Research Article

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Antitubercular and anti-inflammatory

properties screening of natural products
from Plectranthus species
Joana M Andrade1 , Luı́sa Custódio2 , Alessandra Romagnoli3 , Catarina P Reis1,4 , Maria J
Rodrigues2 , Catarina Garcia1 , Elisa Petruccioli3 , Delia Goletti3 , Célia Faustino4 , Gian M
Fimia3,5 & Patrı́cia Rijo*,1,4
1
Universidade Lusófona’s Research Center for Biosciences and Health Technologies (CBIOS), Campo Grande 376, 1749-024
Lisbon, Portugal
2
Center for Marine Sciences (CCMAR), University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
3
National Institute for the Infectious Diseases ‘Lazzaro Spallanzani’, Via Portuense, 292, 00149 Rome, Italy
4
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon (ULisboa), Av. Prof. Gama Pinto,
1649-003 Lisbon, Portugal
5
Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, Lecce, Italy
*Author for correspondence: Tel.: +351 217 515 500; patricia.rijo@ulusofona.pt

Aim: Confirm the use of Plectanthus spp. plants in traditional medicine, particularly as anti-inflammatory
and anti-infective agents. Materials & Methods: Compounds previously isolated from Plectranthus spp.
were studied for their anti-inflammatory activity using the SNAP assay and RAW 264.7 cells, by the quan-
tification of nitric oxide. An halimane diterpene and its derivatives were tested in infected macrophages
with M. tuberculosis H37Rv, using CFU counts assay, at their minimum inhibitory concentration values. Re-
sults: The isolated compounds tested at noncytotoxic concentrations, did not reveal nitric oxide scaveng-
ing in the S-nitroso-N-acetylpenicillamine and the cellular assays. On the other hand, promising results
were obtained regarding one semisynthetic halimane derivative (11R*,13E)-halima-5,13-diene-11,15-diol),
previously prepared (2.1 × 105 CFU/mL), with an effect similar to the antitubercular drugs ethambutol
(2.0 × 105 CFU/mL) and isoniazid (1.2 × 105 CFU/mL). Conclusion: The present report demonstrates the
relevance of Plectranthus spp. in medicinal chemistry drug development for TB and other infective respi-
ratory complaints. Also, this work suggests that further studies involving other inflammatory mediators
are needed to validate the anti-inflammatory use of these medicinal plants.

First draft submitted: 12 February 2018; Accepted for publication: 27 April 2018; Published online:
29 June 2018

Keywords: anti-inflammatory • Mycobacterium tuberculosis • Plectranthus spp. • SNAP • terpenoids • tuberculosis

The genus Plectranthus (Lamiaceae family), widely distributed in tropical Africa, Asia and Australia, comprise many
plants of medicinal interest with a rich diversity of ethnopharmacological uses, including skin, respiratory and
gastrointestinal complaints, pain, infections and fever [1]. The isolation of the bioactive metabolites from Plectranthus
spp. is important to validate the popular use of these plants. The constituents of Plectranthus plants are mainly
abietane, kaurane, clerodane and labdane diterpenoids, together with ursane, oleanane and lupine triterpenoids,
phytosterols, flavonoids and other polyphenolic compounds [1]. These constituents have been reported in several
scientific journals, and also including in the European Scientific Cooperative on Phytotherapy for their biological
activities. They show a wide range of biological activities, such as analgesic [2], anti-inflammatory [2,3], cytotoxic [4,5]
and antimicrobial [3,4,6,7]. Some of these compounds (Figure 1), previously isolated by our research group, are
herein studied as anti-inflammatory and antimycobacterial agents.
The causative agent of TB, Mycobacterium tuberculosis (Mtb), has evolved elaborate survival mechanisms in
the human host, allowing it to remain in a clinically latent infection state before eventually progressing to the
active disease [8,9]. Hence, TB remains one of the major causes of death worldwide from an infectious disease,
according to the WHO [10]. Moreover, TB treatment regimens require a minimum of 6 months of therapy with

10.4155/fmc-2018-0043 
C 2018 Newland press Future Med. Chem. (2018) 10(14), 1677–1691 ISSN 1756-8919 1677
Research Article Andrade, Custódio, Romagnoli et al.

O-
COOH
O P O- OH
O OH
P O
-O O
AcO
H H H H

11R*-acetoxyhalima-5,13E-dien-15-oic acid
TPP TOH iso-TOH

Figure 1. Chemical structures of (+)-tuberculosinyl diphosphate, (+)-tuberculosinol, (±)-iso-tuberculosinol and

11R*-acetoxyhalima-5,13E-dien-15-oic acid.
iso-TOH: (±)-iso-tuberculosinol; TOH: Tuberculosinol; TPP: Tuberculosinyl diphosphate.

