Key words
Loganiaceae -
Buddleja coriacea
- urinary infections - cystitis - antibacterial - anti-inflammatory - secondary metabolites
AcOEt ethyl acetate
ACT actinomycin D
CC50 cytotoxic concentration 50%
COPD chronic obstructive pulmonary disease
DCM dichloromethane
DH2O distilled water
Et2O diethyl ether
HEX n-hexane
IKKαβ suppresses the degradation of
Iκβα and inhibits the translocation of p65 to
the nucleus
IL-1β interleukin-1β
IL-6 interleukin-6
LDH lactate dehydrogenase
LPS lipopolysaccharides
MIC minimum inhibitory concentration
NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells
QTOF quadrupole time-of-flight
rt room temperature
TLR Toll-like receptor
TMS tetramethylsilane
UTI urinary tract infection
ZI zone of inhibition
Introduction
The excretory system is fundamental for health, as it is responsible for the
elimination of all kinds of unnecessary and detrimental substances and waste. In
this context, UTIs are one of the most frequent infections among the adult
population [1]. However, the increasing
prevalence of antibacterial resistance among common uropathogens requires research
on new therapeutic treatments [2] given that
UTIs are diseases caused by pathogens, predominantly bacteria (such as
Escherichia coli, Staphylococcus aureus, and Klebsiella
pneumoniae) [3].
Infections begin with periurethral contamination of a pathogen that resides in the
intestine that is followed by the pathogen’s colonisation of the urethra and
bladder. In the bladder, pathogens proliferate and can ascend to the kidneys and
reach the vascular system, triggering more serious problems [4]. In this sense, TLRs trigger inflammatory
reactions and bind primarily to LPS found in gram-negative bacteria [5], generating bacterial resistance and the
chronicity of the disease. The chronicity of the disease implies that the
inflammatory response can be prolonged over time, damaging the bladder and mucous
membranes, which is detrimental to the patient's life quality [6].
Inflammation is a common physiological process in many diseases that involves tissue
damage and is often followed by pain [7].
Inflammation plays a major role in UTIs due to continuous exposure to noxious
stimuli in the urine [8]. The protein complex
NF-κB is a cytokine implicated in several inflammatory diseases,
such as rheumatoid arthritis and inflammatory bowel disease. In this sense,
NF-κB cytokine is involved in kidney inflammation caused by UTIs
[9].
In response to different cellular stimuli, NF-κB plays a complex role
in different cell types and different disease stages [10]. NF-κB is activated during
infection and promotes the transcription of inflammatory cytokines such as
TNF-α and IL-6 [4] that are
overexpressed in bladder infections [11].
Another factor to control during an infection is the contact with microorganisms,
such as bacteria, fungi, viruses, and parasites. During infection, TLRs detect the
presence of bacterial components and activate macrophages that produce a series of
proinflammatory cytokines (e. g., TNF-α,
IL-1β, IL-6) that act as a defensive response against the existence
of pathogens [12].
Based on the above, the treatment of UTIs can be facilitated by natural products with
medicinal properties [13]. Many studies have
been conducted on the treatment of urinary problems using natural products with
antibacterial and anti-inflammatory action [14]. These medicinal properties can be attributed to the bioactive
phytochemical constituents of various chemical families such as alkaloids, essential
oils, flavonoids, tannins, diterpenes, and phenolic compounds [14]
[15].
In this context, the Buddleja genus has been used in traditional Chinese
medicine for its anti-inflammatory properties [16]. Within this genus, Buddleja coriacea Remy, known as
“Kiswara or Quishuar” and belonging to the Loganiaceae family, is
native to Bolivia and has been traditionally used in the form of an infusion for the
treatment of ulcers, stomach pain, liver problems, urinary tract inflammation, and
rheumatism [17]
[18]
[19].
The B. coriacea species has been reported to contain flavonoid glycosides,
tannins, alkaloids, and diterpenes [20].
Regarding the pharmacological activities of this plant species, it has been reported
that the DCM/hydroalcoholic and hydroalcoholic extracts have presented
anti-inflammatory activity [20].
This manuscript aims to provide a scientific support for the traditional use of B.
coriacea as a remedy for UTI treatment. For this purpose, a bioguided
phytochemical study has been carried out to identify those compounds present in the
aqueous extract of B. coriacea with antibacterial and anti-inflammatory
activities.
Determination of the antibacterial capacity of the aqueous extract, subextracts,
fractions, and compounds of isolates of B. coriacea was carried out against
K. pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa,
and Serratia marcescens bacteria. On the other hand, for the
determination of the anti-inflammatory capacity has been determined by measuring the
inhibition of NF-κB production in the RM-2 (mouse prostate cell) and
MM14.Ov (mouse ovary cell) cell lines.
