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An in vitro study of anti-inflammatory activity of standardised Andrographis paniculata extracts and pure andrographolide

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The anti-inflammatory activity of Andrographis paniculata (Acanthaceae), a traditional medicine widely used in Asia, is commonly attributed to andrographolide, its main secondary metabolite. Commercial A. paniculata extracts are standardised to andrographolide content. We undertook the present study to investigate 1) how selective enrichment of andrographolide in commercial A. paniculata extracts affects the variability of non-standardised phytochemical components and 2) if variability in the non-standardised components of the extract affects the pharmacological activity of andrographolide itself. We characterized 12 commercial, standardised (≥30% andrographolide) batches of A. paniculata extracts from India by HPLC profiling. We determined the antioxidant capacity of the extracts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, oxygen radical antioxidant capacity (ORAC) and a Folin-Ciocalteu (FC) antioxidant assays. Their anti-inflammatory activity was assessed by assaying their inhibitory effect on the release of tumor necrosis factor alpha (TNF-α) in the human monocytic cell line THP-1. The andrographolide content in the samples was close to the claimed value (32.2 ± 2.1%, range 27.5 to 35.9%). Twenty-one non-standardised constituents exhibited more than 2-fold variation in HPLC peak intensities in the tested batches. The chlorogenic acid content of the batches varied more than 30-fold. The DPPH free radical scavenging activity varied ~3-fold, the ORAC and FC antioxidant capacity varied ~1.5 fold among batches. In contrast, the TNF-α inhibitory activity of the extracts exhibited little variation and comparison with pure andrographolide indicated that it was mostly due to their andrographolide content. Standardised A. paniculata extracts contained the claimed amount of andrographolide but exhibited considerable phytochemical background variation. DPPH radical scavenging activity of the extracts was mostly due to the flavonoid/phenlycarboxylic acid compounds in the extracts. The inhibitory effect of andrographolide on the release of TNF-α was little affected by the quantitative variation of the non-standardised constituents.
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R E S E A R C H A R T I C L E Open Access
An in vitro study of anti-inflammatory activity of
standardised Andrographis paniculata extracts
and pure andrographolide
Mitchell Low
1
, Cheang S Khoo
1
, Gerald Münch
1,2
, Suresh Govindaraghavan
3
and Nikolaus J Sucher
4*
Abstract
Background: The anti-inflammatory activity of Andrographis paniculata (Acanthaceae), a traditional medicine widely
used in Asia, is commonly attributed to andrographolide, its main secondary metabolite. Commercial A. paniculata
extracts are standardised to andrographolide content. We undertook the present study to investigate 1) how selective
enrichment of andrographolide in commercial A. paniculata extracts affects the variability of non-standardised
phytochemical components and 2) if variability in the non-standardised components of the extract affects the
pharmacological activity of andrographolide itself.
Methods: We characterized 12 commercial, standardised (30% andrographolide) batches of A. paniculata extracts
from India by HPLC profiling. We determined the antioxidant capacity of the extracts using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) free radical scavenging, oxygen radical antioxidant capacity (ORAC) and a Folin-Ciocalteu (FC) antioxidant assays.
Their anti-inflammatory activity was assessed by assaying their inhibitory effect on the release of tumor necrosis factor
alpha (TNF-α) in the human monocytic cell line THP-1.
Results: The andrographolide content in the samples was close to the claimed value (32.2 ± 2.1%, range 27.5 to 35.9%).
Twenty-one non-standardised constituents exhibited more than 2-fold variation in HPLC peak intensities in the tested
batches. The chlorogenic acid content of the batches varied more than 30-fold. The DPPH free radical scavenging activity
varied ~3-fold, the ORAC and FC antioxidant capacity varied ~1.5 fold among batches. In contrast, the TNF-αinhibitory
activity of the extracts exhibited little variation and comparison with pure andrographolide indicated that it was mostly
due to their andrographolide content.
Conclusions: Standardised A. paniculata extracts contained the claimed amount of andrographolide but exhibited
considerable phytochemical background variation. DPPH radical scavenging activity of the extracts was mostly due to
the flavonoid/phenlycarboxylic acid compounds in the extracts. The inhibitory effect of andrographolide on the release
of TNF-αwas little affected by the quantitative variation of the non-standardised constituents.
Keywords: Andrographolide, Andrographis paniculata, Anti-inflammatory, Antioxidant, TNF-α,Phytochemistry
Background
Chronic inflammation is thought to be a contributing
factor to many prevalent ageing-related diseases, such as
acute and chronic neurodegenerative diseases, degen-
erative musculoskeletal diseases, cardiovascular dis-
eases, diabetes, and cancer [1-5]. Other common chronic
inflammatory conditions include asthma, rheumatoid
arthritis, and inflammatory bowel disease. To date,
pharmacotherapy of inflammatory conditions is based
mainly on the use of non-steroidal anti-inflammatory
drugs (NSAIDs), steroids and more recently tumor ne-
crosis factor alpha (TNF-α) inhibitors [6-8]. However, the
prolonged use of NSAIDs can cause serious gastrointes-
tinal toxicity [9]. Some NSAIDs have also been linked to
increased blood pressure, greatly increased risk of con-
gestive heart failure and occurrence of thrombosis [10].
These findings illustrate the need to develop novel and
safe anti-inflammatory medicines [11].
* Correspondence: nsucher@rcc.mass.edu
4
Science Department, Roxbury Community College, 1234 Columbus Avenue,
Roxbury Crossing, MA, 02120, USA
Full list of author information is available at the end of the article
© 2015 Low et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Low et al. BMC Complementary and Alternative Medicine (2015) 15:18
DOI 10.1186/s12906-015-0525-7
Andrographis paniculata (Acanthaceae), which is en-
dogenous to South India and South East Asia, is used
as an herbal medicine in both traditional Indian and Chin-
ese medicine (where it is known as kalmegh and chuan-
xinlian, respectively) as well as in Malaysia and Thailand
[12,13]. A. paniculata extracts exhibit anti-inflammatory
activity [13] that is commonly attributed to the
ent-labdane diterpenoid andrographolide, its charac-
teristic and main secondary metabolite [14-30].