four antitubercular drugs, and the emergence and spread of Mtb multidrug-resistant strains represents a major
challenge to the global control of the disease [8,11]. Under these circumstances, the identification of new therapeutic
agents remains nowadays an essential achievement in order to control Mtb infection.
Royleanone abietanes found in several Plectranthus spp. are highly active against multidrug-resistant-Mtb strains,
showing very low minimum inhibitory concentration (MIC) values, namely 7α-acetoxy-6β-hydroxyroyleanone
1 (MIC 0.0083 μM) and 6,7-dehydroroyleanone 2 (MIC 0.039 μM), when compared with first-line anti-TB
drugs, isoniazid (INH; MIC 0.292 μM) and rifampicin (MIC 0.019 μM) [6]. Although their mode of action is
not yet fully understood, some works tried to give a clue about this issue, which it is probably associated with the
electrophilic nature of the quinone moiety typical of the royleanone skeleton and its ability to generate reactive
oxygen species that can damage the bacterial cytoplasmic membrane [6,12].
The H37Rv is the most commonly used laboratory strain of Mtb for the studies of virulence properties in human
macrophages and animal models. Recent findings reported the existence of two genes found in a small operon,
Rv3377c and Rv3378c, specific to virulent Mycobacterium strains [13,14] that encode two enzymes involved in
virulence factor formation, essential for bacterial survival inside macrophages. The Rv3377c enzyme is a diterpene
cyclase that catalyzes the conversion of geranylgeranyl diphosphate into tuberculosinyl diphosphate, while Rv3378c
enzyme is a diterpene synthase that converts tuberculosinyl diphosphate into tuberculosinol and iso-tuberculosinols,
bicyclic diterpenes responsible for the inhibition of phagolysosome maturation and macrophage phagocytosis [15,16].
Interestingly, the structure of these compounds is very similar to the halimane diterpene 8 isolated from Plectranthus
ornatus (Figure 1) [7], hence in the present study, compound 8 and several semisynthetic derivatives 9–11 were
tested ex vivo, in peripheral blood mononuclear cell (PBMC)-derived macrophages infected with Mtb H37Rv.
Several Plectranthus spp. have also been referred for their anti-inflammatory activity [1,2]. Among the bioactive
constituents, forskolin-like diterpenoids such as 5–7 are particularly promising [3,17]. Additionally, other secondary
metabolites commonly found in Plectranthus spp., namely triterpenes belonging to the oleanane and ursane series
(like α-amyrin 12 and β-amyrin 13), phytosterols (including β-sitosterol 14 and stigmasterol 15) and polyphenols
(such as rosmarinic acid 16), have been validated for anti-inflammatory purposes [18,19].
Many inflammatory conditions are associated with increased production of prostaglandins from arachidonic
acid, catalyzed by the cyclooxygenase (COX) enzyme, which has two isoforms. The constitutive enzyme, COX-1,
is mainly responsible for the synthesis of cytoprotective prostaglandins in the GI tract, and thromboxane in blood
platelets, while the inducible enzyme COX-2, which is overexpressed mainly in macrophages and monocytes in
response to inflammatory stimuli, is involved in the synthesis of proinflammatory prostaglandins associated with
pain and fever [20].
Nitric oxide (NO), formed by oxidation of L-arginine catalyzed by NO synthase (NOS), is also produced in large
amounts during inflammation by overexpression of inducible NOS in macrophages and neutrophils, in response
to proinflammatory mediators [21,22]. The cross-talk between the NOS and COX pathways has been reported in
the literature and it has been demonstrated, both in vitro and in vivo, that NO activates the COX enzymes leading
to increased production of prostaglandins [19].

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 Antitubercular & anti-inflammatory properties screening of natural products from Plectranthus species Research Article

Conventional nonsteroidal anti-inflammatory drugs are nonselective inhibitors of the COX enzyme, being
associated with several adverse side effects, including GI disturbances, inhibition of platelet aggregation, liver injury
and kidney toxicity [20]. Although bleeding and gastric ulceration have been attributed to intense inhibition of
COX-1, especially with regular uptake of nonsteroidal anti-inflammatory drugs in chronic inflammatory diseases
(e.g., rheumatoid arthritis, ostheoarthritis), some selective COX-2 inhibitors have been associated with severe
cardiovascular side effects due to an imbalance between prostacyclin and thromboxane production [20]. Oleanolic
acid, a pentacyclic triterpene isolated from Plectranthus ecklonii, was found to selectively inhibit COX-2 with an IC50
of 87 μM [23], highlighting the need to further investigate Plectranthus plants to identify potential anti-inflammatory
agents.
In the present study, the anti-inflammatory effect of diterpenes and triterpenoids (Figure 2) previously isolated
from Plectranthus spp. on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was assayed in vitro by NO
quantification. This particular cell line was chosen since it expresses the TLR4 receptor that triggers production
of NO (among other inflammatory mediators) upon incubation with the bacterial endotoxin LPS, a recognized
inducer of inflammation responsible for the development of systemic inflammatory response syndrome in the course
of sepsis due to Gram-negative bacteria [24]. In this context, a reduction in NO production after treatment with
the selected compounds is indicative of their potential to attenuate an inflammatory response. In addition, the NO
scavenging potential can be assessed using the S-nitroso-N-acetylpenicillamine (SNAP) assay. In this complementary
in vitro evaluation, it is possible to determine whether isolated compounds act as anti-inflammatory agents, by
scavenging NO produced upon an inflammation process.

Materials & methods

Reagents & materials
LPS from Escherichia coli, staurosporine from Streptomyces staurosporeus, sodium nitrite, sulfanilamide,
N-(1-naphtyl)-ethylenediamine dihydrochloride (NED), SNAP, quercetin, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), ω-nitro-L-arginine methyl ester hydrochloride (L-NAME), epimuqubulin
A, ethambutol (ETAN), INH, Triton X-100, Tween 80 and phosphate-buffered saline (PBS) and Rosmarinic acid
were purchased from Sigma-Aldrich (MO, USA). Phosphoric acid and DMSO were purchased from Merck (NJ,
USA). RPMI medium, fetal bovine serum, trypsin, L-glutamine and penicillin/streptomycin were from Ionza,
while RPMI 1640 medium was from BioWhittaker (MD, USA). Middlebrook 7H9 broth was from Difco, while
10% middlebrook oleic acid albumin dextrose catalase-enriched medium was from Becton-Dickinson (NJ, USA).
Anti-CD14-conjugated magnetic microbeads were provided by Miltenyi (Bergisch Gladbach, Germany). Lym-
phoprep was obtained from Cedarlane. Middlebrook 7H10 agar plates were from BD Bioscience (CA, USA),
262710. CytoTox R
96 nonradioactive cytotoxicity assay kit (G1780) was from Promega (WI, USA). Murine
leukemic monocyte-macrophage cell line RAW 264.7 (Biologic Safety Level 2) was obtained from the Center for
Neurosciences and Cell Biology from the University of Coimbra, Portugal.

Plant material
The Plectranthus spp. medicinal plants were original from South Africa, and were cultivated in Instituto Superior
de Agronomia campus (Lisbon, Portugal). Seven Plectranthus spp. were used in this study: P. grandidentatus Gürke,
P. ecklonii Benth., P. ornatus Codd., P. madagascariensis (Pers.) Benth. P. porcatus van Jaarsv. and P.J.D.Winter,
P. neochilus Schltr. and P. prostratus Gürke. The plants names have been checked with www.theplantlist.org.
Voucher specimens identified by ES Martins are deposited in the Herbarium of the Instituto de Investigação Cientı́fica
Tropical, Lisbon, Portugal, (ref. C. Marques S/N◦ LISC).

