In this study, Fourier Transform Infrared (FTIR)-, 1H Nuclear Magnetic Resonance (NMR)-spectroscopy and multivariate statistical analysis were used for the screening of in vitro anti-inflammatory activity on three different germplasm accessions namely 11,341 (P), 11,248 (T) and 11,265 (H) of Malaysian Andrographis paniculata ( A. paniculata) leaf. The anti-inflammatory activity by nitric oxide (NO) inhibition assay in relation to the different harvesting ages and times of A. paniculata leaf was determined through comparison by partial least-squares analysis (PLS) using SIMCA-P. The PLS allowed the separation and correlation between the NO assay with the phytochemical present due to the effects of different harvesting ages and times. From the PLS plots, conclusions were drawn with respect to the correlation between A. paniculata leaf metabolites with the anti-inflammatory results which showed that 180 days after transplanting (DAT) of morning session for accessions T and H, and evening for P gave the highest anti-inflammatory activity.
Andrographis paniculata (Burm. F.) Nees or “Hempedu Bumi” belongs to the family Acanthaceae which is widely grown in tropical areas of Asia such as Malaysia, India, Pakistan and Sri Lanka [
Recently, several reports on the bioactivities of A. paniculata particularly anti-in- flammation have been published [
Deuterated methanol-d4 (CH3OH-d4), non-deuterated KH2PO4, sodium deuterated oxide (NaOD), trimethylsilyl propionic acid-d4 sodium salt (TSP), deuterium oxide (D2O) and methanol were purchased from Merck (Darmstadt, Germany). Liquid nitrogen was supplied by MOX-Linde Sdn. Bhd. (Petaling Jaya, Malaysia). Standard compounds such as andrographolide, neoandrographolide and deoxyandrographolide were previously isolated by Professor Dr. Johnson Stanslas from Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. RAW264.7 cells were purchased from American Type Culture Collection (ATCC, Rockville, USA) under the catalogue number of ATCC® TIB-71TM. Dulbecco’s Modified Eagle’s Medium (DMEM) with and without phenol red, fetal bovine serum (FBS), penicillin streptomycin (Pen Strep) and TrypLETM Express were purchased from Gibco by Life Technologies Inc. (Eggenstein, Germany). Phosphate buffer saline (PBS) was purchased from Invitrogen by Life Technologies. Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 and interferon-gamma (IFN-γ) were purchased from Sigma Aldrich Co. (St. Louis, MO). DMSO was purchased from Merck (Darmstadt, Germany). PBS and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen by Life Technologies Inc. (Eggenstein, Germany) and DMSO was purchased from Merck (Darmstadt, Germany).
The A. paniculata seeds from three germplasm accessions of Malaysia (Accession 11,265, 11,341 and 11,248 which will be designated as H, P and T, respectively) were collected in July 2011 from Ladang Dua, Universiti Putra Malaysia (UPM). The germination and cultivation procedure in this study was performed according to Yusof et al. (2014) [
The FTIR sample preparation and measurement in this study were performed according to Yusof et al. (2014) [
Freeze-dried plant leaf (20 mg) was transferred to a 2.0 mL Eppendorf tube. A volume of 1.5 mL of a mixture of KH2PO4 buffer (pH 6.0) in D2O containing 0.05% trimethylsilylpropionic acid sodium salt (TSP, w/w) and methanol-d4 (1:1) was added to the plant sample. The extraction procedure in this study was performed according to Kim et al. (2010), with a minor modification [
Andrographolide and neoandrographolide were selected from the 1H NMR spectra and their concentrations are listed in
Metabolite | Chemical shift (δ) | p value | ||
---|---|---|---|---|
H | P | T | ||
120 vs 150 vs 180 DAT | 120 vs 150 vs 180 DAT | 120 vs 150 vs 180 DAT | ||
Andrographolide (A) (1) | 6.91 (m), 4.54 (dd, J = 5.0, 10.0 Hz), 4.09 (d, J = 10.0 Hz), 1.92 (s), 1.32 (d, J = 7.50 Hz), 1.16 (s), 0.72 (s) | >0.05 | <0.05 | >0.05 |
Deoxyandrographolide (D) (2) | 4.16 (d, J = 6.5 Hz) | ND | ND | ND |
