molecules
Article
Ursane-Type Triterpenes, Phenolics and Phenolic Derivatives
from Globimetula braunii Leaf
Ayodeji Oluwabunmi Oriola 1, * , Adetunji Joseph Aladesanmi 2 , Thomas Oyebode Idowu 3 ,
Florence O. Akinwumi 4 , Efere Martins Obuotor 5 , Temilolu Idowu 6 and Adebola Omowunmi Oyedeji 1
1
2
3
4
5
6
*
Citation: Oriola, A.O.; Aladesanmi,
A.J.; Idowu, T.O.; Akinwumi, F.O.;
Obuotor, E.M.; Idowu, T.; Oyedeji,
A.O. Ursane-Type Triterpenes,
Phenolics and Phenolic Derivatives
from Globimetula braunii Leaf.
Molecules 2021, 26, 6528. https://
doi.org/10.3390/molecules26216528
Academic Editor: Guy P.P. Kamatou
Department of Chemical and Physical Sciences, Faculty of Natural Sciences, Walter Sisulu University,
Mthatha 5099, South Africa; aoyedeji@wsu.ac.za
Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife 220005, Nigeria;
jaladesa@yahoo.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife 220005,
Nigeria; thomasidowu2010@yahoo.com
Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife 220005, Nigeria;
fakinwumi@oauife.edu.ng
Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-Ife 220005, Nigeria;
efereo@yahoo.com
Department of Chemistry, Parkers Building, University of Manitoba, Winnipeg, MB R3T 2N2, Canada;
tidowu01@mail.ubc.ca
Correspondence: aoriola@wsu.ac.za; Tel.: +27-655934742
Abstract: Globimetula braunii is a hemi-parasitic plant used in African ethnomedicine for the management of microbial infections, rheumatic pain and tumors amongst others. We report the isolation
and characterization of eight compounds with their antioxidant and antimicrobial activities. The
air-dried powdered leaf was macerated in EtOH/H2 0 (4:1). The extract was solvent-partitioned
into n-hexane, EtOAc, n-BuOH and aqueous fractions. The fractions were screened for their antioxidant properties, using DPPH, FRAP, TAC and FIC assays. Antimicrobial analysis was performed
using the micro-broth dilution method. The active EtOAc fraction was purified for its putative
compounds on a repeated silica gel column chromatography monitored with TLC-bioautography.
The isolated compounds were characterized using spectroscopic methods of UV, FT-IR, NMR and
MS. Eight compounds (1–8) were isolated and characterized as 13,27-cycloursane (1), phyllanthone
(2), globraunone (3), three phenolics: methyl 3,5-dihydroxy-4-methoxybenzoate (4), methyl 3-methyl4-hydroxybenzoate (5) and guaiacol (6), as well as two phenol derivatives: 4-formaldehyde phenone
(7) and 6-methoxy-2H-inden-5-ol (8). The study identified 4 and 6 as natural antioxidant compounds
with potential as antimicrobial agents.
Received: 7 October 2021
Accepted: 25 October 2021
Published: 28 October 2021
Keywords: Globimetula braunii; Loranthaceae; ursane-type triterpenes; phenolics; antioxidant;
antimicrobial
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1. Introduction
Globimetula braunii (Engl.) Van Tiegh. (Family Loranthaceae) is a hemi-parasitic and
epiphytic plant that derives water and mineral nutrients from its hosts by means of a
specialized root system called “haustorium” [1]. It is commonly called “African Mistletoe”
and locally called “Afomo Onisano” in Southwest Nigeria [2]. The plant is mostly found on
dicot trees, such as Piliostigma thonningii, Leucena leucocephala and Theobroma cacao, where it
becomes bushy and woody, growing up to 5 ft in diameter until the host tree withers [3].
At maturity, it produces reddish to reddish brown inflorescence with yellow patches in the
form of a match sticks. It is widely distributed across tropical West African countries, such
as Nigeria, Ghana, Benin Republic and Cameroun [4].
G. braunii is implicated in the African ethnomedicine for the management of microbial
infections, wounds, cholera, hypertension, diabetes, rheumatism, ulcers and tumors [3,5,6].
Molecules 2021, 26, 6528. https://doi.org/10.3390/molecules26216528
https://www.mdpi.com/journal/molecules
Molecules 2021, 26, 6528
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Biological studies have shown the antioxidant, antimicrobial and cytotoxic potentials of
the leaf extract and its ethyl acetate fraction [7–9]. The hepatic, hematologic, anti-lipidemic,
oxytocic, anti-hyperglycemic and anti-cancer activities of the plant’s alcoholic, aqueous
and ethyl acetate extracts have also been reported [5,6,10–12] with a paucity of information
on its chemistry. Preliminary phytochemical reports showed that the plant contained
phenolics, terpenes, flavonoids and sterols, while compounds, such as globrauneine AF, lupeol, lupeol palmitate, β-sitosterol, friedelin, octanoic acid, lactones and flavonoids
(quercetin, quercitrin, catechin, rutin and avicularin) have been reported in the plant [13,14].
We report, for the first time, the bioactivity-guided isolation and characterization of
three ursane-type triterpenes, three phenolics and two phenol derivatives from the leaf of
G. braunii along with their antioxidant and antimicrobial activities.
2. Results and Discussion
2.1. Spectra Data
Compound 1 (21 mg): 13,27-cycloursane, isolated as white amorphous powder, m.p.
151–152 ◦ C; ESI-MS: [M]+ at m/z 410.0, consistent with the molecular formula C30 H50
[M − C23 H29 ]+ at m/z 105.3; 1 H-NMR (300 MHz, CDCl3 ) δ ppm: 0.75 (3H, s, H-28), 0.89
(3H, d, J = 6.0 Hz, H-30), 0.98 (3H, s, H-26), 1.03 (3H, d, J = 3.0 Hz, H-29), 1.07 (3H, s, H-25),
1.20 (3H, s, H-24), 1.28 (3H, s, H-23); 13 C-NMR (75 MHz, CDCl3 ) δ ppm: 39.27 (C-1), 22.30
(C-2), 29.71 (C-3), 30.02 (C-4), 59.51 (C-5), 18.26 (C-6), 41.32 (C-7), 39.72 (C-8), 58.25 (C-9),
37.47 (C-10), 30.52 (C-11), 41.55 (C-12), 38.32 (C-13), 42.16 (C-14), 36.03 (C-15), 32.46 (C-16),
53.12 (C-17), 28.19 (C-18), 42.82 (C-19), 35.04 (C-20), 35.65 (C-21), 32.80 (C-22), 32.11 (C-23),
31.80 (C-24), 20.27 (C-25), 17.96 (C-26), 35.36 (C-27), 14.68 (C-28), 18.68 (C-29), 6.84 (C-30).