Compounds isolation & hemisynthesis

The studied Plectanthus compounds (Figure 2) were previously isolated from Plectranthus spp. acetone extracts, and
obtained by a phytochemical study that was previously performed in the described plants, using bioguided studies.
The structural elucidation of the bioactive metabolites was based in physicochemical data (melting point, specific
rotation), mostly spectroscopic data ultraviolet (UV), infrared (IR), 1D- and 2D- 1 H- and 13 C-nuclear magnetic
resonance (NMR) spectra, mass spectra, elementary analysis and comparison with bibliographic data.
The compounds are further denoted for their sample number described in Table 1. For evidence of purity
(13 C/1 H NMR profiles) of all compounds tested.

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Research Article Andrade, Custódio, Romagnoli et al.

16
OH
OH O
12
O 15
11 17 O 12 HO HO 11 12
4'
20
13
14
1 9
O 9 1' 7'
O 9
14
8
O 2
7
6
4
O 7 O 5
7

H 6
6
19 18 OH H
O 3
1 2

O O
17 16 16

OH 14
14
O O
15
12 O 11 O 11 13
O H 20 17
13
15 20 17 15
11 13 9
20 O O
H 16 1 1
14 8 10 8 O
1
H R
8 O 5 7
4 4 6
6
7 O
4 6 H H
OH
H 19 18 O 19 18 O
HO 19 18 OH
6, R = H
4 5 O 7, R = OH O

15
R2 30 29 30

16 14
20 29
13
1
OR H
19

H 12 18 H
12
11
H 25 26
COOH
9
1
17 28 COOH
28

20 H 27
5
3 H
4 3
6 HO
H HO
19
H
18
24 23 12 13
8, R1 = COCH3, R2 = COOH
9, R1 = COCH3, R2 = COOCH3
10, R1 = H, R2 = CH2OH
11, R1 = H, R2 = CH2OCO(CH2)2CH3
H
29
21 H 28
22
22

18 20 23
12
23
24
26 H
H 25
17
19
H
H
27

H H
H H 3
3 5
5 HO 15
HO 6
14

OH

O OH OH
O

HO 16

OH

Figure 2. Studied natural product compounds isolated from Plectranthus spp., showing the numeration of the terpenoid skeletons.
 Antitubercular & anti-inflammatory properties screening of natural products from Plectranthus species Research Article

Table 1. Terpene, phytosterols, and polyphenols isolated from the Plectranthus spp. plants used in this study.
Plant material Isolated compounds Ref.
P. grandidentatus 7␣-Acetoxy-6␤-hydroxyroyleanone (1) [6,12]
6,7-Dehydroroyleanone (2) [12]
P. ecklonii Parvifloron D (3) [25]
1:1 Mixture of ␤-sitosterol (14) and stigmasterol (15)
P. porcatus (13S,15S)–6␤,7␣,12␣,19-Tetrahydroxy-13␤,16-cyclo-8- [25]
abietene-11,14-dione
(4)
P. ornatus (11R*,13E)-11-Acetoxyhalima-5,13-dien-15-oic acid (8) [26]
Plectrornatine C (5) [27,28]
(11R*,13E)-11-Acetoxyhalima-5,13-dien-15-oic methyl [7,28]
ester (9)
(11R*,13E)-15-Butyryloxyhalima-5,13-dien-11-ol (11)
(11R*,13E)-Halima-5,13-diene-11,15-diol (10)
1:1 Mixture of 1,6-di-O-acetylforskolin (6) and [17]
1,6–di-O-acetyl-9-deoxyforskolin (7)
P. neochilus 3:1 Mixture of ␣-amyrin (12) and ␤-amyrin (13) [17]
Plectranthus spp. Rosmarinic acid (16) [29]

The semisynthetic compounds (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid methyl ester (9) and

(11R*,13E)-halima-5,13-diene-11,15-diol (10) were obtained from naturally occurring halimane 8 by esterifi-
cation with dimethyl sulfate or reduction with lithium aluminum hydride, respectively, while (11R*,13E)-15-
butyryloxyhalima-5,13-dien-11-ol (11) was obtained from compound 10 by reaction with butyric anhydride, as
fully described elsewhere [7]. The chemical structures of these compounds (Figure 2) have been established by
comparing their spectral data with those in the literature and/or with authentic samples.

In vitro anti-inflammatory activity evaluation

The anti-inflammatory activity was performed in two different assays considering NO as the inflammatory target
under study. First, we developed a chemical assay in vitro by taking advantage of the SNAP molecule that has
the ability to release NO into the medium, in the presence of a good anti-inflammatory compound. This process
evaluates the NO scavenging properties of each compound, which is directly related with their anti-inflammatory
activity. Afterward, we evaluated the compounds activity in the NO production inhibition in macrophages under
inflammation process by LPS stimulation.

Chemical NO scavenging quantification

The NO scavenging assay was performed using SNAP as a NO donor [25]. Briefly, 997 μl of the noncytotoxic
compounds according to cytotoxicity results on following section MTT cytotoxic evaluation (100 μg/ml dissolved
in bidistilled water) was incubated with 3 μl of SNAP at 100 mM in plastic cuvettes, whereas L-NAME was used
as a positive control (10 mM). The cuvettes were incubated at 37◦ C for 3 h in the dark. At the end of the reaction
time, 500 μl of the reaction mixtures was collected to new cuvettes and 500 μl of Griess reagent (sulfanilamide
0.5% [w/v], phosphoric acid 2.5% [v/v] and N-(1-naphtyl)-ethyldiamine 0.5% [w/v]) was added and left to
stand for 15 min in the dark, at room temperature. Absorbances were read at 550 nm on a spectrophotometer,
against a negative control (DMSO 1% in bidistilled water [v/v] and reaction mixtures without SNAP). The nitrite
concentration was determined from a linear regression analysis using serial dilutions of sodium nitrite as standard
(1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μM). All determinations were made in triplicate. Results were expressed as
NO release quantification (μM) of the noncytotoxic compounds.

Cell culture
The murine leukemic monocyte-macrophage cell line (RAW 264.7) was kindly provided by the Faculty of Pharmacy
and Center for Neurosciences and Cell Biology (University of Coimbra, Coimbra, Portugal). Cells were maintained
in Roswell Park Memorial Institute (RPMI) culture medium supplemented with 10% fetal bovine serum, 1%
L-glutamine (2 mM) and 1% penicillin (50 U/ml)/streptomycin (50 μg/ml), at 37◦ C in a CO2 humidified
atmosphere of 5%.

