Key words
Loranthaceae - Tripodanthus acutifolius - flavonoids - anti-inflammatory - anti-arthritic - TNF-α
AcOEt ethyl acetate
ACT actinomycin D
Aq aqueous
BW body weight
C87
3-phenyl-1-(4-phenyl-2-thiazolyl)-1H-pyrazole-4,5-dione-4-[2-(4-chloro-3-nitrophenyl)-hydrazone]
CARR carrageenan
CC50 cytotoxic concentration 50%
CFA complete Freund’s adjuvant
cLogP n-octanol/water partition coefficient
CMC carboxymethylcellulose
CRP C-reactive protein
DCM/MeOH dichloromethane/methanol
DH2O distilled water
HEP n-heptane
ID50 dose of a drug that causes 50% of the maximum
possible
IND indomethacin
LDH lactate dehydrogenase
LPS lipopolysaccharide
NF-κB nuclear factor kappa-light-chain-enhancer of activated B
cells
RA rheumatoid arthritis
TOFMS time-of-flight mass spectrometry
Introduction
Inflammation is a response of self-protection from the body against the harmful
agents that are involved in damaging living cells and tissues [1]. It provides assistance to the body to get
rid of harmful agents and helps in initiating the process of healing [2]. Inflammation is usually manifested by
redness, swelling, heat, pain, and dysfunction (alteration or functional deficiency
of an organ), in which many kinds of cells (mostly macrophages and dendritic cells)
and active ingredients (cytokines, chemokines, and bioactive amines) participate
[3].
RA is a prevalent form of inflammatory autoimmune disease that affects
1–2% of the world population [4]. RA is an autoimmune disease characterised by chronic inflammation and
proliferation of synovial tissue with invasion of immune cells into the interface
between bone and cartilage [5]. The
inflammatory synovium elicits the generation of a pannus structure, an abnormal
layer of granulation tissue, causing progressive bone erosion and cartilage
destruction [6]. In particular, a
proinflammatory cytokine-mediated inflamed synovium is crucial for early RA disease
progression [7].
The proinflammatory agent TNF-α is a key cytokine of inflammation,
which further manifests in tissue destruction [8]. TNF-α is secreted by the monocytes and macrophages in
response to inflammatory agents, cachexia, and septic shock [9]. In addition to this, the proliferation of
synovial fibroblasts occurs and contributes to the secretion of cytokines and
matrix-degrading proteases, thus leading to pannus formation [10]. This causes the loss of joint function and
mobility, progressive degradation of the cartilage, and deterioration of the bone
[11]. Additionally, the overexpression of
TNF-α and proliferation of synovial cells are main features of
RA, thus being the potential therapeutic targets for managing this condition [12].
Commonly used medications against RA include nonsteroidal anti-inflammatory drugs,
glucocorticoids, and biopharmaceuticals (primarily tumour necrosis factor
inhibitors) [13]. Currently, the treatment of
RA has been revolutionised by the discovery of the role of certain cytokines, in
particular TNF-α, in the pathogenesis of the disease [14]
[15].
The approach of targeting TNF-α has considerably improved the
treatment of RA [16]. Since the first
development of TNF-α inhibitors in RA in the 1980s, different drugs
(infliximab, adalimumab, etanercept, golimumab, and certolizumab pegol) have shown
excellent efficacy [17]
[18]. However, the needs of patients with RA are
still unmet because of the severe side effects, inability to achieve a permanent
cure, and the high expenditure of these drugs [19]. Thus, it has become important and essential to develop safer and
more cost-effective TNF-α inhibitors [20].
Many natural compounds belonging to various classes have been found to reduce
TNF-α levels [21]
[22]. These natural compounds have been found to
interfere with various proinflammatory mediators and targets, such as
NF-κB and other signalling molecules, involved in
TNF-α expression and, thus, could provide alternative means for
treating inflammatory diseases by modulating the production of
TNF-α
[ [23]
[24].
In South American countries (Argentina and Bolivia), the infusion of Tripodanthus
acutifolius (Ruiz & Pav.) Tiegh. species is one option for treating
inflammation problems [25], especially those
of the joints, as well as sprains [26],
luxation, rheumatic pains, and bone fractures [27]. It is a species of the Loranthaceae family, popularly known as
“jamillo”, “jamillu”, or “solda solda o
liga” [28]
[29].