Andrographolide appears to be rapidly absorbed [31] and
found to be non-toxic even at very high doses in animals
[32] and is well tolerated by humans with no serious ad-
verse effects at doses in the range of 1 to 2 mg/kg/day
[33,34]. Intriguingly, andrographolide has been reported
to exhibit gastro-protective and ulcer preventive ef-
fects, which combined with its well-documented anti-
inflammatory effects could make it a safe alternative
to traditional NSAIDs [35]. A proprietary A. paniculata
extract (HMPL-004, Hutchison MediPharma) is under
development for the treatment of inflammatory bowel
disease [36,37] and is currently being tested in a global
phase III clinical trial (http://clinicaltrials.gov/show/
NCT01805791).
Commercial A. paniculata tablets standardised to requis-
ite concentrations of andrographolide (5% or 30% w/w) are
used in clinical studies with an assumption of consistency
[38,39]. However with reported innate phytochemical vari-
ation influenced by phytogeographical and spatiotemporal
factors [40-44], it is not known how selective enrichment
(standardization) of andrographolide in commercial prepa-
rations affects the variability of non-standardised phyto-
chemical components. It is also not known if variations
in the non-standardised components affect the anti-
inflammatory activity of the extracts. We undertook the
present study to investigate 1) how selective enrichment
of andrographolide in commercial A. paniculata extracts
affects the variability of non-standardised phytochemical
components and 2) if variability in the non-standardised
components affects the pharmacological activity of the ex-
tracts. To this end, we profiled the phytochemical com-
position and antioxidant capacity of standardised A.
paniculata extracts and compared the activity of the ex-
tracts and purified andrographolide in an assay relevant to
their anti-inflammatory activity.
Anti-inflammatory activity of andrographolide has
been studied using a number of in vivo and in vitro ex-
perimental paradigms including human whole genome
DNA microarrays [20]. The most commonly implicated
molecular mechanism underpinning the anti-inflammatory
and immunomodulatoy effects of andrographolide is inhib-
ition of the mitogen-activated protein kinase/extracellular
signal-regulated kinase (MAPK/ERK) signalling (specifically
p38 MAPK/ERK1/2) pathway and downstream transcrip-
tion factors such as nuclear factor kappa B (NF-κB) and
nuclear factor of activated T cells (NFAT) [24,45-50]. An
exemplary experimental in vitro model where this mechan-
ism has been implicated is the inhibition by andro-
grapholide of the release of TNF-αfrom LPS stimulated
macrophages [22,51-54]. Therefore, we chose TNF-α
release from LPS stimulated monocytic leukaemia cells
(THP-1) as a model to study anti-inflammatory activity
of the extracts and purified andrographolide.
Methods
Extract provenance and preparation
Twelve commercial batch samples of A. paniculata
extracts (extract ratio 14:1) standardised to 30% andro-
grapholide were kindly provided to us by LIPA Pharma-
ceuticals Ltd (NSW, Australia). The extracts conform to
the TGA guidelines for incorporation in herbal medicines
manufactured in Australia. The whole plant starting ma-
terial for each batch was sourced (during 2004 2008)
and the extracts manufactured in India. A systematic bot-
anist authenticated each batchs starting material and the
manufacturer provided traceability documents for each
extract. All the samples were re-analysed by high-
performance liquid chromatography (HPLC) with photo-
diode array detection (PDA) for andrographolide content
to reconfirm the manufacturers certificate of analysis.
Phytochemical analysis
We used HPLC to profile the phytochemical compos-
ition of the A. paniculata extracts. Andrographolide
(14.3 mg in 10 ml) and extract samples (125 mg in
50 ml) were dissolved by sonication in methanol. HPLC
analysis of andrographolide and the extract samples was
performed using a Varian Inc. (USA) HPLC system
equipped with ProStar 335 photodiode array detector
(PDA) and 1200 L quadrupole tandem mass spectrom-
etry (MS/MS) detector. An Alltech Alltima (Alltech
Australia) reverse phase C18 column (46 × 150 mm I.D.,
5μm) with a Phenomenex (California, USA) Security
C18 guard column (20 mm × 4 mm, 5 μm) were used in
these experiments.
We generated HPLC-PDA and HPLC-MS/MS profiles
using a 5 μl injection of extract samples. The mobile
phase consisted of 0.1% (v/v) aqueous formic acid
(mobile phase A) and 0.1% (v/v) formic acid in acetonitrile
(mobile phase B). The mobile phase gradient was 10% B
for 10 min with a linear increase; to 50% B at 63 min, 70%
B at 72 min and then 100% B (wash) for 8 min before
equilibrating at the starting composition for 5 min. Mobile
phase flow rate was maintained at 1 ml/min. The post col-
umn flow was split to send 80% to the PDA (200500 nm)
and 20% to the MS.
The MS conditions were adapted from the work of
Dong et al. [55]. Ionization was achieved in positive elec-
trospray ionization mode, scanning between 70700 m/z
Low et al. BMC Complementary and Alternative Medicine (2015) 15:18 Page 2 of 9
with the needle voltage 5000 V at 13 μA; nebulization
gas (nitrogen) temperature of 350°C at 20 psi; shield
voltage 175 V; capillary voltage 53 V and the detector
voltage was 1600 V.
We quantified the andrographolide (0.14 - 1.4 mg/ml)
content of the extracts at a detection wavelength of
240 nm and the chlorogenic acid (6.6 132.4 μg/ml)
content at 330 nm, using five-point linear calibration
curves. We generated chromatograms at 227 nm for the
detection of diterpenes and at 261 nm and 330 nm for
the detection of flavonoids and phenyl carboxylic acids,
respectively.
While the PDA was used to quantify andrographolide
and chlorogenic acid, the MS detector was used to con-
firm the identity of these peaks by comparing the MS/
MS obtained for the sample and reference standard
peaks. MS/MS data was also used to tentatively identify
other diterpenes by comparison to published MS data.
The UV spectra were used to assign tentatively some of
the observed peaks as flavonoids, phenylcarboxylic acids
or diterpenes as shown in Table 1.
We used the package msProcess[56] (R Project) for
Statistical Computing [57] to remove instrumental noise
and baseline drift from the chromatograms as described
in detail previously [58].