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Research Article Andrade, Custódio, Romagnoli et al.

MTT cytotoxic evaluation

Exponentially growing cells were plated at a density of 1.0 × 103 cells/well in 96-well plates and incubated overnight
in RPMI medium. Then, 100 μl of the compounds 1–8 and 12–16 was applied at different concentrations (100,
50, 25, 12.5, 10, 5, 2.5 and 1 μM) for 24 h. Control cells were treated with DMSO 0.5% (v/v) and RPMI medium
(100% cell viability). Epimuqubulin A (51 μM) was used as positive control [26]. Cell viability was evaluated by
the MTT assay as previously described [27]. In short, 20 μl of MTT (5 mg/ml in PBS buffer) was added to each
well. After 2 h of incubation, formazan crystals were dissolved by addition of 150 μl of DMSO and absorbance was
measured at 590 nm in a microplate reader. Results were expressed as cell viability (%) in comparison to controls.
Samples were considered noncytotoxic when cell viability was up to 80%, compared with negative controls, and
used in the assays regarding NO quantification.

Quantification of the NO production under inflammation

The anti-inflammatory properties of selected compounds at noncytotoxic concentrations were accessed as previously
described, using NO inhibitor L-NAME (IC50 = 29 μg/ml) as positive control [27]. Briefly, RAW 264.7 cells were
plated in a 96-well microplate at a density of 2.5 × 105 cells/well with RPMI medium. After 24 h, 50 μl of LPS
(100 ng/ml) and 50 μl of the testing compounds (10–100 μM, according to cytotoxicity results on MTT cytotoxic
evaluation) were dissolved in a serum- and phenol-free RPMI medium, and added to each well. Then, 100 μl
of the supernatants was removed and mixed with 100 μl of Griess reagent (1% [w/v] sulfanilamide, 0.1% NED
and 2.5% [v/v] phosphoric acid), after 24 h incubation at 37◦ C in a humidified atmosphere with 5% CO2 . After
20 min of incubation in the dark, at room temperature, the absorbances were measured at 540 nm on a microplate
reader. The NO concentration was determined from a calibration curve obtained with several solutions of sodium
nitrite (1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μM). Results were expressed as NO production (μM) relative to
LPS-stimulated cells.

Ex vivo antimycobacterial activity

The antimycobacterial activity of the Plectranthus spp.-isolated compounds 5 and 8–11 under study was assessed
after infecting primary human macrophages with Mtb H37Rv. Then, the cells were treated with the compounds
and evaluated the bacterial growth inhibition by colony forming unit (CFU) counts.

PBMC isolation
The PBMC isolation was performed as previously described [28]. Briefly, PBMCs were isolated from freshly
collected buffy coats obtained from healthy voluntary blood donors by Ficoll density gradient centrifugation using
Lymphoprep. Monocytes were purified by positive sorting using anti-CD14-conjugated magnetic microbeads. The
recovered cells were more than 99% CD14+ as determined by flow cytometry with anti-CD14 antibody. Monocytes
were cultured in 48-well plates at 5.0 × 105 cells/ml in ex vivo medium supplemented with 2% human serum and
incubated for 5 days at 37◦ C in a CO2 humidified 5% atmosphere. No antibiotics were added to the cultures.

Lactate dehydrogenase cytotoxic evaluation

The cytotoxicity of compounds 5 and 8–11 to exposed human macrophages was evaluated by the lactate dehydro-
genase (LDH) assay using the CytoTox 96 R
Non-Radioactive Cytotoxicity Assay Kit (Promega G1780). After 48 h
of cell adhesion, the cells were treated with the compounds at concentrations corresponding to their MIC values
against the Mtb H37Rv strain (0.060 μM for compound 5, 0.071 μM for compound 8, 0.110 μM for compound
9, 0.131 μM for compound 10 and 0.106 μM for compound 11), which have been previously determined [6,7].
The antitubercular drugs ETAN (0.196 μM) and INH (0.073 μM), were used as positive controls in the Mtb
infection assay, and were also tested at a concentration tenfold to their MIC values. Besides the kit lysis solution
consisting of 9% (v/v) Triton X-100 in water, corresponding to 100% LDH release, staurosporine (2 μM) was also
used as positive control. The supernatants were prepared in two dilutions (1:10 and 1:100). Release of LDH was
measured by supplying lactate, NAD+ and iodonitrotetrazolium violet as substrates in the presence of diaphorase.
The substrate mixture was prepared using the assay buffer (PBS containing 0.2 g/l KCl, 8.0 g/l NaCl, 0.2 g/l
KH2 PO4 , 1.15 g/l Na2 HPO4 and 1% [w/v] bovine serum albumin) according to fabricant instructions and 50 μl
was added to each well and incubated for 30 min in the dark. After incubation, 50 μl of stop solution (1 M acetic
acid) was added and quantification of the red formazan product was performed from absorbance measurements

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 Antitubercular & anti-inflammatory properties screening of natural products from Plectranthus species Research Article

at 490 nm in a microplate reader. A maximum LDH release control was also performed by freezing the plates at
-80◦ C and heating at 50◦ C.

Macrophage infection with Mtb H37Rv & treatment with selected compounds
Mtb H37Rv (ATCC 27294) was grown with gentle agitation (80 rpm) in Middlebrook 7H9 broth supplemented
with oleic acid albumin dextrose catalase (bovine albumin fraction V, dextrose and catalase; Microbiol [Cagliari,
Italy]), glycerol (0.5% v/v) and 0.05% Tween 80 until the late log phase. One milliliter aliquots of culture
supplemented with 10% glycerol was stored at -80◦ C until use. Vials were thawed and bacterial viability was
determined by CFU counting on Middlebrook 7H11 agar plates.
Macrophages isolated from PBMC were infected in a Biosafety Level 3 laboratory, according to previous described
procedures [28]. Macrophages were infected with a multiplicity of infection of two bacteria/cell. Treatment with
compounds 5 and 8–11 at a concentration corresponding to their MIC values (0.060 μM for compound 5,
0.071 μM for compound 8, 0.110 μM for compound 9, 0.131 μM for compound 10 and 0.106 μM for
compound 11) started 2 h after infection and was kept until the end of the experiment. ETAN (0.196 μM) and
INH (0.073 μM) were used as positive controls at a concentration tenfold to their MIC values. After 48 h of
treatment with the selected compounds, the infected cell cultures were lysed in PBS containing 0.1% Triton X
for the release of intracellular Mtb. Serial dilutions were prepared in PBS containing 0.05% Tween 80. Aliquots
(50 μl) of each Mtb dilution were plated (in triplicate) to determine the intracellular bacterial load by CFU counts
on Middlebrook 7H11 agar plates. Viable bacteria were evaluated after 13 days of incubation at 37◦ C. Values are
expressed as the logarithm of CFU/ml, at 10-3 dilution. The intracellular amount of CFU at time zero was assessed
to ensure that all wells were infected with the same bacterial concentration.

