Ballodiolic acid a and b: Two new ros, (? oh), (onoo? ) scavenging and potent antimicrobial constituents isolated from ballota pseudodictamnus (l.) benth.

Article abstract of DOI:10.3390/pharmaceutics13030402

Bioassays guided phytochemical investigations on the ethyl acetate-soluble fraction of the root material of Ballota pseudodictamnus (L.) Benth. led to the isolation of two new compounds, ballodiolic acid A (1) and ballodiolic acid B (2), along with three known compounds ballodiolic acid (3), ballotenic acid (4), and β-amyrin (5), which were also isolated for the first time from this species by using multiple chromatographic techniques. The structures of the compounds (1–5) were determined by modern spectroscopic analysis including 1D and 2D NMR techniques and chemical studies. In three separate experiments, the isolated compounds (1–5) demonstrated potent antioxidant scavenging activity, with IC₅₀ values ranging from 07.22–34.10 μM in the hydroxyl radical (^? OH) inhibitory activity test, 58.10–148.55 μM in the total ROS (reactive oxygen species) inhibitory activity test, and 6.23–69.01 μM in the peroxynitrite (ONOO^? ) scavenging activity test. With IC₅₀ values of (07.22 ± 0.03, 58.10 ± 0.07, 6.23 ± 0.04 μM) for^? OH, total ROS, and scavenge ONOO^?, respectively, ballodiolic acid B (2) showed the highest scavenging ability. Antibacterial and antifungal behaviors were also exposed to the pure compounds 1–5. In contrast to compounds 4 and 5, compounds 1–3 were active against all bacterial strains studied, with a good zone of inhibition proving these as a potent antibacterial agent. Similarly, compared to compounds 3–5, compounds 1 and 2 with a 47 percent and 45 percent respective inhibition zone were found to be more active against tested fungal strains.

Full text of DOI:10.3390/pharmaceutics13030402

pharmaceutics
Article
•
−
Ballodiolic Acid A and B: Two New ROS, ( OH), (ONOO )
Scavenging and Potent Antimicrobial Constituents Isolated
from Ballota pseudodictamnus (L.) Benth.
1
1
2,
2
3
4,
5
Fozia , Asmat Shaheen , Ijaz Ahmad *, Syed Badar Amin , Nisar Ahmad , Riaz Ullah * , Ahmed Bari
Muhammad Sohaib 6 , Hafiz Majid Mahmood 7 and Abdulrahman Alobaid 5
,
1
Biochemistry Department, KMU Institute of Medical Sciences, Kohat 26000, Pakistan;
drfoziazeb@yahoo.com (F.); asmatshaheen@yahoo.com (A.S.)
Department of Chemistry, Kohat University of Science & Technology, Kohat 26000, Pakistan;
2
syedbadaramin@gmail.com
Department of Botany, Kohat University of Science & Technology, Kohat 26000, Pakistan;
3
ahmed_kust@yahoo.com
Department of Pharmacognosy (MAPPRC), College of Pharmacy, King Saud University,
4
Riyadh 11451, Saudi Arabia
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
5
Riyadh 11451, Saudi Arabia; abari@ksu.edu.sa (A.B.); aalobaid1@ksu.edu.sa (A.A.)
Department of Soil Science, College of Food and Agriculture Sciences, King Saud University,
6
P.O. Box 2460, Riyadh 11451, Saudi Arabia; msohaib@ksu.edu.sa
Department of Pharmacology, College of Pharmacy, King Saud University,
7
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Riyadh 11451, Saudi Arabia; harshad@ksu.edu.sa
Correspondence: drijaz_chem@yahoo.com (I.A.); rullah@ksu.edu.sa (R.U.); Tel.: +92-335-194-9080 (I.A.)
ꢀ
ꢁꢂꢃꢄꢅꢆ
*
Citation: Fozia; Shaheen, A.; Ahmad,
I.; Amin, S.B.; Ahmad, N.; Ullah, R.;
Bari, A.; Sohaib, M.; Mahmood, H.M.;
Alobaid, A. Ballodiolic Acid A and B:
Abstract: Bioassays guided phytochemical investigations on the ethyl acetate-soluble fraction of
the root material of Ballota pseudodictamnus (L.) Benth. led to the isolation of two new compounds,
ballodiolic acid A (
acid ( ), ballotenic acid (
species by using multiple chromatographic techniques. The structures of the compounds (
were determined by modern spectroscopic analysis including 1D and 2D NMR techniques and
chemical studies. In three separate experiments, the isolated compounds ( ) demonstrated potent
M in the hydroxyl
1) and ballodiolic acid B (
2), along with three known compounds ballodiolic
•
−
Two New ROS, ( OH), (ONOO
)
3
4
), and -amyrin ( ), which were also isolated for the first time from this
β
5
Scavenging and Potent Antimicrobial
Constituents Isolated from Ballota
pseudodictamnus (L.) Benth.
1–5)
1–5
Pharmaceutics 2021, 13, 402. https://
doi.org/10.3390/pharmaceutics13030402
antioxidant scavenging activity, with IC values ranging from 07.22–34.10
µ
50
•
radical ( OH) inhibitory activity test, 58.10–148.55
µ
M in the total ROS (reactive oxygen species)
−
Academic Editor: Javier Garcia-Pardo
inhibitory activity test, and 6.23–69.01
µM in the peroxynitrite (ONOO ) scavenging activity test.
•
With IC50 values of (07.22
±
0.03, 58.10
±
0.07, 6.23
±
0.04
µ
M) for OH, total ROS, and scavenge
Received: 4 February 2021
Accepted: 11 March 2021
Published: 17 March 2021
−
ONOO , respectively, ballodiolic acid B (
2
) showed the highest scavenging ability. Antibacterial and
. In contrast to compounds and
were active against all bacterial strains studied, with a good zone of inhibition
proving these as a potent antibacterial agent. Similarly, compared to compounds , compounds
and with a 47 percent and 45 percent respective inhibition zone were found to be more active
against tested fungal strains.
antifungal behaviors were also exposed to the pure compounds
, compounds
1
–5
4
5
1–3
3–5
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1
2
Keywords: Ballota pseudodictamnus; Lamiaceae; phytochemicals; antioxidative and antimicrobial activities
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1
. Introduction
A variety of foods have been found to contain antioxidants that scavenge active
oxygen species (free radicals) and are generally referred to as scavengers [ ]. In order
1
to protect plants that are exposed to sunlight and live under severe oxygen stress, many
oxidants are phytochemicals and play an important role. Antioxidants also play an im-
portant role in human health, because the biologic protective system cannot work under
extreme oxygen stress. According to recent studies, activated oxygen is thought to be
4
.0/).
Pharmaceutics 2021, 13, 402. https://doi.org/10.3390/pharmaceutics13030402
https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 402
2 of 15
a major factor in ageing, hardening of the arteries, diabetes, cancer, and tissue in injury
skin [
In the inflammatory process, cardiovascular disease [
toid arthritis, neurodegenerative disease and the ageing process, and free radicals have
important significance [ ]. In the etiology of a number of human degenerative diseases,
free radicals and ROS or RNS (reactive nitrogen species), including H O , O , OH,
2
]. Indeed, approximately 90% of age-related diseases are linked to activated oxygen.
3], arteriosclerosis, malaria, rheuma-
4
•
2
2− •
2
•
−
NO , and ONOO , play a significant role. As a consequence of aerobic metabolism, these
reactive species are produced in the body and harm all intracellular components, such
as nucleic acids, proteins, and lipids. ROS are also involved in both ageing and multiple
degenerative diseases [3,4].
The genus Ballota (Lamiaceae or Mint family) is a medium-sized genus comprising
approximately 33–35 species found mainly in the Eurasian and Mediterranean regions [5].
Various members of the genus Ballota are widely found in Khyber Pakhtunkhwa and the
lower hills of West Punjab in Pakistan. Four species, namely Ballota psedodictamnus, Ballota
aucheria, Ballota nigra, and Ballota limbata [6] are present in Pakistan. Plants of the genus
Ballota have been used historically in treatment of burns, wounds, preventing coughs, upper
respiratory system inflammation, antispasmodic, neurosedative, diuretic, antiulcer, nausea,
antihemorrhoidal, vomiting, and nervous dyspepsia and also are used as antioxidant,
antimicrobial, anti-inflammatory, antiemetic, and as hepato-protective [7,8].
Previous phytochemical studies of the Ballota genus have led to the isolation of nu-
merous compounds, including diterpenoides such as balloaucherolide, hispanone, 7α-
acetoxymarrubiin, ballonigrinone, hispanolone, ballonigrin, and rupestralic acid, and
flavonoids like luteolin, salvigenin, nuchensin, ladanein, tangeretin, verbascoside, and
apigenin [9–19].
The literature survey showed that no phytochemical research was done on the Ballota
pseudodictamnus (L.) Benth. plant, which prompted us to conduct phytochemical research
on this species. Previous biological evaluation of Ballota pseudodictamnus (L.) Benth. by
our group confirmed that compared to leaves and stem extracts, the ethyl acetate soluble
fraction of root of this plant have more potency against different bacterial and fungal
strains [20]. Further, among the all tested solvent soluble fractions, the ethyl acetate soluble
fraction of root of the title plant was found to be the most promising for the antioxidant
activity and is therefore selected for further phytochemical investigations.
Herein in this article, we report the isolation and structural elucidation of two new
compounds, ballodiolic acid A (
1
) and ballodiolic acid B (2), along with three known
phytochemicals ballodiolic acid (
3
), ballotenic acid ( ), and
4
β
-amyrin ( ), which were iso-
5
lated for the first time from Ballota pseudodictamnus (L.) Benth. and were characterized
by various 1D and 2D NMR techniques (Figure 1). The antioxidant activities of the com-
pounds (
1
–5) against four different tests along with antibacterial and antifungal activities
of the compounds (
1
–
5) were performed. Though the structures of the compounds (3–5)
were published previously from other plant species, their antimicrobial and antioxidative
activities were not.

