Inducible nitric oxide synthase downregulation and cellular antioxidant enzyme enhancing potential of oleanolic acid from Isodon wightii (Bentham) H. Hara against lipopolysaccharide-induced liver damage in bagg albino strain C mice
Madhusudhanan Gogul Ramnath1, Ramaraj Thirugnanasampandan1, Sadasivam Mathusalini2, Gunasekaran Bhuvaneswari1, Selvaraj Vasantharani1, Palathurai Subramaniam Mohan2
1 Department of Biotechnology, Laboratory of Plant Tissue Culture and Molecular Bioprospection, Kongunadu Arts and Science College, G.N. Mills, Coimbatore, Tamil Nadu, India
2 Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India
|Date of Submission||21-Oct-2018|
|Date of Decision||04-Dec-2018|
|Date of Web Publication||26-Apr-2019|
Department of Biotechnology, Laboratory of Plant Tissue Culture and Molecular Bioprospection, Kongunadu Arts and Science College, G.N. Mills, Coimbatore - 641 029, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Isodon wightii (Bentham) H. Hara is an aromatic medicinal herb belongs to Lamiaceae which has been considered as a prolific source of diterpenoids with diverse structural and medicinal values. Objective: The objective of the study is to evaluate in vivo anti-inflammatory activity of oleanolic acid from I. wightii against lipopolysaccharide-induced liver inflammation in Bagg albino strain C mice. Materials and Methods: Fractions were obtained using silica gel 60–120 mesh column chromatography, and structural elucidation was done using spectroscopic studies. Hepatoprotection, inducible nitric oxide synthase (iNOS) gene downregulation and the levels of cellular antioxidant enzymes with their respective gene expression were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR). Results: White amorphous powder (58 mg) was isolated from petroleum ether extract (15 g) eluted with petroleum ether: ethyl acetate mixture (90.5:9.5), and structure has been elucidated as oleanolic acid. 50 μg of oleanolic acid showed effective hepatoprotection and multiple fold increase in the level of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase. RT-PCR analysis revealed that oleanolic acid had a remarkable effect on iNOS downregulation and antioxidant genes upregulation at 50 μg concentration. Conclusion: The present study proves that oleanolic acid possesses liver protecting activity with a positive effect on increased antioxidant enzymes production. The triterpenoid acid and oleanolic acid could be a suitable natural source for preparing hepatoprotective tonics.
Keywords: Antioxidant enzymes, hepatoprotection, inducible nitric oxide synthase, Isodon wightii, oleanolic acid
|How to cite this article:|
Ramnath MG, Thirugnanasampandan R, Mathusalini S, Bhuvaneswari G, Vasantharani S, Mohan PS. Inducible nitric oxide synthase downregulation and cellular antioxidant enzyme enhancing potential of oleanolic acid from Isodon wightii (Bentham) H. Hara against lipopolysaccharide-induced liver damage in bagg albino strain C mice. Phcog Mag 2019;15, Suppl S1:168-72
|How to cite this URL:|
Ramnath MG, Thirugnanasampandan R, Mathusalini S, Bhuvaneswari G, Vasantharani S, Mohan PS. Inducible nitric oxide synthase downregulation and cellular antioxidant enzyme enhancing potential of oleanolic acid from Isodon wightii (Bentham) H. Hara against lipopolysaccharide-induced liver damage in bagg albino strain C mice. Phcog Mag [serial online] 2019 [cited 2021 May 10];15, Suppl S1:168-72. Available from: http://www.phcog.com/text.asp?2019/15/62/168/257277
- Oleanolic acid isolated from Isodon wightii showed a remarkable downregulation of inducible nitric oxide synthase gene expression and protects liver damage induced by lipopolysaccharide
- Hepatoprotection of oleanolic acid was found to be highly associated with the increased production of cellular antioxidant enzymes and their respective genes upregulation.
Abbreviations used: iNOS: Inducible nitric oxide synthase; LPS: Lipopolysaccharide; BALB/c: Bagg albino strain C mice; Nrf2: Nuclear factor erythroid 2-related factor 2; NF-κB: Nuclear factor kappa B.
| Introduction|| |
Lipopolysaccharide (LPS) is the major cause of the liver injury which initiates inflammation followed by liver damage and cancer induction. The mechanism of liver damage induced by LPS is either by activation of nuclear factor Kappa B (NF-κB) or producing reactive oxygen species. Therefore, the drug which has the capacity of inhibiting NF-κB and reducing oxidative stress may act as an effective hepatoprotectant.