Compound 2 (20 mg): 13,27-cycloursan-3-one (Phyllanthone) isolated as white amorphous powder, m.p. 154–155 ◦ C; ESI-MS: [M]+ at m/z 424.3 consistent with the molecular
formula C30 H48 O, [M − CH2 ]+ at m/z 410.0, [M − C23 H29 ]+ at m/z 105.3; UV (CHCl3 )
λmax: 270.50 nm; IR (KBr) υmax cm−1 : 2948.3 (SP3 C-H), 2836.5 (SP3 C-H), 1681.0 (C=O
of ketone); 1 H-NMR (300 MHz, CDCl3 ) δ ppm: 0.75 (3H, s, H-28), 0.89 (3H, d, J = 6.0 Hz,
H-30), 0.98 (3H, s, H-26), 1.03 (3H, d, J = 3.0 Hz, H-29), 1.07 (3H, s, H-25), 1.20 (3H, s, H-24),
1.28 (3H, s, H-23); 13 C-NMR (75 MHz, CDCl3 ) δ ppm: 39.27 (C-1), 22.30 (C-2), 213.18 (C-3),
30.02 (C-4), 59.51 (C-5), 18.26 (C-6), 41.32 (C-7), 39.72 (C-8), 58.25 (C-9), 37.47 (C-10), 30.52
(C-11), 41.55 (C-12), 38.32 (C-13), 42.16 (C-14), 36.03 (C-15), 32.46 (C-16), 53.12 (C-17), 28.19
(C-18), 42.82 (C-19), 35.04 (C-20), 35.65 (C-21), 32.80 (C-22), 32.11 (C-23), 31.80 (C-24), 20.27
(C-25), 17.96 (C-26), 35.36 (C-27), 14.68 (C-28), 18.68 (C-29), 6.84 (C-30).
Compound 3 (28 mg): Globraunone, isolated as white amorphous powder, m.p.
220–222 ◦ C; ESI-MS: [M]+ at m/z 554.2, consistent with the molecular formula C37 H62 O3 ,
base peak M+ at m/z 554.2, [M + H]+ at m/z 555.0, [M − CH3 ]+ at m/z 539.1, [M − C7 H13 O3 ]+
at m/z 409.9, [M − C14 H25 O3 ] + at m/z 313.6; UV (CHCl3 ) λmax: 229.00 nm, 282.00 nm; IR
(KBr) υmax cm−1 : 3332.2 (OH, broad), 2974.4 (SP3 C-H), 1656.8 (C=O, weak), 1381.0–1274.7
(C–O). 1 H-NMR (300 MHz, CDCl3 ) δ ppm: 0.75 (3H, s, H-28), 0.89 (3H, d, J = 6.0 Hz, H-30),
0.97 (3H, s, H-26), 1.02 (3H, d, J = 3.0 Hz, H-29), 1.07 (3H, s, H-25), 1.20 (3H, d, J = 3.0 Hz,
H-7′ , of C-24), 1.28 (3H, s, H-23); 13 C-NMR (75 MHz, CDCl3 ) δ ppm: 39.27 (C-1), 22.30 (C-2),
213.19 (C-3), 30.02 (C-4), 59.51 (C-5), 18.26 (C-6), 41.32 (C-7), 39.72 (C-8), 58.25 (C-9), 37.47
(C-10), 30.52 (C-11), 41.55 (C-12), 38.32 (C-13), 42.16 (C-14), 36.03 (C-15), 32.46 (C-16), 53.12
(C-17), 28.19 (C-18), 42.82 (C-19), 35.04 (C-20), 35.65 (C-21), 32.80 (C-22), 32.11 (C-23), 20.27
(C-25), 17.96 (C-26), 35.36 (C-27), 14.68 (C-28), 18.68 (C-29), 6.84 (C-30). C-24 Side Chain:
41.74 (C-1′ ), 72.77 (C-2′ ), 30.66 (C-3′ ), 15.80 (C-4′ ), 35.57 (C-5′ ), 35.21 (C-6′ ), 31.80 (C-7′ ).
Compound 4 (1.22 g): methyl 3,5-dihydroxy-4-methoxybenzoate, isolated as ash
amorphous powder, m.p. 160–161 ◦ C; ESI-MS: m/z 198.0 [M]+ consistent with the molecular
formula C9 H10 O5 , loss of methoxy at m/z 167.3 [M − 31]+ , m/z 154.3 [M − 44]+ , loss of
methyl ethanoate at m/z 135.2 [M − 61]+ , loss of both methoxy and methyl ethanoate
at m/z 107.1 [M − 91]+ ; UV-Vis (MeOH) λmax: 210 nm, 232 nm, 253.0 nm; 1 H-NMR:
(300 MHz, MeOD) δ ppm: 3.85 (3H, s, H-1a), 3.91 (3H, d, J = 3.0 Hz, H-4a), 4.89 (1H, s,
H-3, H-5), 7.36 (1H, s, H-2, H-6); 13 C-NMR: (75 MHz, MeOD) δ ppm: 55.26 (C-4-methoxy),
Molecules 2021, 26, 6528
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59.72 (C-1-methoxy), 106.80 (C-3, C-5), 125.72 (C-1), 142.41 (C-4), 152.90 (C-2, C-6), 168.05
(C-1-ester).
Compound 5 (26 mg): methyl 3-methyl-4-hydroxybenzoate, isolated as yellow semisolid; ESI-MS: [M + H]+ at m/z 167.7, consistent with the molecular formula C9 H10 O3 , base
peak M+ at m/z 149.2, [M − 61]+ at m/z 105.2; UV-Vis (MeOH) λmax: 212.50 nm, 242.00
nm, 266.50 nm; IR (KBr) υmax cm−1 : 3385.0 (Phenolic OH, broad), 2939.0 (C-H stretch),
1715.0 (C=O, strong), 1586.0 (C=C aromatic), 1336.3–1123.8 (C–O stretch, strong); 1 H-NMR:
(300 MHz, MeOD) δ ppm: 2.05 (3H, s, Me), 3.90 (3H, s, -OCH3 ), 4.88 (1H, s, -OH), 6.83
(1H, d, 3 J = 9.0 Hz, H-5), 7.21 (1H, s, H-2), 7.46 (1H, d, J = 6.0 Hz, H-6); 13 C-NMR: (75 MHz,
MeOD) δ ppm: 20.35 (C-3, Me), 55.38 (C-1, -OCH3 ), 110.74 (C-2), 114.30 (C-5), 116.27 (C-6),
122.43 (C-1), 144.62 (C-3), 150.09 (C-4), 168.74 (C-1-carbonyl ester).
Compound 6 (29 mg): Guaiacol, isolated as a reddish-brown semisolid, ESI-MS: m/z
125.1 [M + H]+ consistent with the molecular formula C7 H8 O2 ; UV-Vis (MeOH) λmax:
214.5 nm, 241.5 nm, 271.0 nm; IR (KBr) υmax cm−1 : 3339.7 (Phenolic OH, strong), 1638.2
(C=C aromatic, medium), 1094.0 (C–O, weak); 1 H-NMR: (300 MHz, MeOD) δ ppm: 3.91
(3H, s, H-2a), 4.87 (1H, s, H-1a), 6.73 (1H, dd, 3 J = 9.0 Hz, H-3), 6.98 (1H, t, J = 3.0 Hz, H-4,
H-5), 7.10 (1H, t, J = 3.0 Hz, H-6); 13 C-NMR: (75 MHz, MeOD) δ ppm: 55.34 (C-6a), 102.52
(C-4), 108.17 (C-5), 114.83 (C-3), 129.39 (C-6), 144.55 (C-2), 147.65 (C-1).