Statistical analysis
Data comparison was conducted with one-way analysis of variance followed by post hoc Tukey honest significant
difference test, for pairwise comparisons, using GraphPad Prism software version 5 (GraphPad Software, Inc., CA,
USA). Values of p < 0.05 were considered statistically significant. All assays were performed at least in triplicate
and results are presented as mean ± standard deviation, or as median ± standard deviation for antimycobacterial
analysis.

Results & discussion

In vitro anti-inflammatory activity
Secondary metabolites 1–8 and 12–16 (Figure 2) isolated from Plectranthus spp. were tested for their ability to
decrease NO production after LPS-induced inflammation in the macrophage RAW 264.7 cell line. With this assay,
we aimed to scientifically validate the anti-inflammatory ethnopharmacological and traditional uses of Plectranthus
spp. plants. Previous cytotoxicity screening of isolated compounds on RAW 264.7 cells was performed by the
MTT assay [27], and results are shown in Figure 3. Only the noncytotoxic compounds were selected for further
anti-inflammatory assays in vitro.
According to Figure 3, at the highest concentration tested (100 μM), rosmarinic acid (16) was the only compound
holding 79.5% of cell viability, followed by the halimane (8), the Plectrornatine C (5), the cycloabietane (4), the 1:1
mixture of β-sitosterol (14) and stigmasterol (15), and the 3:1 mixture of α-amyrin (12) and β-amyrin (13), which
were nontoxic at 50 μM. Regarding dehydroroyleanone (2) and the 1:1 mixture of forskoline-like diterpenoids
(6) and (7), they only held up to 86.1 and 94.3% of cell viability at concentrations lower than 10 and 12.5 μM,
respectively. On the other hand, Parvifloron D (3) and royleanone (1) exhibited low cell viability, being safe only
at 1 μM (87.2%) and 5 μM (80.2%), respectively. Due to high cytotoxicity, compounds 1 and 3 did not proceed
for further anti-inflammatory testing.
To the best of our knowledge, this is the first report of cytotoxicity values for RAW 264.7 cells with the
tested compounds from Plectranthus spp.; the IC50 values obtained from the MTT assay, corresponding to the
concentration of compound required for 50% inhibition in vitro, are expressed in Table 2, in comparison with the
norsesterterpene peroxide epimuqubilin A, considered as the positive control [26].
The lowest IC50 values belong to Parvifloron D (3) (IC50 = 7.41 μM) and royleanone (1) (IC50 = 9.40 μM). In
comparison with the compound with lowest viability, dehydroroyleanone (2) (IC50 = 19.2 μM), compounds 1 and
3 were almost twofold more toxic. Therefore, these two compounds were not selected for the anti-inflammatory
assay due to their high toxicity for RAW 264.7 cells.

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Research Article Andrade, Custódio, Romagnoli et al.

Compound 1 Compound 2 Compound 3 Compound 4

150 150 200
100

% cellular
% cellular

% cellular
viability
150

% cellular
viability

viability
100 100 80

viability
60 100
50 50 40
20 50
0 0 0 0
DMSO 0.5%
100
50
25
12.5
10
5
2.5
1

DMSO 0.5%
100
50
25
12.5
10
5
2.5
1

DMSO 0.5%
100
50
25
12.5
10
5
2.5
1

DMSO 0.5%
100
50
25
12.5
10
5
2.5
1
Compound Compound Compound Compound
concentration (μM) concentration (μM) concentration (μM) concentration (μM)

Compound 5 Compound 6 Compound 8 Compound 12–13

150 150 140 150
120
% cellular

% cellular
% cellular

% cellular
viability

viability
viability

viability
100 100 100 100
80
60
50 50 40 50
20
0 0 0 0
DMSO 0.5%
100
50
25
12.5
10
5
2.5
1

DMSO 0.5%
100
50
25
12.5
10
5
2.5
1

DMSO 0.5%
100
50
25
12.5
10
5
2.5
1

DMSO 0.5%
100
50
25
12.5
10
5
2.5
1
Compound Compound Compound Compound
concentration (μM) concentration (μM) concentration (μM) concentration (μM)

Compound 14–15 150 Compound 16

150
% cellular
viability
% cellular

100
viability

100
50
50

0 0
DMSO 0.5%
100
50
25
12.5
10
5
2.5
1
DMSO 0.5%
100
50
25
12.5
10
5
2.5
1

Compound Compound
concentration (μM) concentration (μM)

Figure 3. Cytotoxicity evaluation of isolated compounds from Plectranthus plants on RAW 264.7 cells. Black bars represent the
concentration at which cell viability was higher than 80% in comparison with the negative control. For chemical structures and
compound number assignment, refer to Figure 2.

Table 2. Effect of the application of selected compounds isolated from Plectranthus spp. on RAW 264.7 cells, expressed as
IC50 values (μM).
Compounds IC50 /μ M
Epimuqubilin A† 37.1
1 9.40‡
2 19.2
3 7.41‡
4 ⬎100
5 64.8
6–7 43.5
8 47.8
12–13 ⬎100
14–15 ⬎100
16 ⬎100
†
Epimuqubulin A was used as the positive control. For chemical structures and compound number assignment, refer to Figure 1.
‡ The two most cytotoxic compounds.