Antioxidant assays
We performed the DPPH (2,2-di(4-tert-octylphenyl)-1-
picrylhydrazyl) radical scavenging capacity, oxygen rad-
ical absorbance capacity (ORAC) and the total phenol
assay (Folin-Ciocalteu assay; FC) on the extracts as re-
ported in detail previously [58,59].
Cell culture and tumor necrosis factor α(TNF-α) assay
We cultured human monocytic leukaemia cells (THP-1;
American Type Culture Collection, Manassas, VA, USA)
in RPMI (Roswell Park Memorial Institute) media con-
taining 4.5 g/l D-Glucose and supplemented with 2 mM
GlutaMax, 100 U/ml penicillin, 100 μg/ml streptomycin,
and 10% foetal bovine serum at 37°C, 5% CO
2
in 95%
air. Cells (passage number between 10 and 25) were
seeded at a density of 1 × 10
5
cells/well and incubated
for 48 h in phorbol-12-myristate-13-acetate (PMA;
100 nM). Non-adherent cells were removed by washing
with fresh medium. The remaining cells were pre-
incubated with different concentrations of extracts or
andrographolide for 1 h and stimulated by lipopolysac-
charide (LPS; 50 ng/ml) and interferon gamma (50 units)
for 24 h. Andrographolide and extracts were prepared
in DMSO and added at a final concentration of 0.1%
DMSO. We determined the concentration of TNF-αin
the THP-1 culture supernatant by a commercial sand-
wich ELISA following the manufacturers instructions
(PeproTech Inc., Rocky Hill, NJ, USA).
Potential cytotoxicity of the extracts and andrographolide
was investigated using the MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay. No toxic ef-
fects of the extracts or andrographolide were observed at
the tested concentrations (data not shown).
Statistical data analysis
All data is reported as mean ± standard deviation of the
average of three replicates in experiments performed on
three separate days. The TNF-αdose response data were
fitted with a log (inhibitor) vs. normalized response
with variable slope model using Prism 5 for Mac OSX
(GraphPad Software, La Jolla, CA). IC
50
values were
calculated from the fitted curves. F-test was used to
compare if the best-fit values differed between the data
sets. Differences were considered statistically signifi-
cant if p<0.05.
Results and discussion
The phytochemical composition of 12 A.paniculata ex-
tracts was characterized by HPLC-PDA (Figure 1A).
Consistent with the intended standardization, to contain
30% andrographolide, the content was on average close
to this value (32.2 ± 2.1%, range 27.5 to 35.9%; n= 12;
peak #12 in Figure 1B). Correlation analysis of the
andrographolide concentration versus storage time re-
vealed that there was no observable loss in andrographo-
lide concentration due to storage of the dry extracts
(r
2
= 0.03; not illustrated). Non-standardised constitu-
ents other than diterpenes (e. g. peaks 111), varied
to a greater degree between the batches (Table 1). For ex-
ample, the chlorogenic acid content exhibited >30 fold
variation between batches (0.1 to 3.5 mg per g of extract;
mean 1.4 ± 1.1 mg/g). The maximum variation observed
between the chromatograms is illustrated by the red curve
in Figure 1B.
The phytochemical analysis of the extracts revealed
considerable variability in the peak intensities of the
non-standardised constituents, some of which belonged
to the flavonoid and phenylcarboxylic acid class. Flavo-
noids and phenolic acids are known to be good free rad-
ical scavengers and antioxidants [60]. Oxidative stress is
believed to contribute to inflammatory tissue damage
and play a role cytokine signalling [61]. We therefore
wondered to what degree the observed phytochemical
variation might be reflected in the antioxidant capacity
of the extracts [62]. We used three non-cellular assays to
measure the DPPH radical scavenging capacity, oxygen
radical absorbance capacity (ORAC) and total phenol
content (FC assay) [58]. The average DPPH radical scaven-
ging capacity of the total extracts was 81.6 ± 30.9 μmol/g
gallic acid equivalent and varied 3 fold. DPPH reactivity
was correlated with the variation in chlorogenic acid con-
tent (r
2
= 0.8). Online-HPLC revealed that the DPPH
Low et al. BMC Complementary and Alternative Medicine (2015) 15:18 Page 3 of 9
scavenging was mainly due to peaks 111 (flavonoids
and phenylcarboxylic acids) while the diterpenes
(peaks #12, 13, 20, 24 and 25) were virtually devoid of
DPPH radical scavenging capacity under the conditions
of our experiments (Figure 2). The average ORAC of the
extracts was 1.05 ± 0.16 mmol/g gallic acid equivalent
and varied 1.6 fold. The average antioxidant activity of
the extracts in the FC assay was 0.40 ± 0.05 mmol/g gallic
acid equivalent and varied 1.5 fold.