Compound 7 (20 mg): 4-methyl-4-formaldehyde phenone, isolated as brown semisolid; ESI-MS: m/z 136.0 [M] + consistent with the molecular formula C8 H8 O2 ; m/z 119.2
[M − 17]+ , m/z 106.8 [M − 29]+ , m/z 104.3 [M − 32]+ ; UV-Vis (MeOH) λmax: 234.5 nm,
254.5 nm, 278.50 nm, 291.5 nm; IR (KBr) υmax cm−1 : 2926.0 (C-H stretch), 1716.4 (C=O),
1459.3 (C=C aromatic); 1 H-NMR: (300 MHz, MeOD) δ ppm: 1.81 (3H, s, H-1a), 7.21 (1H,
d, J = 3.0 Hz, H-2, H-6), 7.74 (1H, d, J = 3.0 Hz, H-3, H-5), 8.48 (1H, s, H-1b); 13 C-NMR:
(75 MHz, MeOD) δ ppm: 22.29 (C-4b), 54.45 (C-4), 125.56 (C-2, C6), 127.19 (C-3, C-5), 176.60
(C-4a), 199.17 (C-1).
Compound 8 (26 mg): 6-methoxy-2H-inden-5-ol, isolated as yellow semi-solid; ESIMS: m/z 162.0 [M]+ consistent with the molecular formula C10 H10 O2 ; loss of -OH at m/z
145.0 [M − 17]+, loss of -OCH3 at m/z 131.0 [M − 31]+, m/z 114.3 [M − 48]+ ; UV-Vis
(MeOH) λmax: 212 nm, 242 nm, 266 nm; IR (KBr) υmax cm−1 : 3367.6 (Phenolic OH), 2931.6
(SP3 − CH), 1586.0 (C=C aromatic, stretch), 1468.0 (C=C, stretch, 5-member ring), 1336.3
(C–O, of an alcohol), 1213.2 (C–O of an alkoxy); 1 H-NMR: (300 MHz, MeOD) δ ppm: 1.31
(2H, brs, H-1), 3.90 (3H, s, H-6), 4.88 (1H, s, H-5), 7.08 (1H, d, H-4b), 7.21 (1H, t, H-2a, H-2b),
7.46 (1H, dd, H-4a); 13 C-NMR: (75 MHz, MeOD) δ ppm: 29.35 (C-1), 55.24 (C-6′ ), 104.89
(C-2), 110.77 (C-9), 120.56 (C-3), 116.81 (C-8), 116.23 (C-4), 108.91 (C-7), 144.63 (C-6), 147.63
(C-5).
2.2. Structure Elucidation
The 13 C NMR spectra of 1 and 2 revealed C-30 compounds, while that of 3 showed
37 signals. The DEPT-135 experiment of 1 showed twelve methylene carbons, twelve
methyl and methine carbons and six quaternary carbons. That of 2 differs from 1 by having
eleven methylene carbons (one carbon less) attributed to the ketone substituent at δC
213.18 ppm. 1 H NMR spectra of the three compounds showed seven methyl protons at
δH 0.75–1.28 ppm, methylene at δH 1.50–2.32 ppm and methine at δH 2.50–3.50 ppm. The
methyl signals resonated as five singlets and two doublets, which is typical of an α-amyrin
(ursane-type) of triterpene.
The signal at δC 213.18 ppm on both the spectra of 2 and 3 confirmed the presence of a ketone. While the C=O attachment at the C-3 position was based on the
HMBC experiment. Upon consideration of the spectra of 1–3 and in comparison with
spectra data on similar compounds reported in the literature, they were identified as
13,27-cycloursane (1), 13,27-cycloursan-3-one previously identified as phyllanthone (2)
and hexadecahydro-8-hydroxy-9-(2-hydroxy-6-methylheptyl)-1,2,6a,6b,9,12a-hexamethyl6bHcyclopropa[q]picen-10(11H,12bH,15H)-one (3), named globraunone [15–17].
Molecules 2021, 26, 6528
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The 1 H NMR of 4 showed four signals, which were two aromatic methoxy at δH
3.85 and 3.91 ppm, hydroxy at δH 4.89 ppm and a singlet at δH 7.36 ppm indicating an
aromatic proton. The 13 C NMR showed nine signals, indicating a C9 compound. The
signal at δC 168.05 ppm indicated a carbonyl ester, two pairs of signals, each at 106.80 for
olefinic carbons (C-2/C-6) and 152.90 ppm for phenolic carbons (C-3/C-5), typical of an
AABB para-substitution pattern [18]. The IR spectrum further corroborated 4 as a hydroxy
benzoate with strong bands at 3367.6 cm−1 (OH) and 1586.0 cm−1 (R-O-C=O). Based on
the available spectra data and in comparison, with literature data, 4 was identified as
methyl-3,5-dihydroxy-4-methoxybenzoate, previously isolated from Sacoglottis gabonensis
stem bark [19].
The 1 H NMR of 5 showed three aromatic protons at δH 6.83, 7.21 and 7.46 ppm: a
phenolic OH at δH 4.88 ppm and aromatic methoxy at δH 3.90 ppm. A de-shielded methyl
group at δH 2.01 ppm confirmed that it is directly attached to an aromatic ring. There were
nine carbon signals on the 13 C NMR spectrum. The most de-shielded signal resonated
at δC 168.74. IR spectrum showed having a broad phenolic OH band at 3385.0 cm−1 , a
strong carbonyl C=O band at 1715.0 cm−1 , C=C aromatic band at 1586.0 cm−1 and C–O
stretching band at 1336.3–1123.8 cm−1 . UV absorption at 267 nm showed the excitation of
a benzoate skeleton (tabulated as 268 nm). The spectra data of 5 was compared with that of
methyl-4-hydroxybenzoate, a bacterial inhibitor previously reported in the bark of Tsuga
dumosa [20,21], and it was characterized as methyl-3-methyl-4-hydroxybenzoate.
The 13 C NMR spectra of 6 showed seven signals. Based on the HSQC experiment, the
key functional groups identified include aromatic methoxy at δC 55.34 and δH 3.91 ppm,
four olefinic protons at δH 6.73, 6.98, 7.10 and 7.20 ppm, with carbon signals at δC 114.83,
108.17, 129.39 and 110.97 ppm, respectively. The NMR data of 6 agreed with that of guaiacol
reported by Kitanovski et al. [22].
1 H NMR of 7 showed a singlet signal at δ 8.48 representing an aldehyde carbonyl,
H
two olefinic protons at 7.21 and 7.74 ppm, typical of an A2 B2 para-substitution pattern
and a singlet at 1.82 ppm. The aldehydic (1716.4 cm−1 ) and olefinic (1459.3 cm−1 ) bands
were prominent on the IR spectrum, while the UV-Vis spectrum showed λmax of 254.5 nm,
indicative of a benzene nucleus. Based on the comparison of its spectra data with literature
data, 7 was identified as 4-methyl-4-formaldehyde phenone [18].
Compound 8 was showed ten signals on the 13 C NMR as a C-10, characterized as
δC 55.24 (methoxy), one methylene carbon at δC 29.35, four methine carbons at δC 104.89,
108.91, 110.77 and 116.31 and four quaternary carbons at δC 116.81, 120.56, 144.63 and
147.63 ppm, based on the DEPT135 experiment. HSQC experiment showed that the carbon
signals at δC 104.89 and 110.77 ppm were directly attached to the proton signal at δH 7.21,
while the carbon signals at δC 116.81 and 108.91 ppm were directly attached to the protons
at δH 7.46 and 7.08 ppm, respectively, thus, confirming an AABC ring system [23].
