1, a high-resolution UPLC-MS analysis of E3 afforded a single
product. Similar to the analysis of E1, a high‐resolution UPLC‐MS analysis of E3 afforded
compound peak at Rt time of 13.93 min and a high-resolution m/z ratio of 347.2564. From
a single compound peak at Rt time of 13.93 min and a high‐resolution m/z ratio of
the fragmentation pattern of E3, the major daughter ions were 67, 81, 95, 223, and 235. One
347.2564. From the fragmentation pattern of E3, the major daughter ions were 67, 81, 95,
could then infer that these daughter ions were typical for a loss of carboxylic acid (m/z = 45),
223, and 235. One could then infer that these daughter ions were typical for a loss of
a phenolic group (m/z = 95), or acetylic acid group (m/z = 58). These fragmentation groups
carboxylic acid (m/z = 45), a phenolic group (m/z = 95), or acetylic acid group (m/z = 58).
also tended to highlight the fact that E3 contained such groups as part of its structure.
These fragmentation groups also tended to highlight the fact that E3 contained such
groups
2.3.2. as part
One- and of its structure.
Two-Dimensional NMR Analysis of E3
From the C-13 experiment on E3, twenty (20) carbon signals were observed, thus
2.3.2. One‐ and Two‐Dimensional NMR Analysis of E3
implying that E3 consisted of 20 carbon atoms. Because C-20 compounds are usually
From the
commonly foundC‐13in experiment on acids
plants as fatty E3, twenty
or their(20) carbon
esters, E3 signals were to
was thought observed,
be one suchthus
implying that
compound. E3 consisted
These carbon peaksof 20were
carbon atoms.
signaled at Because
13 C NMR C‐20
(101compounds
MHz, CDClare 3 ) δ usually
207.02,
commonly
173.98, 171, found
147.09,in139.03,
plants124.47,
as fatty123.99,
acids or their 114.08,
119.09, esters, E3 was39.89,
60.16, thought to 31.44,
34.42, be one30.94,
such
compound. These carbon peaks were signaled at 13C NMR (101 MHz, CDCl3) δ 207.02,
29.67, 28.96, 24.81, 22.70, 22.6, and 14.21 ppm. Based on the C-13 NMR interpretation,
173.98,
the 171,atoms
carbon 147.09, 139.03, 14.0
between 124.47,
and123.99, 119.09,
30.0 were 114.08, 60.16,
representative 39.89, 34.42,
of saturated 31.44,groups
carbon 30.94,
29.67,
or 28.96,
their 24.81, 22.70,
derivatives. The22.6, andat14.21
carbon ppm.was
60 ppm Based on thetoC‐13
thought be an NMR interpretation,
O-substituted the
carbon
carbona atoms
atom, methoxy between
group 14.0
otherand 30.0 were representative
electronegative or electron-richof saturated
atoms or carbon
group as groups
presentor
their
in E3.derivatives.
Whereas the The carbonatat114.09,
carbons 60 ppm was thought
119.09, to be124.47
123.99, and an O‐substituted
were typicalcarbon atom,
of a phenyl
a methoxy
ring moiety,group
those other
at 138electronegative
and 147.09 ppmorsuggested
electron‐rich
that atoms
E3 shouldor group
have anas O-substituted
present in E3.
phenyl ring system. The carbons at 171.00, 173.98, and 207.02 were also
Whereas the carbons at 114.09, 119.09, 123.99, and 124.47 were typical of a phenyl indicative of three
ring
carbonyl carbon
moiety, those atunits and a
138 and possible
147.09 ppmpresence
suggestedof a that
carboxylic acid unit
E3 should haveinan agreement with
O‐substituted
the information
phenyl fromThe
ring system. thecarbons
HPLC-PDA and 173.98,
at 171.00, UPLC-MS. and The
207.02number of hydrogens
were also indicative present
of three
in E3 wascarbon
carbonyl 24 in total.
units andTheacarbon
possibleand protonofsignals
presence of E3 acid
a carboxylic as well
unitas
in the multiplicity
agreement with
of
thethese signals are
information from summarized
the HPLC‐PDA in Table
and2.UPLC‐MS.
To determine the protonated
The number carbonspresent
of hydrogens in E3,
two-dimensional experiments were conducted. Among these experiments were the HSQC
and the DEPT experiments.
Molecules 2023, 28, 899 6 of 22

Table 2. The carbon-12 and proton (H-1) signals for E3.

Position C-δ (ppm) H-δ (ppm), J (Hz)

1 60.16 4.13 (q, H, J = 7.2)
2 39.89 2.02 (dd, 2H, J = 11.8, 7.0),
3 28.96 1.63 (m, H)
3α 34.42 2.33 (dd, 2H, J = 11.7, 7.2),
4 31.44 2.19 (m, H)
4α 139.03 Cq
5 147.09 Cq
6 114.08 Cq
7 119.09 Cq
8 124.47 7.38 (s, H)
8α 123.99 Cq
9 30.94 1.5 (dd, 2H, J = 13.3, 3.6)
10 24.81 1.30 (m, 2H)
10α 29.68 2.02 (m, H, J = 10.8, 7.6)
10 207.02 Cq
20 171.00 Cq
30 22.63 1.27 (s, 3H)
40 173.98 Cq
50 22.71 0.88 (s, 3H),
60 14.21 0.87 (s, 3H)
Cq = quaternary carbons.

From the DEPT experiments, one is able to tell the number of CH3 , CH2 , and CH atoms
from the quaternary carbons. Whereas CH3 and the CH usually appear as positive signals
and facing up, the CH2 carbons appeared as negative signals and facing down while the
quaternary carbons were not featured in this experiment. From the DEPT experiment of E3,
a saturated methyl group (CH3 ) was signaled at δ 14.21, 22.61, and 22.70 ppm. The methyl
groups at 22.70 and 22.61 were thought to be further downfield because they were placed
in a chemical environment that was electron-rich. As for the methylene (CH2 ) groups, four
of them were present in E3 at δ 24.81 (CH2 ), 28.96 (CH2 ), 29.6 (CH2 ), and 30.94 (CH2 ) ppm.
Whereas three saturated methylene groups appeared at δ 31.44 (CH), 34.42 (CH), and 39.89
(CH), there were two additional ones, δ 60.16 (O=CH) and 124.47 (Ph-CH) ppm, to amount
to a total of five methylene (CH) groups in E3.
As mentioned earlier on, carbon atoms at 207.02, 173.98, 171.00, 147.09, 139.03, 123.99,
119.09, and 114.08 ppm did not appear on the DEPT experiment and are indicative of
the quaternary carbons. Two-dimensional heteronuclear single quantum spectroscopy
(HSQC) is a NMR experiment that is useful in determining all the protonated carbons
present in an organic compound. In the compound E3, the experiment confirmed that
it comprised a total of twelve protonated carbons at 124.47:7.38, 60.16:4.13, 39.89:2.20,
34.42:2.30, 31.44:2.19, 30.94:2.06, 29.68:2.02, 28.96:1.63, 24.81:1.30, 22.70:1.29, 22.63:1.27,
and 14.21:0.87 and eight non-protonated carbons at 207.02, 173.98, 171.19, 147.68, 139.03,
123.99, 119.09, and 114.08 ppm, thus confirming the information obtained from the DEPT
experiment. At this juncture, it must be re-emphasized that E3 consisted of an O-substituted
phenyl, a carbonyl, a carboxylic, and a methyl as well as saturated hydrocarbon moieties.
For the structure of E3 to be fully elucidated, it was imperative to establish how the different
moieties of E3 were connected. The heteronuclear multiple quantum (HMBC) experiment
is very useful in this regard because it reveals both long and short coupling connectivity
of the different units that make a proposed structure. From the HMBC experiment on E3,
there were key diagnostic signals that indicated the connectivity of the different moieties
of E3. These signals occurred between methylic protons at the 0.80 ppm 1-J coupling
to the phenanthrene C-3. In like manner, the phenanthrene H-2 protons at δ 0.88 ppm
3-J were coupled to the carbonyl carbon of the carboxylic acid moiety at 207.02 ppm.
Another connection was that between the 6,7-diacetyl-5-hydroxyphenyl unit of E3 and the
phenanathree-1carboxylic acid fragment through a 3J coupling of H-4 proton at δ 1.29 to
 connectivity of the different units that make a proposed structure. From the HMBC exper‐
iment on E3, there were key diagnostic signals that indicated the connectivity of the dif‐
ferent moieties of E3. These signals occurred between methylic protons at the 0.80 ppm 1‐
J coupling to the phenanthrene C‐3. In like manner, the phenanthrene H‐2 protons at δ
Molecules 2023, 28, 899 0.88 ppm 3‐J were coupled to the carbonyl carbon of the carboxylic acid moiety at 207.02 7 of 22
ppm. Another connection was that between the 6,7‐diacetyl‐5‐hydroxyphenyl unit of E3
and the phenanathree‐1carboxylic acid fragment through a 3J coupling of H‐4 proton at δ
1.29
the to the 6,7‐diacetone‐5‐hydroxyphenyl
6,7-diacetone-5-hydroxyphenyl unit carbon
unit carbon at 147.09
at 147.09 ppm.
ppm. The The skeletal
skeletal struc‐
structure and
ture and the HMBC correlations of E3 are displayed in Figure 3A,B
the HMBC correlations of E3 are displayed in Figure 3A,B respectively.respectively.