Next, we assayed the activity of the extracts and puri-
fied andrographolide on the inhibition of TNF-αrelease
by LPS stimulated THP-1 cells. For this experiment, we
obtained dose response curves of purified andrographo-
lide and the extracts #3 and #9, the extracts with highest
and lowest DPPH free radical scavenging activity. The
results revealed that the dose response curves obtained
upon application of pure andrographolide or the extracts
were very similar (Figure 3), when normalized to
Table 1 Tentative assignment of HPLC chromatogram peaks as flavonoids, phenylcarboxylic acids or diterpenes based
on UV absorbance and MS fragmentation patterns
Peakidentitycor number Rt (min) % Area
3
Fold variation
2
UV peaks
1
(nm) MS fragmentation (m/z)
5
Tentative assignment
4
Chlorogenic acid 12.2 1.0 34.3 327, 218, 235 Not determined (ND) Phenyl carboxylic acid
Isoquercetin 28.7 0.9 5.1 204, 255, 353 ND Flavonol glycoside
Peak 3 29.2 0.6 9.8 323, 219, 234 ND Phenyl carboxylic acid
Peak 4 29.7 3.5 3.8 325,219,235 ND Phenyl carboxylic acid
Peak 5 31 1.4 3.4 326, 219, 271 ND Phenyl carboxylic acid
Peak 6 32.2 4.1 6.1 324, 219, 234 ND Phenyl carboxylic acid
Peak 7 32.4 3.3 4.5 325, 220 ND Phenyl carboxylic acid
Peak 8 32.8 3.3 4.7 346, 224, 256 ND Phenyl carboxylic acid
Peak 9 33.7 2.1 4.5 204, 336, 266 ND Flavone
Peak 10 36.8 1.3 6.6 327, 219, 235 ND Phenyl carboxylic acid
Peak 11 37.7 0.8 4.4 327, 271, 223, 204 ND ND
Andrographolide 38.4 ND ND 227 351 [M+H]
+
, 333 [M-H
2
O]
+
,
315 [M-2H
2
O]
+
, 297 [M-3H
2
O]
+
Diterpene
Andropanoside 39.8 1.4 4.8 207 535 [M+K]
+
Diterpene
Peak 14 40.2 8.0 7.1 225 ND ND
Peak 15 40.7 4.3 3.7 202 ND ND
Apigenin 42.2 2.6 6.7 211, 337, 267 ND Flavone
Wogonoside 45.2 0.7 2.3 265, 212 ND O-methylated flavone
glycoside
Peak18 46.6 0.7 4.6 ND ND ND
Peak 19 46.8 0.8 2.5 ND ND ND
Neoandrographolide 47.7 9.3 2.7 201 503 [M+Na]
+
,519 [M+K]
+
,
319[M+H-Glu]
+
Diterpene
Peak 21 48.6 0.5 3.5 ND ND ND
Peak 22 49.7 1.5 5.3 ND ND ND
Peak 23 50.2 0.76 7.7 ND ND ND
Deoxyandrographolide 52.1 6.7 3.7 200 357 [M+Na]
+
, 317 [M+H-H
2
O]
+
,
299 [M+H-2H
2
O]
+
Diterpene
Dehydroandrographolide 52.6 35.1 2.1 200, 249 355 [M+Na]
+
, 315 [M+H-H
2
O]
+
,
297 [M+H-2H
2
O]
+
Diterpene
Peak 26 53.6 0.8 2.8 229 ND ND
Peak 27 54.3 2.0 6.1 229 ND ND
Peak 28 54.6 2.8 3.0 200, 263 ND ND
1
UV peaks are listed in order of intensity.
2
Fold variation = (Max. peak .area) / (Min. peak area).
3
Percent (%) area = average peak area from the 12 batches / total combined area × 100. Andrographolide was excluded from the % area calculations.
4
Assignments based on UV spectrum, MS fragmentation and/or comparison to reference standard RT.
5
MS fragments are listed in order of intensity.
Low et al. BMC Complementary and Alternative Medicine (2015) 15:18 Page 4 of 9
andrographolide concentration. The half maximal inhibi-
tory concentration (IC
50
) of pure andrographolide was
21.9 μM(n= 3; 95% confidence interval: 18.1 - 26.5 μM)
compared to 16.4 μM(n= 3; 95% confidence interval:
13.8 - 18.8 μM) and 18.7 μM(n= 3; 95% confidence
interval: 14.9 - 23.4 μM) for extracts #3 and #9,
respectively. Thus, the phytochemical background vari-
ation of the extracts appeared not to influence significantly
the activity of andrographolide in this in vitro assay.
We characterized the phytochemical composition of
12 batches of commercial A. paniculata extracts stan-
dardised for andrographolide content (30% w/w). We
confirmed that the extracts contained the specified
amount of andrographolide but observed substantial
variation in the non-standardised components of the ex-
tracts. Chlorogenic acid exhibited maximal (~30 fold)
variation. This acid is one of the most abundant phen-
olic compounds in the human diet and is present in sig-
nificant amounts in coffee [63]. Various pharmacological
effects of chlorogenic acid have been described and re-
cent interest has focused on its effects on glucose and
lipid metabolism [63]. Hypoglycaemic and hypocholes-
terolemic effects of water and ethanol A. paniculata ex-
tracts have been reported but the role of chlorogenic
acid and its other phytochemical constituents and the
molecular mechanisms underpinning these effects re-
main to be established [64]. Patients with diabetes taking
A. paniculata extracts may therefore need extra moni-
toring and dietary counselling upon commencing or
changing A. paniculata extract containing medications
due to batch-to-batch variability of chlorogenic acid and
its potential effects on blood glucose levels.
Our results revealed that of the commonly used
in vitro antioxidant assays the DPPH assay was a more
Figure 2 Online DPPH assay. Representative chromatograms of
A. paniculata extract #9. The chromatogram at 227 nm (black line)
is contrasted with the DPPH absorbance at 529 nm (red line).
Compounds that have DPPH antioxidant activity are observed as a
negative peak at 529 nm. Numbered peaks were identified as
chlorogenic acid (#1), andrographolide (#12), andropanoside (#13),
neoandrogrpapholide (#19), deoxyandrographolide (#23), and
14-deoxy-11,12-didehydroandrographolide (#24).
A
B
Figure 1 Chromatograms of standardised A. paniculata extracts.
A) The chromatograms of twelve batches (#1 at the bottom, #12 at the
top) standardised to contain 30% (w/w) andrographolide were recorded
at 227 nm. B) Composite chromatograms illustrating the variation
between the HPLC profiles of the 12 batches. The black chromatogram
was generated using the highest peaks from each of the 12 extracts.
The red chromatogram was generated using the smallest peaks. Vertical
blue lines indicate the retention time of the peaks that were used for
the quantitative analysis of batch-to-batch variation (see Table 1).
Numbered peaks were identified as chlorogenic acid (#1), isoquercetin
(#2), andrographolide (#12), andropanoside (#13), apigenin (#15),
wogonoside (#16), neoandrogrpapholide (#19), deoxyandrographolide
(#23), and 14-deoxy-11,12-didehydroandrographolide (#24).
Low et al. BMC Complementary and Alternative Medicine (2015) 15:18 Page 5 of 9
sensitive indicator of the phytochemical background
variation in the standardised extracts than the ORAC
and FC assays. The absolute increase in the activity of
the extracts and reduced inter-extract variation observed
in the ORAC and FC assays may be due to the activity
of andrographolide and other diterpenes (and possibly
additional unidentified compounds), which did not ex-
hibit activity in the DPPH assay. The results suggest that
at least under the conditions of our experiments the di-
terpenes mainly functioned via hydrogen atom transfer
(HAT) but not electron transfer (ET) in contrast to the
flavonoids and phenyl carboxylic acids which exhibited
activity in both the HAT and ET mechanisms [58]. The
DPPH reactivity was correlated with the variation in
chlorogenic acid (r
2
= 0.8), illustrating that chlorogenic
acid contributed significantly to the ET antioxidant ac-
tivity of the extracts. The chlorogenic acid quantity
showed no correlation with the ORAC or FC results,
despite chlorogenic acid being a HAT antioxidant; this is
likely due to andrographolide masking chlorogenic acids
contribution to the total HAT activity as andrographo-
lide is in much greater abundance (~100 times).
