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Home > Pharmacological Basis for the Use of Ephedra Gerardiana and Ribes Orientale in Rheumatioid Arthritis: Evaluation of Toxicological and Phytochemcial Profile

Pharmacological Basis for the Use of Ephedra Gerardiana and Ribes Orientale in Rheumatioid Arthritis: Evaluation of Toxicological and Phytochemcial Profile

Thesis Info

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Author

Uttra, Ambreen Malik

Program

PhD

Institute

University of Sargodha

City

Sargodha

Province

Punjab

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Pharmacology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/12321/1/Ambreen%20Malik%20Uttra%202019%20Pharmacology%20uos%20sargoda%20prr.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726876645

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Rheumatoid arthritis (RA) is a chronic inflammatory, autoimmune and multisystem illness. Owing to continued researches in this area, a large number of disease modifying antirheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and immunosuppressive drugs have become available for the therapy of various types of arthritic diseases. Nevertheless, it is requisite to unveil such natural products which may be cost effective and superior to already available synthetic antiarthritic drugs. Many indigenous medicinal plants have been found to contain important active principles, which make these plants helpful to treat many diseases. The present research work aimed to scientifically validate the traditional claim of Ephedra gerardiana Wall ex. Stapf and Ribes orientale Desf for rheumatoid arthritis. The antiarthritic activity of aqueous ethanolic extract and fractions (ethyl acetate, n-butanol and aqueous) of Ephedra gerardiana and aqueous ethanolic extract and fractions (n-butanol and aqueous) of Ribes orientale was evaluated by employing in-vitro and in-vivo methods. The in-vitro inhibition of protein (egg albumin and bovine serum albumin (BSA)) denaturation and human red blood cell (HRBC) membrane stabilization assays were performed at 50, 100, 200, 400, 800, 1600, 3200 and 6400 µg/ml concentrations of extracts and fractions. The in-vivo formaldehyde induced arthritis study was carried out in rats for 10 days at 50, 100 and 200 mg/kg doses of extracts/fractions and rat paw volume/diameter was measured. The in-vivo Freund Complete Adjuvant (FCA) induced arthritis study was performed in rats for 28 days at the dose that produced maximum effect in formaldehyde model i.e., 200 mg/kg of extracts and fractions. In FCA model, paw volume/diameter, arthritic index, body weight, hematological/biochemical parameters and radiographic/histopathological analysis was carried out. The mRNA expression levels of inflammatory mediators (TNF-α, IL-1β, IL-6, NF-kB, COX-2, IL-4 and IL-10) was appraised using RT-PCR and serum PGE2 and TNF-α levels were estimated using ELISA. Moreover, in-vitro anti-oxidant activity of Ephedra gerardiana aqueous ethanolic extract and fractions (ethyl acetate, n-butanol and aqueous) and Ribes orientale aqueous ethanolic extract and fractions (n-butanol and aqueous) at 50, 100, 200, 400, 800, 1600, 3200 and 6400 µg/ml concentrations was appraised by DPPH free radical scavenging assay and ferric reducing power assay. The acute, sub-acute, sub-chronic toxicity tests and resazurin cytotoxicity assay of biologically most active fraction i.e., aqueous fractions of Ephedra gerardiana and Ribes orientale were also conducted. In acute toxicity study, aqueous fractions of both plants were administered once orally to mice at 10, 100, 1000, 1600, 2900 and 5000 mg/kg doses and mice were observed for mortality/significant behavioral changes for 7 days post-treatment and LD50 was calculated. In sub-acute toxicity study, rats were administered 300, 600 and 900 mg/kg doses of aqueous fractions of Ephedra gerardiana and Ribes orientale orally for 14 days. In sub-chronic toxicity study, 50, 100 and 200 mg/kg doses of Ephedra gerardiana and