Phytochemical studies of T. acutifolius revealed the presence of several
phenolic acids, hydroxycinnamic acids, tannins, flavonoids, and tripodantosides
[30]
[31]
[32]. Apaza et al. [33] suggested that
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone,
6,2’,4’-trimethoxyflavone,
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone, and
5,4’-dihydroxy-6,7,8-trimethoxyflavone were the main compounds responsible
for the anti-inflammatory action of T. acutifolius. It was also observed that
flavonoids isolated from the leaves of T. acutifolius, like
3-O-diglucosyl-5,7,3’,4’-tetrahydroxy-8-metoxyflavone, among
others, possess antioxidant activity [30].
Thus, T. acutifolius shows an abundance of metabolites that are
pharmacologically active.
Taking this information into account, the present work was carried out to evaluate
the anti-inflammatory and anti-arthritic activities in mice paw oedema of the Aq
extract and four flavonoids isolated from T. acutifolius.
Results and Discussion
The NMR and MS analysis results confirmed the presence of the flavonoids
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone (1),
6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) in the DCM/MeOH
fraction of the Aq extract ([Fig. 1]). All
compounds were identified based on NMR and MS spectroscopic data interpretation with
the compounds described by Apaza et al. [33].
Fig. 1 Chemical structure of
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone
(1), 6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) from T.
acutifolius.
The Aq extract of T. acutifolius did not show statistically significant
cytotoxic effects (CC5098.33 μg/mL, p0.074) when
compared to the positive control (ACT,
CC5010 µg/mL) in the RAW 264.7 cell line
([Fig. 2]). Regarding the cytotoxicity of
the fractions of the Aq extract, the results showed that the HEP
(CC5091.07 μg/mL), DCM/MeOH
(CC5096.92 μg/mL), and Aq
(CC5098.18 μg/mL) fractions did not show relevant
cytotoxicity (p0.074) when compared to the positive control (ACT,
CC5010 µg/mL) in the RAW 264.7 cell
line ([Fig. 2]).
Fig. 2 CC50 values of the LDH (cytotoxicity) assays
calculated for the extract (Aq.E) and fractions (HEP.F, DCM/MeOH.F,
and Aq.F) from T. acutifolius were calculated with Prism v9.0.0
(GraphPad Software) using nonlinear regression, dose-response curves. CI
95%: confidence interval 95%/Tukey’s
multiple comparisons test. (* *p=0.01,
***p=0.001, ###)
The Aq extract of T. acutifolius showed an anti-inflammatory capacity
(inhibition of TNF-α) at an IC5058.39
μg/mL (p0.074) when compared to the positive control (C87,
IC500.055 µg/mL) in the RAW 264.7 cell
line ([Fig. 3]). Concerning the
anti-inflammatory capacity of the fractions from the Aq extract, the results showed
that the DCM/MeOH (IC5043.95 μg/mL)
fraction presented a higher inhibitory activity of TNF-α than the
fractions HEP (IC5078.70 μg/mL) and Aq
(IC5084.70 μg/mL) in the RAW 264.7 cell line
([Fig. 3]).
Fig. 3 Comparative study of the extract (Aq.E) and fractions (HEP.F,
DCM/MeOH.F, and Aq.F) from T. acutifolius with the C87
positive control on TNF-α inhibition
(* p=0.1, * *p=0.01,
***p=0.001).
As shown in [Fig. 4a], the subplantar
injection of CARR induced an oedema that thickened slowly. The oedema size variation
follows a polynomial function. In fact, 2 h after CARR injection, the size of the
oedema reached a maximum, which indicates an acute inflammation. Treatment with the
Aq extract of T. acutifolius (5 mg/kg BW) significantly decreased the
paw oedema circumference size (97.7% of oedema inhibition). Likewise, the
mice treated with the DCM/MeOH fraction (5 mg/kg BW) showed
increased anti-inflammatory activity (97.7% of oedema inhibition) compared
to the HEP (89.31%) and Aq (89.31%) fractions when reaching the 5th
hour after the CARR injection. As expected, the IND positive control (5
mg/kg) also caused a significant inhibition of oedema (57.67%).
Fig. 4
a, b Oedema size evolution over time in the different groups
of mice treated with the extract (Aq.E) and fractions (HEP.F,
DCM/MeOH.F, and Aq.F) from T. acutifolius. Controluntreated
mice. Each value represents the mean±SEM of results from six
animals. **P<0.01 and
***
p<0.001highly
significant difference in comparison with inflamed mice treated with
indomethacin (IND group) (one-way ANOVA followed by Tukey’s multiple
comparisons test).