There are many anti-inflammatory compounds re-
ported in A. paniculata [52,65,66] but andrographolide
is the most abundant [55]. In this study the most dis-
similar extracts in terms of DPPH activity and chemical
profile (#3 and #9) were compared to pure andrographo-
lide to assess their inhibition of TNF αrelease from LPS
stimulated macrophages. Our comparison between the
purified andrographolide and extracts containing parallel
amounts of andrographolide in this assay found very
similar dose response curves. Thus, at this level of ana-
lysis, there was no evidence for antagonistic, additive or
synergistic effects between andrographolide and other
phytochemical constituents of the extracts. The extract
activity was almost entirely accounted for by the andro-
grapholide content, thus the contribution of the other
anti-inflammatory compounds present was minor, likely
due to their lower abundance.
The IC
50
of andrographolide (21.9 μM) was com-
parable to values obtained in a similar assay using
mouse peritoneal macrophages [51]. A number of
additional in vitro studiesusingvariousexperimental
paradigms have all reported effective concentrations
of pure andrographolide in the range of 7 to 35 μM
[20,24,45,47,48,66-68]. The concentration reported in
ours and other in vitro studies are nominal concentra-
tions and the actualconcentration at the site of
interaction between andrographolide and its potential
(extra- and/or intracellular) target proteins (receptor(s)
or enzymes) has not been determined. Nonetheless, it is
noteworthy that the IC
50
of andrographolide in our
in vitro assay was ~50 times higher than the maximal
plasma concentration (0.5 μM) achieved following oral
administration of 50 mg andrographolide in healthy hu-
man volunteers [69], although significantly higher steady
state blood concentrations (1.9 μM) have been reported
in humans taking ~1 mg andrographolide per kg body
weight per day [31]. In a prospective clinical study for the
relief of rheumatoid arthritis symptoms, Burgos and col-
leagues administered 3 times per day 100 mg of A. pani-
culata extract standardised to 30% andrographolide [38].
Although these authors observed some positive effects,
the in vitro data suggest that the administered dose might
have been at the low end of the effective dose range and
future clinical studies should consider testing higher
doses. It will be interesting to investigate whether or not
the absorption, distribution, metabolism and excretion of
andrographolide alone or andrographolide administered
as A. paniculata extract (with variable phytochemical
background) differ or not.
Conclusion
Supported by a considerable body of published evidence,
standardisation of A. paniculata extracts for andro-
grapholide content is based on the notion that this
Figure 3 Dose response curves for batches #9 (blue, squares),
#3 (red, circles) and andrographolide (green, triangles). IC
50
of
pure andrographolide: 21.9 μM(n= 3; 95% confidence interval:
18.1 - 26.5 μM), extract #3: 16.4 μM(n= 3; 95% confidence interval:
13.8 - 18.8 μM), extract #9: 18.7 μM(n= 3; 95% confidence interval:
14.9 - 23.4 μM). The concentrations have been normalized to
concentration of andrographolide in the batches. The data were
fitted with a log (inhibitor) vs. normalized response with variable
slope model. The IC
50
was calculated from the fitted curve. F-test
indicated that all data could be equally fitted with a single curve with
IC
50
=18.5μM(p= 0.51; 95% confidence interval: 16.7 to 20.7 μM).
Low et al. BMC Complementary and Alternative Medicine (2015) 15:18 Page 6 of 9
compound accounts for the pharmacological effects of the
complex extracts. To the best of our knowledge, however,
our data represent the only quantitative direct comparison
of the efficacy of pure andrographolide and A. paniculata
extracts. Thus, our results support the development of
andrographolide or andrographolide-derived compounds
as anti-inflammatory drugs. Interestingly, A. paniculata
related drug development efforts presently include both
herbal medicine based approaches in the form of stan-
dardised complex extracts as well as orthodox, synthetic
drug based approaches [40,70-79]. We believe that well
characterized standardised A. paniculata extracts present
an excellent opportunity to further investigate the advan-
tages and disadvantages of the herbal vs. synthetic ap-
proach in the treatment of inflammatory conditions.
Competing interests
The authors declare that they have no competing interests.
Authorscontributions
ML carried out the cell culture experiments and phytochemical analysis,
participated in data analysis and manuscript writing. CK participated in the
phytochemical and data analysis. GM, SG and NJS conceived the study, and
collaborated in its design and coordination. NJS wrote the initial and final
draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We thank Mr. Dusko Pejnovic, Chief Executive Officer of LIPA Pharmaceuticals
Ltd. for his support, and LIPA Pharmaceuticals for providing samples of
A. paniculata extracts. This study was partially supported by a Research
Partnership Grant from the University of Western Sydney and LIPA
Pharmaceuticals (NJS) and a UWS RGS grant (NJS and GM).
Author details
1
National Institute of Complementary Medicine, School of Science and Health,
University of Western Sydney, Locked Bag 1797, Penrith, N.S.W. 2751,
Campbelltown, Australia.
2
Department of Pharmacology and Molecular
Medicine Research Group, School of Medicine, University of Western Sydney,
Campbelltown, Australia.
3
Network Nutrition-IMCD Australia, Unit 9, 7 Meridian
Place, Bella Vista, NSW 2153, Australia.
4
Science Department, Roxbury Community
College, 1234 Columbus Avenue, Roxbury Crossing, MA, 02120, USA.