Pharmaceutics 2021, 13, 402
3 of 15
Figure 1. Structures of Compounds 1–5.
2. Materials and Methods
2.1. General Experimental Procedures
To assess the UV spectrum of compounds, (Shimadzu UV-240, Hitachi U-3200, Kyoto,
Japan) UV-240 spectrophotometers were used. On IRA-I and JASCO-320-A spectropho-
tometers, respectively, IR spectra have been reported. On Bruker AM-400, AM-500, and
AM-600 spectrometers (Thermo Fisher Scientific, Waltham, MA, USA) with 400, 500, and
1
13
6
00 aspect data systems, the H-NMR and C-NMR spectra were observed at 400, 500,
and 600 MHz z (instrument used Bruker Biospin, Karlsruhe, Germany). As an internal
guide, TMS (tetramethylsilane) was used. The JMS-HX-110 data system spectrometer was
used to measure the EI-MS spectra of compounds. The Jasco-DIP-360 digital polarimeter
(
JASCO, Tokyo, Japan) was used to determine the optical rotation of the compounds. TLC
was performed with G-25-UV254 pre-coated silica gel plates, and detection was carried
out at 254 nm and 10% H SO by ceric sulphate. For column chromatography and flash
2
4
chromatography, respectively, silica gel (E. Merck,70–230 mesh) and silica gel (E. Merck,
230–400 mesh) were used.
2.2. Plant Material
Ballota pseudodictamnus (L.) Benth. plant sample from Latamber, Karak District, Khyber
Pakhtunkhwa (Pakistan) was collected in adequate quantity and was described by Dr.
Nisar Ahmad, Assistant Professor, Department of Botany, Kohat University of Science and
Technology, Kohat, Pakistan.