Regarding the anti-arthritic activity, the subplantar injection of CFA led the paw to
swell gradually for more than 14 days. The curves of the oedema rate versus time
could be divided into two phases. In the first phase, the oedema rate of the
injected footpad increased and reached a peak during the first 3 days. Thereafter,
the swelling slowly subsided until the 7th day, when the paw began to swell again
and peaked in the 2nd week (second phase). The administration of the Aq extract of
T. acutifolius (5 mg/kg BW) significantly inhibited the
development of joint swelling induced by CFA when compared to the IND positive
control (5 mg/kg) as early as 1 day after CFA injection ([Fig. 4b]). Finally, the DCM/MeOH
fraction (5 mg/kg BW) showed higher anti-arthritic activity (97.7%
of oedema inhibition) when compared to the HEP (89.31%) and Aq
(89.31%) fractions during the 1st day after CFA injection, and this higher
potential was maintained until the experiment was terminated on the 14th day.
The subplantar injection of CARR led to a significant increase in the
TNF-α level in serum when reaching the 5th hour
(***p<0.001). However, the Aq extract
of T. acutifolius (5 mg/kg) decreased the TNF-α level
in serum when reaching the 5th hour after CARR injection
(***p<0.001) when compared to the IND
group (5 mg/kg) ([Fig. 5a]). However,
the subplantar injection of CFA led to a significantly higher increase in the
TNF-α level in serum during the first 3 days
(***p<0.001). Yet, the Aq extract of
T. acutifolius (5 mg/kg) decreased the TNF-α level
in serum during the 1st day after the CFA injection
(***p<0.001) when compared to the IND
group (5 mg/kg) ([Fig. 5b]).
Fig. 5
a, b The effects of the extract (Aq.E) and fractions (HEP.F,
DCM/MeOH.F, and Aq.F) from T. acutifolius on
TNF-α induced by CARR and CFA. Controluntreated mice.
Each value represents the mean±SEM of results from six animals.
**P<0.01 and
***p<0.001highly significant
difference in comparison with inflamed mice treated with indomethacin (IND
group) (one-way ANOVA followed by Tukey’s multiple comparisons
test).
Regarding the CRP level and the fibrinogen rate, a highly significant increase was
observed in the CARR group when compared to the control group
(***p<0.001). Yet, the CRP level
significantly decreased (***p<0.001) in
the groups treated with the Aq extract of T. acutifolius (11.77
mg/mL), with the DCM/MeOH fraction (9.72 mg/mL), and IND
(12.94 mg/mL) when compared to the CARR group (23.34 mg/mL) ([Fig. 6a]). Also, the fibrinogen rate decreased
significantly (***p<0.001) in the groups
treated with the Aq extract of T. acutifolius (4.08 g/L), with the
DCM/MeOH fraction (3.76 g/L), and IND (3.31 g/L) when
compared to the CARR group (5.70 g/L) ([Fig. 6b]).
Fig. 6
a, b Study of CRP levels and fibrinogen in mice treated with
the extract (Aq.E) and fractions (HEP.F, DCM/MeOH.F, and Aq.F) from
T. acutifolius induced by CARR. Values represent the
mean±SD (n=6) in each group.
Finally, when compared to the control group, a highly significant increase was
observed in the CFA group (***p<0.001)
regarding the CRP level and fibrinogen rate. Yet, in the groups treated with the Aq
extract of T. acutifolius (21.50 mg/mL), with the DCM/MeOH
fraction (19.45 mg/mL), and IND (25.04 mg/mL), the CRP levels
decreased significantly (***p<0.001) when
compared to the CFA group (33.07 mg/mL) ([Fig. 7a]).
Fig. 7
a, b Study of CRP levels and fibrinogen in mice treated with
the extract (Aq.E) and fractions (HEP.F, DCM/MeOH.F, and Aq.F) from
T. acutifolius induced by CFA. Values represent the
mean±SD (n=6) in each group.
Moreover, the fibrinogen rate decreased significantly
(***p<0.001) in the groups treated
with the Aq extract of T. acutifolius (13.81 g/L), with the
DCM/MeOH fraction (13.49 g/L), and IND (13.04 g/L) when
compared to the CFA group (15.43 g/L) ([Fig. 7b]).
(E)-2’,4’-Dihydroxy-6’-methoxy-chalcone (1),
6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) exhibited a cytotoxic
effect through the LDH assay, with CC50 values of 56.50, 59.80, 89.20,
and 92.01 μM, respectively, in the RAW 264.7 cell line ([Fig. 8]).