Results
An aqueous extract was prepared from dry leaves of B. coriacea in order to
reproduce the traditional use of the plant species [18]. Subsequently, from this aqueous extract, other subextracts were made
in solvents of different polarity to study which compounds are responsible for its
medicinal potential. In order to know the presence of natural metabolites and the
complexity of the initial aqueous extract and subextracts, NMR spectroscopy was
used.
The evaluation of the initial aqueous extract revealed a series of aromatic signals
with low intensity at around δ
H 6.0-8.0 ppm that indicate
the possible presence of compounds containing aromatic rings such as polyphenolic
compounds, flavonoids, or alkaloids. Likewise, the presence of a large group of
signals between δ
H 0-4.0 ppm (aliphatic region) was
observed, which may correspond to protons close to alcohols, ethers, or ketones or
the presence of aliphatic chains or essential oils ([Fig. 1]).
Fig. 1 Comparison of 1H NMR spectra of the aqueous extract
and subextracts of B. coriacea.
The 1H NMR spectrum of the HEX subextract showed a pattern of aromatic
compounds. Around δ
H 7.0 ppm and
δ
H 5.5 ppm, signals of aromatic and vinyl hydrogens
were found. Signals were observed at δ
H 3.8 ppm,
corresponding to neighbouring hydrogens to a heteroatom such as N-CH3 or
O-CH3. Finally, around 1.3–0.9 ppm, typical aliphatic signals
of simple hydrocarbons were observed that did not overlap with the HEX solvent
([Fig. 1]).
Analysing the spectra of the methanolic subextract, we observed that a group of low
intensity signals appeared between δ
H 6.7–7.5 ppm,
corresponding to aromatic signals. Likewise, signals at δ
H
5.08–5.36 ppm (minisignals) corresponding to the presence of an olefinic
bond were observed ([Fig. 1]).
In addition, the study carried out by 1H NMR together with the polarity of
the used solvents allowed to affirm, based on the observed signals, that the HEX
subextract contains more free structures, while the methanolic extract contains more
molecules linked to sugars since the signals between δ
H
2.0-4.0 ppm are more intense, which correspond to flavonoid glycosides.
Next, a comparison was made between the spectra of the HEX subextract and the three
compounds isolated from it, observing how all the signals present in the compounds
are also found in the subextract, although these are of less intensity and are not
well defined since the subextract is a complex mixture of compounds ([Fig. 2]).
Fig. 2 Comparison of 1H NMR spectra of the HEX subextract
and the isolated compounds of B. coriacea.
Regarding the antibacterial capacity, the HEX subextract showed an MIC on the K.
pneumoniae, P. mirabilis, P. aeruginosa, and S. marcescens strains of
37.51–37.92 μg/mL. These results were better
(statistically significant, ***p<0.001)
than those of the aqueous extract and the other subextracts of B. coriacea
([Table 1]).
Table 1 Minimum inhibitory concentration (MIC) of
B.
coriacea extract and subextracts against different
strains of bacteria.
|
Extracts
|
MIC (µg/mL) at 48 h (CI
95%, R2)
|
|
K. pneumoniae
|
P. mirabilis
|
P. aeruginosa
|
S. marcescens
|
|
Ofloxacin
|
10.01 (5.09 to 15.62, 0.9836)
|
10.01 (5.28 to 15.18, 0.9599)
|
10.01 (5.74 to 15.38, 0.9523)
|
10.01 (5.16 to 15.14, 0.9532)
|
|
Aq extract
|
53.54 (48.36 to 58.50, 0.9615)
|
53.19 (48.93 to 58.05, 0.9968)
|
53.60 (48.81 to 58.82, 0.9825)
|
53.84 (48.84 to 58.90, 0.9507)
|
|
HEX subextract
|
37.55 (31.84 to 41.61, 0.9528)
|
37.51 (31.08 to 41.49, 0.9624)
|
37.80 (31.43 to 41.56, 0.9576)
|
37.92 (31.90 to 41.79, 0.9533)
|
|
DCM subextract
|
81.26 (76.92 to 86.86, 0.9853)
|
81.11 (76.34 to 86.94, 0.9944)
|
81.89 (76.78 to 86.04, 0.9526)
|
81.95 (76.58 to 86.31, 0.9649)
|
|
MeOH subextract
|
67.28 (62.20 to 72.63, 0.9672)
|
67.12 (62.17 to 72.94, 0.9558)
|
67.40 (62.37 to 72.74, 0.9964)
|
67.51 (62.36 to 72.21, 0.9529)
|
MIC was calculated using Prism v9.0.0 (GraphPad Software) using nonlinear
regression, dose-response curves. CI 95%: confidence interval
95%/Tukey's multiple comparisons test
(***p<0.001)
These results were confirmed by the ZI assay where the HEX subextract showed a ZI of
33.39–33.90 mm, which was better (statistically significant,
***p<0.001) than the values of the
aqueous extract and the other B. coriacea subextracts ([Table 2]).