Received: 7 April 2014 Accepted: 15 January 2015
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Low et al. BMC Complementary and Alternative Medicine (2015) 15:18 Page 9 of 9
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Les maladies inflammatoires chroniques de l'intestin (MICI) regroupent la rectocolite hémorragique (RCH) et la maladie de Crohn qui sont considérées comme des pathologies multifactorielles, résultant de l'inflammation chronique le long de la muqueuse digestive ou sur des zones précises. Ces maladies s'imposent aujourd'hui comme un véritable problème de santé publique. Les symptômes digestifs de la RCH sont principalement constitués d'une diarrhée sanglante, douloureuse et fréquemment accompagnée de glaires mélangées ou non aux selles. Et si de nombreuses recherches scientifiques s'intéressent à ces maladies, aucune thérapeutique n'offre, actuellement, des possibilités de guérison aux patients. Pourtant, de nombreuses pistes naturelles se dessinent pour apporter des réponses en complément ou en lieu et place des traitements pharmacologiques. Les patients atteints de MICI sont classés parmi les plus grands consommateurs de thérapies complémentaires et alternatives. Ces dernières deviennent actuellement très populaires y compris dans les pays développés. C'est pourquoi, afin d'améliorer la qualité de vie, patients et médecins sont de plus en plus nombreux à se pencher sur des approches complémentaires aux traitements classiques. De nombreuses études ont montré, sur des modèles animaux d'inflammation intestinale, les effets protecteurs des fines herbes et certaines plantes aromatiques et médicinales. Notre travail se veut une revue de synthèse avec comme objectif de faire le point sur les données de la littérature concernant l'apport préventif de la phyto-aromathérapie sur terrain des MICI, et de les discuter par rapport aux tests in vivo et aux études cliniques réalisées, et ce pour leur éventuelle intégration dans l'arsenal préventif-thérapeutique des pathologies inflammatoires digestives. ABSTRACT Chronic inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease which are considered to be multifactorial pathologies, resulting from chronic inflammation along the digestive mucosa or in specific areas. These diseases are emerging as a real public health problem. The digestive symptoms of UC are mainly bloody, painful diarrhea that is frequently accompanied by mucus. While several studies are interested in these diseases, no potent therapy BOUKHATEM & BELKADI. (Maladies inflammatoires chroniques de l'intestin : Quelle place pour la phyto-aromathérapie ?) Page 60 currently offers a cure for patients. However, many natural products are emerging to provide answers instead of pharmacological drugs. Patients with IBD are classified among the highest consumers of complementary and alternative medicines. These are currently becoming very popular, including in Western countries. In order to improve the quality of life, patients and doctors are trying to test complementary and alternative medicines. Different reports and investigations have shown the protective effects of aromatic herbs and medicinal plants, in vivo, using animal models of intestinal inflammation. Our article is intended to be a comprehensive review with the objective of taking stock of the data in the literature concerning the preventive-therapeutic effects of phytomedicine and aromatherapy against IBD, and to discuss them in relation to in vivo assays and clinical studies.
... THP-1 (RRID:CVCL_0006) is a permanent human monocytic cell line derived from an acute monocytic leukemia patient (Abrink et al., 1994;Tsuchiya et al., 1980). Previously, preparations or modifications of Andro were found to be toxic for THP-1 cells (Habtemariam, 2003;Lee et al., 2012), to enhance the cells' expressions of cytokine IFNγ and of stress-protein GRP-78 (Gupta et al., 2020), and to interfere with their functional properties such as the (immune-induced) activation and/or production of transcription factor NF-κB, matrix metalloproteinase-9, and various cytokines (Gupta et al., 2020;Lee et al., 2012;Low et al., 2015;Nie et al., 2017), and their migration in a chemotaxis assay (Zhang et al., 2019). An analogue of Andro,14-Deoxy-11,12-didehydroandrographolide (AND2), induced apoptosis in THP-1 cells (Raghavan et al., 2014), but-to the best of our knowledge-the present study is first to address how Andro itself induces apoptosis in these cells. ...
Article
Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. Results: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. The apoptotic activities of Andro were largely suppressed by N-acetyl-L-cysteine (NAC), an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Furthermore, whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. Conclusions : Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. Notably, the high sensitivity of H929 cells is encouraging for further studies on the use of Andro against multiple myeloma.
... It also showed both non-specific as well as antigen/antibody-dependent lung inflammation as an effective anti-inflammatory drug (Abu-Ghefreh et al., 2009). The commercial extracts and compound 21 also showed an inhibitory effect on the release of TNF-α in the human monocytic cell line THP-1 (Low et al., 2015). ...
Article
Ethnopharmacological relevance Andrographis paniculata (Burm.f.) Nees is a medicinal herb of the Asian countries used in many traditional medicinal systems for the treatment of diarrhea, flu, leprosy, leptospirosis, malaria, rabies, upper respiratory infections, sinusitis, syphilis, tuberculosis and HIV/AIDS etc. Aim of the study This review aims to provide comprehensive, accurate and authentic information on traditional uses, photochemistry, and pharmacological properties of various extracts/fractions as well as phytocostituents of A. paniculata. In addition, this review also aims to provide advance and sensitive analytical methods along with chemical markers used in the standardization of herbal products for quality control (QC)/quality assurance (QA). Materials and methods All relevant publications were considered within the years 1983–2020. The publications were searched from Google Scholar, PubChem, Chemspider, PubMed, Elsevier, Wiley, Web of Science, China Knowledge Resource Integrated databases and ResearchGate using a combination of various relevant keywords. Besides, relevant published books and chapters were also considered those providing an overview of extant secondary literature related to traditional knowledge, phytochemistry, pharmacology and toxicity of the plant. Results and Discussion In this review, 344 compounds, including, terpenoid lactones, flavonoids, phenolic acids, triterpenes and volatile compounds were summarized out of which more than half of the compounds have no reported pharmacological activities yet. Terpenoid lactones and flavonoids are the major bioactive classes of compounds of A. paniculata which are responsible for pharmacological activities such as anticancer and antioxidant activities, respectively. Biosynthetic pathways and active sites for target proteins of both terpenoid lactones and flavonoids were considered. Analgesic, anticancer, antidiabetic, antifertility, antiinflammatory, antimalarial, antimicrobial, antioxidant, antipyretic, antiviral, antiretroviral, antivenom, cardioprotective, hepatoprotective, immunomodulatory and neuroprotective activities have been also reported. Andrographolide is a major characteristic active principle and responsible for most of the pharmacological activities. Therefore, andrographolide has been selected as a marker for the standardization of raw and marketed herbal products by TLC, HPTLC, HPLC, GC-MS, HPLC-MS and HPLC-MS/MS methods for QC/QA. Conclusions Conclusive evidence showed that the pharmacological activities reported in crude extracts and chemical markers are supporting and provides confidence in the traditional use of A. paniculata as a herbal medicine. The andrographolide could be used as a chemical marker for the QC/QA of raw and A. paniculata derived herbal products. Lactone ring in terpenoid lactone is an active site for targeted proteins. More efforts should be focused on the identification of the chemical markers from A. paniculata to provide a practical basis for QC/QA. Several aspects such as the mechanism of therapeutic potential, molecular docking technology and multi-target network pharmacology are very important for drug discovery and needed more investigation and should be considered. This compilation may be helpful in further study and QC/QA.