Fig. 8 CC50 values of the LDH (cytotoxicity) assays
calculated for the compounds
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone
(1), 6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) from T.
acutifolius. CC50 values were calculated with Prism
v9.0.0 (GraphPad Software) using nonlinear regression, dose-response curves.
CI 95%: confidence interval 95%/Tukey’s
multiple comparisons test (**p<0.01 and
***p<0.001).
Based on the results obtained, only compounds 1 and 2 cause a loss of
cell membrane integrity, causing necrosis of RAW 264.7 cells [34]
[35].
The cLogP of compounds 1, 2, 3, and 4 were 3.62, 3.54,
1.54, and 1.23, respectively. cLogP is a measure that determines the lipophilic
ability of compounds to cross cell membranes. The fact that this value is greater
than 1 means that the compounds have a greater affinity for the octanol phase than
for the aqueous phase and, by extension, the compounds will tend to move through the
epidermal lipid layer. The penetration of the more lipophilic compounds will
generate a greater concentration of them within the cell, which can generate
cytotoxicity.
Analysing the cLogP of the compounds, a relationship between lipophilicity and
cytotoxicity was observed, and this is justified by the fact that increases in
lipophilicity lead to higher cytotoxicity of the compounds.
The flavonoids (E)-2’,4’-dihydroxy-6’-methoxy-chalcone
(1), 6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) inhibited the production
of TNF-α in the RAW 264.7 cell line, with IC50 values of
0.78, 1.43, 5.73, and 9.71 μM, respectively ([Fig. 9]).
Fig. 9 Comparative study of the compounds
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone
(1), 6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) from T.
acutifolius with the C87 positive control on TNF-α
inhibition.
Previous studies indicated that compound 1 inhibited TNF-α
production in B16-F10 (IC500.60 µM), THP-1
(IC501.12 µM), and RAW 264.7
(IC504.6 µM) cells [33]
[34]
[35]
[36]. Regarding compounds 2, 3,
and 4, they inhibited the production of TNF-α with
IC50 values of 1.32, 5.63, and 3.77 μM, respectively,
for B16-F10 cells and 2.38, 12.36, and 8.09 μM, respectively, for
THP-1 cells [33].
Our study confirms that compounds 1 and 2 have higher biological
activity (inhibition of TNF-α production) than compounds 3 and
4. This effect is due to the lipophilic capacity of compounds 1
and 2. However, the smaller TNF-α inhibition potential of
compounds 3 and 4 is due to the degree of hydroxylation, with compound
3 being more active than compound 4 ([Fig. 1]).
All flavonoids decreased the TNF-α level in serum when reaching the
5th hour after the CARR injection
(***p<0.001). Nevertheless,
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone (1)
and 6,2’,4’-trimethoxyflavone (2) showed a higher decrease in
the TNF-α level in serum when reaching the 5th hour after the CARR
injection (***p<0.001) when compared to
the IND group (5 mg/kg) ([Fig. 10a]).
Additionally, all flavonoids (5 mg/kg) decreased the TNF-α
level in serum during the first 3 days after the CFA injection
(***p<0.001). Yet,
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone (1)
and 6,2’,4’-trimethoxyflavone (2) decreased the
TNF-α level in serum after the 1st day following the injection of
CFA (***p<0.001) when compared to the IND
group (5 mg/kg) ([Fig. 10b]).
Fig. 10
a, b The effects of compounds
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone
(1), 6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) from T.
acutifolius on TNF-α induced by CARR and CFA. Control
(untreated control mice). Each value represents the mean±SEM of
results from six animals. **P<0.01 and
***p<0.001highly significant
difference in comparison with inflamed mice treated with indomethacin (IND
group) (one-way ANOVA followed by Tukey’s multiple comparisons
test).
Regarding compound 1, our results are comparable to those described by
Takahashi et al. [37], who mention that
compound 1 decreased TNF-α levels in murine model assays at
concentrations between 0.02–2 mg/kg BW.
The CARR and CFA assays have long been accepted as useful tools to investigate the
development of new drugs with anti-inflammatory and anti-arthritic activity [38]. Recent studies revealed that cytokine
production plays an essential role in the development of RA [39]. The complicated network of cytokines
involved in RA is formed by mutual regulation in the synthesis and secretion of
various cytokines, with TNF-α being the most representative cytokine
that exerts a synergistic effect and facilitates the immune pathological process of
RA [40]
[41].
Statistical analysis revealed that compounds 1 and 2 and IND
significantly inhibited the development of oedema when reaching the 5th hour after
treatment (***p<0.001). Both showed
anti-inflammatory effects on the paw of mice with CARR-induced oedema. Likewise,
compounds 1 and 2 and IND significantly
(***p<0.001) inhibited the
development of oedema in the first 3 days after the injection with CFA in the paws
of mice.