Table 2
In vitro culture plates (agar cup plate method)
of B. coriacea extract and subextracts showing the zone of
inhibition (ZI) concentration against different strains of
bacteria.
|
Extracts
|
ZI (mm) at 48 h (CI 95%, R2)
|
|
K. pneumoniae
|
P. mirabilis
|
P. aeruginosa
|
S. marcescens
|
|
Ofloxacin
|
27.58 (22.92 to 32.71, 0.9833)
|
27.98 (22.89 to 32.88, 0.9593)
|
27.58 (22.65 to 32.93, 0.9919)
|
27.50 (22.89 to 32.81, 0.9991)
|
|
Aq extract
|
64.30 (59.68 to 69.42, 0.9929)
|
64.55 (59.10 to 69.93, 0.9556)
|
64.17 (59.93 to 69.43, 0.9663)
|
64.14 (59.43 to 69.34, 0.9668)
|
|
HEX subextract
|
33.53 (28.92 to 38.35, 0.9828)
|
33.90 (28.21 to 38.23, 0.9949)
|
33.50 (28.52 to 38.90, 0.9748)
|
33.39 (28.50 to 38.90, 0.9743)
|
|
DCM subextract
|
85.88 (80.50 to 90.64, 0.9553)
|
85.89 (80.97 to 90.90, 0.9801)
|
85.61 (80.67 to 90.55, 0.9625)
|
85.06 (80.86 to 90.31, 0.9657)
|
|
MeOH subextract
|
81.17 (76.55 to 86.98, 0.9948)
|
81.91 (76.51 to 86.06, 0.9714)
|
81.12 (76.03 to 86.88, 0.9711)
|
81.10 (76.58 to 86.79, 0.9713)
|
IC50 values of the ZI was calculated using Prism v9.0.0 (GraphPad
Software) using nonlinear regression, dose-response curves. CI 95%:
confidence interval 95%/Tukey's multiple comparisons
test (***p<0.001)
Concerning the cytotoxicity of the B. coriacea extracts, the results showed
that the initial aqueous extract
(CC50=91.31–93.86 μg/mL)
was less cytotoxic (nsp=0.074) than the negative control
(untreated cells) in all cell lines (RM-2 and MM14.Ov). This result supports the
traditional use of an aqueous infusion. Additionally, the HEX subextract
(CC50=86.13–86.73 μg/mL)
was less cytotoxic (p=0.0637) than the negative control (untreated cells)
([Table 3]). Finally, the DCM and MeOH
subextracts were discarded for subsequent fractionation because they presented
statistically significant higher cytotoxicity
(***p<0.001) than the negative
control (untreated cells).
Table 3 CC50 values of the LDH (cytotoxicity)
assays calculated for the extract and subextracts from B.
coriacea
.
|
Extracts
|
Cytotoxicity (CC50
µg/mL) at
48 h (CI 95%, R2)
|
|
RM-2
|
MM14.Ov
|
|
Untreated cells
|
97.92 (92.48 to 102.53, 0.9454)
|
98.35 (93.70 to 103.09, 0.9901)
|
|
DMSO
|
20.15 (15.29 to 25.18, 0.9759)
|
20.19 (15.44 to 25.80, 0.9872)
|
|
Actinomycin D
|
0.01 (−3.991 to 3.357, 0.9503)
|
0.01 (−3.725 to 3.233, 0.9844)
|
|
Aq extract
|
91.31 (86.45 to 96.88, 0.9576)
|
93.86 (88.45 to 98.48, 0.9994)
|
|
HEX subextract
|
86.13 (81.89 to 91.94, 0.9891)
|
86.73 (81.47 to 91.58, 0.9932)
|
|
DCM subextract
|
77.71 (72.78 to 82.97, 0.9765)
|
79.37 (74.23 to 84.88, 0.9659)
|
|
MeOH subextract
|
56.68 (51.34 to 61.07, 0.9986)
|
58.56 (53.55 to 63.85, 0.9712)
|
CC50 was calculated using Prism v9.0.0 (GraphPad Software) using
nonlinear regression, dose-response curves. CI 95%: confidence
interval 95%/Tukey's multiple comparisons test
(***p<0.001)
Regarding the anti-inflammatory capacity, the results showed that the HEX subextract
(IC50=35.29–36.06 μg/mL)
presented a higher inhibitory activity of the production of NF-κB
(***p<0.001) than the aqueous
extract and the other subextracts of B. coriacea ([Table 4]).