Three ursane-type triterpenes (1–3), three phenolics (4–6) and two phenolic derivatives
(7 and 8) were isolated and identified in our study of G. braunii living on Leucena leucocephala
(Fabaceae), its host, which marked the first report of these compounds in the plant and
the family Loranthaceae. Previous phytochemical studies on the plant have shown the
presence of tannins, phenolics, flavonoids, terpenoids and sterols [8,10], while compounds
reportedly isolated include lupeol-type triterpenes (globrauneine A-F, lupeol and lupeol
acetate), lactones and flavonoids (quercetin, quercitrin, rutin and avicularin), identified in
the G. braunii living on Piliostigma thonningi (Fabaceae). Perhaps, this new additions to the
repository of compounds in G. braunii might have occurred because of plant-host specificity,
which was reported to play a critical role in the quality and quantity of constituents elicited
by Mistletoes as well as its influence on their biological properties [4].
Structures of the isolated compounds 1–8 are presented in Figure 1.
Molecules 2021, 26, 6528
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30
29
13
30
25
30
29
27
29
10
3
13
25
26
27
25
28
26
14
10
3
10
8
OH
HO
3
5
O
23
24
1
24'
24'
24
2
O
O
CH3
O
O
3
O
CH3
OH
HO
H3C
4
6
23
28
O
OH
O
28
14
14
8
5
23
27
17
8
4
O
13
17
17
26
CH3
HO
H3C
O
OH
5
CH3
6
7
H3C
O
8
Figure 1. Structures of isolated compounds from the EtOAc fraction of G. braunii leaves.
2.3. Evaluation of the Biological Activities of G. braunii Leaves
TLC-bioautography was used throughout the study. This is a reliable and cost-effective
technique to isolate lead compounds by employing a suitable chromatographic process,
followed by a biological detection system [24]. The TLC-bioautography antioxidant (DPPH)
method was reported to demonstrate an interplay of hydrogen atom transfer (HAT) and
single electron transfer, which are important mechanisms in understanding the antioxidant
properties of natural products [25]. It was used in this study as a guide for rapid and easy
identification and isolation of the free radical scavenging compounds present in the leaf
extract of G. braunii. The result presented in Figure 2 showed that fractionation enhanced
the antioxidant property of the plant. The EtOAc fraction G, bleached the purple DPPH free
radical solution immediately, compared with the n-hexane and aqueous fractions, which
exhibited a bleaching effect after 5 and 20 min, respectively. The intensity of bleaching
(scavenging property) improved with further chromatographic separation.
Quantitative assessments of the antioxidant activity (AOX) of the plant by DPPH,
FRAP, TAC and FIC colorimetric assay methods are presented in Table 1. The results
showed both concentration-dependent and purification-enhanced increase in the AOX
of the plant. The EtOAc fraction exhibited the best AOX among the partition fractions
with significant (p < 0.05) IC50 values of 8.58 and 154.87 µM in the DPPH and FIC assays,
respectively.
Molecules 2021, 26, 6528
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Figure 2. Qualitative antioxidant activity of G. braunii leaves by TLC-bioautography against purple
DPPH radical solution. Key: G1–G6: first column bulk fractions, G3a–G3d: second column bulk
fractions, 3–8: isolated compounds.
Table 1. Antioxidant activities of G. braunii leaves.
Sample
DPPH
(µM)
FRAP
(mgAAE/g)
TAC
(mgAAE/g)
FIC
(µM)
EtOH extract
n-Hexane
EtOAc
n-BuOH
Aqueous
1
2
3
4
5
6
7
8
Positive control
31.21 ± 1.11 g
75.89 ± 5.05 i
8.58 ± 1.39 c, ***
16.06 ± 0.52 f
86.97 ± 24.74 j
>100 k
>100 k
61.53 ± 1.01 h
6.38 ± 0.48 b, ***
15.78 ± 0.41 e
0.86 ± 0.37 a, ***
>100 k
70.64 ± 2.90 i
11.38 ± 0.45 d
109.30 ± 0.76 c
172.88 ± 1.12 d
178.64 ± 2.04 f
175.38 ± 0.97 e
49.94 ± 18.23 b
<10 a
347.26 ± 1.43 g
651.77 ± 7.98 h
702.89 ± 3.09 i
752.76 ± 13.51 k
720.47 ± 10.08 j
764.09 ± 10.12 k
634.84 ± 20.31 h
-
178.15 ± 3.54 i
<10 a
485.81 ± 50.41 k
283.83 ± 23.01 j
<10 a
12.02 ± 1.37 b
43.91 ± 1.20 c
77.72 ± 0.39 d
115.23 ± 4.12 f
121.87 ± 2.73 g
161.57 ± 3.79 h
81.12 ± 2.22 e
115.76 ± 3.65 f
-
281.10 ± 12.09 e
>450 h
154.87 ± 6.54 b
298.79 ± 32.51 e
>450 h
>450 h
>450 h
≥450 h
410.64 ± 8.62 g
389.92 ± 4.76 f
199.63 ± 5.67 c
>450 h
255.53 ± 11.71 d
13.21 ± 2.56 a
n = 3, values presented as the mean ± SEM, *** significant (p < 0.0001) compared with positive control. Values
with different alphabets in superscript are significantly different at p < 0.05; isolated compounds (1–8).
The former was significantly (p < 0.05) better than L-ascorbic acid (AA) with an IC50
value of 11.38 µM. The power (FRAP) and capacity (TAC) of the EtOAc fraction as an
antioxidant were one-sixth and halved, respectively, when compared with AA. This implies
that the fraction was able to perform its antioxidant role by hydrogen atom transfer (HAT)
to the stable DPPH free radical, single electron transfer (SET) in the FRAP and TAC assays
and by metal (Fe2+ ) chelation as in the case of FIC assay [25,26]. These findings corroborate
the reported in vivo antioxidant activity of the EtOAc fraction in mice [10].
As presented in Table 1, compounds 1–3 had low AOX, while 4–8 were active. The
ketone group at C-3 position in phyllanthone 2 might have helped to confer one-third
fold FRAP activity, while globraunone 3 proved to be the most active among the terpenes
isolated partly due to the presence of ketone and hydroxyl groups. This finding is in
consonance with that of Baccouri and Rajhi [27] on the significance of hydroxyl, carbonyl
and olefine to the antioxidant activities of compounds as lead molecules for drug discovery.
Guaiacol 6 demonstrated the best AOX among the isolated compounds. Its AOX was
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12 times better as a hydrogen-atom-donor (HAT) than AA in the DPPH assay, while, in the
FRAP assay, it was 0.76-times as good as a single-electron-donor (SET) when compared
with AA.
This could be due to its pi electron-rich benzene ring and hydroxyl group, which can
exhibit both HAT and SET mechanisms of antioxidant action. HAT is a one-step reaction
by phenolic O-H to effect bond dissociation enthalpy (BDE). SET is a sequential two-step
reaction, which entails proton-loss followed by electron-transfer (SPLET), thus, leading to
proton affinity (PA) and electron transfer enthalpy (ETE). These mechanisms have been
reported to be responsible for the high antioxidant activities of many simple phenolics
including phenolic acids [28].
The micro-broth dilution method of microbial susceptibility testing was adopted in
the study. It is an objective, high-throughput, cost-effective and quantitative method. It
offers high reproducibility, fast generation of MICs, convenience of having pre-prepared
panels and the economy of reagents and space that occurs due to the miniaturization of the
test, which all make it suitable for the antimicrobial analysis of plant samples. It is also able
to assist in generating computerized reports if an automated panel reader is used [29,30].