Neoandrographolide (N) (3) | 7.40 (s), 4.27 (d, J = 10 Hz), 1.00 (s), 0.64 (s) | >0.05 | >0.05 | >0.05 |
α-Glucose (a-Glc) | 5.20 (d, J = 4.0 Hz) | ND | ND | ND |
β-Glucose (b-Glc) | 4.60 (d, J = 8.0 Hz) | ND | ND | ND |
Sucrose (S) | 5.40 (d, J = 3.9 Hz) | ND | ND | ND |
Choline (C) | 3.20 (s) | ND | ND | ND |
Alanine (Ala) | 1.48 (d, J = 7.5 Hz) | ND | ND | ND |
from the A. paniculata was based on the mean peak area of the 1H NMR signal of interest after binning using Chenomx software. Data was analyzed using the Minitab Statistical Software 14.12.0, © 2004 Minitab Inc. PA 16801-9928, USA. Among the groups, significance test was performed using the one-way ANOVA at 5% significance level.
The anti-inflammation activity of A. paniculata crude extracts was evaluated through Nitric oxide (NO) suppression activity based on Lee’s method with some modifications [
DMSO of 0.1% in DMEM without phenol red was utilized as a control vehicle in the serial dilutions in order to maintain the final concentration of 0.1% DMSO in each well. These various concentrations were prepared separately in the aseptic microcentrifuge tubes before being dispensed into the wells.
The RAW264.7 murine macrophages were cultured in 75 cm2 plastic culture flask in DMEM with phenol red containing HEPES, L-glutamine supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO2 atmosphere at 37˚C. After the cells have reached the confluency of 80% - 90%, they were detached from the flask wall by rinsing off the flask with 5 ml of PBS solution followed by 2 ml of TrypLETM Express before incubated for 10 minutes. DMEM with phenol red of 4 mL was used to rinse out the detached cells which then centrifuged at 3000 rpm, 4˚C for 10 minutes by using Rotina 35R (Andreas Hettich GmbH & Co., Tuttlingen, Germany). The supernatant was then removed and the cells were re-suspended with fresh DMEM containing HEPES, and L-glutamine with 5% FBS but without phenol red. The cells were counted by using a standard trypan blue cell counting technique.
The cell concentration was adjusted to 1 × 105 cells/mL in the same medium (DMEM without phenol red) before seeded in a 96-well flat bottom tissue culture plates at initial seeding densities of 5 × 104 cells/50 μL per well. The ready plates with seeded cells were incubated at 37˚C (5% CO2 atmosphere) overnight to allow anchoring of cells to the wells.
The treatment of the cells was done by replenishing the medium in each well with 50 μL DMEM without phenol red containing triggering agent of 1 ng/mL recombinant murine IFN-γ and 1 μg/mL LPS. For each dilution of the plant extracts, 50 μL was added into the appropriate wells in four replicates. The assay plate was incubated for 17 hours at 37˚C in 5% CO2 atmosphere. There were five kinds of controls prepared in duplicates for each plate. The controls were media only without cells (blank control), cells in media only, cells in media containing triggering agent plus 0.1% DMSO (negative control), cells in media containing triggering agent only, and cells containing triggering agents plus curcumin (positive control).
Nitrite concentration was determined by Griess assay through the reaction of 50 μL of cell culture supernatant and 50 μL of Griess reagent (1% sulphanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% phosphoric acid) at room temperature. The optical density was measured at 550 nm after 5 minutes of incubation at room temperature by Spectramax Plus (Molecular Devices) UV/Vis microplate reader. The accumulated NO release was determined spectrophotometrically by the total nitrite in the medium. The nitrite concentration in the samples was determined by regression analysis using serial dilutions of sodium nitrite as a standard. The percentage inhibition was calculated based on the ability to inhibit nitrite below the level produced by cells cultured in the presence of triggering agents and DMSO, which was considered as 0% inhibition.