Table 4 IC50 values of the inhibition of
NF-κB production calculated for the extract and
subextracts from B. coriacea.
|
Extracts
|
Inhibition of NF-κB production
(IC50
µg/mL) at
48 h (CI 95%, R2)
|
|
RM-2
|
MM14.Ov
|
|
Untreated cells
|
19.72 (14.68 to 24.87, 0.9647)
|
19.84 (14.45 to 24.64, 0.9984)
|
|
Celastrol
|
3.33 (-2.23 to 8.32, 0.9888)
|
3.34 (-2.47 to 8.90, 0.9868)
|
|
Aq extract
|
53.27 (48.93 to 58.78, 0.9946)
|
54.16 (49.14 to 59.35, 0.9674)
|
|
HEX subextract
|
35.29 (30.93 to 40.47, 0.9968)
|
36.06 (31.34 to 41.44, 0.9644)
|
|
DCM subextract
|
90.72 (85.05 to 95.55, 0.9937)
|
90.76 (85.18 to 95.38, 0.9938)
|
|
MeOH subextract
|
66.79 (61.07 to 71.35, 0.9563)
|
67.04 (62.17 to 72.05, 0.9789)
|
IC50 was calculated using Prism v9.0.0 (GraphPad Software) using
nonlinear regression, dose-response curves. CI 95%: confidence
interval 95%/Tukey's multiple comparisons test
(***p<0.001)
In relation to the antibacterial capacity of the fractions obtained from the HEX
subextract, fractions F3 and F4 showed an MIC of 30.06–30.25
and 30.01–30.52 μg/mL, respectively (Table
1S, Supporting Information) and values of ZI of 34.03–34.21 and
35.16–35.85 mm, respectively (Table 2S, Supporting Information).
These values were better
(** p<0.05) than those of the
other fractions. Regarding the antibacterial activity of the F1 fraction, it
did not present statistically significant differences (nsp=0.065)
when compared to the other fractions.
On the other hand, regarding the cytotoxicity of the fractions obtained from the HEX
subextract (Table 3S, Supporting Information), a trend of increasing cytotoxicity
was observed as the fractionation proceeded, the first fractions hardly showed any
cytotoxicity while the last fractions turned out to be much more cytotoxic. In this
sense, the fractions that have not shown a statistically significant higher
cytotoxicity (nsp=0.073) than the negative control
(untreated cells) in the LDH assay were F1
(CC50=86.77–86.95 μg/mL),
F3
(CC50=81.57–81.66 μg/mL),
and F4
(CC50=80.14–81.11 μg/mL).
Finally, the anti-inflammatory capacity of the fractions obtained from the HEX
subextract are shown in Table 4S, Supporting Information, observing that fractions
F3
(IC50=30.75–31.44 μg/mL)
and F4
(IC50=30.17–30.41 μg/mL)
presented higher inhibitory activities of the production of NF-κB in
all cell lines than the other fractions
(***p<0.001). Regarding fraction
F1
(IC50=39.33–39.68 μg/mL),
although it presented an inhibitory activity of NF-κB, this was not
higher (not statistically significant, p=0.0246) when compared to the
other fractions.
Considering these results, we can affirm that the fractions of the HEX subextract are
safe for humans and that they present anti-inflammatory activity, although it is
still far from the positive control, celastrol
(IC50=3.32–3.34 μg/mL).
In this sense, fractions F1, F3, and F4 were selected for their
better antibacterial and anti-inflammatory activities and lower cytotoxicity,
focusing on the subsequent isolation and purification of the compounds.
Subsequently, the chromatographic separation of fraction F1 (obtained from the
HEX subextract) was carried out, obtaining a total of six subractions
(F1a–F1f), with fraction F1f (compound 1)
being the most active (Tables 5S–8S, Supporting Information).
Likewise, the chromatographic purification of F3 and F4 (obtained from
the HEX subextract) was carried out, obtaining five and four subfractions,
respectively. Subfractions F3b (compound 2) (Tables
9S–12S, Supporting Information) and F4c (compound
3) (Tables 13S–16S, Supporting Information) were
the most active.
The isolated compounds were identified as
(4aR,4bS,5 S,6aS,6bS,9aR,10aS,10bS)-6b-glycoloyl-5-hydroxy-4a,6a-dimethyl-8-propyl-4a,4b,5,6,6a,6b,9a,10,10a,10b,11,12-dodecahydro-2H-naphtho
[2',1':4,5] indeno [1,2-d][1]
[3] dioxol-2-one (1),
3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl) acrylamide
(2), and
(1β,11β,12α)-1,11,12-trihydroxy-11,20-epoxypicrasa-3,13(21)-diene-2,16-dione
(3) ([Fig. 3]). Compounds 1
and 2 were described as synthesis products by Ryrfeld et al. [21]. and Barf et al. [22], respectively. However, there are no
reports on their chemical characterisation. Finally, in relation to compound
3, it has been isolated from different plant species, such as
Ailanthus altissima
[23] and
Eurycoma longifolia
[24].
Fig. 3 The chemical structures of compounds from B. coriacea:
1
((4aR,4bS,5 S,6aS,6bS,9aR,10aS,10bS)-6b-glycoloyl-5-hydroxy-4a,6a-dimethyl-8-propyl-4a,4b,5,6,6a,6b,9a,10,10a,10b,11,12-dodecahydro-2H-naphtho[2’,1’:4,5]indeno[1,2-d]
[1]
[3] dioxol-2-one), 2
(3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl)acrylamide),
and 3
((1β,11β,12α)-1,11,12-trihydroxy-11,20-epoxypicrasa-3,13(21)-diene-2,16-dione).