Skeletalstructure
Figure3.3.Skeletal
Figure structure(A)
(A)and
andHMBC
HMBCcorrelations
correlations(B)
(B)ofofE3.
E3.

Collating all the chromatographic and spectrometric interpretations afforded the

Collating all the chromatographic and spectrometric interpretations afforded the elu‐
elucidation of E3 as 6,7-diacetyl-5-hydroxyphenyl-3-methylphenanthrene-1-carboxylic acid
cidation of E3 as 6,7‐diacetyl‐5‐hydroxyphenyl‐3‐methylphenanthrene‐1‐carboxylic acid
(Figure 4). In order to established if compounds of E3 type had been previously reported
(Figure 4). In order to established if compounds of E3 type had been previously reported
from plants or other natural products, a thorough literature search was carried out. The
from plants or other natural products, a thorough literature search was carried out. The
results of the search proved that analogues of E3 had indeed been identified in Moringa
results of the search proved that analogues of E3 had indeed been identified in Moringa
oleifera leaves in particular (Lin et al., 2019) [25]. The analogue of E3 identified in Indian
oleifera leaves in particular (Lin et al., 2019) [25]. The analogue of E3 identified in Indian
M. oleifera leaves was identified as ajugaside A. Even though E3 and ajugaside A have a
similar phenanthrene derivative, that is the 5-hydroxyl phenyl phenanthrene carboxylic
acid (5-hydroxylphenyl-di-cyclohexane), there was, however, modification between both
compounds. At C-1 of ajugaside, it appeared as though there would have been a bio-de-
glycosylation of its 1-methylacetyl glucose to a carboxylic acid group detected in E3.
 M. oleifera leaves was identified as ajugaside A. Even though E3 and ajugaside A have a
M. oleifera
similar leaves was derivative,
phenanthrene identified as ajugaside
that A. Even though
is the 5-hydroxyl phenylE3phenanthrene
and ajugasidecarboxylic
A have a
similar phenanthrene derivative, that is the 5‐hydroxyl phenyl phenanthrene
acid (5-hydroxylphenyl-di-cyclohexane), there was, however, modification between both carboxylic
acid (5‐hydroxylphenyl‐di‐cyclohexane),
compounds. At C-1 of ajugaside, it appeared thereaswas, however,
though modification
there would between
have been both
a bio-de-
Molecules 2023, 28, 899 8 of 22
compounds. At C‐1 of ajugaside, it appeared as though there would have been
glycosylation of its 1-methylacetyl glucose to a carboxylic acid group detected in E3. a bio‐de‐
glycosylation of its 1‐methylacetyl glucose to a carboxylic acid group detected in E3.

Figure 4. Structure of compound E3 (6,7-dipropanone-5-hydroxyphenyl-3-methylphenanthrene-1-

Figure4.4.Structure
Figure
carboxylic Structure
acid). ofofcompound
compoundE3
E3(6,7-dipropanone-5-hydroxyphenyl-3-methylphenanthrene-1-
(6,7‐dipropanone‐5‐hydroxyphenyl‐3‐methylphenanthrene‐1‐
carboxylic acid).
carboxylic acid).
An alternative biomodification E3 is a bio-carboxylation using the acetyl glucose and
An
An alternative
rearrangement biomodification
biomodification
and migration E3isisa abio‐carboxylation
E3
of the methyl bio-carboxylation
group from position using
using
C-1theinthe acetyl
acetyl
ajugaside glucose
glucose and
to posi-
and
tion rearrangement
rearrangement and migration
and migration
C-3 in E3. Whereas there was of
of the the methyl
methylmethyl
another group
group group from
from position position
at C-4 ofC‐1 C-1 in ajugaside
in ajugaside
ajugaside that was to posi‐to
ab-
position
tion in
sent C‐3 C-3
E3,inwein E3.
E3.propose Whereas
Whereas there
there was
a second was another methyl
another methyl group
bio-de-glycosylation group
at C-6atof at
C‐4 C-4 of ajugaside
of ajugaside
ajugaside that
that was
and further wasab‐
rear-
absent
sent in in
E3,E3,
we we propose
propose a a second
second bio-de-glycosylation
bio‐de‐glycosylation at at C-6
C‐6 of of ajugaside
ajugaside and and further
further rear‐
rangement of 2-methylpropanol to a diacetyl moiety at positions 2′ and 04′ in E3. In terms
rearrangement
rangement of 2-methylpropanol to a to a diacetyl moiety at positions 2 4′
and 40 inInE3. In
of biologicalofactivities
2‐methylpropanol
of both compounds, diacetyl
none moiety at positions
is available 2′ and
in the literature in
forE3. terms
ajugaside.
terms
of of biological
biological activitiesactivities
of both of both compounds,
compounds, none is none is available
available in the in the literature
literature for ajugaside. for
We herein report that E3 is characterized by antioxidant activity against the DPPH (1,1-
ajugaside.
We herein We reportherein
thatreport that E3 is characterized
E3 isradical
characterized by antioxidant by antioxidant
activity activity
against the against
DPPH the
diphenyl-2-picrylhydrazyl) with a DPPH free radical scavenging activity of IC(1,1‐
50 of
DPPH (1,1-diphenyl-2-picrylhydrazyl)
diphenyl‐2‐picrylhydrazyl) radical radical
with a DPPH with a
freeDPPH free
radical radical
scavenging scavenging activity
0.67 mg/mL, better than ascorbic acid with IC50 of 0.88 mg/mL, but less activity
than that of of
IC50 bu-of
of IC50
0.67 of 0.67better
mg/mL, mg/mL, than better thanacid
ascorbic ascorbic
with acid
IC with IC50 of 0.88but
50 of 0.88 mg/mL,
mg/mL,
less thanbutthat
lessofthan bu‐
tylated hydroxyl toluene with IC5O value 0.08 mg/mL in vitro. To the best of our
that of butylated
tylated hydroxyl hydroxyl toluene with IC5O0.08value 0.08 mg/mL in vitro. Tobest
the best of
knowledge, this is toluene
the firstwith
time IC value
an5Oajugaside-type mg/mL
compoundin vitro. To the
has been reported offrom
our
our knowledge,
knowledge, this
thisleaves is the
is the first timeananajugaside‐type
ajugaside-type compound has been reported from
Moringa oleifera offirst time
the South African ecotype. compound has been reported from
Moringa oleifera
Moringa oleifera leaves of the South Africanecotype.
leaves of the South African ecotype.
2.4. Charaterization
2.4. Charaterization and and Structural
Structural Elucidation
Elucidation of of Isolated
Isolated Compound
Compound Ra Ra
2.4. Charaterization and Structural Elucidation of Isolated Compound Ra
2.4.1. Structural Characterization and Elucidation of Compound Compound Ra Ra
2.4.1. Structural Characterization and Elucidation of Compound Ra
The importance of absorbance maxima ((ʎMAX) values in structural elucidation of com- com-
pounds
pounds Thehas
importance
has already been
already of absorbance
been emphasized.
emphasized. maxima
As for
As MAX) values in structural elucidation of com‐
for(ʎthe
the isolate Ra,
isolate Ra, it
it was
was 196
196 nm.nm. Other possible
pounds has already
absorbances of Ra been emphasized.
Ra appeared
appeared atat251
251andandAs
261 for
261 nm.theThe
nm. isolate
TheHPLC Ra,analysis
HPLC it was 196
analysisof nm.
Ra Other
resolved
of Ra possible
resolved as the
as
absorbances
the
majormajor
peak peakof Ra
at aat appeared
a retention
retention at
timetime251
of 3ofand 261
3 min.
min. nm. The
In order
In order HPLC analysis
to further
to further confirm
confirm of Ra
the the resolved
purity
purity of theasthe
of the
Ra,
major
Ra,
1.0 1.0 peak
mg/mL
mg/mL at aanalyzed
was retention
was analyzedtime using
using of 3 min.
UPLC-MS In
inorder
UPLC-MS intopositive
positive further
mode. The confirm
mode. theobtained
The
results purity of
results the Ra,
obtained
revealed
a1.0 mg/mL
revealed
single peakwasat analyzed
a single retention using
peak at retention
time of UPLC‐MS
time of
14.05 min, in whereas
14.05positive mode.
min, whereas The
theresults
the molecular (m/zobtained
molecular (m/z
ratio) ofrevealed
ratio)
Ra was of
a
Ra single
was peak
355.0740,at retention
implying time
that of
the14.05 min,
actual whereas
molecular the
mass molecular
of Ra
355.0740, implying that the actual molecular mass of Ra should be 356.0740 since the anal- should(m/z beratio) of
356.0740 Ra was
since
355.0740,
the
ysis analysisimplying
was conducted that
in a the
was conducted actual
in
positive molecular
a positive
mode. mode.
As formass of
As for
the Ra
theshould bepattern
356.0740
fragmentation
fragmentation of since
pattern ofthe
Ra, the Ra, anal‐
the
major
ysis
major was
daughter conducted
daughter
ion was ion266 in
was a
and positive
266
theand mode. As
thefragments
other for
other fragments the fragmentation
were 281, were
250,281, pattern
207,250,
191,207, of
147,191, Ra,
133,147, the
89, 133,major
89,
and 73.
daughter
and 73. ion was 266 and the other fragments were 281, 250, 207, 191, 147, 133, 89, and 73.
2.4.2. One- and Two-Dimensional NMR Analysis of Ra
2.4.2.
2.4.2. One-
One‐ and
and Two-Dimensional
Two‐DimensionalNMR NMRAnalysis
Analysisof ofRa
Ra
From the C-13 experiment on Ra, 24 carbon signals were observed, thus implying
From
that RaFrom the
consist C-13
the C‐13 experiment
of 24experiment
carbon atoms. on Ra,
on Ra, 24
These carbon
24 carbon signals
carbon peaks were
signalswere observed,
weresignaled
observed, at thus
thus
13 implying
C NMRimplying
(126
that Ra consist of 24 carbon atoms. These carbon peaks were signaled at 13 C NMR
that Ra 24 carbon atoms. These carbon peaks were signaled at C NMR (126 13