Isodon wightii is a tall perennial herb with white flowers and serrated leaves with numerous trichomes. I. wightii is a rich source of bioactive terpenoids including melissoidesin, abietic acid, mono, and sesquiterpenes. Oleanolic acid, a pentacyclic triterpenoid widely distributed in leaves, fruits, and vegetables, has wide spectrum of medicinal properties. To the best of our knowledge, no previous reports are available on anti-inflammatory property of oleanolic acid from natural source. Hence, oleanolic acid isolated from the leaves of I. wightii was used in this study.
| Materials and Methods|| |
I. wightii (Bentham) H. Hara leaves were collected from Coonoor, Tamil Nadu. Authenticated voucher specimen (BSI/SC/5/23/06-07-Tech. 881) has been deposited in the herbarium of Botanical Survey of India, Southern Circle, Coimbatore, Tamil Nadu, India.
Extraction and isolation
Petroleum ether extract of leaf powder (715 g) was obtained using Soxhlet apparatus at room temperature. After evaporating the solvent in vacuo at 45°C, 15 g of crude extract was obtained and subjected to silica gel (60–120 mesh size, Ranbaxy Fine Chemicals Limited, New Delhi, India) column chromatography (15:45) and eluted with petroleum ether: ethylacetate mixture in the ratio of 90.5:9.5 v/v (Ranbaxy Fine Chemicals Limited, New Delhi, India). Further, the fractions were analyzed by thin-layer chromatography (TLC) coated with silica gel G-60 (Himedia Laboratories Pvt. Ltd., Mumbai, India). The eluted fraction was white amorphous powder. Further, structural elucidation of isolated compound was carried using spectroscopic studies.
1 H (400MHz), 13C (100MHz) (Bruker Avance-400), and 2D NMR (Bruker Avance-400MHz) spectra including 1H=H correlated spectroscopy, heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) were recorded on Bruker Avance-400 spectrometer in CDCl3. 13C NMR chemical shifts are reported in parts per million from tetramethylsilane with the solvent resonance as the internal standard. Infrared (IR) spectrum was recorded on a Nicolet Avatar Model FT-IR (4000–400 cm−1) spectrometer using KBr pellets. Shimadzu LC-MS was used for recording EI-MS. Data are reported as follows: chemical shift, multiplicity (s¼singlet, brs¼broad singlet, d¼doublet, t¼triplet, q¼quartet, and m¼multiplet), coupling constants (Hertz), and integration.
Animals and experimental design
Based on the regulations of Council Directive CPCSEA no: 08/2016/IAEC/KASC regarding good laboratory practice on animal experimentation, animals were maintained. Adult Bagg albino strain C (BALB/c) mice were purchased from Small Animal Breeding Centre, Kerala Veterinary and Animal Sciences University, Mannuthy, Kerala, India. Micro isolators with autoclaved bedding, cages, food pellets, and deionized water were used for animal maintenance. Standard conditions of humidity, temperature (25 ± 2°C), and light (12 h light/dark) were also provided. Aseptic condition was set up to avoid the entry of harmful microbes. Adult BALB/c mice weighing 30 g were used and randomly assigned into three groups normal group (n = 3), LPS treated (n = 3) and experimental group (n = 6) (oleanolic acid + LPS). Mice were placed on restricted, once a day diet and given 25 and 50 μg concentrations of oleanolic acid/30 g body weight followed by LPS (1.5 μg/30 g body weight). After treatment, animals were sacrificed and liver was excised. A part of liver fixed in 10% formalin was used for histopathological analysis, and another part of the liver was taken for antioxidant enzyme and gene expression studies.
The liver tissue was impregnated with histology grade paraffin wax (melting point 58–60°C) at 60°C for two changes of 1 h each. The wax impregnated tissues were embedded in paraffin blocks, mounted, and cut with rotary microtome at 3 μM thickness. The sections were stained for 8 min with Ehrlich's hematoxylin (0.75%). Further, they were counterstained in 1% aqueous eosin (1 g in 100 mL water) for 1 min, and the excess stain was washed in tap water and allowed to dry. The dried sections were mounted in Distrene, Plasticiser, and Xylene. The sections were wetted in xylene, and the mount was inverted to place over a coverslip. Low-power and high-power dry objective lens with × 10 magnification were used for observing the hepatocyte architecture and injury.
Extraction and estimation of cellular antioxidant enzymes
Excised liver was homogenized using 0.1M ice cold sodium phosphate buffer (10% w/v) and then centrifuged at 10,000 rpm for 15–20 min at 4°C, and finally, the supernatant was collected and used for subsequent enzyme assays. The total protein was assayed with bovine serum albumin as the standard. Antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were estimated.