The antimicrobial activity of the G. braunii as shown in Table 2 showed the EtOAc fraction
with the best activity among the fraction, based on its lowest MIC range (0.63–5.00 mg/mL)
and broad-spectrum activity against the test organisms, which justified our focus on the
EtOAc fraction. The tested microorganisms were not strongly susceptible to the terpenes
(1–3) isolated. Globraunone (3) was only inhibitory against B. subtilis at 2.50 mg/mL and
fairly against C. albicans at 5.00 mg/mL. This could be due to the ursanoid (α-amyrin),
carbonyl and hydroxyl moieties, all playing key roles in the inhibitory action 3 against C.
albicans. Similar natural compounds, such as ursolic acid and its derivatives, have been
reported to exhibit inhibitory actions against B. subtilis, MRSA, P. aeruginosa and C. albicans
at 0.10–0.25 mg/mL [31].
Table 2. Antimicrobial activities of G. braunii leaves.
Concentration (mg/mL)
Sample
Negative control
EtOH extract
n-Hexane
EtOAc
n-BuOH
Aqueous
1
2
3
4
5
6
7
8
Positive control
E. coli
P.
aeruginosa
MRSA
B. subtilis
C. albicans
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MFC
20.00
2.50
5.00
20.00
10.00
2.50
5.00
1.25
10.00
2.50
5.00
20.00
20.00
5.00
10.00
5.00
5.00
0.25
20.00
5.00
2.50
20.00
5.00
10.00
2.50
20.00
5.00
10.00
10.00
20.00
5.00
20.00
0.50
5.00
20.00
1.25
2.50
20.00
10.00
20.00
2.50
20.00
2.50
20.00
2.50
5.00
5.00
5.00
0.50
20.00
0.63
5.00
10.00
20.00
20.00
2.50
1.25
2.50
0.63
10.00
2.50
2.50
20.00
10.00
5.00
10.00
1.25
5.00
0.25
10.00
1.25
1.25
20.00
5.00
2.50
5.00
1.25
20.00
5.00
20.00
2.50
2.50
20.00
10.00
20.00
5.00
10.00
0.10
MIC—Minimum Inhibitory Concentration, MBC—Minimum Bactericidal Concentration, MFC—Minimum Fungicidal Concentration; Positive control—Ciprofloxacin (MBC) & Ketoconazole (MFC); isolated compounds (1–8);
negative control = 50% Aq. MeOH; No activity at >20 mg/mL (-).
On the other hand, the phenolic compounds (4–6) and their derivatives (7 and 8)
demonstrated remarkable antimicrobial properties. Guaiacol 6, a methoxyphenolexhibited the strongest inhibitory activities against B. subtilis (0.63 mg/mL) and C. albicans (1.25 mg/mL) among the compounds. This was closely followed by dihydroxy-4methoxybenzoate (4) with MIC range of 2.50 and 5.00 mg/mL. However, ciprofloxacin and
ketoconazole were 2.5- and 12.5- times better antimicrobial agents compared with guaiacol.
Based on the observed antimicrobial activities of the isolated compounds, ranking can be
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done as follows: Ketoconazole, Ciprofloxacin > Guaiacol 6 > 4 > 8 > 5 > 7 > Globraunone 3
> Phyllanthone 2 > 1 > 50% Aq. MeOH.
Phenolics and its derivatives are referred to as products of the phenylpropanoid
pathway, many of which possess significant biological properties. Generally, phenolics with
less complex structures (simple phenolics) have been shown to demonstrate remarkable
bactericidal and fungicidal activities. These include catechol, caffeic acid, resveratrol and
gallic acid amongst others. They all have the phenolic OH and/or other substituents,
such as ester, carboxylic, amine, amide and thiol, which have significant antimicrobial
properties [32]. Methyl-4-hydroxybenzoate from the bark of Tsuga dumosa is a known
bacterial inhibitor [20,21], while bulbiferate A and B from Microbulbifer spp. are phenolic
esters, structurally like compounds 4 and 5.
According to a report, bulbiferates inhibited the growth of E coli and methicillinsensitive Staphylococcus aureus (MSSA) at 0.20 mg/mL [33]. In the same vein, the antioxidant
and antimicrobial potentials of natural methoxyphenols, such as eugenol, capsaicin and
vanillin have been reported. They exhibited an IC50 range of 0.68–1.38 mg/mL against S.
aureus, 1.21 mg/mL (capsaicin) against P. aeruginosa and 2.70 mg/mL (eugenol) against E.
coli [34].
These leave more to be desired on the biological potentials of natural methoxyphenols
and hydroxybenzoates, especially as antioxidant and antimicrobial agents; thus, guaiacol
(O-methoxyphenol) (6) and methyl 3,5-dihydroxy-4-methoxybenzoate (4), which were
the considerably active antioxidant and antimicrobial compounds identified in this study,
could be candidate leads in this respect.
3. Materials and Methods
3.1. Plant Material
Globimetula braunii was collected during the wet season at the Obafemi Awolowo
University (OAU), Ile-Ife Campus, Nigeria (GPS Coordinates: Latitude 7.520767, Longitude
4.530315; DMS Lat 7◦ 31′ 14.7612′′ N). It was found parasitizing Leucena leucocephala (Lam)
De Wit. (Family Fabaceae), its host tree. It was authenticated at the Ife Herbarium, OAU,
Ile-Ife, where a herbarium specimen was deposited with Voucher number IFE 17229.
3.2. Plant Extraction and Fractionation
The leaves of G. braunii were dried at room temperature (25–27 ◦ C). They were
powdered (5.0 kg) and macerated with 25 L of EtOH-H2 O (4:1) at room temperature
for 72 h with frequent agitation. This extraction method was a follow-up process to the
previous study on the plant [8]. The filtrate was concentrated to dryness in vacuo on
a Heidolph RE 400 Rotary Evaporator set at 45 ◦ C and 100 rpm. The extract (300 g)
was suspended in distilled water (300 mL) and successively partitioned with n-Hexane
(600 mL × 4), EtOAc (600 mL × 7) and n-BuOH (300 mL × 2).
3.3. Qualitative Antioxidant Screening
2,2-Diphenyl-1-picrylhydrazyl (DPPH) Rapid Radical Scavenging Test
Thin-layer chromatography (TLC) bioautography method was used according to
Wang et al. [35]. This involved TLC development of the fractions in the appropriate solvent
systems in duplicate. The TLC chromatograms were sprayed with 0.5 mg/mL DPPH in
MeOH and 10% sulfuric acid. The bleaching effect of the purple DPPH solution by the
spots was indicative of the antioxidant potential of the fractions.
3.4. Quantitative Antioxidant Screening
All the chemical reagents, solvents and standards used for antioxidant screening were
purchased from Sigma Aldrich (St. Louis, MO, USA).
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3.4.1. DPPH Spectrophotometric Assay
The DPPH spectrophotometric assay was carried out according to Xiao et al. [36]. A
1 mL DPPH solution in methanol (0.05 mg/mL) was added to 1 mL samples ((positive
controls: L-ascorbic acid) and (plant fractions/isolated compounds)) at varying concentrations: 50.00, 25.00, 12.50, 6.25 and 3.13 µg/mL. The experiment was carried out in triplicate.
The samples were incubated in the dark room for 30 min after which the absorbance was
measured at 517 nm on a CamSpec M 107 Spectrophotometer (Spectronics Camspec Ltd.,
Leeds, UK), where methanol (negative control) was used as the blank. The percentage
inhibition of DPPH by each test sample was calculated thus:
% Inhibition of sample =
Abscontrol − Abssample
× 100
Abscontrol
(1)
where Abscontrol = Absorbance of negative control, Abssample = Absorbance of test sample.
The result was expressed as % inhibition and/or IC50 .