(126 MHz, CDCl3 ) 124.48, 123.96, 119.10, 114.03, 68.32, 68.04, 34.86, 34.52, 33.79, 31.91, 31.42,
30.21, 30.02, 29.67, 29.63, 29.49, 29.33, 29.14, 28.94, 28.55, 27.07, 22.66, 18.81, and 14.06 ppm.
Based on the C-13 NMR interpretation, the carbon atoms at δ 124.48, 123.96, 119.10, and
114.06 ppm suggested Ra consisted of two olefinic (double) bonds groups. In addition,
carbons at δ 68.32 and 68.04 were signals typical of two O-substituted carbons as an integral
part of Ra. Furthermore, the carbons at δ 34.86, 34.52, 33.79, and 31.91 indicated Ra was
Molecules 2023, 28, 899 9 of 22

likely to have four methylene (-CH) units while those at δ 31.42, 30.21, 30.02, 29.67, 29.63,
29.49, 29.33, 29.14, 28.94, 28.55, 27.07, and 22.66 could either be protonated methylene
(CH2 ) groups or quarternary carbons. Lastly, Ra was found to have two saturated methyl
(-CH3) groups that resonated at δ 18.81 and 14.03 ppm. The proton NMR experiment on Ra
revealed that the protons in Ra integrated to 36 protons in total. From the two-dimensional
HSQC experiment it was deduced that Ra was characterized by 20 protonated and four
quaternary carbons (Cq) at δ 119.90, 29.67, 30.21, and 123.96 ppm. Table 3 summarizes
the carbon-13, proton, multiplicity, and coupling constants as well as the protonated and
quarternary carbons characteristic of Ra. The diagnostic long J-coupling correlations from
the HMBC experiment interpretation revealed the different moieties that made the struc-
ture of Ra and are displayed in Figure 5B, while the proposed carbon skeleton of the
structure of Ra is represented by Figure 5A. From Figure 5B, the connection between the
five-cyclic, otherwise called pentacyclic, ring system that made up Ra is displayed. The
diagnostic 3J couplings include the H-12 proton at 7.52 and the C-11 and C-18 at 68.03 and
34.86 ppm. This indicates the linkage between lower (ring AB) and upper bicyclic (DE)
systems by ring C.

Table 3. The carbon-12 and proton (H-1) signals for Ra.

Position C-δ (ppm) H-δ (ppm), J (Hz)

1 22.66 1.36 (2H, dd, J = 10, 4)
2 29.33 1.14 (2H, td, J = 12, 4)
3 68.32 3.87 (H, m)
4 34.49 1.23 (2H, d, J = 8)
5 119.90 Cq
6 124.48 7.37 (H, m)
7 29.63 1.30 (2H, m)
8 29.67 Cq
9 31.42 2.02 (H, m)
10 30.21 Cq
11 68.03 3.59 (H, td, J = 12, 4)
12 114.03 7.51 (H, m)
13 123.96 Cq
14 31.91 2.06 (H, t, J= 8)
15 29.49 1.31 (2H, td, J = 8, 4)
16 28.94 1.15 (2H, dt, J = 16, 4)
17 34.52 1.30 (H, m)
18 34.86 2.00 (H, m)
19 29.14 1.99 (2H, m)
20 28.55 1.29 (2H, m)
21 27.07 1.13 (2H, tt, J = 16, 4)
22 30.02 1.26 (2H, td, J = 8, 4)
23 18.81 1.24 (3H, s)
24 14.04 0.93 (3H, s)
Cq = quaternary carbons.

Combining the one-dimensional with the two-dimensional experiments (COSY, DEPTH,

HSQC, and HMBC) as well as the UPLC-MS data of Ra, its structure (Figure 6), was eluci-
dated as hexademethylated 3β,11β-dihydroxyfriedelane. This compound is a pentacyclic
triterpene of the type isolated and reported from Maytenus robusta by Sousa and co-workers
in 2012 [26] and by Salimi et al. (2019) [27] from Indonesian Moringa oleifera Lam. leaves’
hexane extract.
Molecules 2023, 28, x FOR PEER REVIEW
Molecules 2023, 28, 899 10 of 22

Figure 5. Skeletal structure of (A) HMBC connectivity of the different moieties (B) of Ra isolated from
Figure 5. Skeletal
the hexane extract of structure of (A) HMBC connectivity of the different moieties (B) of Ra
M. oleifera leaves.
from the hexane extract of M. oleifera leaves.

Combining the one‐dimensional with the two‐dimensional experiments (

DEPTH, HSQC, and HMBC) as well as the UPLC‐MS data of Ra, its structure (Fig
was elucidated as hexademethylated 3β,11β‐dihydroxyfriedelane. This compou
pentacyclic triterpene of the type isolated and reported from Maytenus robusta by
and co‐workers in 2012 [26] and by Salimi et al. (2019) [27] from Indonesian Morin
era Lam. leaves’ hexane extract.
Molecules 2023,
Molecules 2023, 28,
28, 899
x FOR PEER REVIEW 11
11 of
of 22

6. Proposed
Figure 6. Proposedstructure
structureofofRaRaisolated from
isolated thethe
from hexane extract
hexane of M.
extract of oleifera leaves
M. oleifera and elucidated
leaves and eluci‐
dated
as as hexademethylated
hexademethylated 3β,11β‐dihydroxyfriedelane.
3β,11β-dihydroxyfriedelane.