Gene expression profiling
Total RNA extraction and cDNA synthesis
Total RNA was extracted from excised liver tissue using Trizol reagent (GeNei, Bengaluru). Synthesis of cDNA from isolated total RNA was performed using a cDNA kit (HELINI, Chennai). Briefly, 20 μL reaction mixture was prepared through the addition of 6 μL of cDNA reaction mix, 1 μL of oligodT, 1 μL of random hexamer, 2 μL of enzyme mix, 2 μL of isolated RNA, and 8 μL of nuclease-free water in a 0.2 mL polymerase chain reaction (PCR) tube. Reverse transcription was carried out for one cycle at 42°C for 30 min and inactivation at 95°C for 2 min.
The PCR analysis was performed on the aliquots of prepared cDNA for detecting the expression patterns of genes such as inducible nitric oxide synthase (iNOS), SOD, CAT, GPx, and GST. The sequence of oligonucleotide primers for iNOS-F (5'-ATG GAC CAG TAT AAG GCA AGC-3'), iNOS-R (5'-GCT CTG GAT GAG CCT ATA TTG-3'), SOD-F (5'-GAC CTG CCT TAC GAC TAT GG-3'), SOD-R (5'-GAC CTT GCT CCT TAT TGA AGC-3'), CAT-F (5'-GAA CGA GGA GGA GAG GAA AC-3'), CAT-R (5'-TGA AAT TCT TGA CCG CTT TC-3'), GPx-F (5'-CCT CAA GTA CGT CCG ACC TG-3'), GPx-R (5'-CAA TGT CGT TGC GGC ACA CC-3'), GST-F (5'-TGG CCG GAA GCA CAA CCT G-3'), GST-R (5'-CCC CAC CAA CAC CGG CAC-3'), β-actin-F (5'-TGG AAT CCT GTG GCA TCC ATG AAA-3'), and β-actin-R (5'-TAA AAC GAG CTC AGT AAC ATC CG-3'). Amplification reactions were performed in a gradient thermocycler (Applied Biosystems, California) as follows: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 92°C for 30s, annealing at 58°C (iNOS and SOD), 55°C (CAT), 50°C (GPx), and 54°C (GST) for 1 min, and extension at 72°C for 1 min followed by final extension at 72°C for 10 min. PCR reactions were carried out in a volume of 10 μL containing 5 μL of PCR master mix (2×) (Merck Specialties, Mumbai) solution, 1 μL of forward and reverse primer, 1.5 μL of cDNA, and 1.5 μL of nuclease-free water.
Mean ± standard deviation (n = 3) was calculated to analyze the data obtained from the antioxidant enzyme study.
| Results|| |
Compound isolation and structure elucidation
Compound isolated as white amorphous powder and observed on TLC as a light-yellow fluorescent spot under ultraviolet (UV) light (366 nm) and showed UV absorption maxima at 281 and 232 nm. The IR spectrum displayed intense absorption at 3415.31 (COOH), 1715.37 (C = O), 1621.00 (C = C), and 1453.1 cm−1 (Ar). A combination of 13C-NMR and HSQC spectral data suggested that seven methyl, five methane, and ten methylene groups. The proton and 13C-NMR spectrum showed signals at δ5.21 (1H, s, H-12), 3.80 (1H, s, H-3), 1.57 (1H, d, H-18) along with seven singlets at δ1.08, 0.76, 0.95, 0.93, 1.14, 0.99, and 1.50 and another one signal at δ9.80 (1H, br, s) denoted the presence of an carboxylic group. The interpretation of HMBC correlations showed the correlations of H-29 (δ0.99) to C-21 (δ31.50), H-27 (δ1.14) to C-27 (22.06), C-8 (39.00), C-14 (43.00), and C-16 (20.27). The HMBC also shows a correlation between methyl protons with carbon signals, H-23 (δ1.08) to C-23 (24.54), H-24 (δ0.76) to C-24 (19.51), H-25 (δ0.95) to C-25 (16.06), and H-26 (δ0.93) to C-26 (17.40) [Table 1]. Based on mass spectral data, molecular weight has been determined as 457.73. The spectral studies support the compound as oleanolic acid (C30H48O3) [Figure 1].
