Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/12321
Title: Pharmacological basis for the use of Ephedra gerardiana and Ribes orientale in rheumatioid arthritis: evaluation of toxicological and phytochemcial profile
Authors: Uttra, Ambreen Malik
Keywords: Pharmacology
Pharmacy
Issue Date: 2019
Publisher: University of Sargodha, Sargodha.
Abstract: 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-𝛼 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.
Gov't Doc #: 19950
URI: http://prr.hec.gov.pk/jspui/handle/123456789/12321
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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