Comparing
Comparing the the structure
structure of of Ra
Ra and
and its its analogue
analogue 3β,11β‐dihydroxyfriedelane
3β,11β-dihydroxyfriedelane revealed revealed
the
the major differences between the two structures. Whereas Ra consisted of two methyl
major differences between the two structures. Whereas Ra consisted of two methyl
groups, 3β,11β-dihydroxyfriedelanehad
groups, 3β,11β‐dihydroxyfriedelane hadsix sixadditional
additionalmethylmethylgroups.
groups.It It
is is also
also logical
logical to
to
saysay
thatthat
thethe
C‐5C-5andandC‐12 C-12
methyl methyl groups
groups in 3β,11β-dihydroxyfriedelane
in 3β,11β‐dihydroxyfriedelane couldcould
have have
been
been biosynthetically
biosynthetically converted
converted to the two to the two olefinic
olefinic groupsgroups
in Ra. With in Ra. With
such such similarities
similarities between
between these compounds, we propose that Ra may be
these compounds, we propose that Ra may be called hexademethylated 3β,11β‐dihy‐ called hexademethylated 3β,11β-
dihydroxyfriedelane. With a proposed molecular formula
droxyfriedelane. With a proposed molecular formula of C24H36O2 and a calculated molec‐ of C 24 H 36 O 2 and a calculated
molecular
ular mass of mass of 356.27,
356.27, the high-resolution
the high‐resolution mass of mass of 356.0740
356.0740 obtained obtained from UPLC-MS
from UPLC‐MS analy‐
analysis of Ra hereby confirms the proposed structure.
sis of Ra hereby confirms the proposed structure. Furthermore, the fragmentation Furthermore, the fragmentation
patterns
patterns
of 281, 266,of 250,
281, 207,
266,191,
250,147,
207,133, 191,89,147,
and133, 89, and 73 corresponded
73 corresponded to (M‐C20H31O), to (M-C
(M‐C 20 H31OO),
17H30 2),
(M-C H30OO2), 17
(M‐C17 16H25 2 ),(M‐C
(M-C HH
1316 252O
21O
), 12
), 2(C (M-C H21
H19O132), (CO H),16(C
112 (CH
), 12 HO
1019 2 ),C(C
17), 5H11
H16 ),
13O), and(C10
(CH4H9),O),
C5respec‐
H13 O),
and (C4 H9 O), respectively, as the daughter ions in the compound.
tively, as the daughter ions in the compound.
2.5. Qualitative Antimicrobial Assay
2.5. Qualitative Antimicrobial Assay
An in vitro qualitative antimicrobial assay was performed only for compounds Ra
and E3Anduein vitro qualitativeofantimicrobial
to insufficiency assay wasactivity
E1. The antimicrobial performed only was
screening for compounds
determined for Ra
and E3 due to insufficiency of E1. The antimicrobial activity screening was
the four extracts and pure compounds. The screening revealed that S. aureus, S. pyogenes, P. determined for
the four extracts
aeruginosa, E. coli,and
andpure compounds.
N. gonorrhoeae Theall
were screening
resistantrevealed that S. aureus,
to the extracts. OjiakoS. pyogenes,
(2014) [28]
P. aeruginosa,
and Okorondu E.etcoli,
al. and N. [29]
(2013) gonorrhoeae
previouslywerefound
all resistant
that M.to the extracts.
oleifera Ojiako
leaf extracts (2014)
were [28]
potent
and Okorondu et al. (2013) [29] previously found that M. oleifera leaf extracts
against E. coli and S. aureus when extracted with both the non-polar solvent hexane and the were potent
against
polar E. coliethanol.
solvent and S. aureus when
However, noextracted with both
action against the non‐polar
the organisms solvent
was found inhexane and
the current
the polar solvent ethanol. However, no action against the organisms
investigation. This could be due to a variety of reasons, including genetic background and was found in the
current
the investigation.
concentration of theThis could
extract be due
utilized. Into a variety
addition, of reasons,
Semenya et al.including
(2020) [30]genetic
conductedback‐ a
groundinvestigation
similar and the concentration of the extract
that corroborated utilized.ofInthis
the findings addition, Semenya
study. The et al.of(2020)
presence [30]
bioactive
conducted metabolites
secondary a similar investigation that corroborated
on the developed chromatograms the findings of this by
was detected study. The pres‐
the thin-layer
ence of bioactive secondary metabolites on the developed chromatograms
chromatography (TLC) agar overlay bioautography assay. Contamination is a disadvantage was detected
by the thin‐layer chromatography (TLC) agar overlay bioautography
of antimicrobial secondary metabolite detection. The agar overlay TLC–bioautography assay. Contamina‐
tion isisathe
assay disadvantage
most dependable,of antimicrobial secondary
cost-effective, simple,metabolite
and sensitive detection. The agarwith
assay available, overlay
the
TLC–bioautography
added assay antimicrobial
benefit of detecting is the most dependable,
metabolites cost‐effective, simple,that
in microbial extracts andare sensitive
viable
assay available,
against bacteria with the added
and fungi. The benefit
coloredofbackground
detecting antimicrobial
of formazanmetabolites
is producedininmicrobial
the agar
extracts TLC–bioautography
overlay that are viable againstdue bacteria
to theand fungi. The colored
dehydrogenase background
activity of formazan
of microorganisms thatis
producedvital
converts in the
dyes agar
intooverlay TLC–bioautography
a chromogenic product by the due to the dehydrogenase
reduction activity
process. The extracts of
and
microorganisms
isolated compounds thathad
converts vital dyes
no activity into a chromogenic
and therefore the plates hadproduct by the
a purple reduction
color pro‐
zone which
cess. Thethat
denoted extracts
thereandwasisolated compounds
no inhibition of test had no activity
pathogens [31]. and therefore the plates had a
purple color zone which denoted that there was no inhibition of test pathogens [31].

2.6. Qualitative Antioxidant Evaluation

Molecules 2023, 28, 899 12 of 22
Molecules 2023, 28, x FOR PEER REVIEW 12 of 22

2.6. Qualitative Antioxidant Evaluation

M. oleifera leaf extracts were tested against the stable DPPH (2,2‐diphenyl‐1‐picryl‐
M. oleifera leaf extracts were tested against the stable DPPH (2,2-diphenyl-1-picryl-
hydrazyl‐hydrate) free‐radical technique to assess antioxidant activity. In these investiga‐
hydrazyl-hydrate) free-radical technique to assess antioxidant activity. In these investiga-
tions, the ability to scavenge DPPH radicals was assessed by the staining of the solution.
tions, the ability to scavenge DPPH radicals was assessed by the staining of the solution.
DPPH is a free radical that generates a violet solution in methanol and is stable at room
DPPH is a free radical that generates a violet solution in methanol and is stable at room tem-
temperature. When a free radical interacts with an antioxidant, it loses its free radical
perature. When a free radical interacts with an antioxidant, it loses its free radical property
property and turns light yellow because of the chain breaking. The extracts that exhibited
and turns light yellow because of the chain breaking. The extracts that exhibited yellow
yellow creamy bands against the purple background in the DPPH free radical scavenging
creamy bands against the purple background in the DPPH free radical scavenging capacity
capacity experiment by TLC were regarded as having antioxidant potential. Antioxidants
experiment by TLC were regarded as having antioxidant potential. Antioxidants showed
showed as yellow creamy bands on a light purple background because of this procedure
as yellow creamy bands on a light purple background because of this procedure [32,33].
[32,33]. The extracts of M. oleifera leaves (n‐hexane, dichloromethane (DCM), EtOAc, and
The extracts of M. oleifera leaves (n-hexane, dichloromethane (DCM), EtOAc, and EtOH)
EtOH) were spotted and developed using various solvent systems with different ratios,
were spotted and developed using various solvent systems with different ratios, including
including
n-hexane: n‐hexane:
DCM: EtOAc DCM: EtOAc
(6:2:0.5 w/v); (6:2:0.5 w/v);DCM:
n-hexane: n‐hexane:EtOAc DCM:
(6:2:1EtOAc (6:2:1 v/v/v);
v/v/v); n-hexane: n‐
DCM:
hexane: DCM: EtOAc (6:2:2 v/v/v); n‐hexane: DCM: EtOAc (6:2:3 v/v/v);
EtOAc (6:2:2 v/v/v); n-hexane: DCM: EtOAc (6:2:3 v/v/v); acetic acid: n-hexane (1:9 v/v) acetic acid: n‐hex‐
ane
and(1:9 v/v) and
n-hexane: n‐hexane:
DCM: EtOAcDCM: (6:2:0.8 EtOAc
v/v/v).(6:2:0.8 v/v/v). of
The purpose The purpose of experimenting
experimenting with different
with different solvent systems was to achieve better resolution
solvent systems was to achieve better resolution of the various antioxidant compoundsof the various antioxidant in
compounds in the four extracts. The dried developed plates
the four extracts. The dried developed plates were sprayed with 0.2% solution of were sprayed with 0.2% so‐
DPPH.
lution
Figureof7DPPH.
shows Figure 7 showsin
the difference thethedifference
numberin ofthe number of
substances substances
extracted from extracted
the leaves fromof
the leaves of M. oleifera and separated by TLC analysis to observe
M. oleifera and separated by TLC analysis to observe antioxidant compounds. The hexaneantioxidant compounds.
The hexane
extract had extract
as many hadasassixmany as six antioxidant
antioxidant bands at Rfbands at Rf of 0.01–0.75.
of 0.01–0.75. In addition, In addition,
the DCM
the DCM
extract extract displayed
displayed three antioxidant
three antioxidant bands at Rf bands at Rf of 0.58–0.75,
of 0.58–0.75, thus highlighting
thus highlighting it contained it
contained similar antioxidant
similar antioxidant compounds compounds to those
to those present present
in the hexanein the hexane
extract. Onextract.
the other Onhand,
the
other hand,EtOAc
the polar the polar EtOAc
extract extractone
revealed revealed
band one
withband with potential
potential antioxidant antioxidant
activity activity
at Rf of
at Rf of 0.78. However, based on the very slight intensity of the
0.78. However, based on the very slight intensity of the cream stain against the purple cream stain against the
purple background, one can say that the compound in the EtOAC
background, one can say that the compound in the EtOAC extract had minimal antioxidant extract had minimal
antioxidant activityto
activity compared compared to the DCM
the non-polar non‐polar
and DCM
hexane and hexane
extract. Asextract.
for theAs for the
EtOH EtOHa
extract,
extract, a single
single band at Rfband
value atof
Rf0.05
value of 0.05 strong
indicated indicated strong antioxidant
antioxidant potential.the
potential. Whereas Whereas
spot on
the
the spot
EtOH onappeared
the EtOHasappeared
a dot plot,asdue a dot plot,mobile
to the due tophase
the mobile phase Hex:DCM:EtOAc
Hex:DCM:EtOAc (6:2:0.8 v/v/v)
(6:2:0.8
used forv/v/v) used for the
the analysis, we analysis,
propose that we propose
the bandsthat the bands
could appearcould
moreappear
if a moremore if a more
solvent was
solvent
used aswasthe used
mobile as phase.
the mobile
Evenphase.
though Even
the though the dichloromethane
dichloromethane extractthe
extract indicated indicated
highest
the highest
quantity of quantity
antioxidantof antioxidant
constituentsconstituents fromleaves,
from M. oleifera M. oleifera
herballeaves, herbalofdecoction
decoction the plant
prepared
of the plant with a consumable
prepared alcohol such
with a consumable as ethanol
alcohol such should furnish
as ethanol shouldthefurnish
body ofthe thebody
user
with
of theantioxidants.
user with antioxidants.