Analysing the anti-inflammatory action that many flavonoids possess, we observed that
they are related to the inhibition of various enzymes involved in the inflammatory
process. Polyhydroxylated flavonoids have been reported to act preferentially via
the 5-lipoxygenase pathway in in vitro assays, while the less hydroxylated
ones fundamentally inhibit the cyclooxygenase pathway. However, in in vivo
assays, there are reports that they behave as dual inhibitors of both pathways. This
difference in behaviour, not exclusive to flavonoids, is due to the
biotransformation (methoxylation) they undergo in the body [42].
In our case, we observed that the anti-inflammatory activity of flavonoids not only
depends on the degree of hydroxylation, but also on the lipophilic capacity. Our
results show that compound 1 has a greater in vitro and in vivo
anti-inflammatory effect when compared to compounds 2, 3, and 4
due to its lipophilic capacity (cLogP3.62), degree of hydroxylation (presence of 2
hydroxyl groups), and chemical structure (open ring C). However, when we compare the
anti-inflammatory activity of compound 2 with compounds 3 and
4, we observe that the activity of compound 2 is mainly due to its
lipophilic capacity (cLogP3.54), since it does not have hydroxyl groups. Finally, in
relation to compounds 3 and 4, we observed that there is a correlation
between the lipophilic capacity and the degree of hydroxylation, with compound
3 being more active than compound 4.
In relation to the CRP level, this decreased significantly
(***p<0.001) in the groups treated
with (E)-2’,4’-dihydroxy-6’-methoxy-chalcone (8.12
mg/mL), 6,2’,4’-trimethoxyflavone (10.17 mg/mL), and
IND (12.85 mg/mL) compared to the CARR group (23.37 mg/mL) ([Fig. 11a]). Lastly, with respect to the
fibrinogen rate, this decreased significantly
(***p<0.001) in the groups treated
with (E)-2’,4’-dihydroxy-6’-methoxy-chalcone (2.36
g/L), 6,2’,4’-trimethoxyflavone (2.68 g/L), and IND
(3.34 g/L) when compared to the CARR group (5.67 g/L) ([Fig. 11b]).
Fig. 11
a, b Study of CRP levels and fibrinogen in mice treated with
the compounds
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone
(1), 6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) from T.
acutifolius by CARR. Values represent the mean±SD
(n=6) in each group.
On the other hand, the CRP level decreased significantly
(***p<0.001) in the groups treated
with (E)-2’,4’-dihydroxy-6’-methoxy-chalcone (14.86
mg/mL), 6,2’,4’-trimethoxyflavone (16.91 mg/mL), and
IND (25.12 mg/mL) when compared to the CFA group (33.95 mg/mL)
([Fig. 12a]). Finally, with respect to
the fibrinogen rate, this decreased significantly
(***p<0.001) in the groups treated
with (E)-2’,4’-dihydroxy-6’-methoxy-chalcone (11.19
g/L), 6,2’,4’-trimethoxyflavone (11.51 g/L), and IND
(13.07 g/L) when compared to the CFA group (15.64 g/L) ([Fig. 12b]).
Fig. 12
a, b Study of CRP levels and fibrinogen in mice treated with
the compounds
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone
(1), 6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) from T.
acutifolius by CFA. Values represent the mean±SD
(n=6) in each group.
Haematological changes that occur after the administration of CARR or CFA confirm the
resulting acute or chronic inflammation. These changes led to an impressive increase
in blood cells and platelets at the site of inflammation due to the release of
inflammatory cytokines. Likewise, the inhibition of inflammation in the mice treated
with the compounds (5 mg/kg) was confirmed by the significant decrease in
fibrinogen and CRP levels compared to mice treated with the synthetic drug
(IND).
In conclusion, we found that in assays of short duration, such as CARR-induced
oedema, as well as in assays of longer duration, such as CFA-induced arthritis, the
Aq extract from T. acutifolius at a concentration of 5 mg/kg
significantly inhibited the swelling of the mice feet. These results suggest that
this plant has anti-inflammatory effects on acute experimental arthritis.
