|
 
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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 8
| Issue : 2 | Page : 124-133 |
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Preconditioning with Partharishta reduces the oxidative stress associated with myocardial ischemic reperfusion injury in H9c2 cells
M Gowri, Rajmohan Velayudhan Pillai, S Thara Lakshmi
Department of Rasashastra and Bhaishajya Kalpana, Government Ayurveda College, Trivandrum, Kerala, India
| Date of Submission | 25-Jul-2021 |
| Date of Acceptance | 23-Dec-2022 |
| Date of Web Publication | 31-Mar-2023 |
Correspondence Address:
Dr. M Gowri
Department of Rasashastra and Bhaishajya Kalpana, Government Ayurveda College, Trivandrum, Kerala
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/jdras.jdras_25_21
BACKGROUND: Myocardial Ischemia Reperfusion (I/R) injury- a paradoxical challenge associated with contemporary methods of successful resuscitation triggers uncontrolled apoptosis induced by the concomitant oxidative stress. Adaptive mechanisms incorporating the upregulation of cytoprotective genes like Nrf2 protects cells from apoptotic changes and dysfunction under stress conditions. Partharishta an Ayurvedic formulation with all its individual ingredients being potent antioxidants, is thought to bring a positive result in reducing oxidative stress in cardiac tissues. Therefore the study aims to evaluate the antioxidant activity of the formulation Partharishta via the expression of Nrf2 gene in ischemia induced H9c2 rat cardiomyoblast cells when preconditioned with Partharishta. METHODS: Partharishta was prepared in accordance with the Ayurvedic Formulary of India (AFI). To evaluate the extent of cell viability the Partharishta pretreated H9C2 cell lines were subjected to MTT Assay. As confirmatory assays for antioxidant activity, an invitro ROS measurement assay and Reverse transcriptase PCR Analysis to confirm the expression of Nrf2 gene against oxidative stress was conducted. RESULTS: On MTT Assay the cells pretreated with 25µg/ml of dessicated Partharishta provided an average cell viability of 85.12%. The confirmatory Assays conducted in the cells at this concenteration showed that the ROS production is relatively low and the Nrf2 gene against oxidative stress was expressed in these cells. CONCLUSION: These findings of the present in-vitro study reveal that the formulation Partharishta possess significant antioxidant as well as the free radical scavenging activity and therefore may be considered as one of the good sources of natural cardioprotective formulation. Keywords: Antioxidant activity, Partharishta, Nrf2gene expression, oxidative stress
How to cite this article:
Gowri M, Pillai RV, Thara Lakshmi S. Preconditioning with Partharishta reduces the oxidative stress associated with myocardial ischemic reperfusion injury in H9c2 cells. J Drug Res Ayurvedic Sci 2023;8:124-33 |
How to cite this URL:
Gowri M, Pillai RV, Thara Lakshmi S. Preconditioning with Partharishta reduces the oxidative stress associated with myocardial ischemic reperfusion injury in H9c2 cells. J Drug Res Ayurvedic Sci [serial online] 2023 [cited 2023 Jun 10];8:124-33. Available from: http://www.jdrasccras.com/text.asp?2023/8/2/124/373017 |
| Introduction | |  |
Myocardial ischemic reperfusion injury (IRI) also termed as Myocardial Stunning was first postulated in 1960 by Jennings et al and its pathology was first described by Murray in 1986.[1],[2] The condition is defined as a paradoxical challenge associated with restoration of blood flow in the oxygen deprived tissues of myocardium associated with conventional therapies in case of Ischemic heart disease (IHD).[3] IHD, being one of the major cause of CVD, has alone caused more than 9 million deaths worldwide in 2016.[4] The current mortality rate of IHD in India is approximately 13.6%.[5] Thus today IHD has turned to be the largest single contribution to global mortality and is sure to dominate the mortality trends in the future too.[6] In this scenario, after an acute myocardial infarction, early and successful myocardial reperfusion is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome. However, the process of restoring blood flow to the so far oxygen deprived ischemic myocardium can induce myocardial and vascular injury as a result of inflammation and oxidative damage through the induction of oxidative stress by the generation of large amount of free radicals, rather than restoration of normal function. This phenomenon, can paradoxically reduce the beneficial effects of myocardial reperfusion and lead to lethal damage to myocardium by the trigerring of apoptosis.[7] Thus this neglected therapeutic phenomenon has raised doubt about the clinical relevance of the therapy.