Figure
Figure7.7.Developed
DevelopedTLC TLCplates of of
plates hexane, dichloromethane
hexane, dichloromethaneextracts (A),(A),
extracts and and
ethylethyl
acetate and eth‐
acetate and
anol extracts (B). Plates were stained with 10% DPPH solution and visualized under visible
ethanol extracts (B). Plates were stained with 10% DPPH solution and visualized under visible light.
light.
Molecules 2023,28,
Molecules2023, 28,899
x FOR PEER REVIEW 13 13
of of
2222

2.7.
2.7. Quantitative Antioxidant
Antioxidant
2.7.1. DPPH Free Radical Scavenging
2.7.1. DPPH Free Radical Scavenging Assay
Assay
The
The reaction between the
reaction between the DPPH
DPPH andand the
the plant
plantextracts
extractsororisolated
isolatedcompounds
compoundswas was
tested
tested in 96‐well plates. Yellow coloration of the solutions in the plates (Figure 8) was anan
in 96-well plates. Yellow coloration of the solutions in the plates (Figure 8) was
indication
indication ofof positive good radical
positive good radical scavenging
scavengingpotential
potentialofofthe
theplant
plantextracts
extractsand
andisolated
isolated
compounds E3 and Ra. DPPH is a stable free radical that accepts an electron
compounds E3 and Ra. DPPH is a stable free radical that accepts an electron or a hydrogenor a hydrogen
radical
radical to
to form
form aa stable diamagnetic molecule
stable diamagnetic moleculethatthatisiswidely
widelyusedusedininresearch
researchononradical
radical
scavenging
scavenging activity. In the
activity. In the DPPH
DPPH radical
radicalscavenging
scavengingassay,
assay,antioxidants
antioxidantsreact
reactwith
withDPPH
DPPH
(deep
(deep violet
violetcolor)
color)totoproduce
produceyellow
yellowcolored
coloredα,α, α‐diphenyl‐β‐picrylhydrazine.
α-diphenyl-β-picryl hydrazine.The
The degree
de‐
of discoloration
gree indicates
of discoloration strong
indicates ability
strong of the
ability of extract or isolated
the extract compound
or isolated compound to scavenge
to scav‐
free
engeradicals [34]. [34].
free radicals

Figure 8.
Figure 8. DPPH
DPPH radical
radical scavenging
scavenging activity
activity of
of M.
M. oleifera
oleiferaextracts
extractsand
andpure
purecompounds.
compounds.Data
Dataare
are
presented as the percentage of DPPH radical scavenging. Each value is expressed as mean ± stand‐
presented as the percentage of DPPH radical scavenging. Each value is expressed as mean ± standard
ard deviation (n = 3).
deviation (n = 3).

It is
It is of
of paramount
paramount importance
importance to to know
know which
whichextracts
extractsor orcompounds
compoundsfrom fromextracts
extracts
have the best free radical scavenging activity, both nutritionally and
have the best free radical scavenging activity, both nutritionally and clinically. Furthermore,clinically. Further‐
more,
the the concentration
concentration of aextract
of a plant plant extract or phytochemical(s)
or phytochemical(s) required required
to inhibitto inhibit or scav‐
or scavenge 50%
enge 50% of the free radical in vitro is a useful tool when developing
of the free radical in vitro is a useful tool when developing herbal medicines. Figure 8 de- herbal medicines.
Figure
picts M.8oleifera
depictsLam’s
M. oleifera
DPPH Lam’s DPPH
radical radical scavenging
scavenging activity. The activity.
extractsThe andextracts and pure
pure compounds
compounds were able to neutralize the DPPH free radicals by donating
were able to neutralize the DPPH free radicals by donating hydrogen to a specific extent. hydrogen to a
specific extent. As can be seen in Figure 8, the ethanol extract revealed
As can be seen in Figure 8, the ethanol extract revealed an optimal antioxidant potential an optimal antiox‐
idant
at potential at aofconcentration
a concentration approximately of approximately
0.30 mg/mL followed0.30 mg/mL followed
by that by that ofRa
of compounds com‐
and
pounds Ra and E3 and lastly, the dichloromethane extract. Because
E3 and lastly, the dichloromethane extract. Because it contains more phenolic compounds, it contains more phe‐
nolic compounds,
ethanol inhibits free ethanol
radicalsinhibits
morefree radicalsthan
effectively moren-hexane.
effectivelyThe than n‐hexane.DPPH
decreasing The de‐ ab-
creasing DPPH absorbance of the test samples and controls were translated
sorbance of the test samples and controls were translated to percentage DPPH free radical to percentage
DPPH free radical
scavenging (% DPPH scavenging (% DPPH
antioxidant), as shownantioxidant),
in Figureas shown Using
8 above. in Figure 8 above.analysis,
regression Using
regression analysis, the IC 50 values (Table 1) for test and control samples were calculated.
the IC50 values (Table 1) for test and control samples were calculated. Compound Ra and
Compound
ethanol Ra andhigh
exhibited ethanol exhibitedactivity
antioxidant high antioxidant activity at
at concentrations asconcentrations
low as ≈0.28 mg/mL as low asin
ൎ0.28
comparison with n-hexane extract, compound E3, ascorbic acid, and butylatedand
mg/mL in comparison with n‐hexane extract, compound E3, ascorbic acid, bu‐
hydroxy
tylated hydroxy toluene standards. The radical scavenging activity of
toluene standards. The radical scavenging activity of almost all M. oleifera plant extracts almost all M. oleifera
plant extracts
against DPPH against DPPH at
was observed was observed
0.28 mg/mL;athowever,
0.28 mg/mL; however,
the highest the highest
activity activityat
was observed
wassame
the observed at the same
concentration forconcentration
ascorbic acidfor andascorbic
BHT with acidaand
lowBHTIC50 with
valuea oflow IC50mg/mL,
0.08 value
of 0.08 mg/mL, and compound Ra and ethanol with a low IC 50 of 0.4 mg/mL, respectively.
and compound Ra and ethanol with a low IC50 of 0.4 mg/mL, respectively. As illustrated
AsFigure
in illustrated in Figure
8, DPPH 8, DPPHincreased
scavenging scavenging in aincreased in a concentration‐dependent
concentration-dependent manner. man‐
ner.
Molecules 2023, 28, x FOR PEER REVIEW 14 of 22
Molecules 2023, 28, 899 14 of 22

2.7.2. Hydrogen
2.7.2. Hydrogen Peroxide
Peroxide Free
FreeRadical
Radical Scavenging
Scavenging Assay
Assay
Hydrogen peroxide (H 2O2) can penetrate cellular membranes; as a result, H2O2 is ex‐
Hydrogen peroxide (H2 O2 ) can penetrate cellular membranes; as a result, H2 O2 is
tremely important
extremely importantin cellular metabolism.
in cellular metabolism. H2OH2 is not particularly reactive, but it can be
2 O2 is not particularly reactive, but it can be
hazardous
hazardous to cells when it produces hydroxyl radicals(OH
to cells when it produces hydroxyl radicals (OH)−in) in
− thethe
cell. In In
cell. thethe
presence of of
presence
oxygen, the lipid radical will launch a chain reaction, resulting in lipid
oxygen, the lipid radical will launch a chain reaction, resulting in lipid peroxide, which thenperoxide, which
then breaks
breaks downdown to malondialdehyde
to malondialdehyde aldehydes
aldehydes [31].[31]. In this
In this study,
study, the maximum
the maximum hy‐
hydrogen
drogen
peroxideperoxide scavenging
scavenging activityactivity
was found wasatfound at 0.28 concentration
0.28 mg/mL mg/mL concentrationwith 82%with 82%
scavenging
scavenging
activity foractivity for the
the ethanol ethanol
extract andextract and E3. Ra
E3. However, However,
indicated Raaindicated
relativelyabetter
relatively bet‐
scavenging
teractivity
scavenging activity at a concentration of 0.42 mg/mL at 89%, while
at a concentration of 0.42 mg/mL at 89%, while the hexane extract exhibited the the hexane extract
exhibited the least
least activity activity in agreement
in agreement with the
with the results resultsfor
obtained obtained
the DPPH for the DPPH
assay. Theassay. The
IC50 values
ICfor
50 values for the H O assay (Table 1) were also in agreement with
the H2 O2 assay (Table 1) were also in agreement with the trend in the free radical
2 2 the trend in the free
radical scavenging
scavenging activity
activity observed
observed in theintwo
theassays
two assays
(Figure(Figure
9). 9).