In order to avoid any false positive result of NO inhibition, the treated cells were also submitted for MTT assay as to measure any cytotoxic effect on the cells by using the Heras’s method [
Data were analyzed by using GraphPad Prism software version 5.01 (GraphPad Software Inc., San Diego, USA). The graph of half maximum inhibitory concentration (IC50) for each sample was obtained from logarithm graph plot. In which each graph showed the goodness of fit, R2 > 0.95 and within the 95% of the confidence interval with p-values < 0.05 were considered significant. All results were expressed as the mean ± S.E.M of n, where n represented the number of wells replication. Differences between the mean values for different groups were analyzed by Analysis of variance (ANOVA) using SPSS 16.0 (Statistical Package for the Social Sciences).
Multivariate data analysis by partial least-squares analysis (PLS) was performed with the SIMCA 13.0 software using scaling based on Variance.
In the present work, metabolomics analysis was applied to discriminate the metabolite variation among the three accessions of P, T and H. Metabolites from this plant including the major andrographolide, deoxyandrographolide, neoandrographolide and carbohydrates (α-glucose, β-glucose and sucrose) as well as amino acids (alanine and choline) were successfully identified.
neoandrographolide signals were also observed in the aromatic region of δ 6.91 and 7.40 respectively.
Relative quantification of the identified major compounds which are andrographolide and neoandrographolide in A. paniculata extracts was based on the mean peak area of the 1H NMR signals from the three different accessions (P, T and H), which were harvested at three different ages (120, 150 & 180 DAT) and two different time sessions of morning and evening. A. paniculata accessions from different ages were able to be differentiated by the intensities of andrographolide (binned at 0.74 ppm) and deoxyandrographolide (binned at 0.62 ppm). The significance (p < 0.05) difference between the samples at different age for each accession was conducted by One-Way ANOVA using MINITAB 14. Morning and evening session data for 120, 150 & 180 DAT of three different accessions (P, T and H) were combined in this relative quantification analysis.
The concentration of andrographolide at 180 DAT of P accession was significantly higher (p < 0.05) as compared to 120 and 150 DAT as shown in
DATs (
Investigation on the in vitro anti-inflammatory effect of the A. paniculata accessions methanol crude extracts was carried out via nitric oxide assay. A. paniculata has been used traditionally for inflammation since ages [
Since the A. paniculata leaf extracts have shown promising results at the screening stage, the IC50 values were then further determined as summarized in
Age | 120 DAT | 150 DAT | 180 DAT | ||||||
---|---|---|---|---|---|---|---|---|---|
Accessions | P | T | H | P | T | H | P | T | H |
Morning | |||||||||
IC50/μM | 32.35 ± 6.46 | 29.21 ± 4.73 | 20.47 ± 3.51 | 54.56 ± 4.85 | 45.83 ± 6.16 | 56.20 ± 5.84 | 42.94 ± 4.65 | 10.61 ± 0.97 | 13.99 ± 2.14 |
Evening | |||||||||
IC50/μM | 35.40 ± 4.60 | 14.74 ± 1.04 | 18.71 ± 2.35 | 48.02 ± 5.88 | 21.95 ± 3.27 | 47.00 ± 5.02 | 18.51 ± 1.62 | 35.98 ± 5.24 | 49.60 ± 4.02 |
Standard: IC50 curcumin = 14.69 μM; P = Pasir Puteh, T = Tanjung Ipoh, H = Harapan.
were assessed from the inhibition of the NO production without the induction of cell death. Thus, another test was carried out in order to determine the cell viability of the induced macrophages.
