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Home > Bioassay Guided Isolation and Characterization of Secondary Metabolites from Isodon Rugosus Wall. Ex Benth. and Their Anti-Nociceptive Activities

Bioassay Guided Isolation and Characterization of Secondary Metabolites from Isodon Rugosus Wall. Ex Benth. and Their Anti-Nociceptive Activities

Thesis Info

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Author

Zeb, Anwar

Program

PhD

Institute

University of Malakand

City

Malakand

Province

KPK

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Applied Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/9048/1/Anwar_Zeb_Pharmacy_HSR_2018_UoM_Malakand_05.04.2018.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676725580519

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Isodon rugosus Wall.ex Benth. is widely employed in folk medicine to cure various types of pain conditions like dental, gastric, abdominal, ear pain and rheumatism. In this thesis, we have explored the analgesic potential of Isodon rugosus, to isolate the active compounds responsible for analgesic activity and to figure out its possible mechanism of action. The bioassay guided isolation was followed to obtain analgesic compound from Isodon rugosus. Preliminary phytochemical evaluation was carried out for the presence of various secondary metabolites in Isodon rugosus. Initially, the analgesic activity was performed using methanolic extract and subsequent fractions. The chloroform fraction was sorted out as most active following various analgesic activities. The isolation of analgesic compounds was carried out from chloroform fraction by using gravity column chromatography, in which thin layer chromatography and various stains were employed for confirmation of single and pure spots of compounds. At the end, three (3) compounds (AZ-1, AZ-2 and AZ-3) were isolated from chloroform fraction of Isodon rugosus in pure form. The pure compounds were then subjected to various analytical techniques for identification purposes including 1HNMR, 13CNMR and MS. The compound AZ-1 and AZ-2 were in sufficient quantity and were subjected to evaluate their analgesic potential following various standard protocols. In acetic acid induced writhing test, the compound AZ-1 showed 61% inhibition whileAZ-2 showed 4% inhibition of pain. In formalin test, AZ-1at 50 mg/kg displayed 69 and 63% inhibitions at phase-I and phase-II respectively. Similarly, the compound AZ-2at a similar dose, i.e. 50 mg/kg displayed 8 and 13% inhibitions at early and late phase respectively. In hot plate test,AZ-1 and AZ-2 exhibited average reaction times of 8 min and 3 min respectively. The possible mechanism of action of AZ-1 was figured out using different receptors including opioid, dopaminergic and adrenergic receptors. Moreover, the AZ-1 2 was also assessed against enzymes lipoxygenase and cyclooxygenase, which have a prominent role in nociception. The mechanistic study revealed that AZ-1 was actively involved in the anti-nociception through opioid receptors. The possible mechanism of analgesic action was confirmed to be the central pathway using opioid receptors, because the mice showed activity as that of morphine after the administration of opioid antagonist naloxone. The involvement of opioids receptors is evident from both hot plate and formalin assays in which nalaxone was used to figure out the involvement of opioids receptors. The AZ-1 was also involved in anti-nociception through adrenergic receptor and showed the reaction time of 5.35± 0.66, 5.17± 0.53, 6.53± 0.68, 5.90± 0.58 and 4.63±0.56 at the interval of 15, 30, 45, 60 and 90 min respectively. The involvement of adrenergic receptor shown by the pre-treatment of adrenergic antagonist (yohimbine) which reversed the activity of AZ-1. Moreover,AZ-1 was also active against cyclo-oxygenase and lipoxygenase enzymes. The positive control used was Zileuton in lipoxygenase assay, in which the AZ-1 demonstrated 49.40± 0.65, 42.37± 0.72, 36.87± 1.04, 31.80± 0.55 and 26.97± 1.49% inhibitions at concentrations of 1000, 500, 250, 125 and 62.5 μg/ml respectively having IC50of 1024 μg/ml. The celecoxib was used as positive control in cyclo-oxygenase in which AZ-1 showed 42.77± 0.82, 38.87± 0.70, 32.73± 0.95, 28.60± 1.19 and 21.70± 1.57% inhibitions at concentration of 1000, 500, 250, 125 and 62.5 μg/ml respectively with IC50 1423μg/ml. The antioxidant properties of Isodon rugosus was also explored using various free radicals assays such as DPPH, H2O2 and ABTS. In DPPH, H2O2 and ABTS assays, AZ-1 revealed 56.33± 0.89, 49.33± 0.33 and 59.33± 0.33% inhibitions respectively at 1000 μg/ml concentration each. Additionally, the essential oil of Isodon rugosus (Ir.Eo) was also isolated and was subjected to GC coupled MS analysis. A total of 141 compounds found in the essential oil of Isodon rugosus. 3 The essential oil was also subjected to anti-nociceptive and antioxidant activity to find out it role in pain. The Ir.Eo showed a comparative response of the sample (57.14%) with the positive control (70.29%) at the dose of 100 mg/kg. The Ir.Eo in combination with naloxone reduced 09.01and 07.95% pain in phase I and II respectively. Mean reaction time for a sample of Ir.Eo and naloxone at a dose ratio of 50 + 1 mg/kg body weight was figured out as 05.28 ±0.13 min. Similarly, in DPPH and ABTS assay Ir.Eo revealed IC50 of <0.1 μg/ml. In brief, AZ-1 was revealed as an active anti-nociceptive compound of Isodon rugosus. This compound should further be subjected to various investigations which can help in drug discovery.
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مولانا سید اسعد مدنی