Likewise, compounds
(E)-2’,4’-dihydroxy-6’-methoxy-chalcone (1),
6,2’,4’-trimethoxyflavone (2),
5,3’,4’-trihydroxy-6,7,8-trimethoxyflavone (3), and
5,4’-dihydroxy-6,7,8-trimethoxyflavone (4) isolated from T.
acutifolius have a potent suppressive effect in proinflammatory responses,
proving to be an alternative for the treatment of inflammatory diseases. Among the
four compounds, the chalcone appears as the most promising compound in terms of
pharmacological activity, and this is due to the fact that it misses the C ring.
Similarly, our results support the traditional use of T. acutifolius as an
anti-inflammatory treatment, showing evidence that the compounds isolated from the
Aq extract inhibit the production of the TNF-α cytokine. In this
sense, our study contributes towards the discovery of compounds capable of
modulating the activities of TNF-α.
Materials and Methods
Plant material
The leaves of T. acutifolius were collected from the Titicani-Tacaca
community, Ingavi province, La Paz, Bolivia
(16°41’45.6"S 68°51’04.5"W), in
July 2018, at an altitude of 3901 m. The botanical identification was confirmed
by the National Herbarium of Bolivia (No. 12779).
General experimental procedures
TLC was performed using Merck silica gel 60-F254 plates. The chromatograms thus
obtained were visualised by UV absorbance (254 nm) and through heating a plate
stained with 5% of phosphomolybdic acid
(H3PMo12O40). Open column chromatography
was performed with silica gel (20–45 and 40–63
μm), and the indicated eluent was in accordance with standard
techniques.
NMR experiments were performed on a Bruker Analytische Messtechnik GmbH
spectrometer operating at 500 MHz (1H) or 125 MHz (13C)
and a Bruker BioSpin GmbH operating at 700 MHz (1H) or 175 MHz
(13C). The deuterated solvents were MeOD-d4,
CDCl3-d1 and D2O-d2. Spectra
were calibrated by the assignment of the residual solvent peak to
δ
H 7.26 ppm and δ
C 77.16
ppm for CDCl3-d1, δH 3.31 ppm and
δc 49.0 ppm for MeOD-d4 and
δH 4.79 ppm for D2 O-d2. The
complete assignment of protons and carbons was done by analysing the correlated
1H-1H COSY, HSQC and HMBC spectra. HREIMS analyses
were performed using a QSTAR XL quadrupole TOFMS. MS samples were prepared in
MeOH.
Preparation of aqueous extract of Tripodanthus acutifolius and
isolation of compounds
Air-dried leaves (500 g) were extracted by decoction (30 min with boiling
DH2O; 500 g). The resulting Aq solution was placed in glass
containers weighing approximately 100 g per tray. The glass containers were
placed in a lyophilisation tunnel for their respective drying for a period of
2–3 h at a pressure of 0.3–1 torr with an initial temperature of
25 ℃ until reaching a temperature of 60 ℃
(approximately).
Subsequently, the Aq extract (240 g) was further extracted with HEP,
DCM/MeOH, (1:1) and DH2O. As a result, three fractions of 15,
70, and 135 g, respectively, were obtained. Each fraction was evaluated for its
cytotoxicity and anti-inflammatory activities.
The DCM/MeOH fraction (50 g) was selected as the most active one and was
fractionated using a chromatographic column (silica gel; 40–63
μm) using a step gradient of HEP/AcOEt
(7:1→3:1) to produce 13 fractions (I-XIII). Fractions X (2.5 g),
XI (3.7 g), and XII (2.8 g) were observed to be the most
active (anti-inflammatory activity).
The chromatographic profile analysis of these fractions (X, XI, and
XII) showed that they were similar, therefore they were pooled. A
second chromatographic column (silica gel; 20–45 μm) was
performed using a step gradient HEP/AcOEt (3:0→0:3). Ten
fractions (1–10) were obtained. All ten fractions showed
anti-inflammatory activity, but fractions 2, 5, 8, and
9 were the most active.
The analysis of the chromatographic profile of fractions 2, 5,
8, and 9 showed that they were pure (> 99%).
They were identified as
(E)-2’4’-dihydroxy-6’-methoxy-chalcone (1 g)
(1), 2’,4’,6-trimethoxyflavone (0.93 g) (2),
3’,4’,5-trihydroxy-6,7,8-trimethoxyflavone (0.75 g) (3),
and 4’,5-dihydroxy-6,7,8-trimethoxyflavone (0.53 g) (4),
respectively.
Cell lines and culture conditions
The used cell line was RAW 264.7 (Mus musculus macrophages, SC-6003). The
cell line was obtained from ATCC. The cells were cultivated in DMEM
(Sigma-Aldrich) containing 2 mM L-glutamine (Applichem), supplemented with
10% FBS (Sigma), 100 units/mL of penicillin (Austria), and
100 µg/mL of streptomycin (Austria) in
culture flasks in a CO2 incubator with a humidified atmosphere
containing 5% CO2 in air at 37°C.