The concept of IR injury was first postulated when the scientists noted the production of a large fraction of cellular enzymes during reperfusion, causing a high degree of myocardial injury resulting in extensive tissue damage, greater than that caused during hypoxia.[8] The pathology involved was the production of excess Reactive Oxygen Species (ROS) which resulted in an imbalance in the generation of ROS and the antioxidant defence systems in the body. The resultant phenomenon is termed as oxidative stress which caused excessive inflammation and Ca2+ overload in the cells thereby resulting in the triggering of apoptosis. Most cells including cardiomyocytes contain enzymatic antioxidant defense mechanisms that quickly convert ROS to water. But when the proportion of ROS production exceeds the manageable levels of these antioxidant system of the cells, it triggers the redox sensitive transcription factor that upregulate cytoprotective and antioxidant genes expression to protect against IR injury.[9]
Adaptive mechanisms incorporating the upregulation of cytoprotective genes like Nrf2 protects cells from apoptotic changes and dysfunction under stress conditions.[10] The Nuclear factor erythroid 2 related factor 2 (Nrf2)is a member of Cap n collar subfamily of basic region leucine zipper transcription factors.[10] The core regulation of cellular responses to electrophilic/ oxidative stress is controlled by Nrf2. Under basal conditions, Nrf2 is sequestered by cytoplasmic Kelch – like ECH- associated protein 1(Keap 1) and targeted to proteasomal degradation.[10],[11] This Nrf2 – Keap 1 interaction is disrupted under conditions of oxidative stress and leads to translocation of Nrf2 to the nucleus where it activates transcription of antioxidant and detoxifying genes by binding it to the Antioxidant Response elements (AREs).[11],[12],[13] Thus upregulation of Nrf2 gene act as a major sensor of oxidative stress in the cell. In most of the cases the myocardial cells which was in a hypoxic state fails to upregulate the Nrf2 gene to sufficient levels so as to protect these cardiac cells from oxidative stress and thereby results in an array of cell damage.[10],[11],[12],[13] So the only possible intervention suggested in the conventional system of medicine, is to make the myocardial tissues potent with antioxidant drugs to upregulate the Nrf2 gene and resist the oxidative stress to the maximum extent and thereby control the adverse effects.[14],[15]
Extensive researches in different perspectives have been pursued to explore the free radicle scavenging and antioxidant activity of various bioactive phytochemicals and plant derived compounds as Nrf2 inducers.[10] Natural compounds like Resveratrol, Flavanoids, Curcumin, Quercetin predominate such researches[16],[17]
Partharishta, a medicinal preparation mentioned in Hridroga Chikitsa prakarana of Bhaishajya Ratnavali[18] made by soaking the specified raw drugs, either in powder form or in the form of decoctions in a solution of jaggery, for a specified period of time, during which it undergoes a process of fermentation generating alcohol, thus facilitating the extraction of the active principles contained in the drug.[19] The ingredients of Partharishta with its scientific name and proven activities are mentioned in [Table 1] and corresponding images in [Figure 1]. Being a self generated alcohol preparation,the formulation extracts the alcohol soluble contents[19] of the ingredients which are potent and proven antioxidants and free radicle scavengers.[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31] Flavanoids and resveratrol which have a proven history of Nrf2 gene upregulation[16],[17] are also found to be the active constituents of the ingredients in this formulation. Though the individual ingredients of Partharishta were proven for its antioxidant action, no studies were so far initiated regarding the antioxidant action or Nrf2 gene expression activity of Partharishta as such. Thereby on evaluating the cell viablity,extent of Reactive Oxygen species(ROS) production and expression of Nrf2 gene against oxidative stress of Partharishta treated cells, the effectiveness of the formulation in myocardial reperfusion injury could be inferred. Further being an invitro evaluation, of the cardioprotective activity of Partharishta in reperfusion injuries, permit an enormous level of simplification of the system under study.