Figure 9. Scavenging activity (%) of H2 O2 of M. oleifera extracts and pure compounds. Data are
Figure 9. Scavenging activity (%) of H2O2 of M. oleifera extracts and pure compounds. Data are pre‐
presented as the percentage of H2 O2 radical scavenging. Each value is expressed as mean ± standard
sented as the percentage of H2O2 radical scavenging. Each value is expressed as mean ± standard
deviation (n = 3).
deviation (n = 3).
2.7.3. Ferric Reducing Power Assay
2.7.3. Ferric Reducing Power Assay
The reducing power activity of the extracts was determined by their capacity to
The reducing
contribute power
electrons activitythe
to enable of reduction
the extractsof was
ferricdetermined
ions (Fe3+) by their capacity
to ferrous ions (Feto2+con‐
). The
tribute electrons to enable the reduction of ferric ions (Fe 3+) to ferrous ions (Fe2+). The ab‐
absorbance at 700 nm was determined after different quantities of sample extracts were
sorbance
chargedatwith
700 Fe
nm 3+ was determined
solutions. after different
This absorbance quantities
reflects of sample
the quantity extracts
of Fe were
2+ in solution;
charged with Fe3+ solutions. This absorbance reflects the quantity of Fe2+ in 2+
therefore, the greater the absorbance, the higher the concentration of Fe and the capacity solution; there‐
fore, theanalyte
of the greatertothe absorbance,
donate thei.e.,
electrons; higher the concentration
the higher of Fe2+ and
the extract’s reducing the capacity
power. of
The stronger
thethe
analyte to donate
antioxidant electrons;
activity, i.e., the
the greater thehigher the extract’s
reducing power. The reducing
reducingpower.
power Theofstronger
M. oleifera
theleaves
antioxidant
extractsactivity,
and purethecomponents
greater the reducing power.
are displayed in The
Figure reducing
10. power of M. oleifera
leaves extracts and pure components are displayed in Figure 10.
As evident in Figure 10, the antioxidant activity of all the extracted and isolated
compounds peaked at the same concentration range of 0.28 mg/mL, like that in the DPPH
and free H2 O2 assays. However, a slight deviation was observed for the hexane extract that
matched that of the other extracts and isolated compounds. In addition, all the extracts
and isolated compounds performed better than the ascorbic acid and BHT, which were
employed as reference medicines. Overall, our findings indicate that M. oleifera leaves
extracts and isolated compounds have potential as a natural antioxidant, as previously
described [35]. As a result, the plant extracts may be effective in the treatment of oxidative
stress-related ailments such as atherosclerosis, chronic obstructive pulmonary disease,
Alzheimer’s disease, and cancer [36].
Molecules 2023,28,
Molecules2023, 28,899
x FOR PEER REVIEW 15 15
of of
2222

Figure 10.
Figure 10. %
% Reducing
Reducing power
power of
of M.
M.oleifera
oleiferaextracts
extractsand
andpure
purecompounds.
compounds.The Theresults
resultsare
arepresented
presented
as the percentage reducing power free radical scavenging activity. Each value is expressed
as the percentage reducing power free radical scavenging activity. Each value is expressed as meanas
± standard deviation (n = 3).
mean ± standard deviation (n = 3).

As evident
2.8. Statistical in Figure 10, the antioxidant activity of all the extracted and isolated com‐
Analysis
pounds peaked at
The IC50 results thepresented
same concentration rangestatistically
in Table 4 were of 0.28 mg/mL, like that
analyzed in the DPPH
to compare and
the signifi-
free H 2O2 assays. However, a slight deviation was observed for the hexane extract that
cance of the antioxidant assay methods. The results obtained indicated that IC50 values of
matched
the DPPHthat andofFRAP
the other extracts
assays and isolated
were significant compounds.
with a p-value ofIn0.03.
addition, all the extracts
and isolated compounds performed better than the ascorbic acid and BHT, which were
employed
Table asvalues
4. IC50 reference medicines.
(mg/mL) of M. Overall, our findings
oleifera extracts indicate
and pure that M.inoleifera
compounds DPPH leaves ex‐
scavenging,
tracts and isolated compounds have potential
hydrogen peroxide, and reducing power assays.
as a natural antioxidant, as previously de‐
scribed [35]. As a result, the plant extracts may be effective in the treatment of oxidative
stress‐related ailments such DPPH as atherosclerosis,
IC50 chronic
H2 O2 obstructive
IC50 pulmonary
FRAP IC disease,
50
Analyte
Alzheimer’s disease, and cancer (mg/mL)
[36]. (mg/mL) (mg/mL)
BHT 0.08 Nd 0.323
2.8. Statistical Analysis
Ascorbic acid 0.88 0.443 0.421
n-Hexane
The IC50 results presented 0.761 0.639 analyzed to compare
in Table 4 were statistically 0.211 the sig‐
nificance of the antioxidant assay methods. The results obtained indicated that0.249
Ethanol 0.435 0.541
IC50 values
Compound E3 0.671 0.559 0.208
of the DPPH and
Compound Ra
FRAP assays were
0.475
significant with a p‐value
0.689
of 0.03. 0.213
Nd = Not determined. Each value is expressed as mean ± standard deviation (n = 3).
Table 4. IC50 values (mg/mL) of M. oleifera extracts and pure compounds in DPPH scavenging, hy‐
drogen peroxide, and reducing power assays.
3. Discussion
TheAnalyte DPPH
plant leaves’ ethanol extract IC50 strong scavenging
showed H2O2 IC FRAP
of50DPPH at 65%, IC50
hydrogen
peroxide with a percentage inhibition (mg/mL) (mg/mL)
of 89%, and reducing power with a(mg/mL)
percentage
inhibition BHT
of more than 85%. In some 0.08 cases, the extracts showed
Nd the lowest 0.323
percentage
Ascorbic
inhibition of anyacid
experiment compared 0.88 to the standards (ascorbic
0.443 0.421 This
acid and BHT).
does notn‐Hexane
preclude the plant leaves0.761 0.639
from being used as an antioxidant 0.211
substitute because
activity exists,
Ethanolalbeit at a lower level0.435
than expected. Factors such
0.541as plant location0.249
or storage,
drying procedure,E3solvent polarity0.671
Compound (methanol instead of ethanol),
0.559 and the contribution
0.208 of
carbohydrates
Compound in the
Ra extracts could 0.475
have influenced the results. Ascorbic acid showed
0.689 0.213 more
activity
Nd = Notagainst DPPH
determined. Eachcompared to ethanol
value is expressed extract,
as mean which deviation
± standard has high(npolarity.
= 3). In general,
the plant leaves exhibited concentration-dependent but significant scavenging activity at
the least concentration of 0.28 mg/mL. The results for both DPPH and hydrogen peroxide
are motivating because traditional healers use water or alcohol to prepare decoctions from
leaves and other parts. The observation that the plant leaves’ ethanol extract exhibited
the best free radical activity agrees with a previous report by Nobossé and co-workers
Molecules 2023, 28, 899 16 of 22

(2018) [37]. This group also found that ethanolic extract had the highest DPPH scavenging
activity for Moringa leaves from Cameroon. The agreement between our results and those of
other researchers is beneficial to South Africans in the area of solvent of choice for the best
extraction of antioxidants from M. oleifera. Furthermore, it demonstrates that traditional
healers will extract more antioxidants using alcohol, which can scavenge both DPPH
and hydrogen peroxide. This plant has little potential to be the source of antibacterial
agents that could be used to manage bacterial infections. The isolated compounds E3
and Ra, now known as 6,7-diacetyl-5-hydroxyphenyl-3-methylphenanthrene-1-carboxylic
acid and hexademethylated 3β,11β-dihydroxyfriedelane, respectively, equally indicated
concentration-dependent antioxidant activity. This implies that these compounds could
serve as lead compounds that can be synthesized or form part of a library of compounds
that may be developed into antioxidant drugs that could replace those that have not been
as effective hitherto. The main limitation to this study was the low concentration of 1
mg/mL of extraction solution that was used to investigate the antimicrobial potentials of
the extracts. This we thought was the reason we could not confirm activity for the extracts
consistent with some other reports.