Pharmaceutics 2021, 13, 402
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2.3. Extraction and Isolation of compounds
At room temperature, the shade-dried and grinded root material (04 Kg) of Ballota
pseudodictamnus (L.) Benth. was exhaustively extracted with methanol. The extract was
dried and measured in the (55 g). The crude extract was suspended in water and then
was successively partitioned between n-hexane (11 g), chloroform (7 g), ethyl acetate (8 g),
n-butanol (12 g), and aqueous extracts (16 g). Ethyl acetate-soluble fraction of the title plant
was found to be the most promising for the said activities and is therefore selected for
further phytochemical investigation. The fraction of ethyl acetate was subjected to silica gel
chromatography using n-hexane with an ethyl acetate gradient of up to 100 percent. Four
F1, F2, F3, and F4 sub-fractions were obtained from the compilation process. Furthermore,
sub-fraction F1 was subjected to column chromatography and eluted with EtOAc: hexane
(
1:9) for purification of compound 5 [21]. Similarly, sub-fraction F2 was further referred to
column chromatography by eluting the fraction with n-hexane-ethyl acetate (6:4). From this
fraction, on successive chromatography with n-hexane-ethyl acetate (2:8 and 1:9) elution,
two pure compounds 2 [22] and 3 [22] were extracted, respectively. The sub-fraction F3
was further subjected to column chromatography by carry-out elution with n-hexane-ethyl
acetate (5:5). Two pure compounds 1 and 2 were supplied by the fraction. On TLC plates,
the purity of the compounds was confirmed.
2.4. Ballodiolic Acid A (1)
Light yellow oil (21 mg); molecular formula C H O ; molecular weight m/z 466.5048
2
9
41
6
+
(
1
Calcd 466.6037 for C H O -H O) [M-H O] ; IR (KBr) νmax: 3634, 2930, 1720, 1715, 1625,
29 40 6 2 2
−
1
23
◦
590 cm ; UV (MeOH) λmax: 216 (3.12) nm; [α]D −20.7 (c = 0.155, CHCl ).
3
2.5. Ballodiolic Acid B (2)
Pale yellow oil (19 mg); molecular formula C H O ; molecular weight m/z 482.5544
2
9
41
7
+
(
1
Calcd 482.6058 for C H O -H O) [M-H O] ; IR (KBr) νmax: 3630, 2925, 1722, 1715, 1628,
29 40 7 2 2
−
1
23
◦
591 cm ; UV (MeOH) λmax: 216 (3.12) nm; [α]D −20.3 (c = 0.150, CHCl ).
3
2.6. Ballodiolic Acid (3)
Colorless oil (17 mg); molecular formula C H O ; molecular weight m/z 320.2722.
2
0
34
4
+
−1
;
(
Calcd 320.4618 for C H O -H O) [M-H O] ; IR (KBr) νmax: 3632, 3590, 2934, 1688 cm
UV (MeOH) λmax: 214 (3.5) nm, [α]D −19.6 (c = 0.170, CHCl ) [22].
20
34
4
2
2
23
◦
3
2.7. Ballotenic Acid (4)
Colorless oil (15 mg); molecular formula C H O ; molecular weight m/z 316.2240
2
0
30
4
+
−1
(
Calcd 316.4306 for C H O -H O) [M-H O] ; IR (CDCl ) cm 1765, 1675 and 2925;
20
30
4
2
α
2
3
23
◦
0.51 (c = 0.103, CHCl ). The H- and C-NMR
3
1
13
UV (MeOH) λmax 213 nm (3.8); [
]D
−
data of the compound were in complete agreement with those reported in literature for
ballotenic acid [22].
2
.8. β-amyrin (5)
◦
Needle-like crystal (25 mg); melting point 197–198 C; molecular formula C H O;
3
0
50
−
1
1
13
molecular weight m/z 426.38; IR (KBr) νmax: cm 3510, 1636. The H- and C-NMR data of
the compound was in complete agreement to those reported in literature for -amyrin [21].
β
2.9. Alkaline Hydrolysis of Compounds 1 and 2
The Compound (10 mg) in benzene (25 mL) was refluxed with 5% methanolic potas-
1
sium hydroxide (15 mL) for 4 h, concentrated in vacuo, diluted with water (30 mL), and
extracted with ether. The aqueous layer was acidified with dil. hydrochloric acid and
extracted with dichloromethane. The residue recovered from the organic phase was charac-
◦
terized as p-hydroxy cinnamic acid, colorless crystals from H O, mp 212 C (characterized
2
through m.m.p., Co-TLC, superimposable IR). Corresponding hydrolysis of
2 afforded
3,4-dihydroxy cinnamic acid (characterized through m.m.p., IR, Co-TLC).

Pharmaceutics 2021, 13, 402
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2.10. Evaluation of Antioxidative Activity
2.10.1. Measurement of Total ROS Generation Inhibition
Rat kidney homogenates prepared from the kidneys of newly killed male Wistar rats,
30–180 g in weight, were mixed with or without the suspension of extracts or compounds,
which were dissolved in 10% EtOH (final concentration: 0.4%). The mixtures were then
1
0
0
incubated with 12.5 mM 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular
Probes Inc., Eugene, Oregon), which was dissolved in 100% EtOH (final concentration:
◦
0
.2%), at 37 C for 30 min. A 50 mM phosphate buffer (Wako Pure Chemical Industries,
Osaka, Japan) solution at pH 7.4 was also used. DCFH-DA is a stable compound that
is hydrolyzed by intracellular esterase to create a reduced, nonfluorescent compound of
0
0
,7 -dichlorodihydrofluorescein (DCFH). The ROS produced by the homogenates oxidizes
2
0
0
the DCFH to a highly fluorescent 2 ,7 -dichlorofluorescein (DCF). A microplate fluorescence
spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT) with excitation and emission
wavelengths of 460 and 530 nm, respectively, was used to track the fluorescence intensity
of the oxidized DCF [23]. As a positive regulation, Trolox has been used as an important
standard oxidant.
2.10.2. Measurement of Hydroxyl Radical Generation inhibition
First, 1 mM H O and 0.2 mM FeSO (Fisher Science, Fair Lawn, NJ, USA) were
2
2
4
applied to extracts or compounds dissolved in 10 percent EtOH (final concentration:
◦
0
.4 percent) and incubated at 37 C for 5 min. Then, 2 M DCFH-DA (Molecular Probes
Inc., Eugene, Oregon) treated with esterase in 100 percent EtOH was introduced, and
fluorescence changes were tracked on a microplate fluorescence spectrophotometer (BioTek
Instruments Inc., Winooski, VT, USA) with 460 and 530 nm excitation and emission wave-
lengths, respectively, for 30 min [24]. As a positive regulation, Trolox, an efficient standard
oxidant, was used.
−
2
.10.3. Measurement of ONOO Scavenging Activity
−
The ONOO scavenging activity was calculated by monitoring the oxidation of di-
hydrorhodamine 123 (DHR 123, Molecular Probes Inc., Eugene, OR, USA) using a slight
◦
modification of the reported method [25]. At 80 C, DHR 123 (5 mM) in DMF, purged with
N , was stored as a stock solution. Prior to the analysis, this solution was then put on ice
2
and held in the dark. The buffer consisted of 90 mM NaCl, 50 mM Na PO , 5 mM KCl at
3
4
pH 7.4, and 100 M diethylenetriaminepentaacetic acid (DTPA), each of which was prepared
and purged with N with high quality deionized H2O.The final DHR 123 concentration
2
amounted to 5 M. The context and final fluorescent intensities were assessed with and
−
−
without the authentic ONOO 5 min after treatment. The authentic ONOO oxidized
DHR 123 was rapidly oxidized, and the final fluorescent strength of the oxidized DHR
123 was determined using the FL 500 microplate fluorescence reader (BioTek Instruments
Inc., Winooski, VT, USA) at 480 and 530 nm excitation and emission wavelengths, respec-
tively. For the final fluorescence intensity minus background fluorescence, the results are
expressed as the mean
±
standard error (n = 3). The results are expressed as DHR 123
oxidation percent inhibition, and DLpenicillamine, the normal oxidant, has been used
as a positive regulation. In all three scavenging experiments, the IC50 was classified as
sample concentrations showing 50 percent scavenging activity and was calculated from a
triplicate experiment.
2.11. Antimicrobial Screening of Compounds 1–5
2.11.1. Stock Solution Preparation
First, 30
µg of each compound was dissolved in 1mL of dimethyl sulfoxide oxide
(DMSO) to form a solution of 30
µg/mL concentration in the preparation of the stock
solution. For the present analysis, DMSO was chosen as a solvent, because it shows no
activity against bacteria [26].