|Table 1: Nuclear magnetic resonance spectrum data of oleanolic acid (δ ppm)a|
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1.5 μg LPS induced the liver inflammation with characteristic lymphocytic infiltration near the central vein and severe necrosis [Figure 2]b. Mildly affected hepatic lobule toward the central vein was seen in 25 μg of oleanolic acid-treated hepatocytes [Figure 2]c. Complete reduction in inflammation and revived liver architecture was noticed in 50 μg of oleanolic acid [Figure 2]d similar to normal liver [Figure 2]a and it is considered to be the optimum dose to have high rate of liver protection.
|Figure 2: Hepatoprotective effect of oleanolic acid on lipopolysaccharide-induced Bagg albino strain C mice: (a) normal liver, (b) lipopolysaccharide-induced liver, (c and d) LPS-induced liver treated with 25 and 50 μg of oleanolic acid, respectively|
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Cellular antioxidant enhancement
A reducible amount of antioxidant enzymes was recorded in LPS (1.5 μg)-induced liver whereas fourfold increase in SOD (12.36 ± 0.16 μM/min/mg protein) and fifteen-fold (141.98 ± 2.34 μM/min/mg protein) increase in CAT were observed with 50 μg of oleanolic acid administration. GPx and GST levels were also increased considerably (1.10 ± 0.02 and 0.40 ± 0.07 μM/min/mg protein, respectively) after administering with 50 μg of oleanolic acid. Overall, it is observed that the level of antioxidant enzymes was increased with oleanolic acid treatment [Figure 3]A.
|Figure 3: (A) Effect of oleanolic acid on antioxidant enzymes level in lipopolysaccharide-induced Bagg albino strain C mice liver. Error bar represents mean ± standard deviation (n = 3), (B) Downregulation of inducible nitric oxide synthase and upregulation of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase genes by oleanolic acid in lipopolysaccharide-induced Bagg albino mice strain C mice liver: (a) lipopolysaccharide-induced liver and (b and c) lipopolysaccharide-induced liver treated with 25 and 50 μg of oleanolic acid, respectively. β-actin-Internal marker|
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Inducible nitric oxide synthase downregulation and antioxidant genes upregulation
iNOS mRNA production was high in 1.5 μg LPS-induced hepatocytes [[Figure 3]B Lane a]. 50 μg of oleanolic acid showed good inhibitory effect on iNOS mRNA production [[Figure 3]B Lane c]. LPS inhibited the expression of SOD, CAT, GPx, and GST genes at transcriptional level [[Figure 3]B Lane a]. On treatment with 25 μg of oleanolic acid, the respective antioxidant genes were upregulated; however, 50 μg concentration was found to be highly efficient for upregulating antioxidant genes expression to overcome the oxidative stress caused by LPS [[Figure 3]B Lanes b and c].
| Discussion|| |
Oleanolic acid is a pentacyclic triterpenoid widespread in many food and medicinal plants, and it is also reported from Isodon loxothyrsus, Isodon phyllostachys, and Isodon xerophilus.,, Oleanolic acid isolated from hexane extract of Olea europaea using petroleum ether: ethyl acetate solvent system supports the present study where petroleum ether: ethyl acetate was used for oleanolic acid extraction.
LPS acts on NF-κB/IκB complex by attaching with CD14 receptor and induces phosphorylation and ubiquitination of IκB. Further, NF-κB gets released and upregulates nitric oxide synthase gene. LPS-induced nitric oxide synthase (iNOS) produces nitric oxide that mediates signal transduction to induce inflammatory response and leads to hepatic injury. In this study, it is assumed that iNOS downregulation followed by hepatoprotection effect of oleanolic acid might be associated with the inhibition of NF-κB or prevention of binding of LPS with NF-κB/IκB complex, but the exact mechanism of action remains obscure.
Reactive oxygen species induced by LPS is thought to reduce the level of antioxidant enzymes, namely, SOD, CAT, GPx, and GST in the liver. Results of the present study clearly showed the increased production of antioxidant enzymes in oleanolic acid-treated liver. It is assumed that oleanolic acid could have increased the nuclear accumulation of nuclear factor erythroid 2-related factor 2-dependent genes which play an important role in the protection of liver by regulating antioxidant and detoxifying enzymes. In addition, it is taken into consideration that antioxidant potential of oleanolic acid is also one of the reasons for observed hepatoprotection. This is the first report on in vivo anti-inflammatory activity of oleanolic acid isolated from I. wightii.
| Conclusion|| |
The pharmacological investigation of oleanolic acid isolated from I. wightii may act as a promising phytocompound which could protect the liver from oxidative stress and other damages.
We are grateful to the management of Kongunadu Arts and Science College for the necessary infrastructure to carry out the research work.
Financial support and sponsorship
This work was financially supported by the University Grants Commission, New Delhi, India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]