Thereby the basic objective of the study was to evaluate the antioxidant activity of the formulation Partharishta via the expression of Nrf2 gene in ischemia induced H9c2 rat cardiomyoblast cells when preconditioned with Partharishta.
| Materials and Methods | | |
Preparation of Partharishta
Partharishta used for the study was prepared in the Department of Rasashastra and Bhaishajya Kalpana as per the reference from Bhaishajya Ratnavali. The raw materials for Partharishta except Madhuka Pushpa were collected from its corresponding sources in Thiruvananthapuram. Madhuka Pushpa was collected from the tribal forests in Kandhamal district, Bhubaneswar, Orissa. The genuinity and identity of the samples were confirmed by analysis of its API parameters in Drug Standardization unit of Govt Ayurveda College Thiruvananthapuram. The voucher sample no of these raw drugs are 72,73,74,75 and 76 respectively for Arjuna twak, Draksha, Madhuka Pushpa, guda and Dhataki Pushpa and is available in Department of Rasashastra and Bhaishajya Kalpana, Govt Ayurveda College, Thiruvananthapuram.
Phytochemical analysis of prepared sample
Thin layer chromatography (TLC)
Preparation of the sample
Dry 50 ml of the prepared standard Partharishta in vacuum to remove the self generated alcohol. The extract is partitioned successively with n -hexane (50 ml x 3), chloroform (50 ml x 3) and ethyl acetate (50 ml x 3). Filter and concentrate the ethyl acetate extract under vacuum and weigh. With this extract carry out thin layer chromatography.
Another test solution is prepared similarly with 50ml of the prepared standard Partharishta by drying it in vaccum to remove the self generated alcohol. The extract is then partitioned with ethyl acetate(50mlx3). Filter and concentrate the ethyl acetate extract under vacuum and weigh. With this extract carry out thin layer chromatography.
Mobile phase
Toluene: ethyl acetate: formic acid: methanol (3: 2: 0.1:0.12) was used as the mobile phase
Apply separately 15 µl of both the test solution prepared as above on TLC plate and develop the plate to a distance of 8 cm using the mobile phase. After development, allow the plate to dry in air and derivatized with Natural product reagent and examine under ultraviolet light 256nm and 366 nm.
High performance thin layer chromatography (HPTLC)
Preparation of sample
HPTLC profiling of Two randomly selected market samples of Partharishta procured from two GMP certified companies (M1 and M2) were compared with the prepared standard sample (AC). HPTLC profile of the 3 samples – one prepared standard sample and two market sample were developed by extracting in the following two ways. One set was successively extracted using n- Hexane, chloroform and ethyl acetate and the other was simply extracted in ethyl acetate.
Mobile phase
Toluene: ethyl acetate: formic acid: methanol (3: 2: 0.1:0.12) as mobile phase
Thus a total of 6 samples (S1-successively extracted market sample1, S2- Successively extracted market sample2, S3- Successively extracted prepared sample, S4- ethyl alcohol extracted market sample1, S5- ethyl alcohol extracted market sample2, S6- ethyl alcohol extracted prepared sample) were sprayed at a concenteration of 0.1µg/µlon the HPTLC plates as bands and developed using the mobile phase to produce the HPTLC Profiling.
The whole procedure controlled by WINCATS software The imaging visualized with CAMAG TLC Visualizer and is documented via digital CCD camera connected by USB.
Cell culture and treatment
H9C2, rat cardiomyoblast cells procured from NCCS, Pune, India, was used for the present study. The cells were grown in Dulbecco’s modified eagles medium (Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Grand Island, NY, USA) antibiotic solution containing penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (2.5 μg/mL) and maintained at 5% CO2, 37°C in a humidified condition.
Preparation of extracts of Partharishta
100ml of the prepared sample of Partharishta was filtered through a Whatman filter paper 1 and dessicated on a dessicator, sterilized and applied directly to the cultered cells on 5 different concenterations(as mentioned in the procedure below) to evaluate the cell viablity as per MTT Assay.
Cell viability evaluation by MTT ASSAY(Arung et al., 2000)
Cultured H9C2 cells were evaluated in triplicates. Normal control in which cells were maintained under normal conditions. Ischemic control cells in which cells were treated with ischemic buffer alone and then reperfused with DMEM. Sample treated cells where H9C2 cells were pretreated with dessicated extracts of Partharishta and then induced ischemia and further reperfused with the reperfusion fluid. These cells when reached 70 -80% confluency, were induced ischemia using ischemic buffer for 2 hours and were then reperfused with normal DMEM. The group three cells, were pretreated at five different concentrations of Partharishta extract ranging from 6.25, 12.5, 25, 50 and 100 μg/mL for 24 hours, and the viability was assessed by MTT assay. The evaluation was conducted in triplicates. The optimum concentration which showed maximum average cell viablity was used for conducting the following confirmatory assays.