Pharmaceutics 2021, 13, 402
6 of 15
2.11.2. Anti-Bacterial Assay
To test the antibacterial activities of isolated compounds, the process of Agar diffusion
was used. In the antibacterial bioassay, five bacterial strains were used, i.e., Escherichia coli,
Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis, and Staphylococcus aureus. The
various bacterial strains were sub-cultivated for a second time to obtain new and fresh
culture of each strain in order to test the antibacterial activities of acquired compounds.
The nutrient broth was inoculated with a single colony of each strain, then it was incubated
◦
for 24 h at 37 C. Nineteen grams of nutrient agar was dissolved in 1L of distilled water,
◦
and the resulting solution was autoclaved for 30 min at 121 C. Then, 75 mL of solid media
was prepared after cooling by pouring the media into Petri dishes (14 cm). Four holes
per plate were made with sterile cork borer in these media (8 mm). For inoculation of all
bacterial strains, a single Petri dish was used. DMSO (dimethyl sulfoxide) was prepared in
the stock solution of compounds 1–3. Two solutions of DMSO (negative control) and that of
levofloxacin (positive control) were used in these specific holes for 100
The activity of each hole zone was calculated in mm when the generic drug inhibition zone
Levofloxacin) was compared using the protocol [26].
µL of each solution.
(
2.11.3. Anti-Fungal Assay
For the determination of antifungal activities of isolated compounds, the Disk diffusion
method was used. In an antifungal bioassay of isolated compounds, five fungal strains
were used: Aspergillus flavus, Fusarium solani, Aspergillus fumigatus, Aspergillus niger, and
Canadida glabrata. The old fungal strains were subcultivated using nutrient broth medium
in order to evaluate the antifungal activities of the obtained compounds and to obtain the
◦
fresh culture of each fungal strain. For seven days at 28 C, the new cultures were incubated.
Every fungal strain was inculcated through point inoculation on a separate potato dextrose
agar plate (PDA). Then, 100 µL of stock solution of compounds 1–3, pure DMSO (negative
control) and Miconazole (positive control) were used for experiments. After incubation for
◦
seven days, the activity of each compound was measured at a temperature of 28 C [27].
3. Results and Discussion
The ballodiolic acid A (1) was isolated as light yellow oil with molecular formula
of C H O , which was confirmed through HR-EIMS having molecular ion peak at m/z
29
40
6
4
66.5048 (Calcd 466.6037 for C H O -H O) accompanied by loss of water molecule [M-
29 40 6 2
+
−1
H O] . The IR spectrum for the compound 1 at 3630 cm indicates the presence of free
hydroxyl group (OH);
The IR spectrum revealed the presence of the aromatic group 1590 cm and broad IR band
2
−
1
α
,
β
-unsaturated carbonyls were observed at 1715 and 1689 cm
.
-1
−
1
at 2925 cm confirm the presence hydroxyl (OH) moiety of
acid. For the compound
NMR spectrum at 0.77 (3H, d, J = 5.1 Hz, H-17),
s, H-20); an olefinic proton at 6.82 (1H, br s, H-3); the oxygenated methylene protons
appeared at 3.69–4.1 (2H, m, H-15); and free hydroxyl methylene protons appeared
at and 3.75–3.79 (2H, m, H-16), respectively. The other protons signals appeared at
.79–0.88 (2H, m, H-1), 2.17–2.37 (2H, m, H-2), 2.39–2.40 (2H, m, H-6), 1.38–1.50 (2H,
m, H-7), 1.44 (1H, m, H-8), 1.38 (1H, d, J = 10.3 Hz, H-10), 1.35–1.45 (2H, m, H-11),
.63–1.68 (2H, m, H-12), 1.55 (1H, m, H-13), and 1.20–1.27 (2H, m, H-14), suggesting
that compound
has bicyclical clerodane diterpene skeleton [22]. The 13C NMR spectral
α
,
β
-unsaturated carboxylic
1
1
, three methyl protons signals (Table 1) were observed in H
δ
δ 1.3 (3H, s, H-19), and δ 0.73 (3H,
δ
δ
δ
δ
0
δ
δ
δ
δ
δ
δ
δ
1
δ
δ
1
analysis of compound 1 (Table 1) also confirmed bicyclical clerodane diterpene skeleton
on the basis of distortionless enhancement by polarization transfer (DEPT) and broad
band (BB) NMR experiments. The carboxylic group carbon and two oxygenated methylene
carbon resonated at
carbons shows signals at
tertiary methyl and for one secondary methyl group observed at C-19
and C-17 16.3. For seven upfield methylene carbons and four methines carbons, signals
resonating at 17.9, 27.7, 37.2, 35.3, 27.5, 36.4, 38.5, 46.8, 35.5, 24.8, 39.9, and 29.9 were
δ
172.7, 62.7, and 61.8 for C-18, C-15, and C-16, respectively. The olefinic
141.0 for C-3 and at 141.9 for C-4. The signals for the two
20.6, C-20 18.7,
δ
δ
δ
δ
δ
δ