Regarding antibacterial activity, the MIC assay showed MIC values of
11.64–11.81 (compound 1), 0.17–0.19 (compound 2), and
0.34–0.36 μM (compound 3). The compounds
obtained showed statistically significant higher antibacterial activity
(***p<0.001) compared to the positive
control (ofloxacin,
IC50=27.58–27.65 μM) ([Fig. 4]).
Fig. 4 Minimum inhibitory concentration (MIC) of B. coriacea
compounds against different strains of bacteria at 48 h. MIC was
calculated using Prism v9.0.0 (GraphPad Software) using nonlinear
regression, dose-response curves. CI 95%: confidence interval
95%/Tukey's multiple comparisons test
(***p<0.001).
However, in the ZI assay, no statistically significant difference (p=0.0869)
was observed between the three compounds when compared to the positive control
(ofloxacin, ZI=23.26–24.10 mm) in K. pneumoniae,
P. mirabilis, P. aeruginosa, and S. marcescens bacteria
([Fig. 5]).
Fig. 5
In vitro culture plates (agar cup plate method) of B. coriacea
compounds showing the zone of inhibition (ZI) concentration against
different strains of bacteria at 48 h. IC50 values of the
ZI were calculated using Prism v9.0.0 (GraphPad Software) using nonlinear
regression, dose-response curves. CI 95%: confidence interval
95%/Tukey's multiple comparisons test
(***p<0.001).
In relation to the determination of the cytotoxicity of the compounds, [Table 5] shows that compound 1
(CC50=88.44–89.24 μM) did not
present more cytotoxicity (p=0.0586) than the negative control (untreated
cells). Although compounds 2
(CC50=69.51–70.28 μM) and
3 (CC50=53.95–55.16 µM)
showed cytotoxicity when compared to the negative control; these cytotoxicity values
were lower than the positive control (ACT,
CC50=0.008 µM)
Table 5 CC50 values of the LDH (cytotoxicity)
assays calculated for the compounds from B.
coriacea.
|
Compounds
|
Cytotoxicity (CC50
µM) at 48 h
(CI 95%, R2)
|
|
BEAS-2B
|
IMR-90
|
|
Untreated cells
|
98.45 (93.90 to 103.35, 0.9783)
|
98.97 (93.75 to 103.84, 0.9635)
|
|
DMSO
|
10.05 (5.75 to 15.08, 0.9634)
|
10.05 (5.36 to 15.56, 0.9506)
|
|
Actinomycin D
|
0.008 (-4.867 to 4.338, 0.9625)
|
0.008 (-4.642 to 4.675, 0.9764)
|
|
Compound 1
|
88.44 (83.43 to 93.39, 0.9897)
|
89.24 (84.74 to 94.96, 0.9827)
|
|
Compound 2
|
70.28 (65.71 to 75.54, 0.9607)
|
69.51 (64.94 to 74.45, 0.9522)
|
|
Compound 3
|
53.95 (48.45 to 58.37, 0.9856)
|
55.16 (50.75 to 60.46, 0.9562)
|
CC50 was calculated using Prism v9.0.0 (GraphPad Software) using
nonlinear regression, dose-response curves. CI 95%: confidence
interval 95%/Tukey's multiple comparisons test
(***p<0.001).
Regarding anti-inflammatory activity, compounds 2
(IC50 =0.15–0.16 μM)
and 3 (IC50=0.33–0.36 μM)
showed an inhibitory capacity of NF-κB production that was higher
(statistically significant, ***p<0.001)
than that of the positive control (celastrol,
IC50=7.41 µM). Regarding compound
1
(IC50=11.25–11.34 μM), its
inhibitory activity against the production of NF-κB was similar to
the positive control (p=0.0865) ([Fig. 6]).
Fig. 6 IC50 values of the inhibition of
NF-κB production, calculated for the compounds from B.
coriacea at 48 h. IC50 was calculated using Prism
v9.0.0 (GraphPad Software) using nonlinear regression, dose-response curves.
CI 95%: confidence interval 95%/Tukey's multiple
comparisons test
(***p<0.001).
Discussion
NMR spectroscopy has recently emerged as a very powerful and versatile tool for the
identification of compounds in plant extracts [25]
[26]. Unlike the most widely
used characterisation methods, such as chromatography, NMR allows investigations to
be carried out both in solids and in solution. Furthermore, in recent decades, NMR
hardware, computer technology, and experimental design have been developed
simultaneously, thus producing a modern generation of NMR spectrometers capable of
probing complex structures. This has resulted in substantial improvements in
spectral data processing and visualisation [27]. In this sense, we were able to identify the presence of different
compounds in the extracts of B. coriacea by means of 1H NMR
spectroscopy, focusing on those that had a higher concentration, which were
(4aR,4bS,5 S,6aS,6bS,9aR,10aS,10bS)-6b-glycoloyl-5-hydroxy-4a,6a-dimethyl-8-propyl-4a,4b,5,6,6a,6b,9a,10,10a,10b,11,12-dodecahydro-2H-naphtho
[2',1':4,5] indeno [1,2-d][1]
[3] dioxol-2-one (1),
3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl) acrylamide
(2), and
(1β,11β,12α)-1,11,12-trihydroxy-11,20-epoxypicrasa-3,13(21)-diene-2,16-dione
(3).