4. Materials and Methods

4.1. Sample Collection and Preparation
Moringa oleifera Lam leaves were collected from Ga-Mphahlele, South Africa, Limpopo
province using a convenience sampling method. The leaves were collected in bulk and
air-dried at room temperature, then ground into powder using an electrical grinder and
stored in the laboratory cupboard until used. All solvents used for the extractions were of
analytical grade (AR) and were purchased from Rochelle Chemicals, South Africa. Five
hundred grams (500 g) of the finely ground plant material was mixed with 2300 mL of
dichloromethane in 5000 mL Erlenmeyer flasks. The flasks were placed on an orbital shaker
and the flasks and contents were shaken for 18 h to allow for extraction. The supernatant
post-extraction was filtered using Whatman no. 1 filter paper into a round bottom flask and
the process was repeated two more times. The filtrates were combined and concentrated
using a Stuart rotary evaporator (RE400, Cole-Parmer Ltd. Stones, St15 OSA, UK), to afford
the dried dichloromethane extract. The same procedure was followed using the same plant
residues with 2300 mL of ethanol to give dried ethanol extract. The mass and percentage
yield of each extract was determined using standard protocols.

4.2. Isolation of Compounds from the Hexane Extract

The plant hexane extract was prepared for column chromatography purification by
dissolving 15 g of hexane extract in 100 mL of DCM. The solution was adsorbed with
30 g of dry silica using pestle and mortar and the solvent was allowed to evaporate at room
temperature under a stream of air for 20 min. While the mixture was drying, 80.619 g of
silica was mixed with ethyl acetate to form a homogenous slurry and stirred to eliminate
bubbles using a glass stirring rod. The slurry was poured into a sintered glass column
(C.C. Imenmman PTY, Johannesburg, South Africa) whose outlet narrowly opened to
allow for packing of the silica gel. The adsorbed hexane extract-dry silica mixture was
carefully loaded on the column gel bed. Cotton wool was placed onto the extract sample to
avoid splashing when adding the mobile phase. The column was eluted with n-hexane:
dichloromethane (6:2 v/v; 6: 2: 0.5 v/v/v and 6:2:1 v/v/v) to obtain a total of 530 fractions.
The different fractions were analyzed by TLC, and the compound spots were visualized
under the UV light at 254 and 365 nm and then derivatized with a combination of methanol
and sulfuric acid (9:1 v/v) to expose those compounds that could not be seen with the
naked eye. The fractions that had same retardation factor (Rf) value were bulked together,
leading to 26 (A–Z) fractions. These major fractions were dried in pre-weighed beakers and
their masses determined after air-drying in a fume hood.
Molecules 2023, 28, 899 17 of 22

4.2.1. Bulking of Fractions from Hexane Extract Column

Contents of test tubes (TT) 1–14 were combined to result in major fraction A, since the
compound bands on the TLC plate displayed similar Rf values. In like manner, contents
of test tubes 15–40 were combined to afford fraction B, 41–90 afforded fraction C. Other
fractions were pooled as follows respectively D (TT91-116), E (TT117-160), F (TT161-172),
G (TT173-286), H (TT287-340), I (TT341-263), J (TT364-367), K (TT368), L (TT369-379),
M (TT380-387), N (TT388-405), O (TT406-415), P (TT416-423), Q (TT424-437), R (TT438-439),
S (TT440-450), T (TT451-473), U (TT474-485), V (TT486-495), W (TT496-500), X (TT501-509),
Y (TT510-519), Z (TT520-530).

4.2.2. Re-Chromatography of Major Fraction A

Fraction A was re-chromatographed because its TLC profile was semi-pure with a
single but not compact spot. Silica gel 60 (160.02 g) was mixed with ethyl acetate using
a stirring rod, and loaded into a glass column. The solvent was allowed to flow out so
that the silica gel settled and formed a column bed. Then, 3.75 g of A was dissolved with
40 mL of DCM and loaded onto the gel in the column. The loaded column was eluted using
n-hexane: DCM (6: 1 v/v). Twenty fractions were collected and concentrated using a rotary
evaporator. These fractions were again analyzed using TLC and those that had similar
bands with identical Rf values were combined together. Detailed bulking involved the
combining of contents of test tubes 1 and 2 to give sub-fraction A1 ; contents of test tubes
3–16 were pooled together to yield sub-fraction fraction A2 . Lastly, contents of test tubes
17–20 were combined together to form sub-fraction fraction A3 . Since sub-fractions A1 and
A3 indicated good potential to contain a pure compound because they displayed a single
compact spot from TLC analysis; they were stored in the fridge for characterization. On the
other hand, sub-fraction A2 was stored safely for further purification because it lacked the
characteristics of sub-fraction A1 and A3 .

4.2.3. Re-Chromatography of Major Fraction A2

A glass column of was used for re-purification of sub-fraction A2 . The column was
prepared as earlier described. The sample was prepared by adsorbing 3.01 g of sub-fraction
A2 with 65.0 g of silica gel. The mixture was dried in room temperature for 15 min. The
dried sample was loaded carefully onto the silica gel slurry bed and eluted with pure
n-hexane. One hundred and six (106) fractions were collected, concentrated and analyzed
using TLC. These sub-fractions were bulked as follows: A2.1 (TT1-4), A2.2 (TT5-20), A2.3
(TT21-36), A2.4 (TT37-45), A2.5 (TT46-60), A2.6 (TT61-70), and A2.7 (TT71-106). Based on TLC
profile obtained, A2.2 was further re-purified to A2.2.1 to A2.2.7 and upon re-crystallization
of A2.2.1, Ra and Rb were realized with Ra indicating the best degree of purity.

4.3. Isolation of Compounds from the Ethanol Extract

The plant ethanol extract was prepared for column chromatography purification by
dissolving 7.01 g of ethanol extract in 100 mL of ethanol. The solution was adsorbed with
80.03 g of silica gel using a pestle and mortar and the solvent was allowed to evaporate
at room temperature under a stream of air for 25 min. While the mixture was drying,
silica gel (160.0 g) was mixed with n-hexane to form a homogenous slurry and stirred to
eliminate air bubbles using a glass stirring rod. The slurry was poured into a sintered glass
column and the solvent was allowed to flow out of the column opening so that the gel could
settle. The dried silica gel extract mixture was carefully treated and applied on the column
gel bed. The mobile phase used for eluting the column consisted of hexane:DCM:EtOAc
(6:2:0.5; 6:2:0.8; 6:2:1.5 v/v/v), DCM:EtOAC (8:2; 7:3; 6:4; 1:1; 4:6; 3:7; 2:8 v/v), EtOAc, and
EtOAc:EtOH (8:2 v/v). A total of 880 fractions were collected and concentrated using a
rotary evaporator. These fractions were analyzed by TLC, and the compound spots were
visualized under the UV light at 254 and 365 nm and then derivatized with a combination
methanol and sulfuric acid (9:1 v/v) to enhance visualization. The fractions that had a
similar profile from TLC analysis were bulked together, leading to 57 major fractions from
Molecules 2023, 28, 899 18 of 22

the ethanol extract. These major fractions were dried in pre-weighed beakers and their
masses were determined as well.

4.3.1. Bulking of Fractions from Ethanol Extract Column

Similar to bulking method used during purification of the hexane extract, contents
of test tubes (TT 1-6) were combined to afford major fraction A. The other major fractions
obtained from the chromatography of the ethanol extract were B(TT 7-13), C(TT14-28),
D(TT29-70), E(TT71-80), F(TT81-90), G(TT91-100), H(TT101-110), I(TT111-130), J(1TT31-150),
K(TT151-160), L(TT161-166), M(TT167-175), N(TT176-185), O(TT186-200), P(TT201-214),
Q(TT215-228), R(TT229-242), S(TT244-252), T(TT253-260), U(TT261-280), V(TT281-380),
W(TT381-412), X(TT413-436), Y(TT437-449), Z(TT450-460), AA(TT461-470), BB(TT471-490),
CC(TT491-510), DD(TT511-550), EE(TT551-580), FF(TT581-590), GG(TT591-600), HH(TT601-
610), II(TT611-616), JJ(TT617-620), KK(TT621-628), LL(TT629-632), MM(TT633-636),
NN(TT637-650), OO(TT651-672), PP(TT673-680), QQ(TT681-685), RR(TT686-688), SS(TT689-
720), TT(TT721-754), UU(TT755-788), VV(TT789-810), WWTT(811-812), XX(TT813-818),
YY(TT819-840), ZZ(TT841-848), AAA(TT849-860), BBB(TT861-865), CCC(TT866-870),
DDD(TT871-875), and EEE(TT876-880). The test tube portions were bulked in a round
bottom flask and concentrated under pressure to afford a dry mass labeled sub-fraction
E1(S-U), E2(V-VW), E3(A–R), E4(MM), E5(NN), and E6(OO-EEE). Whereas most of the
sub-fractions revealed more than a spot on TLC analyses and were discarded, sub-fractions
E1 and E3 had single compact spots, thus indicating good purity compounds. The two
compounds were further characterized to examine their purity.