Pharmaceutics 2021, 13, 402
7 of 15
assigned to C-1, C-2, C-5, C-6 C-7, C-8, C-9, C-10, C-11, C-12, C-l3, and C-14, respectively.
The above spectral data were closely related to (ballodiolic acid
having additional characteristic signals for p-hydroxycinnemoyl moiety (aromatic protons
showing AA‘, BB‘ pattern with 7.40 (2H, d, J = 8.1 Hz) and 6.87 (2H, d, J = 8.1 Hz);
trans olefinic protons at 7.55 (1H, d, J = 16.3 Hz) and 6.47 (1H, d, J = 16.3 Hz)), and the
downfield signals in 13C NMR spectra at
166.3 and 117, 144.7 could be assigned to the
3) (Table 1) [22], differing in
δ
δ
δ
δ
δ
cinnamoyl carbonyl group and its olefinic carbons, respectively (Table 1). The presence
of p-hydroxycinnemoyl moiety was further confirmed by electron impact mass spectrum
(
EI-MS) showing intense peaks at m/z 147.
Table 1. H- (400 MHz) and 13C-NMR (100 MHz) assignments (δ/ppm) of Compounds 1–3 in CDCl3.
1
1
2
3
Position
δ(1H)
δ(13C)
δ (1H)
δ(13C)
δ(1H)
δ(13C)[M1]
1
2
3
4
5
6
7
8
9
0.79–0.88 (m)
2.17–2.37 (m)
6.82 (br s)
-
17.9
27.7
141.0
141.9
37.2
35.3
27.5
36.4
38.5
46.8
35.5
24.8
39.9
29.9
62.7
61.8
16.3
172.7
20.6
18.7
166.3
117.0
144.7
125.9
130.2
116.0
159.3
116.0
130.2
0.81–0.90 (m)
2.15–2.33 (m)
6.81 (br s)
-
17.6
27.5
140.8
141.5
37.4
35.5
27.3
36.3
38.4
46.6
35.7
24.7
39.5
29.6
62.3
61.5
16.1
172.5
20.4
18.5
165.9
117.3
144.9
126.4
112.9
148.2
150.3
116.0
124.1
0.80–0.89 (m)
2.14–2.34 (m)
6.79 (br s)
17.5
27.4
140.0
141.3
37.5
35.5
27.2
36.1
38.6
46.6
35.8
24.9
39.8
29.7
66.3
61.1
15.9
172.0
20.5
18.4
-
-
-
-
-
2.39–2.40 (m)
1.38–1.50 (m)
1.44 (m)
2.35–2.38 (m)
1.36–1.47 (m)
1.45 (m)
-
1.35 (d, 10.3)
1.37–1.43 (m)
1.62–1.70 (m)
1.56 (m)
1.22–1.28 (m)
3.67–4.1 (m)
3.73–3.78 (m)
0.78 (d, 5.3)
-
2.35–2.38 (m)
1.38–1.49 (m)
1.46 (m)
-
-
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
1.38 (d, 10.3)
1.35–1.45 (m)
1.63–1.68 (m)
1.55 (m)
1.20–1.27 (m)
3.69–4.1 (m)
3.75–3.79 (m)
0.77 (d, 5.1)
-
1.36 (d, 10.3)
1.36–1.44 (m)
1.61–1.69 (m)
1.58 (m)
1.23–1.29 (m)
3.65–4.0 (m)
3.70–3.77 (m)
0.76 (d, 5.3)
-
1.23 (s)
0.73 (s)
1.24 (s)
0.71 (s)
1.21 (s)
0.70 (s)
0
0
-
-
-
-
-
-
-
-
-
-
-
0
0
0
0
0
0
0
0
6.47 (d, 16.3)
7.55 (d, 16.3)
-
7.40 (d, 8.1)
6.87 (d, 8.1)
-
6.45 (d, 16.2)
7.55 (d, 16.2)
-
7.15 (d, 2.3)
-
-
-
-
-
-
-
-
-
-
6.87 (d, 8.1)
7.40 (d, 8.1)
6.75 (d, 8.1)
7.07 (dd, 8.1, 2.3)
Multiplicities and coupling constants (J = Hz) are given in parenthesis. δ in ppm from TMS.
1
13
All the assignments were further confirmed by H- C multiple bond interaction in
heteronuclear multiple-quantum coherence (HMBC) spectrum of (Figure 2). Beside the
1
1
13
H- C multiple bond correlations established earlier for ballodiolic acid [22], additional
interactions were observed by the oxymethylene protons (H-15 protons), which showed J
39.9), respectively, and are also correlated
166.3). The olefinic proton (H-2 ) at
p-hydroxycinnemoyl moiety showed cross peak with carbonyl carbon (
2
3
and J correlations with C-14 (
with carbonyl carbon of cinnamoyl moiety (
δ
36.4) and C-13 (
δ
0
δ
δ
6.47 of
166.3) and C-3
0
δ
(
δ 144.7). These correlations of HMBC spectrum confirmed the attachment of cinnamoyloxy
methylene at C-14. All these evidences were in accordance to the assigned structure
of (E)-5-(3-(hydroxymethyl)-5-(3-(4-hydroxyphenyl)acryloyloxy)pentyl)-5,6,8a-trimethyl-
3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid as ballodiolic acid A (1).

Pharmaceutics 2021, 13, 402
8 of 15
Figure 2. Important HMBC Correlations of 1.
Ballodiolic acid B (
2
) was obtained as a pale yellow oil, molecular formula C H O
2
9
40
7
+
by [M] peak at m/z 482.5544 in HR-EIMS spectrometry (calcd. for C H O ; 482.6058).
29
40
7
1
13
The UV, IR, H, and C NMR spectra of
2
were almost identical to those of
1, sug-
0
0
gesting that had the same skeleton as that of
2
1
, except the difference due to 6 ,7 -
1
dihydroxylcinnamoyl moiety, which was confirmed by H NMR (ABX system at
δ
7.15
0
0
(
=
1H, d, J = 2.3 Hz, H-5 ),
δ
7.07 (1H, dd, J = 2.3, 8.1 Hz, H-9 ), and
δ
6.75 (1H, d, J
0
13
0
0
8.1 Hz, H-8 )) [28]. The C NMR spectra (Table 1) of
2
δ
also confirmed the 6 ,7 -
150.3 (C-7 ) and
0
dihydroxylcinnamoyl group showing downfield signals at
C-6 ) [28] and was further confirmed by electron impact mass spectrum (EI-MS) show-
δ
148.2
0
(
ing intense peaks at m/z 163. All the above spectral data confirmed the structure of
compound 2 as (E)-5-(5-(3-(3,4-dihydroxyphenyl)acryloyloxy)-3-(hydroxymethyl)pentyl)-
5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid (2).
Alkaline hydrolysis of and provided p-hydroxy cinnamic acid and 3,4-dihydroxy
1
2
cinnamic acid, respectively (characterized through m.m.p., Co-TLC, superimposable IR),
providing conclusive evidence for the presence of these moieties in the respective com-
pounds. The important HMBC correlations were in complete agreement to the assigned
structures of compounds 1 and 2.
For the isolated compounds 1–5 and different solvent soluble extracts, we investigated
the general antioxidant effects of the compounds and extracts to inhibit OH and absolute
−
ROS and to scavenge genuine ONOO . The different solvent soluble fractions of root,
stem, and leave parts of Ballota pseudodictamnus exhibited remarkable anti-oxidant activities
(Table 2). The ethyl acetate soluble fraction of root part of the plant showed stronger
anti-oxidative activity than that of the other fractions in all the three scavenging tests with
•
−
the IC50 values of 19.10
±
0.05, 65.13
±
0.05, and 20.18
±
0.05 for OH, ROS, and ONOO ,
•
respectively. The chloroform soluble fraction of root part of the plant observed the OH,
−
ROS, and ONOO scavenging activities with IC50 values of 47.28
±
0.07, 80.19
±
0.03, and
41.23 ± 0.07 µg/mL, respectively (Table 2).