Regarding the antibacterial activity of B. coriacea, there are no previous
reports, however, there are reports on the antibacterial activity of other species
of the genus Buddleja, e. g., the MeOH extract of Buddleja
saligna showed an MIC of 5 mg/mL against K.
pneumoniae, P. aeruginosa, and S. marcescens. When comparing
this to our results, we can confirm the antibacterial potential of the
Buddleja species against gram-negative bacteria. Moreover, we can also
indicate that the HEX subextract of B. coriacea is more active than the MeOH
extract of B. saligna
[28]. Likewise,
there are reports that indicate that UTIs are usually caused by gram-negative
bacteria, which tend to become multiresistant to conventional antibiotics [29]
[30],
thus confirming the importance of B. coriacea extracts as an alternative
treatment.
Moreover, although no reports were found on the cytotoxicity of the B.
coriacea species, it was found that other species of the genus
Buddleja presented cytotoxicity, e. g., the aqueous extract of
Buddleja globosa presented cytotoxicity at concentrations higher than
50 μg/mL [31]. Analysing our results, the extracts obtained from B. coriacea
were less cytotxic compared to other extracts from other Buddleja
species.
On the other hand, previous studies have shown that the hydroalcoholic and
DCM/hydroalcoholic extracts of B. coriacea presented
anti-inflammatory activity (inhibition of the production of COX-2) in vivo in
a range of concentrations around 3 g/kg b.w. [20].
In our study, in vitro assays of the HEX subextract showed that showed
what? after their production of NF-κB is inhibited in the
RM-2 and MM14.Ov cell lines. In addition, the use of solvents of different polarity
allowed for evaluating compounds of a different nature present in the initial
aqueous extract, indicating that in the case of the HEX subextract, there is a
higher relationship between its anti-inflammatory activity and its cytotoxicity. For
this reason, the HEX subextract was fractionated using HEX/AcOEt as the
mobile phase, producing seven fractions that were subjected to antibacterial,
cytotoxicity, and anti-inflammatory assays.
In relation to the antibacterial activity of the isolated compounds, results on
compound 1 contradict previous studies. Wang et al. [32] reported that compound 1 suppresses
the lung antibacterial defence, increasing the infection caused by P.
aeruginosa. In our study, compound 1 has been reported to have
antibacterial activity against gram-negative bacteria such as E. coli or
P. aeruginosa in a concentration range of
0.01-100 μg/mL [33] and against gram-positive bacteria such as S. aureus in a
concentration range of
16-2000 μg/200 μL. Thus,
these results obtained confirm the antibacterial properties of compound 1
against gram-negative bacteria, contradicting the results of Wang et al.
Regarding the antibacterial activity of compounds 2 and 3, there are no
previous studies. However, the three compounds, especially 2 and 3,
have shown better activity than the positive control, which is why they stand as
potential alternatives for the eradication of those bacterial strains that cause
chronic UTIs.
Analysing the mechanism of action based on the positive control (ofloxacin), we can
indicate that the isolated compounds prevent bacterial division, which is achieved
by inhibiting topoisomerases II and IV and DNA gyrase, which are necessary to
complete this process [34].
Regarding the cytotoxicity activity of compounds isolated from B. coriacea,
through an in silico study, it has been reported that compound 1 does
not present significant toxicity [35]. Our
results support that this compound is not cytotoxic. Concerning compound 2,
no previous cytotoxicity studies have been found. However, the results obtained did
not show significant cytotoxicity when compared with untreated cells
(98.45–98.97 μM). Finally, in relation to
compound 3, there are reports on its cytotoxicity against different tumour
cell lines, with an IC50 values of
0.25–0.52 μM [36]. This result is not confirmed in this study. Yet, these contradictory
results can be due to the fact that the studies have been carried out in different
cell lines [37].
Finally, regarding the anti-inflammatory activity of the isolated compounds, compound
1 is a glucocorticoid with powerful anti-inflammatory effects [38] that is used to treat chronic inflammatory
diseases [39]. However, although compound
1 was isolated as a mixture of diastereoisomers, they do not show
significant structural differences, so the activity of both structures can be
assumed to be similar. Moreover, there are previous studies on diastereoisomeric
mixtures with anti-inflammatory activity [40].
It should be noted that glucocorticoids are used in the treatment of lung diseases
such as asthma and COPD because they inhibit the gene expression dependent on
NF-κB [33] and suppress the
production of proinflammatory cytokines. [32].