4.3.2. Bacterial Culture and Maintenance

The pathogens used were Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC
10536), Pseudomonas aeruginosa (ATCC 9721), Neisseria gonorrhoeae (ATCC 49981), and Strep-
tococcus pyogenes (ATCC 19615), purchased from Sigma-Aldrich (Pretoria, South Africa) at
University of Pretoria. They were selected based on their pathogenicity, clinical relevance
and the literature and were used to evaluate antimicrobial activity of crude extracts and
proposed pure compounds isolated from M. oleifera Lam leaves’ hexane extract as well as
ethanol extract. Stock bacterial cultures were sub-cultured into freshly prepared Mueller–
Hinton agar (MHA) and incubated at 37 ◦ C for 18–24 h to produce fresh bacterial culture.
However, Neisseria gonorrhoeae (ATCC 49981) was incubated in a jar with carbon dioxide at
the same temperature. To keep the bacterial strains alive, glycerol stock cultures of each
organism were prepared and stored at 80 ◦ C until needed.

4.3.3. Preparation of Inoculum

Bacterial colonies were transferred to sterile Mueller–Hinton broth (MHB) which was
stored in the refrigerator before the experiment. This was performed to standardize the
overnight cultures by diluting with MHB until all bacteria had an absorbance (OD600
nm) of 0.08–0.1. The same protocol was applied throughout the experiments for culture
preparation.

4.4. TLC Bioautography

The antimicrobial screening of M. oleifera Lam extracts and isolates were tested using
direct bioautography evaluation on a TLC plate (10 × 20 cm, 0.25 mm thickness, silica
gel G 60 F254, Merck, Darmstadt, Germany). The extracts and isolated compounds were
spotted on ten plates (for five bacteria and executed in duplicate) at a concentration of
5 mg/mL and left to dry for five days at room temperature. The plates were developed with
chloroform: ethyl acetate: formic acid (4:3:1 v/v/v) and hexane: ethyl acetate (9.5:0.5 v/v)
solvent mixtures in a glass chamber for ethanol extracts and isolates and hexane extracts
and isolates, respectively. This process was carried out in a fume hood cabinet (Vivid Air,
manufacturer and suppliers of clean air equipment). The developed air-dried plates were
placed in sterile Petri dishes, and a 10 µL inoculum of all selected bacteria was poured
Molecules 2023, 28, 899 19 of 22

into every 5 mL of melted soft agar (composed of 1.3 g bacteriology agar, 2 g tryptone, 1 g
sodium chloride (NaCl), and 200 mL distilled water (dH2 O) and then distributed over the
plates. The plates were incubated at 32 ◦ C for 24 h after the nutrient agar had solidified.
Following that, the bioautograms were sprayed with a solution of 0.2 mg/mL INT. On the
TLC plate, inhibition zones appeared as distinct spots on a purple background.

4.4.1. In Vitro Qualitative and Quantitative Antioxidant Assay

A solution of 2,2-diphenyl-1-picryl-hydrazyl (DPPH) was used to qualitatively test
the antioxidant activity of each extract. Following the development of each plant extract
solution on TLC, which was allowed to dry, the plate was sprayed with 0.2% DPPH in
methanol solution. As for the quantitative antioxidant potentials of the extracts and isolated
compounds the underlisted protocols were followed.

4.4.2. DPPH Antioxidant Activity Assay

This was accomplished by modifying a method described by (Moyo et al., 2012) [38]
and (Olivier et al., 2017) [39]. To that end, non-polar n-hexane extract, polar ethanol extract,
and isolated compounds (compound E3 and Ra), were prepared in a range of concentrations
(0.2 to 1.0 mg/mL). In a test tube, 1.0 mL of the extract solution was mixed with 1.0 mL of
a DPPH solution containing a concentration of 0.2 mg/mL. The contents of the test tube
were vortexed to thoroughly mix them together before being placed in a dark cardboard for
30 min. An aliquot of 200 µL was transferred into a 96-well plate. Thereafter, the absorbance
of the various concentrations was measured spectrophotometrically at 517 nm using a
96-well microplate-reader spectrophotometer (SprectraMax® , Molecular Devices, San Jose,
CA, USA). As reference standards, the same concentrations of ascorbic acid and butylated
hydroxyl toluene (BHT) were used. The extracts’ percentage radical scavenging activity
was calculated using the equation below.

Acontrol − Asample
% DPPH radical scavenging activity = × 100
Acontrol

where Asample = absorbance of the sample, Acontrol = absorbance of the negative control.

4.4.3. Hydrogen Peroxide Activity Assay

The method described by Olivier et al. (2017) [25] was followed with minor modifica-
tions to assess the hydrogen peroxide (H2 O2 ) scavenging potential of M. oleifera extracts
and isolated compounds. Different concentrations (0.2 to 1.0 mg/mL) of the extracts and
isolated compounds were prepared and 1 mL of each was transferred into a test tube. A
volume of 2 mL hydrogen peroxide (20 mM) prepared in a phosphate buffer saline (pH
7.2) was mixed with 1.0 mL of each of the extracts and isolated compounds from the stock
solutions. The reaction was thoroughly mixed with a vortex at 3000 rpm and incubated for
10 min at room temperature. An amount of 200 µL of each mixture was transferred into a
96-well microtiter plate and the absorbance was measured at 560 nm with a spectropho-
tometer. As positive control standards, BHT and ascorbic acid were used. The extracts’
ability to scavenge H2 O2 was calculated using the equation below.

A0 − As
H2 O2 scavenging activity % = × 100
A0

where A0 = absorbance of negative control and AS = absorbance of sample.

4.4.4. Reducing Power Activity Assay

The method used in this study was adapted from (Moyo et al., 2012) [38] with minor
modifications. The various extracts and compounds were redissolved in the solvents from
which they were extracted. Following that, various concentrations ranging from 0.2 to
1.0 mg/mL were prepared. In a test tube, 2.5 mL of 0.2 M potassium phosphate buffer
Molecules 2023, 28, 899 20 of 22

(pH 7.2) and 2.5 mL of 1% (w/v) potassium ferricyanide (K3Fe(CN)6) were mixed by means
of vortexing at 3000 rpm. After mixing the contents in the test tube, they were incubated
for 20 min at 50 ◦ C. Following that, 2.5 mL trichloroacetic acid (TCA) (10% w/v) was added
to the mixture and centrifuged for 10 min at 3000 rpm. An amount of 2.5 mL of the solu-
tion’s upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of ferric chloride
(FeCl3 ) (0.1% w/v). The absorbance of the mixture was measured using a spectropho-
tometer at 700 nm against blank. The procedure was repeated for the reference standards
ascorbic acid and BHT. The extracts’ percentage reducing power was calculated using the
following equation:

A0 − As
Reducing power activity % = × 100
A0

where A0 = absorbance of negative control, AS = absorbance of sample

4.4.5. Statistical Analysis

A t-test statistical analysis was performed to compare the probability (p-value) of
significance between the three assays used to investigate the antioxidant potential of the
extracts and isolated compounds in Microsoft Excel® .

5. Conclusions
The study has successfully investigated and established the antioxidant and antimi-
crobial potential of South African Moringa oleifera Lam leaves extracts and three isolated
compounds. Even though the study found no antimicrobial activity against Staphylococcus
aureus (ATCC 25923), Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 9721),
Neisseria gonorrhoeae (ATCC 49981), and Streptococcus pyogenes (ATCC 49981) (ATCC 19615),
our results are among the very few to the best of our knowledge that will inform local
farmers and other stakeholders on the possibility of using South African-based Moringa
oleifera leave products that may mitigate oxidative stress and related diseases.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/molecules28020899/s1, Figures S1–S26. HPLC, UPLC-
MS, and NMR data of E1, E3, and Ra.
Author Contributions: Conceptualization, K.B.; methodology, M.K.S.; formal analysis, K.B., S.C. and
M.K.S.; investigation, M.K.S. and S.C.; resources, K.B. and S.C.; data curation, K.B. and S.C.; writing—
original draft preparation, K.B. and M.K.S.; writing—review and editing, K.B.; funding acquisition,
K.B. and S.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the South African National Research Foundation (NRF) and
Chemical Industries Education and Training Authority (CHIETA).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank the National Research Foundation (NRF) and Chemical
Industries Education and Training Authority (CHIETA).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not available.

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