Several natural products from Plectranthus plants have been reported for their anti-inflammatory activity [1–3].
Of these, forskolin-like compounds, such as 5–7, are especially notorious for their anti-inflammatory properties [3].
Additionally, phytosterols, such as β-sitosterol (14) and stigmasterol (15) from P. ecklonii, and triterpenes, like
the mixture of α- and β-amyrins (12–13) isolated from P. neochilus, have been validated for anti-inflammatory
purposes [18]. Rosmarinic acid (16) was expected to show some anti-inflammatory activity, since many polyphenolic

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 Antitubercular & anti-inflammatory properties screening of natural products from Plectranthus species Research Article

Table 3. Nitric oxide scavenging activity of selected compounds isolated from Plectranthus spp.
Compounds NO quantification/μ M
L-NAME† 19.86 ± 0.036
2 1.40 ± 0.006
4 ⬍1.16
5 6.67 ± 0.077
6–7 ⬍1.16
8 ⬍1.16
12–13 ⬍1.16
14–15 ⬍1.16
16 ⬍1.16
Results are expressed as nitric oxide production (μ M).
†
L-NAME was used as positive control.
L-NAME: ␻-Nitro-L-arginine methyl ester hydrochloride; NO: Nitric oxide.

30 ns
No concentration (μM)

NH2 O
O2N • HCI
N N OCH3 20 ns
H
NH2
10
L-NAME (IC50 29 µg/ml) ***
***
0
NC

NC + LPS

L-NAME

12–13

14–15

16

6–7
+ LPS (100 ng/ml)

Figure 4. Nitric oxide production in vitro by lipopolysaccharide-stimulated macrophage RAW 264.7 cells. NC,
NC + LPS (negative control), L-NAME (positive control). Data expressed as the mean ± standard deviation (n = 6). For
chemical structures and compound number assignment, refer to Figure 2.
***p < 0.0001.
L-NAME: ω-Nitro-L-arginine methyl ester hydrochloride; LPS: Lipopolysaccharide; NC: Negative control; ns: Not
significant.

compounds have even been proposed as an alternative natural approach to prevent or treat chronic inflammatory
diseases, due to their anti-inflammatory properties [18].
The NO radical displays important reactivity with certain types of proteins and other free radicals, and its toxicity
becomes adverse when it reacts with superoxide radical, forming a highly reactive peroxynitrite anion (ONOO- ) [29].
Considering this, the noncytotoxic compounds 2, 4–8 and 12–16 were also tested for the NO scavenging activity
assay using SNAP. The NO release quantification from the SNAP corresponds to the ability for the compounds
to make a homolytic cleavage of the S–N bond and release NO, detected with the Griess reagent. Results are
summarized in Table 3.
The positive control L-NAME represents the highest amount of NO scavenged from SNAP (19.9 μM) and only
the compounds 2 and 5 revealed a scavenging activity higher than the negative control of DMSO 1% (1.16 μM).
Thus, similarly to the RAW 264.7 cells’ results (Figure 4), the compounds did not display an anti-inflammatory
capacity by NO scavenging from SNAP, validating the results obtained in the previous cellular assay.
On the following assay, the noncytotoxic compounds 2, 4–8 and 12–16 were evaluated for their effect on in
vitro NO production by LPS-stimulated macrophages, by the Griess assay (Figure 4). This methodology involves
sample treatment with a diazotizing agent, usually sulfanilamide in acidic media, to form a transient diazonium
salt, which is further reacted with a coupling agent, NED to form a stable azo-compound with an intense pink

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Research Article Andrade, Custódio, Romagnoli et al.

color. The compounds were tested at the maximum concentration at which they were noncytotoxic, and results
were compared with L-NAME, an arginine analog competing for NOS and a known inhibitor of NO production
upon inflammation [22].
Treatment of RAW 264.7 cells with LPS resulted in an NO production of 15.7 μM. The tested compounds were
not able to reduce the NO production after LPS-stimulated inflammation (p > 0.05), which was at approximately
the same level (16.1 μM) or slightly raised it to 20.2 μM. This slight increase in NO production after macrophage
treatment with some of the compounds suggests an intensification of the observed inflammation, supporting the
need to better understand the apparent immunostimulation and its possible therapeutic uses in altered innate and
adaptive immune responses.
To the best of our knowledge, this is the first report on the anti-inflammatory effect of such compounds from
Plectranthus spp. tested in vitro with macrophage cells by evaluation of NO production and NO scavenging from
SNAP. Overall, the results suggest that the tested compounds do not exert an anti-inflammatory response through
the NO pathway, but it does not exclude other possible mechanisms of anti-inflammatory action. For sustainable
conclusions, it would be necessary to further evaluate their effect on other inflammation mediators, such as COX-2.
Anti-inflammatory action through COX inhibition has been reported for several secondary metabolites isolated
from medicinal plants [30].
In fact, several diterpenoid compounds have been reported as COX-2 inhibitors [30], while triterpenoids, phy-
tosterols and polyphenolic compounds are known for their anti-inflammatory properties [18].