Invitro ROS measurement using DCFDA staining
The intracellular ROS activity was determined using the extent of fluorescence produced on DCFDA staining. The cells which produced maximum cell viability in MTT assay were washed with PBS and added with 50 µl of DCFDA and incubated for 30 minutes. Excess dye was washed with PBS and the fluorescence was measured using a fluorimeter at 470 nm excitation and emission at 635nm (Qubit 3.0, Life technologies, USA) and expressed in arbitrary units.
Reverse transcriptase polymerase chain reaction(RT PCR)
Total RNA was isolated using the total RNA isolation kit according to the manufacture instruction (Invitrogen – Product code10296010) with Trizol (Life Technologies Thermo Fisher Scientific, Inc) and equal amounts of RNA were reverse transcribed to cDNA using a Thermo Scientific verso cDNA synthesis kit Product code AB- 1453/A. The thermal cycler (Eppendorf Master Cycler) was programmed to undergo cDNA synthesis. The following cycling conditions as in [Table 2] were employed.
The amplification as in [Table 3] was done using Thermoscientific amplification kit. Initial denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 30s, annealing at Tm for 30 s and extension at 72°c for 1 minute which was repeated for 35 cycles and the final extension at 72°C for 5 minutes. After the amplification, the PCR product was separated by agarose gel electrophoresis. The stained gel was visualized using a gel documentation system (E gel imager, Invitrogen).
| Results | | |
Phytochemical analysis
TLC analysis
The Retention factor (Rf) of the compound were calculated with the plates being observed under Ultra violet lamp and also in Iodine vessel. The successively extracted sample showed only three spots two in UV lamp and one in Iodine chamber. The ethyl acetate alone, extracted sample showed 4 spots three in UV lamp and one in Iodine vessel.
HPTLC analysis
The samples showed similar peaks in all the samples and the values of the prepared samples were almost similar in all market samples. The ethyl acetate alone extracted samples showed all the 8 spots in all the three samples while the successively extracted sampled showed only 7 spots in 2 of the samples(S1 and S3). The Rf value of each peak and the net area % is tabulated in [Table 4] and corresponding HPTLC tracks in [Figure 2]. Here no marker compounds were used to detect the components.
The pretreated H9C2 cells imparts increased cell viability after ischemic reperfusion
MTT assay was used to analyse the cytoprotective effect of Partharishta on H9c2 cells. From the data in [Table 5]. it was evident that the pre- treated cells shows an increased percentage in cell viability(at all concenterations) after ischemic reperfusion when compared to the untreated cells. The untreated cells were found to have only an average cell viability of 46.35%. The maximum cell viability was observed when the cells were pretreated with the extract of Partharishta at a concenteration of 25µg/ml, which provides an average cell viability of 85.12%. Thus the cells treated at 25µg/ml were used to perform the confirmatory assays. Illustrations in [Figure 3]
The pretreated cells showed remarkably less ROS production
In DCFDA staining the increased green fluroscence shows the presence of increased ROS activity within the cells and thereby increased oxidative stress. The net production of ROS and thereby the proportional degree of oxidative stress is re expressed in arbitrary units in [Table 6]. Graphical data in [Figure 4] and Illustration in [Figure 5]
The pretreated cells showed significant Nrf2 gene upregulation
The gene expression is expressed as relative band width and intensity in comparison to the House keeping genes for normalization of the samples. The results are further interpreted on this basis and tabulated in [Table 7]. The graphical data is represented in [Figure 6], relative expression in [Figure 7] and illustration in [Figure 8]
| Discussion | | |
The present work was an experimental research to evaluate the antioxidant activity of an Ayurvedic Formulation – Partharishta against the oxidative stress induced in Myocardial ischemic reperfusion injury with H9C2 cell lines as the study model. The study tools utilized in the study provides us the following information.
The increased cell viability of Partharishta treated cells in MTT Assay in comparison to untreated cells denotes the cardioprotective activity of the formulation. The maximum cell viability was observed when the cells were pretreated with the extract of Partharishta at a concenteration of 25µg/ml. The ROS DCFDA staining concludes its free radicle scavenging activity and as Reactive oxygen species being the primary cause for oxidative stress and thereby I/R in cardiac tissues, the remarkably less ROS production in pretreated cells denotes a decrease in oxidative stress in pretreated cells when compared to untreated cells. With this data we could infer that subsequently, the reperfusion damage induced in the Partharishta treated cells will also be relatively less, when compared to the untreated cells. The RT-PCR assay confirmed the upregulation of Nrf2 Gene in the cells pretreated with the extract of Partharishta thereby confirming the action of the formulation against oxidative stress. It was also observed that the gene expression was maximum in the pretreated cells when compared to the normal cells and ischemia induced untreated cells.