Pharmaceutics 2021, 13, 402
9 of 15
Table 2. Antioxidant activity of the solvent fractions of root, stem, and leaves of B. pseudodictamnus.
IC50 [µg/mL] a
Sample
Solvent Fractions
•
OH b
Total ROS c
− d
ONOO
n-hexane
CHCl3
EtOAc
n-BuOH
H O
2
79.17 ± 0.05
47.28 ± 0.07
19.10 ± 0.05
31.51 ± 0.03
97.37 ± 0.05
>300
80.37 ± 0.03
41.23 ± 0.07
20.18 ± 0.05
61.16 ± 0.04
89.12 ± 0.05
80.19 ± 0.03
65.13 ± 0.05
90.41 ± 0.06
>400
Root
n-hexane
CHCl3
EtOAc
n-BuOH
H O
2
83.41 ± 0.03
55.13 ± 0.05
29.15 ± 0.06
40.09 ± 0.01
91.12 ± 0.05
>400
78.25 ± 0.05
50.47 ± 0.06
26.07 ± 0.03
60.49 ± 0.05
87.17 ± 0.06
87.21 ± 0.01
75.09 ± 0.05
99.34 ± 0.03
>400
Stem
n-hexane
CHCl3
EtOAc
77.39 ± 0.06
59.25 ± 0.03
31.15 ± 0.05
45.20 ± 0.01
6.30 ± 0.03
-
>400
85.13 ± 0.07
52.26 ± 0.05
30.18 ± 0.01
67.38 ± 0.07
-
89.45 ± 0.06
73.27 ± 0.03
99.19 ± 0.05
40.05 ±0.07
-
Leaves
n-BuOH
e
Trolox
DL-Penicillamine f
2.07 ± 0.09
a
−
Values of OH, total ROS, and ONOO are expressed as the mean
Inhibitory activity of hydroxyl radical generation in 1.0 mM H O and 0.2 mM FeSO . Inhibitory activity of
2 2 4
total ROS generation in kidney post microsomal fraction. Inhibitory activity of authentic peroxynitrite. Trolox
was used as a positive control. f DL-Penicillamine was used as a positive control.
±
standard error of triplicate experiments.
b
c
d
e
In those three studies, Compounds 2, with one aliphatic hydroxyl, one carboxylic acid,
and 3,4-dihydroxy cinnamoyl moieties and 1, with 3-hydroxy cinnamoyl, one aliphatic
•
hydroxyl, one carboxylic acid moieties, showed greater potential to scavenge OH, total
−
ROS, and ONOO with the IC50 values (07.22
±
0.03, 58.10
M), respectively. Compound
free hydroxyl groups and carboxylic acid moiety in its structure showed good antioxidant
±
0.07, and 6.23 ± 0.04 µM
and 09.15
±
0.07, 64.09
±
0.02, and 8.16
±
0.01
µ
3
with two
activity (Table 2). Lactone and carboxylic acid moiety compounds
4
±
displayed moderate
•
activity with IC50 values (19.08
±
0.05, 69.15
±
0.08, and 21.13
-amyrin (
0.09
µM) for OH,
−
total ROS, and ONOO , respectively, for scavenge.
β
5) with one hydroxyl
moiety showed lower activity, but still significant activity in all the scavenging activity
tests (Table 3).
Table 3. Antioxidative activity of Compounds 1-5.
IC50[µM] a
Total ROS c
Compound
•
OH b
− d
ONOO
1
2
3
4
5
09.15 ± 0.07
07.22 ± 0.03
12.17 ± 0.04
19.08 ± 0.05
34.10 ± 0.07
2.51 ±0.03
-
64.09 ± 0.02
58.10 ± 0.07
69.15 ± 0.08
87.91 ± 0.04
148.55 ± 0.05
35.06 ± 0.07
-
8.16 ± 0.01
6.23 ± 0.04
10.27 ± 0.02
21.13 ± 0.09
69.01 ± 0.05
-
e
Trolox
DL-Penicillamine
f
1.09 ± 0.07
a
−
Values of OH, total ROS, and ONOO are expressed as the mean
Inhibitory activity of hydroxyl radical generation in 1.0 mM H
±
standard error of triplicate experiments.
b
c
2
O
2
and 0.2 mM FeSO
4
.
Inhibitory activity of
d
e
total ROS generation in kidney post microsomal fraction. Inhibitory activity of authentic peroxynitrite. Trolox
was used as a positive control. f DL-Penicillamine was used as a positive control.
The antioxidative activity was also examined in terms of the chemical structures
including those of functional radical and its orientation. Hydroxyl groups in the 1,4- and 1,3-
orientation at cinnamoyle are mainly involved in the scavenging potential of compounds
and . Based on these results, a benzene ring where the hydroxyl radical is in 1,4-orientation
allow the oxygen atom to share a positive charge, thereby causing stabilization through
1
2

Pharmaceutics 2021, 13, 402
10 of 15
delocalization and hence increasing the antioxidant activity as was observed in compound
1
. Because of the electron donating effect of the hydroxyl group in 1,3-orientation, it helps
to stabilize positive charge, and this is taught to influence the scavenging ability. This was
observed in case of compound where further enhanced scavenging activity was observed
2
as this compound bear 3,4-dihydroxy cinnamoyl moieties.
The antibacterial and antifungal activities of compounds (
1–5) against the various
bacterial strains Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis,
and Staphylococcus aureus (Table 4) were observed in this study. Table 4, displays the
compounds (
the examined microorganisms, such as Escherichia coli, Salmonella typhi, and Staphylococcus
aureus, the compounds were found to be prominently active. In contrast to compound
compounds and were active against all bacterial strains studied, with an inhibition zone
of 12 0.03 and 11 0.01 mm, respectively. In the same concentration range, compound
demonstrated good antibacterial activity against the test species. Antifungal activity
of these compounds ( ) against Aspergillus flavus, Fusarium solani, Aspergillus fumigatus,
Aspergillus niger, and Canadida glabrata, as shown in Table 5, with an inhibition zone of 7%
to 47%. Compound showed the highest antifungal activity with a 47% to 25% inhibition
zone, while the other two compounds and showed good antifungal activity against the
1–5) from the test samples in terms of diameters of inhibition zones. Against
3–5,
1
2
±
±
3
1–5
2
1
3
fungal strains listed above. In recent years, while technology and medicine have grown
widely, due to a reduction in natural wealth and disadvantages, some countries have
found it necessary to use natural products for several different purposes. As in several
other nations, plants known to individuals with health benefits are collected and used in
Pakistan for the treatment of various diseases. The antimicrobial and antifungal capabilities
of compounds (1–5) isolated from Ballota pseudodictamnus have been identified in this study.
Owing to the strength of widespread resistance to them, the use of certain antibiotics is
no longer recommended. As shown in Table 4, these compounds can also be used instead
of antibiotics; they have shown visible antibacterial activity against Escherichia coli, Pseu-
domonas aeruginosa, Salmonella typhi, Bacillus subtilis, and Staphylococcus aureus. In addition,
against different strains such as Aspergillus flavus, Fusarium solani, Aspergillus fumigatus,
Aspergillus niger, and Canadida glabrata, the antifungal activity of these compounds was
prominent (Table 5).
Table 4. Antibacterial activities of compounds 1–5 (30 µg/mL) against different bacteria strains.
Zone of Inhibition (mm)
Name of
Standard Drug
Levofloxacin
Bacterial Strain
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
E. coli
P. aeruginosa
S. typhi
11 ± 0.01
8 ± 0.02
13 ± 0.09
5 ± 0.053
7 ± 0.02
12 ± 0.03
9 ± 0.02
11 ± 0.04
7 ± 0.02
9 ± 0.01
10 ± 0.01
7 ± 0.08
9 ± 0.06
4 ± 0.03
6 ± 0.01
8 ± 0.09
5 ± 0.06
2 ± 0.04
2 ± 0.05
3 ± 0.02
7 ± 0.02
0 ± 0.01
6 ± 0.06
3 ± 0.04
5 ± 0.03
16 ± 0.01
19 ± 0.02
24 ± 0.03
22 ± 0.02
20 ± 0.01
B. subtilis
S. aureus
Mean ± standard error of triplicate experiments.