Antimycobacterial activity
Therapeutic approaches targeting virulence factors, such as the tuberculosinols (tuberculosinol and isotubercu-
losinols) in Mtb, have been attracting considerable attention [13–15,31]. Although not essential for bacterial growth
outside the host cells, virulence factors are involved in invasion, persistence, lysis and evasion of innate immune
system defences. Halimane diterpene 8, isolated from P. ornatus and its semisynthetic derivatives 9–11, due to
their bicyclic diterpenoid skeleton, resemble tuberculosinols and are thus interesting compounds to be studied
as potential inhibitors of tuberculosinol synthase (Rv3378c), blocking tuberculosinol production and ultimately
avoiding phagocytosis arrest. The labdane diterpenoid Plectrornatine C 5 was also studied since some labdane-type
diterpenes have also been referred as substrates for tuberculosinol synthase [13]. Due to the structural similarity
between tuberculosinol synthase (Rv3378c) and the cis-decaprenyl diphosphate synthase enzyme (Rv2361c) in-
volved in cell wall biosynthesis, inhibition of both enzymes is possible. This may contribute to the development of
multitarget inhibitors as a novel therapeutic strategy to simultaneously inhibit cell wall biosynthesis and virulence
factor formation inside Mtb-infected macrophages [15].
Quantitative analysis of Mtb growth via the determination of CFUs per unit volume of culture from serially
diluted suspensions was the method of choice to evaluate the anti-Mtb activity of selected diterpene compounds 5
and 8–11. Although time-consuming (it takes ∼2–4 weeks to obtain a primary culture, and antibiotic susceptibility
can only be determined after an additional 2–4 weeks) and requiring a Biologic Safety Level 3 laboratory, the CFU
assay is considered the most reliable method [32].
In order to avoid false positives resulting from cytotoxicity of the compounds to primary human macrophages,
a previous cytotoxicity evaluation was performed. The cytotoxicity assay measured the release of cytosolic enzyme
LDH after treatment of macrophages, derived from PBMC, with the selected compounds. The anti-TB drugs,
INH and ETAN, used as positive controls in the Mtb infection assay, were also tested to guarantee that they were
not toxic to macrophages at a concentration up to tenfold to their MIC values. Staurosporine (IC50 = 250 nM), a
known protein kinase inhibitor that induces apoptosis, was used as the cytotoxicity positive control [33]. The results
obtained are shown in Figure 5.
According to Figure 5, staurosporine used as positive control resulted in macrophage cytotoxicity of 21.9%
(p < 0.0001). On the other hand, the selected compounds tested, including ETAN and INH did not reveal
significant toxicity toward macrophage cells (p > 0.05), since cell viability was similar to values obtained for
untreated cells used as negative control.
After cytotoxicity evaluation, human primary macrophages obtained from PBMC were infected with Mtb as
described in section ‘Macrophage infection with Mtb H37Rv and treatment with selected compounds’, and then
treated with compounds 5 and 8–11 at a concentration of (0.060 μM for compound 5, 0.071 μM for compound
8, 0.110 μM for compound 9, 0.131 μM for compound 10 and 0.106 μM for compound 11), corresponding to
their MIC values against Mtb, which have been previously determined [6]. However, other concentrations of the

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 Antitubercular & anti-inflammatory properties screening of natural products from Plectranthus species Research Article

H 1.0
N O

0.8

LDH cytotoxicity
0.6
N N

H3C O H 0.4
H3C
O *** ns
0.2
HN
CH3
0.0
Staurosporine IC50 = 250 nm

PC

STS

NT

INH

ETAN

10

11
Figure 5. Cytotoxicity of isolated compounds from Plectranthus spp. Data expressed as median ± standard deviation (n = 3). For
chemical structures and compound number assignment refer to Figure 2.
***p < 0.0001.
ETAN: Ethambutol; INH: Isoniazid; NC: Negative control; ns: Not significant; PC: Positive control; STS: Staurosporine (2 μM, PC).

H
O N
NH2

ns
1,000,000 ns
N

Isoniazid * *
Log CFU

*
MIC = 0.1 µg/ml
100,000
OH
H
N
N 10,000
H NT INH ETAN 5 8 9 10 11
HO

Ethambutol
MIC = 4 µg/ml

Figure 6. Effect of selected compounds, at their MIC values, on Mycobacterium tuberculosis viability on infected
peripheral blood mononuclear cell-derived macrophages. Data expressed as mean ± standard deviation (n = 3). For
chemical structures and compound number assignment, refer to Figure 2.
*p < 0.05 comparing treatment versus no treatment.
CFU: Colony forming unit; ETAN: Ethambutol; INH: Isoniazid; MIC: Minimum inhibitory concentration; ns:
Not significant; NT: Not treated.

compounds should be tested since only MIC was tested. The anti-TB drugs INH and ETAN were used as positive
controls, at concentrations tenfold to their MIC values, while untreated cells were the negative control. Viable
bacteria were evaluated after 13 days of incubation at 10-3 dilution and the results obtained, expressed as logarithm
of CFU/mL, are shown in Figure 6.
Interestingly, the normalized CFU counts (at 10-3 dilution) reported in Figure 6 show that compound 10, a
semisynthetic derivative from the halimane diterpene 8 isolated from P. ornatus, significantly decreased Mtb growth
from 6.0 × 105 to 2.1 × 105 CFU/mL, similar to the positive controls ETAN (2.0 × 105 CFU/mL) and INH
(1.2 × 105 CFU/mL), two known drugs used for the treatment of Mtb infections. The remaining compounds

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Research Article Andrade, Custódio, Romagnoli et al.

revealed approximately the same CFU counts as the nontreated control. Regarding the tested semisynthetic halimane
diterpenes 9–11, only minor structural differences were performed from natural-occurring halimane diterpene 8.
The tested halimanes have in common a lipophilic bicyclic system with a hexenyl side chain bearing two
oxygenated functions at C-11 and C-15, thus showing an amphipatic character that can be determinant for their
interaction with the bacterial cytoplasmic membrane. The lipophilicity of these compounds has been previously
reported [7], and according to the estimated log p-values was found to decrease in the order 11 (6.05 ± 0.87) >9
(5.58 ± 0.87) >8 (5.20 ± 0.48) >10 (4.59 ± 0.72). Thus, lipophilicity may play an important role in the
antimycobacterial properties of the halimane derivatives since only the less lipophilic compound 10 was found to
be active [6].
Interestingly, when tested against Gram-positive bacteria, the less lipophilic compounds 10 and 8 were the more
active ones against a collection of both antibiotic-sensitive and antibiotic-resistant strains of Staphylococcus and
Enterococcus, with MIC values of 0.045–0.089 and 0.102–0.204 μM, respectively [7]. The halimane 10, which is
the less lipophilic compound, is the only one with two hydrogen bond donor/acceptor oxygen atoms, arising from
a primary and a secondary hydroxyl groups at C-15 and C-11, respectively, while the halimane template 8, with an
acidic proton, has a lipophilic character mainly dependent upon pH.
Therefore, the hydrogen donor/acceptor features of the halimanes can also contribute to the observed antimy-
cobacterial activity, since among the more lipophilic compounds, 11 and 9, the first is esterified at C-15 but still has
one secondary hydroxyl group at C-11 while the latter, being esterified at both C-11 and C-15, has no hydrogen
bond donor atoms. Hydrogen bond donor/acceptor groups at C-11, as well as the size of the lipophilic ester at
C-15, may also be relevant for interaction with Mtb enzymes, such as the tuberculosinol synthase (Rv3378c), thus
inhibiting the production of tuberculosinols and ultimately avoiding the phagocytosis suppression [13,15,16], which
would translate into the observed decrease in CFU.
In order to elucidate the mechanism of action of compound 10 on anti-Mtb activity in infected cells, further
studies must be undertaken to evaluate whether this compound exclusively targets Mtb enzymes [6] or if it also
impacts on the host immune response to Mtb infection [8].