It could be observed that all the three accessions yielded the highest NO activity at 180 DAT harvesting age of morning session for T and H, and evening session for P with IC50 values of 13.99, 10.61 and 18.51 µg/mL, respectively. In relation to the NO inhibition, P and H accessions gave a significant difference between 120 and 150 DAT, but no significant difference between the morning and evening harvesting times. However, T accession exhibited a significant variation between morning and evening harvesting time for each of the harvesting age.
Generally, the small IC50 value is the lowest concentration that could inhibit the production of NO by at least 50%. However, the statistical computation only recognizes big values in representing a significant activity. Thus, prior to any MVDA computation step, the IC50 values were converted into their antiradical scavenging activity (1/IC50) readings in order to able the statistically translation of the activity.
PLS was applied using a validation model with a degree of over fit between the variables and the responses [
Three clusters were obtained from the PLS bi-plot that combined the score and loading plot of the FTIR data (
three A. paniculata accessions (P, T and H) correlated to NO assay was accomplished (
The other two harvesting ages were projected to the negative side of the plot by PC1 which was opposite of the NO. The peak signals were characterized as those belonging to the major compounds of andrographolide and neoandrographolide. The same data distribution was observed over the ellipse for the T accession of different harvesting ages model (
There was a different observation for H accession, whereby all the three harvesting ages were well-separated in groups (
A. paniculata samples were analyzed with NMR spectrometer to support the data obtained from FTIR analysis. The PLS loading bi-plot data gave 180 DAT of P and T accessions as well separated from 120 and 150 DAT by PC1 (
H accession gave a different observation wherein both 120 and 180 DAT were close to NO assay compared to 150 DAT as shown in
The correlation between anti-inflammatory and metabolite variations among the three different harvesting ages of P, T and H accession using NMR spectroscopy in this study is agreeing with the data obtained from the FTIR analysis. Both of the spectroscopic data are in congruent when correlated to the NO inhibition activity.
PLS model validation was performed by checking the Q2 and R2 accumulative means after cross validation and permutation test with 100 permutations (
the higher significance and lower variation of the NMR-adopted-permutation has helped each model to be validated as a good model. In the current study, the Q2 and R2 values were higher than 0.5 (P accession: R2Y = 0.75833, Q2Y = 0.71733; T accession: R2Y = 0.84973, Q2Y = 0.77553; H accession: R2Y = 0.75833, Q2Y = 0.57511) which suggested that all of the models meet the criteria of the validation and prediction performances.
Utilizing the FTIR and NMR-metabolomics approach has provided a rapid way to study the metabolites variation of A. paniculata in relationship to its different harvesting ages & time with a biological activity of anti-inflammation. The PLS allowed the separation and correlation between the NO assay with the phytochemical present due to the effects of different harvesting ages and times. P (11,341), T (11,248) and H (11,265) accessions exhibited 180 DAT as the best harvesting age possessing the highest anti-inflammatory activity. The accessions at this age also contained higher level of the major compounds which could be attributed to the anti-inflammatory capacity.
Our appreciation goes to Professor Dr. Johnson Stanslas, Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia for providing the andrographolide, deoxyandrographolide and neoandrographolide. The authors also thank Mr. Tajuddin Abd Manap (Biodiversity Unit, Institute of Bioscience, UPM) and Mr. Mohd Norhaizan Saliudin (Department of Crop Science, Faculty of Agriculture, UPM) for guiding in the plant cultivation. This work was supported by Research University Grant Schemes (RUGS) of Universiti Putra Malaysia (grant number 91,968) and Exploratory Research Grant Scheme (ERGS) of Ministry of Higher Education Malaysia (grant number 5,527,124).
Isha, A., Yusof, N.A. and Ismail, I.S. (2016) Harvesting Age and Time Effect of Andrographis paniculata Leaf on Its Anti-Inflammatory Activity. Journal of Biosciences and Medicines, 4, 175-188. http://dx.doi.org/10.4236/jbm.2016.412021