On the other hand, the mechanism of action of compounds 2 and 3 on the
NF-κB pathway is unknown since there are no previous reports
showing anti-inflammatory activity. A possible mechanism of action may be similar to
the one of celastrol (positive control), which is based on the suppression of
phosphorylation of IKKαβ (it suppresses the degradation of
Iκβα and inhibits the translocation of p65 to the
nucleus) [34]. However, this is only a
hypothesis since there are no other studies that analyse the mechanism of action of
the isolated compounds.
Thus, we can conclude that the isolated compounds have anti-inflammatory properties.
Additionally, by predicting the potential pharmacokinetic properties of the three
compounds using SwissADME, it was determined that all three compounds have
high gastrointestinal absorption and are soluble. These results support the
traditional use of the aqueous extract due to its pharmacokinetic properties.
From in vitro assays, it has been confirmed that the three compounds isolated
from B. coriacea leaves have antibacterial and anti-inflammatory activities,
and they are not cytotoxic, being partly responsible for the ethnopharmacological
activity (treatment of urinary infections) of the plant. This is the first time that
compounds 1, 2, and 3 have been studied for the treatment of
UTIs due to their antibacterial and anti-inflammatory properties.
With the present work, it has been established that the isolated compounds could have
potential use for the development of oral treatments for UTIs, offering an
alternative to those already present on the market. In addition, the isolated
compounds may be useful in the future as structures for the synthetic development of
libraries of compounds that may have better activity. However, the continuation of
bioguided isolation in search of new compounds cannot be ruled out, since hardly any
previous studies were found that report the chemical composition of the species
B. coriacea.
Material and Methods
Bacteria, cell lines, biochemicals, and chemicals
Gram-negative bacteria, K. pneumoniae (BAA-3079), P. mirabilis
(BAA-3087), P. aeruginosa (CRM-9027), and S. marcescens (25419),
were purchased from the ATCC. Animal cell lines RM-2 (CRL-3311) and MM14.Ov
(CRL-6383) were purchased from the ATCC.
Cells were cultured in media DMEM (Sigma-Aldrich). FBS (Summit Biotechnology) and
PBS (SAFC Biosciences, Inc.) were used as culture mediums. L-Glutamine was
obtained from Applichem. Penicillin and streptomycin were purchased from Fisher
Scientific. For both the cytotoxicity and activity assays, the compounds were
dissolved in DMSO (Merck) at a concentration of 10 mM, while extracts
and fractions were dissolved at 20 mg/mL in DMSO. The incubation
condition for all cells was in an atmosphere of 95% air and 5%
CO2 at 37°C.
Samples were separated by column chromatography with silica gel (20–45
μm and 40–63 μm; Merck). TLC (Merck
Silica gel 60-F254 plates) was used for the compounds visualisation
by means of UV light [Spectroline E-Series UV lamp with one longwave
(365 nm) and one shortwave (254 nm) tube, 230 V]
and/or by staining the plate with a 5% phosphomolybdic acid
(Merck) solution in EtOH (Merck) followed by heat application. All organic
solvents that were used for isolating the compounds were purchased from
Merck.
Plant material
The aerial parts of B. coriacea were collected from the Pucarani town, Los
Andes province, La Paz, Bolivia (16°24’13.5”S
68°28’37.7”W), in June 2019, at an altitude of
3865 m. The botanical identification was confirmed by the National
Herbarium of Bolivia (No. 14021).
Extraction and isolation
The air-dried leaves (1 kg) from B. coriacea were extracted by
decoction (30 min at boiling point) with 2 L of DH2O.
The resulting aqueous extract was frozen in glass containers at a temperature of
-38ºC and then lyophilised (freeze dryer, Christ alpha 1e2 LD plus) at
-50ºC. Subsequently, 45 g of the aqueous extract were extracted
by repeated maceration (3 times/24 h/rt) with
500 mL of different solvents, increasing the polarity: HEX, DCM, and
MeOH. Next, the subextracts were filtered, and the respective solvents were
removed by vacuum rotary evaporation at rt (25ºC). As a result, three
subextracts of 1.38, 1.77, and 2.67 g, respectively, were obtained.
The HEX subextract (1.77 g) was selected because it was the most active
one, and was subsequently fractionated using a chromatographic column
(2×30 cm) with Si-60 Silica gel (40-63 μm; Merck)
as a stationary phase and a gradient of HEX/AcOEt (49:1→ 5:1) as
the eluent. A total of seven fractions (F1–F7) were
obtained: F1 (0.204 g), F2 (0.047 g), F3
(0.114 g), F4 (0.045 g), F5 (0.112 g),
F6 (0.074 g), and F7 (0.149 g).