Ribes orientale aqueous fractions were administered orally for 30 days to rats. In both sub-acute and sub-chronic studies, body weight of rats was recorded on weekly basis, blood was collected at the completion of study for hematological and biochemical analysis and vital organs (liver, kidney and heart) were removed at the end of study for measuring organ weight and histopathological analysis. In resazurin assay, the effect of 0.5% solution of Ephedra gerardiana and Ribes orientale aqueous fractions on Caco-2 cell viability was evaluated. Additionally, the preliminary phytochemical analysis, determination of total flavonoid/phenolic contents and FTIR and HPLC analysis of most active fraction i.e., aqueous fractions of Ephedra gerardiana and Ribes orientale was performed. In in-vitro anti-arthritic studies, aqueous ethanolic extracts and various fractions of Ephedra gerardiana and Ribes orientale presented a significant concentration dependent increase in percentage protection, with maximum effect obtained at 6400 µg/ml concentration. The percentage inhibition of heat induced egg albumin denaturation by Ephedra gerardiana aqueous ethanolic extract, aqueous, n-butanol and ethyl acetate fractions at 6400 µg/ml was observed as 3219.44%, 2899.30%, 2106.94% and 1533.33%, respectively. While, at 6400 µg/ml, Ribes orientale aqueous ethanolic extract, aqueous and n-butanol fractions showed 3063.88%, 2894.44% and 2557.64% inhibition of egg albumin denaturation, respectively. The standard drug, diclofenac sodium also exhibited 716.66% protection of egg albumin from heat induced denaturation at 6400 µg/ml. Likewise, in case of BSA denaturation inhibition study, the results explicated 99.10%, 98.59%, 92.14% and 85.72% anti-denaturation effect by Ephedra gerardiana aqueous ethanolic extract, aqueous, n-butanol and ethyl acetate fractions at 6400 µg/ml, respectively. Whereas, Ribes orientale aqueous ethanolic extract, aqueous and n-butanol fractions showed 96.88%, 94.74% and 86.14% inhibition of thermally induced BSA denaturation, respectively at 6400 µg/ml. The reference drug, aspirin also exhibited 80.59% protection of BSA from denaturation at 6400 µg/ml. In the same way, aqueous ethanolic extract of Ephedra gerardiana and its aqueous, n-butanol and ethyl acetate fractions at 6400 μg/ml presented 89.36%, 86.35%, 67.14% and 47.09% protection of erythrocyte membrane in hypotonic medium, respectively. While, Ribes orientale aqueous ethanolic extract, aqueous and n- butanol fractions at 6400 µg/ml demonstrated maximum stabilization of HRBC membrane as 90.69%, 86.15% and 71.23%, respectively. Diclofenac sodium (standard drug) at 6400 µg/ml also presented 70.49% protection of erythrocytes membrane against hemolysis. The findings of in-vitro anti-arthritic activities hence, revealed that aqueous ethanolic extracts of both plants exhibited maximum effect and among the fractions, aqueous fractions of both tested plants showed more pronounced activity and their anti-arthritic effects were much closer to their respective aqueous ethanolic extracts. In formaldehyde induced arthritis study, animals treated prophylactically with Ephedra gerardiana and Ribes orientale aqueous ethanolic extracts, and their respective fractions exhibited significant (p<0.001) and dose dependent reduction in formaldehyde injected paw volume and diameter, when compared with arthritic control group. On 10th day of experiment, maximum decline in paw volume and diameter was observed with 200 mg/kg dose of Ephedra gerardiana and Ribes orientale extracts and their fractions. Rats treated with 10 mg/kg piroxicam also unveiled considerable reduction in paw volume and diameter on 10th day. Accordingly, the aqueous ethanolic extracts of both plants proved to be highly efficacious. Interestingly, amid the fractions, aqueous fractions of both tested plants were again found to be the most active ones, as their efficacy was comparable to their respective aqueous ethanolic extract. In FCA model, prophylactic treatment with Ephedra gerardiana