... Comparison with approved drugs can help to explore the exact MoA of a natural product. As an example, andrographolide is the main active ingredient of Andrographis paniculate, which is used as an herbal medicine in both traditional Indian and Chinese medicine (where it is known as kalmegh and chuanxinlian, respectively) [8]. A. paniculata extracts exhibit anti-inflammatory activity that is commonly attributed to andrographolide [9]. ...
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Natural products from traditional medicine inherit bioactivity from their source herbs. However, the pharmacological mechanism of natural products is often unclear and studied insufficiently. Pathway fingerprint similarity based on “drug-target-pathway” heterogeneous network provides new insight into Mechanism of Action (MoA) for natural products compared with reference drugs, which are selected approved drugs with similar bioactivity. Natural products with similar pathway fingerprints may have similar MoA to approved drugs. In our study, XYPI, an andrographolide derivative, had similar anti-inflammatory activity to Glucocorticoids (GCs) and Nonsteroidal Anti-inflammatory Drugs (NSAIDs), and GCs and NSAIDs have completely different MoA. Based on similarity evaluation, XYPI has similar pathway fingerprints as NSAIDs, but has similar target profile with GCs. The expression pattern of genes in LPS-activated macrophages after XYPI treatment is similar to that after NSAID but not GC treatment, and this experimental result is consistent with the computational prediction based on pathway fingerprints. These results imply that the pathway fingerprints of drugs have potential for drug similarity evaluation. This study used XYPI as an example to propose a new approach for investigating the pharmacological mechanism of natural products using pathway fingerprint similarity based on a “drug-target-pathway” heterogeneous network.
... Andrographis paniculata has been used in various traditional medication systems. It has been found to have a wide range of pharmacological applications including antiinflammatory, antioxidant and antimalarial properties among others (4)(5)(6). A. paniculata contains several active constituents include flavonoids, flavonoid glycosides, lactones and diterpenes. Four lactones consists of andrographolide, neoandrographolide, deoxyandrographolide and 14-deoxy-11, 12didehydroandrographolide were isolated from the aerial parts of A.paniculata (7). ...
Article
Background: Placental malaria has ability to upregulate prostaglandin synthesis by increasing cyclooxygenase-2 (Cox-2) enzyme activity. Cox-2 and prostaglandin have a role in causing uterine contraction and therefore can cause abortion or preterm labor. Tablet AS201-01 containing the ethyl acetate fraction of Andrographis paniculata was tested in vivo on pregnant mice infected with Plasmodium berghei. AS201-01 inhibited the growth of P. berghei, increased TGF-β expression, decreased TLR-4 expression and apoptosis index of placental tissue in P. berghei infected pregnant mice and thus prevented placental malaria complications. These effects were correlated with the decrease of Cox-2 and prostaglandin expression. Methods: Twenty-four pregnant mice (Balb/c) were divided into 4 groups (n=6). Mice were maintained at Animal Laboratory of Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia in 2016. G1 were uninfected pregnant mice, G2 untreated infected pregnant mice, G3 infected pregnant mice treated with AS201-01, and G4 infected pregnant mice treated with DHP tablet. All infection groups (G2-G4) were inoculated with 1x106 of P. berghei parasite on day 9 of gestation and treated on day 11. All mice were terminated at day 15 of gestation, and placental tissue was collected. Cytokine expression of Cox-2 and prostaglandin were evaluated using immunohistochemistry. Results: G3 was found to have lower Cox-2 and prostaglandin expression compared to G4 and G2, but higher compared to G1. Cox-2 and prostaglandin expression was significantly different among groups (P<0.001). Conclusion: This study demonstrates the ability AS201-01 tablets have to decrease Cox-2 and prostaglandin expression on placental of P. berghei infected mice and therefore eliminates the adverse effects of placental malaria.
... The higher polarity of solvent mixtures can modify the capability to dissolve selected compounds of antioxidant and thus influenced the estimation of the antioxidant activity in the extracts [24]. DPPH radical scavenging activity of the extracts was mostly due to the flavonoid or phenlycarboxylic acid compounds in the extracts of A. paniculata [25]. ...
Article
Andrographis paniculata (A. paniculata) is widely used as a topical treatment due to its medicinal properties, such as anti-inflammatory, antibacterial and collagen synthesis, which are essential for the wound healing process. Non-ionic surfactant vesicles or niosomes can increase the bioavailability of poorly water-soluble drugs through a biological membrane. Therefore, A. paniculata extract was loaded into the niosome, which acts as a nanocarrier to facilitate the active compound in the delivery system. A standard Soxhlet method was used to extract A. paniculata by using water or ethanol as a solvent. Both extracts were subjected to High Pressure Liquid Chromatography (HPLC), antioxidant activity and cell culture analysis to determine the best concentration for niosome encapsulation. Niosome was prepared by proniosome-derived niosomal dispersion and homogenized for size reduction. The resulted niosome was incorporated into carbopol gel and the wound healing efficacy was analysed in vivo to evaluate the macroscopic and histology of treated rats. Extraction of A. paniculata by ethanol shows higher andrographolide content (52 ppm) and antioxidant activity at 2.5 mg/mL. Entrapment efficiency (EE) showed that the formulation 1 (0.6 mmol Span60, 0.4 mmol Cholesterol, 0.1 mmol Labrasol) had the highest EE for water and ethanol extract by 97.75% ± 1.28 and 97.21% ± 1.89, respectively. The optimized concentration of 100 ppm was loaded into niosomal gel because it stimulates the proliferation and migration of human skin fibroblast cells. Wound treated by niosomal gel containing A. paniculata ethanol extracts exhibited total recovery by 100%. Prominent collagen fibres and formation hair follicle were observed at the end of 14 days. The present study demonstrates a beneficial effect of A. paniculata-loaded niosomal gel which effectively assist in the wound healing process and protects the tissue from oxidative stress.