Cytotoxicity assay
For this assay, the protocol developed by Apaza et al. [33] was used. RAW 264.7 cell proliferation
was analysed using a commercial LDH kit (Innoprot Company). Cells were
subcultured in 96-well culture plates at a density of 1×104
cells/well in 100 μL of DMEM medium and incubated
for 24 h at 37°C. After 24 h incubation, the old medium was
removed, and the cells were filled with 100 μL of fresh
medium and treated with the samples.
Stock solutions were prepared by dissolving the extract and the fractions in DMSO
at a concentration of 20 mg/mL. For the compounds, the concentration was
10 mM. Subsequently, a series of dilutions from the stock solutions were
performed until a final DMSO concentration of 0.1% was obtained in each
of the wells for each of the tested concentrations (100, 50, 25, 12.5, 6.3, 3.1,
1.6, 0.8, 0.4, and 0.2 µg/mL or
μM). ACT (≥ 95%; Sigma-Aldrich, CAS Number
50–76–0) was used as a positive control at a concentration of
10 µg/mL (equivalent to
0.008 µM for the compounds).
The treated plates were incubated in a humidified incubator at
37°C under a 5% CO2 atmosphere for 6 h. After
the treatment with the samples, 100 µL of culture medium
supernatants were collected and incubated in the reaction mixture of the LDH
kit. After 30 min, the reaction was stopped by the addition of 1 N HCl, and the
absorbance at a wavelength of 490 nm was measured using a spectrophotometric
ELISA plate reader, SpectraMax i3; Molecular Devices).
TNF-α inhibition assay
The protocol developed by Tena Pérez et al. [43] was used for this assay. RAW 264.7 cell
(1×104 cells/well) were seeded on 96-well culture
plates and incubated for 6 h. The cells were then pretreated with various
concentrations of the samples (extract, fractions, and compounds) for 2 h before
stimulation with 0.1 μg/mL of LPS with or without
samples for 6 h. Supernatants were then collected and the protein expression
levels of TNF-α were measured by using an ELISA kit according to
the manufacturer’s instructions (Diaclone Company). Absorbance was read
at 450 nm on a spectrophotometric ELISA plate reader (Anthos 2020, Version
2.0.5; Biochrom Ltd.). The percentage of TNF-α inhibition was
calculated from the ratio between the observed TNF-α amount
secreted by treated cells (µg/mL or μM)
and the baseline secretion of TNF-α (pg/mL). C87
(≥ 98%; Sigma-Aldrich, 332420–90–3) was used as
a positive control at a concentration of
0.055 µg/mL for the extract and fractions,
equivalent to 0.11 µM for the compounds. Results were
normalised to the DMSO solvent control (1%).
Animals
Male Swiss mice (25–35 g) housed at 22±2°C and
with access to food and water ad libitum were used in these experiments.
Experiments were performed during the light phase of the cycle. The animals were
allowed to adapt to the laboratory for at least 1 h before testing and were used
only once. Experiments reported in this study were performed after approval,
with protocol no. 127, by the Institutional Ethics Committee of our University
on June 13, 2020 and were carried out in accordance with the “Principles
of Laboratory Animal Care” from NIH publication 85–23.
Anti-inflammatory activity: measurement of paw oedema
The anti-inflammatory activity of the Aq extract, fractions, and compounds of
T. acutifolius was evaluated by the method of CARR-induced
inflammation in the anterior paw of the mice as described by Abd-Allah et al.
[44], with some modifications. Eleven
groups (six mice for group) were selected for this study. Group 1 served as the
negative control and received normal saline solution at a dose of 5
mL/kg (p.o.), group 2 received CARR (300 μg/paw,
50 μL of 0.9% saline solution), group 3 received IND
(98.5–100.5%; Sigma-Aldrich, CAS Number 53–86–1)
(5 mg/kg, p.o.) and served as the positive control, while groups
4–11 were treated with an Aq solution of the extract (5 mg/kg,
p.o.), fractions (5 mg/kg, p.o.), and compounds (5 mg/kg, p.o.)
of T. acutifolius.