and Ribes orientale aqueous ethanolic extracts/fractions at 200 mg/kg dose, significantly (p<0.001) averted an increase in paw volume/diameter of FCA injected rats and extensively reduced primary signs of chronic inflammation on 28th day of study, as compared to arthritic control rats. Also, 10 mg/kg of reference drug (piroxicam) prevented significant (p<0.001) increase in paw volume/diameter on 28th day, with reference to FCA control rats. The results too illustrated that animals treated wih Ephedra gerardiana and Ribes orientale aqueous ethanolic extracts/fractions and piroxicam presented significantly (p<0.001) decreased macroscopic arthritic score and all the treatments notably prevented body weight loss, compared to arthritic control animals except Ephedra gerardiana ethyl acetate fraction. The treatment with extracts and fractions also remarkably prevented abnormal alterations in hematological (WBC, RBC, Hb, ESR and platelets) and biochemical parameters (AST, ALT, ALP, urea, creatinine, RF, CRP). Besides, the serum samples examined on 28th day of study using ELISA shown a significant suppression of PGE2 and TNF-? levels in rats treated with Ephedra gerardiana and Ribes orientale aqueous ethanolic extracts, their fractions and piroxicam, compared to arthritic control rats. Additionally, the outcomes of RT-PCR analysis carried out at the end of study period divulged a significant downregulation of COX-2, IL-1β, IL-6, TNF-α, NF-kB mRNA expression levels and a significant upregulation of IL-4 and IL-10 mRNA expression levels in all treated groups as opposed to disease control group. As well, Ephedra gerardiana and Ribes orientale aqueous ethanolic extracts/fractions and piroxicam averted radiographic modifications and ankle joint histopathological alterations, when paralleled with FCA control group. The afore-mentioned results of FCA induced chronic arthritis experiment validate that aqueous ethanolic extracts of Ephedra gerardiana and Ribes orientale showed maximum protection against FCA induced arthritis for all the tested parameters. Moreover, therapeutic effects exhibited by aqueous fractions of Ephedra gerardiana and Ribes orientale were again comparable to their respective aqueous ethanolic extract, hence suggesting aqueous fraction to be pharmacologically most active one in case of both plants. In in-vitro anti-oxidant assays, it was found that extracts and fractions of Ephedra gerardiana and Ribes orientale possessed appreciable concentration dependent antioxidant potential (p<0.001). The maximum anti-oxidant effect was observed at highest concentration of 6400 µg/ml. In DPPH assay, Ephedra gerardiana aqueous ethanolic extract, aqueous, n-butanol and ethyl acetate fractions at 6400 µg/ml displayed 84.13%, 77.63%, 72.40% and 69.78% anti-radical activity, correspondingly. While, at 6400 µg/ml, Ribes orientale aqueous ethanolic extract, aqueous and n-butanol fractions revealed 82.11%, 80.28% and 73.96% free radical scavenging potential, respectively, compared to standard drug, ascorbic acid (91.02%). Moreover, the reducing potential of Ephedra gerardiana aqueous ethanolic extract, aqueous, n-butanol and ethyl acetate fractions was found to be 1237.23%, 1138.21%, 1035.00% and 863.73%, respectively at 6400 µg/ml. Similarly, Ribes orientale aqueous ethanolic extract, aqueous and n-butanol fractions presented 1157.60%, 1018.54% and 905.99% reducing power, respectively, when compared with reference drug, ascorbic acid (1336.52%) at 6400 µg/ml. In acute toxicity study, no dose prompted mortality/significant behavioral changes in mice and LD50 was >5000 mg/kg for aqueous fractions of Ephedra gerardiana and Ribes orientale. The sub-acute doses (300, 600 and 900 mg/kg) of aqueous fractions of Ephedra gerardiana and Ribes orientale did not cause alterations in body and organ weight. Nonsignificant differences were observed in blood and serum parameters between treatment and control groups. However, with reference to control, considerable variances were seen only in