Conclusion
The selected natural compounds 2, 4–8 and 12–16 isolated from Plectranthus spp. did not reveal measurable
anti-inflammatory properties, as evaluated by the SNAP assay and quantification of NO production and release
by macrophage 264.7 cells after LPS-stimulated inflammation. However, anti-inflammatory action involving
inflammation mediators other than the NO pathway cannot be excluded.
The CFU assay for evaluation of Mtb H37Rv growth after infection of PBMC-derived macrophages and
treatment with selected diterpene compounds revealed very promising results concerning the halimane derivative
10 obtained by hemisynthesis from the naturally occurring halimane diterpene 8 previously isolated from P. ornatus.
Treatment of the infected macrophages with compound 10 led to bacterial growth inhibition with concomitant
decrease of the observed CFU (2.1 × 105 CFU/ml) to values almost identical with the ones obtained with the
antitubercular drugs (2.0 × 105 CFU/ml) and isoniazid (1.2 × 105 CFU/ml). Lipophilicity and the presence of
hydrogen bond donor groups seem to contribute to the observed activity of the halimane derivative 10. Further
studies for better understanding the mechanisms of action of halimane derivative 10 upon anti-Mtb activity are
perspectivated.
According to the references of this study, this is the first report with in vitro anti-inflammatory and an-
timycobacterial properties of Plectranthus spp.-isolated compounds, with scientific validations upon their known
ethnopharmacological uses. Primarily, we could confirm the antimycobacterial activity on M. tuberculosis H37Rv,
but further studies are required to attain sustainable conclusions regarding the anti-inflammatory activity.

Future perspective
According to WHO Global Tuberculosis Report of 2017, the pipelines for new diagnostics, drugs, treatment
regimens and vaccines are slowly progressing. It is required an increase of investment in research and development
by 2025 [10]. Nevertheless, ending the TB epidemic is a target under the sustainable development goals. This requires
implementing a mix of biomedical, public health target and socioeconomic interventions along with research and
innovation [34].

1688 Future Med. Chem. (2018) 10(14) future science group

 Antitubercular & anti-inflammatory properties screening of natural products from Plectranthus species Research Article

Given the recent renewed interest on medicinal plants and their biological properties, it is relevant to further
improve the studies on Plectranthus spp. concerning the scientific validation of their ethnopharmacological uses, their
toxicity profiles for safety and the unravelling of new bioactive compounds with potential therapeutic applications.
The antimycobacterial activity improvement with the derivatives obtained from natural products of Plectranthus
spp. places these medicinal plants on the course of action against tuberculosis.
Due to the recent discovery of Rv3377c and Rv3378c enzymes that yield halimane-type diterpenes (tuberculosinol
and (±)iso-tuberculosinols) [13–15], in the near future, we will synthesize new derivatives using the natural halimane
as starting point and test them against Mtb and on target-based assays on the enzymes involved in tuberculosinol
biosynthesis. The final compounds can be further developed to a pharmaceutical formulation.

Summary points
• Natural products are a unique source of lead compounds for medicinal chemistry drug development, and
Plectranthus spp. are often used in traditional medicine, for anti-inflammatory purposes and TB.
• The cytotoxicity evaluation of the selected compounds for the anti-inflammatory assay exhibited low-cell viability
for Parvifloron D (3) and royleanone (1).
• 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxic evaluation.
• The lactate dehydrogenase (LDH) assay toward primary macrophage cells did not reveal significant toxicity for
the selected compounds tested for Mycobacterium tuberculosis (Mtb).
• LDH cytotoxic evaluation.
• Noncytotoxic-isolated compounds were unable to reveal in vitro anti-inflammatory activity: neither in the
S-nitroso-N-acetylpenicillamine assay nor in lipopolysaccharide-stimulated RAW 264.7 cells.
• Quantification of the nitric oxide production under inflammation.
• Halimane derivatives 8–11 had no considerable cytotoxic effects up to 25 μg/ml.
• LDH cytotoxic evaluation.
• After infection of primary macrophage cells, halimane derivative 10 decreased colony-forming units of Mtb
H37Rv.
• Macrophage infection with Mtb H37Rv and treatment with selected compounds.
• Further studies must be undertaken to evaluate whether halimane derivative 10 exclusively targets Mtb enzymes,
or if it also impacts on the host immune response to Mtb infection.
• According to the references of this study, this was the first report on Plectranthus spp. concerning preliminary
scientific validation of anti-inflammatory applications and TB, which are known ethnopharmacological uses.

Financial & competing interests disclosure

This work was supported in part by FCT – Fundação para a Ciência e Tecnologia (UID/DTP/04567/2016; PEst-
OE/SAU/UI4013/2014, CCMAR/Multi/04326/2013) and funding from Italian Ministry of Health (‘Ricerca Corrente, Linea 4’
and ‘Ricerca Finalizzata, GR-2011–02350886’) and from Italian Ministry of Ministry of Education, Universities and Research (PRIN
20152CB22L). L Custódio was supported by FCT Investigator Programme (IF/00049/2012). Also, part of the work was performed
under a short-term scientific mission supported by the COST Action CM1407 ‘Challenging organic synthesis inspired by nature:
from natural products chemistry to drug discovery’. The research concerning CFU assay for Mycobacterium tuberculosis H37Rv
growth was developed under the short-term scientific mission COST action CM1407 (Natural diterpenoids as potential antitu-
bercular drugs) at the National Institute for the Infectious Diseases Lazzaro Spallanzani, Laboratory of Cell Biology and Electron
Microscopy, Rome, Italy. The authors have no other relevant affiliations or financial involvement with any organization or entity
with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those
disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

Human blood was collected at the Blood Bank of San Camillo Hospital (Rome, Italy), operating under license from the Italian
Ministry of Health, from healthy volunteers after written informed consent.

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Research Article Andrade, Custódio, Romagnoli et al.

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