3.4.2. Ferric Reducing Antioxidant Power (FRAP) Assay
This is based on the reduction of the greenish ferric ion (Fe3+ ) 2,4,6-tri-(2-pyridyl)1,3,5-triazine (TPTZ) to the bluish ferrous ion (Fe2+ ) by natural antioxidants at 593 nm
absorbance measurement. The ferric reducing power of plant extracts were determined
as ascorbic acid equivalent (AAE) from the calibration curve of the positive control (Lascorbic acid) at concentrations 1000.00, 500.00, 250.00, 125.00, 62.50 and 31.25 µg/mL in
methanol [37].
3.4.3. Total Antioxidant Capacity (TAC) Assay
The TAC assay is based on the reduction of Mo6+ to Mo5+ by the plant samples and
subsequent formation of green phosphate/Mo (V) complex at acidic pH according to Prieto
et al. [38]. A 0.3 mL extract was combined with 3 mL of reagent solution (0.6 M sulfuric
acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The absorbance of
the reaction mixture was measured at 695 nm. The calibration curve was prepared by
mixing ascorbic (1000.00, 500.00, 250.00, 125.00, 62.50 and 31.25 µg/mL) with methanol.
Data were expressed as mean ± standard error of mean (SEM). The TAC of each sample
was expressed as the number of gram equivalent of ascorbic acid (AAE/g).
3.4.4. Ferrous Ion Chelating (FIC) Ability
FIC assay was carried out according to the method of Singh and Rajini [39]. Solutions
of 2 mM FeCl2 ·4H2 O and 5 mM ferrozine were diluted 20 times. An aliquot (1 mL) of
different concentrations of extract was mixed with 1mL FeCl2 ·4H2 O. After 5 min incubation,
the reaction was initiated by the addition of ferrozine (1 mL). The mixture was shaken
vigorously, and, after a further 10 min incubation period, the absorbance of the solution was
measured spectrophotometrically at 562 nm. The percentage inhibition of ferrozine–Fe2+
complex formation was calculated by using the formula:
% Chelating ability =
Abscontrol − Abssample
× 100
Abscontrol
(2)
3.5. Statistical Analysis
All quantitative antioxidant data were analyzed using a One-way Analysis of Variance
(ANOVA), followed by the Bonferroni post-hoc test on a GraphPad Prism 9 (GraphPad
Software Inc., San Diego, CA, USA).
3.6. Antimicrobial Test
Micro-Broth Dilution Assay
The assay was carried according to the Clinical and Laboratory Standard Institute [40,41].
Bacteria and fungi used for the antimicrobial screening were obtained from the culture
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collections of the Microbiology Laboratory of the Department of Pharmaceutics, Obafemi
Awolowo University where the experiment was conducted. The bacteria and fungi strains
were isolated on a Nutrient broth and Sabouraud Dextrose broth (Merck KGaA, Darmstadt,
Germany), respectively. The organisms were identified using their morphological characteristics and standard biochemical tests. The reference strains used were Escherichia coli ATCC
25923, Pseudomonas aeruginosa ATCC 10145, Bacillus subtilis NCTC 8236, methicillin-resistant
Staphylococcus aureus ATCC 29213 and Candida albicans ATCC 24433.
Bacteria were maintained on nutrient broth and fungi on Sabouraud Dextrose broth
at 4 ◦ C and sub-cultured regularly. Bacteria were grown for 18 h in Nutrient broth and
culture suspensions of 108 cfu/mL (equivalent of 0.50 Mc Farland standard) were applied
to the dilutions of the fraction/isolates, positive controls (Ciprofloxacin, Ketoconazole) and
negative control 50% aqueous methanol employing a multipoint inoculator. Plates were
incubated at 37 ◦ C for 24 h. for bacteria strains and 25 ◦ C for 72 h for fungal strains, after
which all plates were observed for growth of the microorganisms. The minimum dilution
of fractions completely inhibiting the growth and killing each organism was taken as the
MIC and MBC/MFC. The sample with the lowest range of MIC and the widest spectrum
of activity against bacteria and fungi was taken as the most active.
3.7. Isolation of Compounds
Column Chromatography of the EtOAc Fraction
The EtOAc fraction (G, 30.0 g) was adsorbed unto 30 g silica gel (70–230 ASTM mesh,
Merck KGaA, Darmstadt, Germany), dry-packed on a 600 g silica gel stationary phase
within a 300 cm × 5 cm glass column (L x i.d., Fisher Scientific, Waltham, MA, USA). Mobile
phase comprising solvent systems of increasing polarity was introduced as thus: n-Hex
(100%, 700 mL, de-gas), EtOAc (9:1, 8:2, . . . , 1:9; 500 mL each), EtOAc (100%; 700 mL),
EtOAc-MeOH (95:5, 9:1, 8:2, 1:1; 500 mL) and MeOH (100%; 250 mL). Eluates were collected
in 20 mL test tubes (1–303). They were bulked into six sub-fractions G1–G6 based on their
TLC profiles (SiO2 , Hex-EtOAc 75:25, 1:1, EtOAc-MeOH 1:1, UV 254 and 365 nm, 10%
H2 SO4 spray).
After a 24 h period, sub-fraction G1 (1.2 g) afforded solid deposits a, b and c, while
sub-fraction G2 (3.1 g) gave a solid deposit d. Each deposit was washed with 100 mL
of MeOH (100%), affording compounds 1–4, respectively. The most active sub-fraction
G3 (3.5 g), based on TLC-bioautography was further purified on a Silica gel column with
mobile phase from DCM (100%, 100 mL) to DCM-MeOH (98:2, 96:4, . . . , 80:20; 100 mL
each). The eluates (1–187) were collected in 10 mL test tubes and were subsequently bulked
into four sub-fractions, G3a–G3d, based on their TLC profiles (SiO2 , DCM-MeOH 97:3,
85:15, 1:1, UV 254 and 365 nm, 10% H2 SO4 spray). A preparative TLC separation of G3c,
using DCM-MeOH (96:4) afforded bands i–iv, labelled compounds 5–8.
3.8. Characterization of Isolated Compounds
Thin-layer chromatography (TLC) of compounds was performed on aluminumbacked silica gel 60 F254 GF plates (0.25 mm, Merck KGaA, Darmstadt, Germany). Chemical detection of the class of compound isolated was done by spraying the developed
TLC plates with chromogenic reagents, such as 5% FeCl3 for phenolics and 10% H2 SO4
for terpenes. Melting point ranges of the solid compounds were determined on Gallenkamp MPD350-BM 3.5 electrothermal instrument (Gallenkamp, Kent, UK). The UV-Vis
absorption was determined within 200–800 nm on Shimadzu UV-1800 UV/Visible Scanning Spectrophotometer: 115 VAC (Shimadzu Corporation, Nkagyo-Ku, Kyoto, Japan).
Infrared spectroscopy was done within the 650–4000 cm−1 transmittance on Cary 630
FTIR Spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). 1 H, 13 C and 2D
(DEPT135, COSY, HSQC and HMBC) NMR spectra of compounds were recorded as solutions on Bruker AMX-300 Spectrometer (Bruker Corporation, Bremen, Germany), where
tetramethylsilane was used as the internal standard.