TLC, neutrophils, platelets, ALP, albumin and glucose levels at all sub-acute doses of Ephedra gerardiana aqueous fraction. Whereas, in case of Ribes orientale aqueous fraction, substantial differences were noticed only in the levels of neutrophils and lymphocytes at 900 mg/kg, total protein at 600 and 900 mg/kg and ALP and glucose at all doses, with reference to control. Nonetheless, aforementioned hematological and biochemical changes were not associated with histopathological changes in liver, kidney and heart tissue at all tested doses. The sub-chronic doses (50, 100 and 200 mg/kg) of Ephedra gerardiana and Ribes orientale aqueous fractions neither altered body and organ weight of rats, nor resulted in significant differences in hematology and biochemistry. This was also supported by histopathology (liver, kidney and heart tissue) findings at aforesaid doses. The results from resazurin test displayed that Caco-2 cells treated with 0.5% solution of Ephedra gerardiana aqueous fraction showed cell viability of 90% at 3rd h and 88% at 24th h. While, Caco-2 cells treated with 0.5% solution of Ribes orientale aqueous fraction were associated with only 10% decrease in viability and integrity at 3rd h and only 11% reduction in viability and integrity at 24th h. Hence, this study validated that aqueous fractions from aqueous ethanolic extracts of Ephedra gerardiana and Ribes orientale could be deliberated as reasonably safe of toxicity because they neither resulted in lethality nor brought about any notable hematologic, biochemical and structural side effects in rodents especially at low doses i.e., 50, 100 and 200 mg/kg body weight given orally for 30 days. This further strengthen our study, as we evaluated anti-arthritic effect of Ephedra gerardiana and Ribes orientale at 50, 100 and 200 mg/kg doses, respectively. Nonetheless, the plants should be used with caution at high doses, as they may disrupt certain hematological and biochemical parameters in the long run. In addition, aqueous fractions of both plants at the concentration tested (0.5%) did not show significant toxicity and enhanced the chance of viability on Caco-2 cells after 3 and 24 h of incubation. Moreover, preliminary phytochemistry of aqueous fraction of Ephedra gerardiana and Ribes orientale revealed the presence of flavonoids, phenols, tannins, saponins, alkaloids and glycosides. The FTIR analysis of aqueous fractions of both plants shown the presence of different functional groups. The phenolic profile was further confirmed by HPLC analysis, which revealed quercetin, along with gallic, vanillic, benzoic, chlorogenic and Mcoumaric acids in Ephedra gerardiana aqueous fraction while, quercetin together with pcoumaric, M-coumaric and cinnamic acids in Ribes orientale aqueous fraction. In summary, the results suggest that the mechanism of anti-rheumatic and immunomodulatory effect of Ephedra gerardiana and Ribes orientale may perhaps be due to their ability to inhibit protein denaturation, stabilize lysosomal membrane, down regulate IL-1β, TNF-α, IL-6 and NF-кB and up regulate IL-10 and IL-4 levels, together with dropping the concentrations of inflammatory enzymes COX-2 and PGE2 that could be credited to anti-oxidant, anti-arthritic and immunomodulatory properties of polyphenolics (identified through HPLC), tannins, alkaloids and flavonoid constituents of both plants. The anti-arthritic response of Ephedra gerardiana was in the order of aqueous ethanolic extract > aqueous fraction > n-butanol fraction > ethyl acetate fraction, while, anti-arthritic effect of Ribes orientale was in the sequence of aqueous ethanolic extract > aqueous fraction > n-butanol fraction. However, further studies would be necessarily needed to identify and isolate active principle(s) in these plants and to elucidate the exact mechanism(s) of antiarthritic activity of selected plant and to establish the real efficacy and safety in patients by following the FDA approved protocols." xml:lang="en_US
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۔غزل