Pharmaceutics 2021, 13, 402
11 of 15
Table 5. Antifungal activities of compounds 1–5 (30 µg/mL) against different fungal strains.
Zone of Inhibition (%)
Name of Fungal
Strain
Standard Drug
Miconazole
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
A. flavus
F. solani
45%
30%
33%
21%
25%
47%
34%
39%
25%
29%
35%
27%
30%
17%
22%
22%
9%
25%
15%
17%
11%
7%
100%
100%
A. fumigatus
A. nigar
11%
15%
17%
100%
100%
C. glabrata
100%
The established antimicrobial mechanisms associated with each group of chemicals to
which the isolated compounds belong can explain the antimicrobial potential of Ballota pseu-
dodictamnus solvent soluble fractions and compounds . Disruption of the membrane has
been proposed as one of the possible mechanisms of action [29 30]. The antimicrobial activities
of compounds may also be clarified by this. The bacterial and fungal organisms differed
in their antimicrobial activities. The genetic differences between the microorganisms [29 30
1–5
,
1–5
,
]
may be the explanation for these variations. This is interesting in view of the possibility of
new antimicrobial medicines being produced from natural products.
Based on structure–activity relationships, the hydroxyl substitution pattern on benzene
ring is an indicator of antimicrobial activity for compounds, and the additional hydroxyl
0
group at 4 -position significantly increases the activity, while the methylation of these
hydroxyl groups reduces the antibacterial potential at different levels. Besides this, the
addition of the 3-hydroxyl group seems to enhance the activity [31
reported compounds, and have 3-hydroxyl group and 3,4-dihydroxyl substitution
pattern, respectively, and hence the compound was found more active than compound
followed by compounds 1, 4, and 5 (Tables 4 and 5).
–33]. Here in our
2
3
3
2,
Similarly, the antioxidant properties of the Ballota species growing in Turkey were
investigated by Cito g˘ lu GS et al. The authors recorded the antioxidant properties of ethanol
extracts for superoxide anion formation and lipid peroxidation of 16 species of Ballota culti-
vated in Turkey. The extracts of Ballota glandulosissima, Ballota antalyense, Ballota larendana,
Ballota pseudodictamnus, Ballota macrodonta, Ballota nigra subsp. anatolica, Ballota rotundifolia,
saxatilis, Ballota saxatilis subsp., and Ballota saxatilis subsp. brachyodonta with IC50 values of
0.50 to 0.87 mg/mL exhibited remarkable anti-superoxide anion formation [34].
The in vitro antioxidant function of ethanol extracts extracted from 21 aromatic plants
belonging to the family Lamiaceae has been investigated by Couladis et al. Salvia ringens,
Stachys spruneri, Phlomis lanata, Salvia pomifera, Origanum dictamnus, Ballota pseudodictamnus,
Ballota acetabulosa, Teucrium polium, Calamintha glandulosa, and Micromeria graeca exhibited
the same behavior as
the chemical composition of the Ballota pseudodictamnus essential oil obtained by GC/MS
from the aerial components. Aryophyllene oxide, phytol, and -muurolene were the major
α-tocopherol among the extracts tested [35]. The authors also studied
γ
components of the 52 reported constituents of the crude. In addition, the essential oil for
its antimicrobial activity has been investigated [36].
Previously, our research group reported in vitro antimicrobial, antiprotozoal activities
and heavy metals toxicity of different parts of Ballota pseudodictamnus (L.) Benth. [20]. The
analysis was conducted to verify the function of different sections of Ballota pseudodictamnus
(L.) Benth. in antimicrobial and antiprotozoal terms. These behaviors were then compared
with the toxicity of heavy metals in various parts, plants accumulating in different amounts
in different parts. Ethanolic extract, chloroform, and ethyl acetate fractions in the Ballota
pseudodictamnus (L.) Benth. roots showed antileishmanial activity in in-vitro antileishmanial
results. The fraction of ethanol, n-butanol, and ethyl acetate in the stem showed inhibition
of leishmania amastigote type. Inhibition of the leishmanial parasite was seen in the ethanol