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Signals were recorded in the order of chemical shifts (δ) in part per million (ppm) relative
to the indicated deuterated solvents (CDCl3 , MeOD), integral values (number of protons),
multiplicity (s, singlet; d, doublet; t, triplet; and m, multiplet) and coupling constant (J) in
hertz (Hz). Electrospray Ionization Mass Spectrometry (ESI-MS) was performed on a Varian
500-MS ion trap Mass Spectrometer (Varian, Inc., Palo Alto, CA, USA) for molecular weight
determination, expressed in mass-to-charge ratio (m/z). ESI-MS analysis was performed
at 10 µL/min sample infusion flow rate; 2.56 kV capillary voltage; 3.0 V extraction cone;
475 L/h desolvation-gas flow rate; 80 and 100 ◦ C for the source- and desolvation-gas
temperatures, respectively; and 5.82 mm Vernier-probe-adjuster position. The spectrometer
scan range was 99.5–1500.5 m/z in the positive mode.
4. Conclusions
Our activity-guided study on the leaves of G. braunii led to isolation of eight compounds (1–8) from the most active EtOAc fraction. The compounds were identified based
on their spectroscopic data and in comparison with literature reports. They were ursanetype triterpenes (1–3), phenolics (4–6) and phenolic derivatives (7 and 8), all reported
for the first time in the plant and in the family Loranthaceae. Guaiacol (6) and methyl
3,5-dihydroxy-4-methoxybenzoate (4) were remarkably antioxidant with considerable
antimicrobial potentials.
Author Contributions: Conceptualization, A.O.O. (Ayodeji Oluwabunmi Oriola) and A.J.A.; methodology, A.O.O. (Ayodeji Oluwabunmi Oriola) and A.J.A.; software, A.O.O. (Ayodeji Oluwabunmi
Oriola) and T.I.; validation, A.J.A., T.O.I., E.M.O. and A.O.O. (Adebola Omowunmi Oyedeji); formal
analysis, A.O.O. (Ayodeji Oluwabunmi Oriola), E.M.O., F.O.A. and T.I.; investigation, A.O.O. (Ayodeji
Oluwabunmi Oriola); resources, A.O.O. (Ayodeji Oluwabunmi Oriola), E.M.O. and A.O.O. (Adebola
Omowunmi Oyedeji); data curation, A.O.O. (Ayodeji Oluwabunmi Oriola), E.M.O., F.O.A. and T.O.I.;
writing—original draft preparation, A.O.O. (Ayodeji Oluwabunmi Oriola); writing—review and editing, A.J.A. and A.O.O. (Adebola Omowunmi Oyedeji); visualization, A.J.A., T.O.I., E.M.O. and F.O.A.;
supervision, A.J.A. and T.O.I.; project administration, A.O.O. (Ayodeji Oluwabunmi Oriola), A.J.A.,
T.O.I.; funding acquisition, A.O.O. (Ayodeji Oluwabunmi Oriola) and A.O.O. (Adebola Omowunmi
Oyedeji). All authors have read and agreed to the published version of the manuscript.
Funding: The study received no funding support.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not available.
Acknowledgments: The corresponding author acknowledges the Drug Research and Production
Unit, O.A.U., Ile-Ife, Nigeria, for study leave permission to Walter Sisulu University, South Africa.
Conflicts of Interest: The authors declare no conflict of interest.
Samples Availability: Samples of the compounds 1–8 are available from the authors.
References
1.
2.
3.
4.
5.
6.
Siegfried Didier, D.; Obiang Nestor Laurier, E.; Din, N.; Richard Jules, P.; Victor, T.; Henri, F.; Georges, S.; Alain Didier, M.; Issaka
Joseph, B.; Akoa, A. An Assessment on the Uses of Loranthaceae in Ethno Pharmacology in Cameroon: A Case Study Made in
Logbessou, North of Douala. J. Med. Plants Res. 2009, 3, 592–595.
Ayoola, M.D.; Oriola, A.O.; Faloye, K.O.; Aladesanmi, A.J. Two Antihyperglycaemic Compounds from Globimetula braunii (Engl.)
Van Tiegh (Loranthaceae). GSC Biol. Pharm. Sci. 2020, 2020, 46–054. [CrossRef]
Burkill, H.M. The Useful Plants of West Tropical Africa. Available online: https://agris.fao.org/agris-search/search.do?recordID=
GB9618106 (accessed on 8 August 2021).
Adesina, S.K.; Illoh, H.C.; Johnny, I.I.; Jacobs, I.E. African Mistletoes (Loranthaceae); Ethnopharmacology, Chemistry and
Medicinal Values: An Update. Afr. J. Tradit. Complement. Altern. Med. AJTCAM 2013, 10, 161–170. [CrossRef]
Okpuzor, J.; Kareem, G.; Ejikeme, C. Lipid Lowering Activity of Globimetula braunii. Res. J. Med. Plant 2009, 3, 45–51. [CrossRef]
Okpuzor, J.; Ogbunugafor, H.A.; Kareem, G.K. Hepatic and Hematologic Effects of Fractions of Globimetula braunii in Normal
Albino Rats. EXCLI J. 2009, 8, 182–189.
Molecules 2021, 26, 6528
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
12 of 13
Oriola, A.O.; Aladesanmi, A.J.; Arthur, G. Anticancer Activity of Three African Mistletoes. Niger. J. Nat. Prod. Med. 2018, 22,
129–134. [CrossRef]
Oriola, A.O.; Aladesanmi, A.J.; Akinkunmi, E.O.; Olawuni, I.J. Antioxidant and Antimicrobial Studies of Some Hemi-Parasitic
West African Plants. Eur. J. Med. Plants 2020, 31, 17–26. [CrossRef]
Enehezeyi Aliyu, R.; Alonge, S.; Aliyu, R. Antibacterial Activity of Globimetula braunii Sourced from Five Different Host Trees in
Screening Cowpea Magic Rils for Variation in Agro-Physiological Traits for Tolerance to Biotic and Abiotic Stresses View Project
Antibacterial Activity of Globimetula braunii Sourced from Five Different Host Trees in Samaru, Zaria, Nigeria. Int. J. Curr. Sci.
2015, 18, 117–123.
Okpuzor, J.; Ogbunugafor, H.; Karecm, G.K. Antioxidative Properties of Ethyl Acetate Fraction of Globimetula braunii in Normal
Albino Rats. J. Biol. Sci. 2009, 9, 470–475. [CrossRef]
Erukainure, O.L.; Abovwe, J.A.; Adefegha, A.S.; Egwuche, R.U.; Fafunso, M.A. Antilipemic and Hypocholesteremic Activities of
Globimetula braunii in Rats. Exp. Toxicol. Pathol. 2010, 63, 657–661. [CrossRef]
Ie, O.; Zam, N. Oxytocic Properties of the Aqueous Extract of Globimetula braunii (Loranthaceae). Pak. J. Pharm. Sci. 2008, 21,
356–360.
Muhammad, K.J.; Jamil, S.; Basar, N.; Bakri Bakar, M.; Sarker, S.D.; Flanagan, K.J.; Senge, M.O. Lactones and Flavonoids Isolated
from the Leaves of Globimetula braunii. NPC Nat. Prod. Commun. 2017, 12, 1455–1458.