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راقم نے انہیں کئی بار دیکھا اور اس حال میں دیکھا جب وہ صحت مند، توانا، تروتازہ، متحرک اور جوش عمل سے سر شار تھے لیکن ملاقات کا شرف دو تین بار ہی حاصل ہوا، مارچ ۱۹۸۸؁ء میں ان کے والد بزرگوار مولانا سید حسین احمد مدنیؒ پر مسجد عبدالنبی نئی دہلی میں سمینار ہوا تھا جس کے کنویز ڈاکٹر رشید الوحیدی مرحوم تھے، اس میں پہلی بار ان سے شرف نیاز حاصل ہوا، بڑی بشاشت اور گرم جوشی سے ملے، آخری بار ۱۹۹۲؁ء میں سعودی سفارت خانے سے حج بیت اﷲ کا ویزا لینے گیا تو دفعتاً ان پر نظر پڑی، لپک کر ملا، دریافت کرنے پر اپنے کو بتایا تو قریب کی کرسی پر بیٹھنے کے لیے کہا اور کچھ دیر تک باتیں کیں، غالباً بیماریوں کا سلسلہ شروع ہوچکا تھا، اس لیے مجھے بہت مضمحل اور بجھے بجھے سے نظر آئے۔
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A G R O - Physiological Studies of Wheat Tritic Maestivuml. under Different Management Systems

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In case of interaction between irrigation and seed rate it was observed that maximum (386.25) tillers m -2 , grains spike -1 (70.00), leaf area index (4.78), 1000-grain weight (45.20 g), grain yield (3800.00 kg ha -1 ), biological yield (14287.50 kg ha -1 ), net income (Rs. 54043.00), BCR (4.26 %) and MRR % (426.35) was noted from the plots irrigated with 470 mm water and seed rate was used @ 120 kg ha -1 . Similar trend was observed in 2004-05 and in two years average. Identical results were observed during 2004-05 and mean of both years An experiment entitled “Yield and yield components of wheat as affected by different seed rates and nitrogen levels” was conducted during 2003-04 and 2004-05. Five seed rates (S 1 = 60 kg ha -1 , S 2 = 80 kg ha -1 , S 3 = 100 kg ha -1 , S 4 = 120 kg ha -1 and S 5 = 140 kg ha -1 ) and five nitrogen doses (N 0 = Control, N 1 = 40 kg ha -1 , N 2 = 80 kg ha -1 , N 3 = 120 kg ha -1 and N 5 = 140 kg ha -1 ) were applied. Analysis of the data recorded from the irrigation treatments showed that maximum (334.85) tillers m -2 , leaf area index (4.50), grains spike -1 (60.80), 1000-grain weight (41.91 g), grain yield (3957.64 kg ha -1 ), biological yield (10172.40 kg ha -1 ), protein in gains (7.13 %), protein (6.90 %), net income (Rs. 50225.00), BCR (4.28 %) and MRR % (428.27) was noted from the plots which were seeded with 120 kg ha -1 . Identical results were observed during 2004-05 and mean of both years. Statistical analysis of the data also viirevealed maximum (348.00) tillers m -2 , leaf area index (4.42) grains spike -1 (63.90), 1000-grain weight (42.19 g), grain yield (4145.14 kg ha -1 ), biological yield (10579.75 kg ha -1 ), protein in gains (9.87 %), protein (8.09 %), net income (Rs. 52831.00), BCR (4.46 %) and MRR % (446.21) was noted from the plots fertilized with 120 kg N ha -1 . The same trend was followed during 2004-05 and pooled data. 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It can be inferred from the data showed that maximum ((369.20) tillers m -2 , grains spike -1 (65.30), leaf area index (4.03), 1000- grain weight (45.30 g), grain yield (4222.80 kg ha -1 ), biological yield (15030.53 kg ha -1 ), net income (Rs. 62119.00), BCR (5.18 %) and MRR % (518.03) was noted from the plots seeded with 120 kg ha -1 . In 2004-05 and in two years average, almost the same trend was observed. In case of interaction between varieties and seed rate it was observed that maximum (420.25) tillers m -2 , leaf area index (4.79), grains spike -1 (74.00), 1000-grain weight (47.33 g), grain yield (4676.25 kg ha -1 ), biological yield (19535.16 kg ha -1 ), net income (Rs. 66863.00), BCR (5.47 %) and MRR % (546.95) viiiwas noted from the plots sown with Fakhre Sarhad and seed rate was used @ 120 kg ha -1 . Similar trend was observed in 2004-05 and in two year average. Identical results were observed during 2004-05 and mean of both years.