Pharmaceutics 2021, 13, 402
12 of 15
extract, chloroform, and n-butanol fraction in the leaves. Results have shown that different
parts of the plant have different properties of inhibition [20].
Diterpenes and diterpenoids have antioxidant activity. Carnosol and carnosic acid are
inhibitors of lipid peroxidation; they prevent the oxidation of fatty acids, triglycerides, and
low-density lipoproteins in human aortic endothelial cells [37,38]. Under oxidative stress,
nematodes treated with carnosol had a 21% increase in lifespan compared to controls, and
under heat stress increased worm survival was higher by 9% [39]. The combined action of
carnosic acid and carnosol against ROS and lipid radicals makes this diterpenoid tandem
an effective antioxidant defense. In the Ames test, carnosol was found to have significant
antioxidant and anti-mutagenic activity comparable to ascorbic acid [40]. In a micronucleus
test, it was found that carnosol is even more effective than ascorbic acid in protection
against gamma radiation [41]. Carnosol protects cells from eco-toxicants [42].
Diterpenes and diterpenoids are reported for antiviral, antibacterial, antiparasitic,
antifungal, and antiprotozoal action. Some terpenoids are toxic to microorganisms and
insects and play an important role in plant protection [43,44]. Extract of Stevia and its
glycosides (for example, steviol), in addition to their value as sweeteners, has a therapeutic
effect against cystic fibrosis. Carnosic acid and carnosol, isosteviol, andrographolide,
and dehydroabietic acid have several important protective properties, including anti-
tuberculosis and antiseptic, and can be used in the treatment of colds, showing antibacterial
and antiviral activity [45,46].
Kadidiatou O. et al. investigated Plectranthus madagascariensis-isolated antimycobacte-
rial, cytotoxic, and antioxidant activities of abietane diterpenoids. P. madagascariensis’ phyto-
chemical study has resulted in the isolation of five recognized royleanone abietanes, namely
6-β,7- alpha-dihydroxyroyleanone, 7 -alpha-acetoxy-6β-hydroxyroyleanone, horminone,
coleon U quinone, and carnosolone. The first four compounds demonstrated antimycobac-
terial activity against Mycobacterium tuberculosis H Rv (minimum inhibitory concentration
37
for 90 percent inhibition, MIC90 = 5.61–179.60
µM). All the isolated compounds showed
antioxidant activity through single-electron transfer (SET) and/or hydrogen-atom transfer
(
HAT) with compound carnosolon being the most active antioxidant agent [47].
González-Cofrade et al. identified 19 diterpenoids with anti-inflammatory activity.
For their anti-inflammatory activities against NLRP3 inflammasome activation, the authors
assessed a series of dehydrohispanolone derivatives. IL-1 secretion was substantially
inhibited by four derivatives, being the most active (IC50 = 18.7 and 13.8 M, respectively).
Analysis of the expression of IL-1 and caspase-1 showed that these four diterpenoids were
β
µ
β
selective inflammasome NLRP3 inhibitors, confirming the anti-inflammatory properties of
hispanolone derivatives shown earlier [48].
S. Núñez et al. have documented antibacterial activity against three bacteria: Staphy-
lococcus aureus, Escherichia coli, and Mycobacterium smegmatis, two natural diterpenoids
isolated from Azorella compacta together with six semisynthetic derivatives. There was
no antibacterial activity for the natural diterpenoids, mulinolic acid and azorellanol, but
six derivatives, 12-oxo-11,13-alpha, alpha-dihydroxymulin-20-oic acid, 11-oxo-12,13-alpha,
alpha-dihydroxymulin-20-oic acid, 11,12-dioxo-13-alpha-hydroxymulin-20-oic acid, 7-
acetoxymulin-9,12-diene, mulin-9,12-dien-7-ol, and 7-acetoxy-12,13-dihydroxymulin-9-en,
were found to be active against three bacteria tested. They indicated that the antibacterial
activity reported for six semi-synthetic diterpenoids may not be comparable to that reported
for positive control ampicillin, but showed that the biological activity of diterpenoids may
be modified [49].
The efficacy of seven predominant wine terpenoids (i.e., alpha-pinene, limonene,
myrcene, geraniol, linalool, nerol, and terpineol) against foodborne pathogenic bacteria
was determined by Chung-Yi Wang et al., and their antioxidant activities were observed.
Against foodborne pathogenic bacteria, antibacterial activities were observed. The MIC50
and MBC values were 0.420–1.598 and 0.673–3.432 mg/mL for Escherichia coli, Salmonella
enterica, and Staphylococcus aureus, respectively. The fastest free radical scavenging DPPH
(IC50 value = 12.57
±
0.18 mg/mL) and the highest reducing strength (L-ascorbic acid

Pharmaceutics 2021, 13, 402
13 of 15
equivalent 213.7
±
5.27
µg/mL) were shown by the terpenoid alpha-pinene. New possible
sources of natural antibacterial and antioxidant agents for use in the food industry may be
the seven predominant terpenoids in wines that were found in the recorded study [50].
Antibacterial and antioxidant cassane diterpenoids from Caesalpinia benthamiana have
been reported by Rita Akosua Dickson et al. Using the microdilution assay method
and the DPPH spectrophotometric and TBA lipid peroxidation assays, the antibacterial
and antioxidant activities of the isolated cassane diterpenoids were evaluated. For both
Staphylococcus aureus and Micrococcus flavus, Benthaminin 1 was identified as the more active
antibacterial compound with MIC values of 47.8 microM. The more active antioxidant
compound was found to be benthaminin 2 and showed IC50 values of 42.7 microM and
7
4.2 microM for DPPH and TBA assays, respectively. Deoxycaesaldekarin C possessed
both antibacterial and antioxidant activities [51].
Diterpeniod, abietane 7 alpha-acetoxy-6 -hydroxyroyleanone (AHR), obtained from
β
plant extracts, has been stated to be an attractive lead for drug production due to its
established antimicrobial properties by Carlos E et al. [52].
The compliance of the biological effects of terpenoids with the parameters of geropro-
tectors, including primary criteria, was evaluated by Ekaterina Proshkina et al. Among the
different terpenoid groups, the number of substances showing the greatest compliance with
both primary and secondary geroprotector criteria was found. Terpenoids are therefore an
underrated source of potential geroprotectors that can have an effective impact on ageing
and age-related disease mechanisms [53].
4
. Conclusions
The bioassays guided isolation from ethyl acetate soluble fraction of the root mate-
rial of the plant resulted two new compounds ( ) along with three already reported
compounds ( ). The structures of the compounds were elucidated with the help
of 1D and 2D NMR techniques. The compounds were found active against different
1, 2
3–
5
1–5
1–5
microorganisms and exhibited antioxidative properties. The medicinal efficacy of these
compounds will, however, be explored through more in vivo studies.
Author Contributions: Conceptualization: I.A and N.A; methodology: F., A.S., and I.A.; Validation:
F., A.S., A.B., and A.A.; formal analysis: I.A., S.B.A., and R.U.; investigation: F., A.S., I.A., N.A., S.B.A.,
and M.S.; resources: A.B., R.U., and A.A.; data curation: H.M.M., writing—original draft: M.S., N.A.;
writing—review and editing: H.M.M., M.S., I.A., R.U., and A.A.; visualization: I.A., F., A.B., and R.U.;
supervision: N.A. and I.A.; project administration: I.A.; funding acquisition: R.U., A.B., and I.A. All
authors have read and agreed to the published version of the MS. All authors have read and agreed
to the published version of the manuscript.
Funding: Supporting agency is Deanship of Scientific Research King Saud University under Research
Group Number 1440-009.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability: All available data incorporated in the manuscript.
Acknowledgments: All authors wish to thank Deanship of Scientific Research, King Saud University
Riyadh Saudi Arabia via RG 1440-009 for financial support.
Conflicts of Interest: The authors have declared no conflict of interest.
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