Muhammad, K.J.; Jamil, S.; Basar, N.; Sarker, S.D.; Mohammed, M.G. Globrauneine A–F: Six New Triterpenoid Esters from the
Leaves of Globimetula braunii. Nat. Prod. Res. 2019, 34, 2746–2753. [CrossRef] [PubMed]
Ren, F.C.; Li, G.Y.; Nama, N.; Liu, Z.H.; Yang, L.; Zhou, J.; Hu, J.M. 13,27-Cycloursane, Ursane and Oleanane Triterpenoids from
the Leaves of Lucuma nervosa. Fitoterapia 2019, 136, 104178. [CrossRef] [PubMed]
Ndlebe, V.J.; Crouch, N.R.; Mulholland, D.A. Triterpenoids from the African Tree Phyllanthus polyanthus. Phytochem. Lett. 2008, 1,
11–17. [CrossRef]
Lee, T.-H.; Juang, S.-H.; Hsu, F.-L.; Wu, C.-Y. Triterpene Acids from the Leaves of Planchonella duclitan (Blanco) Bakhuizan. J. Chin.
Chem. Soc. 2005, 52, 1275–1280. [CrossRef]
Pavia, D.L.; Lampman, G.M.; Kriz, G.S.; Vyvyan, J.R. Nuclear Magnetic Resonance Spectroscopy Part Five: Advanced NMR
Techniques. In Introduction to Spectroscopy; Thomson Learning Inc.: Boston, MA, USA, 2013; pp. 511–576.
Alade, G.O.; Moody, J.O.; Awotona, O.R.; Adesanya, S.A.; Lai, D.; Proksch, P. Spermicidal Constituents of Ethanolic Extract of
Sacoglottis gabonensis Stem Bark. Folia Med. 2017, 59, 437–442. [CrossRef] [PubMed]
MedChemExpress. Methylparaben—Advanced Dermatology. Available online: https://www.advanced-dermatology.com.au/
methylparaben (accessed on 25 September 2021).
MedChemExpress. Methyl Paraben (Methyl 4-Hydroxybenzoate)|Bacterial Inhibitor|MedChemExpress. Available online:
https://www.medchemexpress.com/Methyl_Paraben.html (accessed on 6 October 2021).
Kitanovski, Z.; Cusak, A.; Grgić, I.; Claeys, M. Chemical Characterization of the Main Products Formed through Aqueous-Phase
Photonitration of Guaiacol. Atmos. Meas. Tech. 2014, 7, 2457–2470. [CrossRef]
Guthrie, R.D. Introduction to Spectroscopy (Pavia, Donald; Lampman, Gary, M.; Kriz, George, S., Jr.). J. Chem. Educ. 1979, 56,
A323. [CrossRef]
Dewanjee, S.; Gangopadhyay, M.; Bhattacharya, N.; Khanra, R.; Dua, T.K. Bioautography and Its Scope in the Field of Natural
Product Chemistry. J. Pharm. Anal. 2015, 5, 75–84. [CrossRef] [PubMed]
Santos-Sánchez, N.F.; Salas-Coronado, R.; Villanueva-Cañongo, C.; Hernández-Carlos, B. Antioxidant Compounds and Their
Antioxidant Mechanism; IntechOpen: London, UK, 2019. [CrossRef]
Siddeeg, A.; AlKehayez, N.M.; Abu-Hiamed, H.A.; Al-Sanea, E.A.; AL-Farga, A.M. Mode of Action and Determination of
Antioxidant Activity in the Dietary Sources: An Overview. Saudi J. Biol. Sci. 2021, 28, 1633–1644. [CrossRef]
Baccouri, B.; Rajhi, I. Potential Antioxidant Activity of Terpenes. In Terpenes and Terpenoids Recent Advances; Intech Open Book
Series; IntechOpen: London, UK, 2021. [CrossRef]
Chen, J.; Yang, J.; Ma, L.; Li, J.; Shahzad, N.; Kyung Kim, C.K. Structure-Antioxidant Activity Relationship of Methoxy, Phenolic
Hydroxyl, and Carboxylic Acid Groups of Phenolic Acids. Sci. Rep. 2020, 10, 2611. [CrossRef] [PubMed]
Chen, S.C.; Liu, J.W.; Wu, X.Z.; Cao, W.L.; Wang, F.; Huang, J.M.; Han, Y.; Zhu, X.Y.; Zhu, B.Y.; Gan, Q.; et al. Comparison of
Microdilution Method with Agar Dilution Method for Antibiotic Susceptibility Test of Neisseria gonorrhoeae. Infect. Drug Resist.
2020, 13, 1775–1780. [CrossRef]
Reller, L.B.; Weinstein, M.; Jorgensen, J.H.; Ferraro, M.J. Antimicrobial Susceptibility Testing: A Review of General Principles and
Contemporary Practices. Clin. Infect. Dis. 2009, 49, 1749–1755. [CrossRef]
Jesus, J.A.; Lago, J.H.G.; Laurenti, M.D.; Yamamoto, E.S.; Passero, L.F.D. Antimicrobial Activity of Oleanolic and Ursolic Acids:
An Update. Evid.-Based Complement. Altern. Med. 2015, 2015. [CrossRef] [PubMed]
Maddox, C.E.; Laur, L.M.; Tian, L. Antibacterial Activity of Phenolic Compounds Against the Phytopathogen Xyllela fastidiosa.
Curr. Microbiol. 2010, 60, 53–58. [CrossRef] [PubMed]
Jayanetti, D.R.; Braun, D.R.; Barns, K.J.; Rajski, S.R.; Bugni, T.S. Bulbiferates A and B: Antibacterial Acetamidohydroxybenzoates
from a Marine Proteobacterium, Microbulbifer sp. J. Nat. Prod. 2019, 82, 1930–1934. [CrossRef]
Orlo, E.; Russo, C.; Nugnes, R.; Lavorgna, M.; Isidori, M. Natural Methoxyphenol Compounds: Antimicrobial Activity Against
Foodborne Pathogens and Food Spoilage Bacteria, and Roles in Antioxidant Processes. Foods 2021, 10, 1807. [CrossRef]
Molecules 2021, 26, 6528
35.
36.
37.
38.
39.
40.
41.
13 of 13
Wang, J.; Yue, Y.D.; Tang, F.; Sun, J. TLC Screening for Antioxidant Activity of Extracts from Fifteen Bamboo Species and
Identification of Antioxidant Flavone Glycosides from Leaves of Bambusa textilis Mcclure. Molecules 2012, 17, 12297–12311.
[CrossRef] [PubMed]
Xiao, F.; Xu, T.; Lu, B.; Liu, R. Guidelines for Antioxidant Assays for Food Components. Food Front. 2020, 1, 60–69. [CrossRef]
Benzie, I.F.F.; Strain, J.J. [2] Ferric Reducing/Antioxidant Power Assay: Direct Measure of Total Antioxidant Activity of Biological
Fluids and Modified Version for Simultaneous Measurement of Total Antioxidant Power and Ascorbic Acid Concentration.
Methods Enzymol. 1999, 299, 15–27. [CrossRef] [PubMed]
Prieto, P.; Pineda, M.; Aguilar, M. Spectrophotometric Quantitation of Antioxidant Capacity through the Formation of a
Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E. Anal. Biochem. 1999, 269, 337–341.
[CrossRef] [PubMed]
Singh, N.; Rajini, P. Free Radical Scavenging Activity of an Aqueous Extract of Potato. Food Chem. 2004, 85, 611–616. [CrossRef]
Clinical and Laboratory Standard Institute, CLSI. M07-A10 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically; Approved Standard-Tenth Edition; Clinical and Laboratory Standard Institute: Annapolis Junction, MD, USA,
2015.
Clinical and Laboratory Standard Institute, CLSI. Antimicrobial and Antifungal Susceptibility Testing Resources. Available
online: https://clsi.org/about/about-clsi/about-clsi-antimicrobial-and-antifungal-susceptibility-testing-resources/# (accessed
on 22 September 2021).