Article
Andrographis Herba, the aerial part of Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae), has a wide geographic distribution and has been used for the treatment of fever, cold, inflammation, and other infectious diseases. In markets, sellers and buyers commonly inadvertently confuse with related species. In addition, most Chinese medicinal herbs are subjected to traditional processing procedures, such as steaming and boiling, before they are sold at dispensaries; therefore, it is very difficult to identify Andrographis Herba when it is processed into Chinese medicines. The identification of species and processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA barcoding has received considerable attention as a new potential means to identify species and processed medicinal materials. In this study, 17 standard reference materials of A. paniculata, 2 standard decoctions, 27 commercial products and two adulterants were collected. Based on the ITS2 sequence, it could successfully identify A. paniculata and adulterants. Moreover, a nucleotide signature consisting of 71 bp was designed, this sequence is highly conserved and specific within A. paniculata while divergent among other species. Then, we used these new primers to amplify the nucleotide signature region from processed materials. In conclusion, the DNA barcoding method developed in the present study for authenticating A. paniculata is rapid and cost-effective. It can be used in the future to guarantee the quality of Andrographis Herba of each regulatory link for clinical use.
Article
Objective: To explore the influences of andrographolide (Andro) on bladder cancer cell lines and a tumor xenograft mouse model bearing 5637 cells. Methods: For in vitro experiments, T24 cells were stimulated with Andro (0-40 µmol/L) and 5637 cells were stimulated with Andro (0 to 80 µmol/L). Cell growth, migration, and infiltration were assessed using cell counting kit-8, colony formation, wound healing, and transwell assays. Apoptosis rate was examined using flow cytometry. In in vivo study, the antitumor effect of Andro (10 mg/kg) was evaluated by 5637 tumor-bearing mice, and levels of nuclear factor κB (NF-κB) and phosphoinositide 3-kinase/AKT related-proteins were determined by immunoblotting. Results: Andro suppressed growth, migration, and infiltraion of bladder cancer cells (P⩽0.05 or P⩽0.01). Additionally, Andro induced intrinsic mitochondria-dependent apoptosis in bladder cancer cell lines. Furthermore, Andro inhibited bladder cancer growth in mice (P⩽0.01). The expression of p65, p-AKT were suppressed by Andro treatment in vitro and in vivo (P⩽0.05 or P⩽0.01). Conclusions: Andrographolide inhibits proliferation and promotes apoptosis in bladder cancer cells by interfering with NF-κB and PI3K/AKT signaling in vitro and in vivo.
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The medicinal herb Andrographis paniculata containing a bitter principle andrographolide is widely used in indigenous system of medicines for treatment of various ailments in south Asian countries including India. It is found to grow as an understory in its natural habitats. With a view to understand the role of shade on herbage yield, a two-year field study was conducted on sandy loam soil. The crop was grown under 25, 50, 70 and 100% incident photosynthetic photon flux density (PFD) by providing artificial shade nets and observations on plant height, fresh and dry weight of leaf, stem and total plant along with leaf gas exchange parameters. Significant differences were observed for all growth characters between open and shaded plants. Leaf photosynthesis increased from 11.56 to 19.70 mmol m−2s−1 as PFD increased from 25 to 100% and herbage yield increased from 226.70 to 379.45 g. Andrographolide content was not consistent under different PFD levels and highly influenced by the weather parameters. Since the total herbage and andrographolide yield was the highest under open light condition, it was concluded that A. paniculata is suitable for open cultivation.
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A rapid resolution liquid chromatography/time-of-flight tandem mass spectrometry (RRLC-TOF/MS) method was developed for qualitative and quantitative analysis of the major chemical constituents in Andrographis paniculata. Fifteen compounds, including flavonoids and diterpenoid lactones, were unambiguously or tentatively identified in 10 min by comparing their retention times and accurate masses with standards or literature data. The characteristic fragmentation patterns of flavonoids and diterpenoid lactones were summarized, and the structures of the unknown compounds were predicted. Andrographolide, dehydroandrographolide and neoandrographolide were further quantified as marker substances. It was found that the calibration curves for all analytes showed good linearity (R2 > 0.9995) within the test ranges. The overall limits of detection (LODs) and limits of quantification (LOQs) were 0.02 μg/mL to 0.06 μg/mL and 0.06 μg/mL to 0.2 μg/mL, respectively. The relative standard deviations (RSDs) for intra- and inter-day precisions were below 3.3% and 4.2%, respectively. The mean recovery rates ranged from 96.7% to 104.5% with the relative standard deviations (RSDs) less than 2.72%. It is concluded that RRLC-TOF/MS is powerful and practical in qualitative and quantitative analysis of complex plant samples due to time savings, sensitivity, precision, accuracy and lowering solvent consumption.
Article
Objective: To observe the effect of andrographolide on the expression of TNF-α and IL-12 in activated macrophages. Methods: The peritoneal macrophages were harvested from mice intraperitoneally injected with thioglycollate. The macrophages were pretreated with andrographolide and then stimulated with lipopolysaccharide. RT-PCR was used to examine the expression of TNF-α, IL-12a, and IL-12b in the macrophages, and ELISA was used to measure the protein levels of TNF-α and IL-12 in the supernatant. Results: Andrographolide inhibited the mRNA levels of TNF-α, IL-12a, and IL-12b, and the protein levels of TNF-α and IL-12 in activated macrophages, and the inhibition increased with the increase of andrographolide concentration. Andrographolide at 12 μg/ml (P<0.05) and 25 μg/ml (P<0.01) significantly inhibited TNF-α mRNA level in macrophages; andrographolide at 1 μg/ml (P<0.05) and 12 μg/ml (P< 0.01) significantly inhibited TNF-α protein level (P<0.01). Andrographolide at 1 μg/ml significantly inhibited IL-12b mRNA level and at 12 μg/ml significantly inhibited IL-12a mRNA level (P<0.01); and andrographolide at 1 μg/ml also significantly inhibited IL-12 protein level (P<0.01). Conclusion: Andrographolide can inhibit both the expression of TNF-α and IL-12 in activated macrophages.