Based on the biological activity data, we could then conclude that fractions
F1, F3, and F4 had lower cytotoxicity. A second
separation of F1 was carried out by using a chromatographic column
(1×20 cm) with Si-60 Silica gel (40-63 μm; Merck)
as a stationary phase and HEX/AcOEt (2:1) as the mobile phase. A total
of six subfractions (F1.a–F1.f) were obtained: F1.a
(2.8 mg), F1.b (3.9 mg), F1.c (4.7 mg),
F1.d (1.1 mg), F1.e (1.8 mg), and F1.f
(4.8 mg), which was compound 1.
A third separation of F3 was carried out by using a chromatographic column
(1×20 cm) with Si-60 Silica gel (40-63 μm; Merck)
as the stationary phase and Et2O/DCM (1:6) as the mobile
phase. A total of five subfractions (F3.a–F3.e) were
obtained: F3.a (5.4 mg), F3.b (1.4 mg), which was
compound 2, F3.c (1.0 mg), F3.d (3.7 mg),
and F3.e (6.8 mg).
Finally, a separation on a silica gel microcolumn was carried out to separate
F4 using DCM/AcOEt (1:2). A total of four subfractions
(F4.a–F4.d) were obtained: F4.a
(2.4 mg), F4.b (1.1 mg), F4.c (1.2 mg,
compound 3), and F4.d (1.8 mg).
NMR analysis
NMR spectroscopy experiments were carried out on Bruker Advance DRX 300 and 500
spectrometers operating at 300 MHz, 500 MHz (1H), or
75 MHz, and 126 MHz (13C) with TMS as the reference
solvent (δ
H 0 ppm). Spectra were calibrated by
assignment of the residual solvent peak to δ
H 7.26,
δ
H 3.31, and δ
C 77.16,
δ
C 49.00 for CDCl3
(≥99.8%; Sigma-Aldrich, CAS Number 865-49-6) and MeOD
(≥99.8%; Sigma-Aldrich, CAS Number 811-98-3), respectively. The
complete assignment of protons and carbons was done by correlated
1H-1H COSY, HSQC, and HMBC spectra analysis.
MS analysis
HR-EI-MS analyses were performed using a mass spectrometer with a hybrid QTOF
analyser, model MAXIS II from Bruker, S.A. Samples were analysed using the
electrospray ionisation technique, by direct infusion at a flow of
3 μL/min, using MeOH (Merck) with
0.1% formic acid (Merck) as the ionising phase. The source parameters
were as follows: end plate offset: 500 V; capillary: 3500; nebuliser:
0.2 bar; dry gas: 2.0 L/min; dry temp.: 250°C;
and mass range of 50-3000 Da.
Broth microdilution method
The MIC values of the samples against the bacterial strains were determined using
the microdilution method in 96-well plates following the procedure described by
Apaza et al. [34]. Ofloxacin
(≥98%; Sigma-Aldrich, CAS Number 82419-36-1) was used as a
positive control (of 10.01 µg/mL, equivalent to
27.67 µM for the compounds). Each MIC determination
was carried out in triplicate.
Agar well diffusion method
Agar well diffusion method was carried out in accordance with the procedure
described by Apaza et al. [41]. Ofloxacin
(≥98% Sigma-Aldrich; CAS Number 82419-36-1) was used as a
positive control (of 10.01 µg/mL, equivalent to
27.67 µM for the compounds). ZI of each bacterial
strain was measured with a calibrated digital Vernier caliper in triplicate.
Cytotoxicity assay
The samples were tested in a panel of two cell lines (RM-2 and MM14.Ov) by means
of the LDH assay in accordance with the procedure described by Apaza et al.
[34]. ACT (≥95%;
Sigma-Aldrich, CAS Number 50-76-0) was used as a positive control at a
concentration of 0.01 µg/mL (equivalent to
0.008 µM for the compounds).
Nuclear factor kappa-light-chain-enhancer of activated B cells inhibition
assay
In vitro anti-inflammatory activity (NF-κB inhibition
assay) was carried out following the procedure described by Apaza et al. [34]. Celastrol (≥98%;
Sigma-Aldrich, CAS Number 34157-83-0) was used as a positive control at a
concentration of 3.34 µg/mL (equivalent to
7.41 µM for the compounds).
Statistical analysis
Cytotoxic concentration 50% (CC50) and inhibitory
concentration 50% (IC50) values were determined by nonlinear
regression. All experiments were performed in triplicate. One-way ANOVA
statistical analysis (Tukey's multiple comparisons test,
** p<0.05,
*** p<0.001) was performed
to evaluate the significant differences among values. All analyses were
performed using 1994–2020 GraphPad Prism Software LLC version 9.0.0.
(86) for Mac (producer Dennis Radushev www.graphpad.com).
Supporting information
1H- and 13C- NMR, 1H-1H COSY, HSQC,
HMQC, HMBC, and MS spectra for the different fractions assayed in this study and
for the isolated compounds (Figs. 5S–33S) as well as
activity data of the B. coriacea fractions from the different samples
(Tables 1S–16S) are provided as Supporting
Information.