Thirty minutes after intraperitoneal treatment of the animals of groups
3–11 by IND and the samples (extract, fractions, and compounds), the
inflammation was induced by an injection of 50 μL of the CARR
solution (type IV; Sigma Chemical Company) into the right hind paw of each
mouse. The thickness of the paw oedema was measured immediately after the CARR
injection, and during the 6 h following the induction of the oedema at an
interval of 1 h after the administration of the edematogenic agent using a
plethysmometer (model 520; IITC Life Science). All the assessments were
performed by the same investigator in order to reduce any potential
inter-operator differences. The size of the oedema was calculated as
follows:
EOedemaXt − X0
Xt: size of the paw oedema after edematogenic agent (CARR) injection
at ‘t’.
X0: size of the paw oedema before edematogenic agent (CARR)
injection.
The anti-inflammatory activity was calculated as a percentage of inhibition of
oedema by the extract in the treated animals under test in comparison with the
CARR control group.
% Inhibition[((X – Y)/X)] * 100
X: increase in paw oedema of the CARR group.
Y: increase in paw oedema of the treated group.
The animals were sacrificed after 6 h. The CARR-induced oedema feet were
dissected and stored at −70°C. Blood samples were taken
from anesthetised mice 6 h after CARR administration and kept at
−70°C. According to the type of assay, samples were taken in
citrate tubes to be centrifuged for 15 min at 4000 rpm, obtaining the plasma to
analyse TNF-α, in heparin tubes to be centrifuged for 15 min at
4000 rpm, obtaining the plasma to analyse CRP, and in citrate tubes to be
centrifuged for 15 min at 4000 rpm, obtaining the plasma to analyse
fibrinogen.
Inflammatory parameters measurement
Measurement of TNF-α serum: Serum levels of TNF-α
were determined using an ELISA kit according to the manufacturer’s
instructions (Diaclone Company). Absorbance was read at 450 nm on a
spectrophotometric ELISA plate reader (Anthos 2020, Version 2.0.5; Biochrom
Ltd.). TNF-α was determined from a standard curve.
CRP dosage: The CRP was determined by the automatic analyser
“COBAS INTEGRA@ 400 plus analyser” C-Reactive. The results were
automatically calculated in terms of concentration. The CRP level is expressed
in mg/L of serum.
Fibrinogen dosage: The plasma fibrinogen assay was determined according to
Clauss [45] on an STA line machine. The
plasmas to be tested were loaded into the STA line apparatus where they were
diluted with Owren-Koller buffer (pH7.35) (the dilutions were carried out
automatically by the apparatus). Then, the reagent STA-fibrinogen was added. The
level of fibrinogen in the tested samples is expressed in g/L of
plasma.
Anti-arthritic activity: measurement of paw oedema
Experimental arthritis was induced in mice according to the method proposed by
Newbould [46], with some modifications.
Eleven groups (six mice for each group) were selected for this study. Group 1
served as the negative control and received normal saline solution at a dose of
5 mL/kg (p.o.), group 2 received 0.05 mL (s.c.) of CFA (1%
suspension in olive oil; Difco), and group 3 received IND (5 mg/kg,
p.o.) in 1% CMC as a positive control. Mice in groups 4–11 were
treated with an Aq solution of the extract (5 mg/kg, p.o.), fractions (5
mg/kg, p.o.), and compounds (5 mg/kg, p.o.) of T.
acutifolius 24 h before an injection of CFA and then with a daily
treatment until 14 days after the CFA injection. The oedema and inflammatory
parameters were measured with the same methods described above.
Statistical analysis
CC50 and IC50 values were determined by nonlinear
regression. All experiments were performed in triplicate. One-way ANOVA
statistical analysis (Tukey’s multiple comparisons test;
**p<0.01 and
***p<0.001) was performed to evaluate the
significant differences among values. All analyses were performed using GraphPad
Prism, version 9.0.0., software.
Regarding the results of the inflammatory assays in plantar oedema, the data are
presented as means±S.E.M., except for ID50 values
(i. e., the dose of extract, fractions, and isolated compounds from
T. acutifolius reducing the inflammatory response by 50%
relative to the control value), which are reported as geometric means
accompanied by their respective 95% confidence limits. Data were
subjected to Student’s unpaired t-test or to analysis of variance
(ANOVA) complemented by Tukey’s multiple comparisons test post hoc. A
**p<0.01 and
***p<0.001 was considered as
indicative of significance. The ID50 values were determined by linear
regression from individual experiments using GraphPad Prism, version 9.0.0.,
software.
Supporting information
1H- and 13C- NMR, 1H-1H COSY, HSQC, HMQC,
HMBC, and MS spectra for the different fractions assayed in this study together with
isolated compounds are provided as Supporting Information (Fig. 1S–31S).