غزل۔۔۔قاضی اعجاز محور

خاک ہوں آسماں پہ پہنچا دے
میں مرا ہوں تری محبت میں
پیار تو ایک عام سی شے ہے
بن کے جگنو مجھے دکھا رستہ
میں تجھے دیکھ کر غزل لکھوں

 

عشق کا عین مجھ کو پڑھا دے
دل کے آنگن میں مجھ کو دفنا دے
اپنی نفرت کا مجھ کو تحفہ دے
اور رستے سے مجھ کو بھٹکا دے
ایک بوسے کا طرحی مصرعہ دے

EFFECTS OF PHYSICAL THERAPY-BASED MANAGEMENT APPROACHES FOR TENSION TYPE HEADACHE

Background of the Study: Multiple Physical Therapy approaches have recently been developed and reported in the literature for providing better results in the treatment regimens of tension-type headaches. The advancement in the field of Physical therapy towards the treatment approaches of tension-type headaches has become the driving force for writing this article. Methodology: Studies comparing the effects of physical therapy management with conventional treatment approaches are included in the meta-analysis. PRISMA guidelines were used for performing the qualitative analysis and assessment of risk of biases. Results: According to the findings of nine randomized controlled trials, the analysis of the results had revealed that physical therapy intervention demonstrated a significant improvement in reducing headache severity. In a random effect model, the pool effects of physical therapy strategies in terms of Standardized Mean Difference had an impact of 1.41, which according to a Cohen rule of thumb displays a larger effect of physical therapy management in significant decrease in pain intensity among tension-type headache patients Conclusion: The study has concluded that physical therapy-based management strategies as provided in several RCTs analyzed in this review article revealed a pool effect of moderate size in managing the frequency of pain and a larger effect size in managing pain intensity and duration. Further, it was concluded that tension-type headaches can be effectively managed through physical therapy-based approaches.

Combined Ligand and Structure Based Studies on Modulators of Akt Kinases

Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine kinases with three structurally homologous mammalian isoforms, Akt1 (PKBα), Akt2 (PKBβ), and Akt3 (PKBγ). Akt isoforms emerged as anti-cancer drug targets because of their constitutive hyperactivation in various oncological disorders. However, due to high intra-family similarity within ATP binding region, the development of isoform-selective modulators of Akt represent a challenging endeavor and thus, until now only a handful of compounds were selected for the clinical investigation. Yet none of them could reach the market for routine clinical use due to their off-target toxicity and poor pharmacokinetic properties. Recent reports on achieving isoform selectivity by designing inhibitors against less conserved pleckstrin homology (PH) domain offer the opportunity to reduce the major off-target toxic effects of Akt antagonists. Therefore, in this thesis, combined ligand- and structure-based in silico strategies have been utilized to probe the key structural features for the inhibition of the PH domain of Akt2 which is more commonly overexpressed in solid tumors. Toward this end, various predictive 2DQSAR (two-dimensional quantitative structure-activity relationship) and GridIndependent Molecular Descriptor (GRIND) and pharmacophore models using structurally diverse data set of 111 quinoline analogs have been developed. Our key findings demonstrate that the presence of three hydrogen bond donors (D1-D3) at a molecular distance of ~8.4, 21.6, 16.8 Å between D1–D2, D1–D3 and D2–D3, respectively is important for selective inhibition of PH domain of Akt2. In addition, our docking results indicated a crucial role of Lys30 for the optimal fit of quinoline-type inhibitors within the binding cavity of the Akt2 PH domain. Moreover, the structurebased pharmacophore model exhibited three hydrogen bond acceptors (A1-A3) at a distance of 4.05 Å (A1-A2), 11.58 Å (A2-A3) and 15.33 Å (A1-A3) that are complementary to the molecular distances identified by GRIND which further validate the reliability of our developed models. Additionally, identified hits through pharmacophoric-based virtual screening provided a new arsenal of potentially selective chemical scaffolds which have a broad structural diversity and less chemical similarity to any of the other known Akt2-PH domain inhibitors. Subsequently, selectivity profiling with the help of proteochemometric modeling revealed essential substructures such as Nmethylpent-3-en-2-amine for selective inhibition of Akt1, methylene amine, isoproenylterazol and 2H-tetrazole for Akt2, and formaldehyde hydrazine for the Akt3 selective inhibition. The present work also illustrates the substructure based similarity search of ChemBridge database to identify Akt2-selective hit compounds. In the present study, one of the selected carboxamide-type hit showed 1.2 and 2.1 fold selectivity against Akt2 as compared to Akt1 and Akt3, respectively. Overall, the work described in this thesis could pave the way towards the identification of potential modulators of Akt2 against cell proliferation in cancer with high isoform-selectivity and limited side effects.