Even in the lime light of extensive researches and interventions, the medical community has however failed to develop a strategy to minimize the paradoxical cascade of Myocardial reperfusion, when it comes to patient care. Drugs and compounds triggering Nrf2 activation is one of the anticipated areas of research where the medical fraternity could explore the various dimensions to preclude the apprehension raised regarding this conventional therapy. Tremendous researches have been carried out in regards to various natural compounds to elicit the regulation and activation of Nrf2 genes.[16] Unfortunately the Ayurvedic fraternity lacks studies pertaining to the effect of Ayurvedic formulations in the perview of I/R injury. This study bridges the lacunae in this regard and is a preliminary experimental study in this aspect.
Among the studied natural compounds, phytochemicals like Resveratrol, Quercetin and Flavanoids are proven to be active constituents in the alcoholic extract of ingredients of Partharishta.[23],[24],[25],[26],[27],[28],[29],[30],[31] Quercetin – the active constituent in Madhuka Pushpa upregulates Nrf2 via Phosphorylation by p38 and ERK (extracellular signal-regulated kinases)[32] as well as modification of cysteines in Keap1.[33] Resveratrol- the constituent in Draksha activates Nrf2 via general proteasome inhibition and phosphorylation by ERK, [34,35] Flavanoids and polyphenols – are constituents in almost all ingredients of Partharishta, activate the gene by covalent modification of Keap 1[36] The combination of these phyto chemicals in Partharishta might have rendered the formulation to upregulate Nrf2 gene expression in the present study.
While analyzing the method of preparation of the formulation it could be seen that the ingredients which have rich antioxidant contents like phenols, flavonoids and tannins are all kwatha dravyas. These contents on boiling(upto 120◦C) increases its antioxidant activity by producing gallic acid and glucosidase content.[37] On the other hand Dhataki Pushpa which contains rich tannins along with poly phenolic compounds which are susceptible to enzymatic conversion to simple phenols and alcohol during anaerobic fermentation of Arishta preparations are included as prakshepa.[38]Dhataki Pushpa further contain certain yeast cell species which will be destructed on exposure to a temperature above 40◦C thereby placing it as a prakshepa dravya.[39]
Oxidative stress, thus being the basic pathophysiology involved in the reperfusion damages of cardiac tissue, confirming the upregulation of Nrf2 factor in sample pretreated cells proves the antioxidant activity and thereby cardioprotective mechanism of Partharishta in Myocardial Ischemia Reperfusion Injury(IR). This study being an invitro research using RT PCR is just an indicator of Nrf2 gene expression. The underlying mechanism of Nrf2 regulation using Partharishta need to be investigated further and need to be supported with invivo as well as relevant clinical studies.
| Conclusion | | |
Current study was intended to explore the dimensions of Partharishta a herbal Formulation to impede the neglected therapeutic phenomenon. With a significant cell viability observed via MTT Assay in Partharishta preconditioned H9C2 cells induced with ischemia, along with the observation of a remarkably low ROS production in DCFDA staining, and a positive expression of Nrf2 gene in RT PCR, streamlines the antioxidant activity of the formulation and proves its relevance in Oxidative Stress induced by I/R Injury. The present study focusses on the invitro aspects, to further establish its clinical outcome, its activity need to be monitored in animals as Invivo studies and in Humans via Clinical studies. The current study just provides a preliminary data which could be utilized for further investigation. Furthermore clinically, the scenario demands to manage patient with Myocardial reperfusion Injury as a post interventional complication and in most of the cases, the interventions are made on an emergency basis where pretreatment with antioxidants are not practically possible. So the effect of the formulation as a post treatment strategy has to be evaluated and if found effective, the formulation need to be redesigned into a dosage form which can be administered immediately as a post operative drug.
Acknowledgement
Expressing sincere gratitude to Dr Rajesh Ramachandran PhD, Biogenix Research Lab, Thiruvananthapuram, for his valuable suggestions, timely advices and whole hearted support througout the study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]
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