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Home > Optimization of Callus, Cell Suspension and Adventitions Root Cultures for the Production of Active Components in Stevia Rebaudiana

Optimization of Callus, Cell Suspension and Adventitions Root Cultures for the Production of Active Components in Stevia Rebaudiana

Thesis Info

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Author

Naveed Ahmad

Program

PhD

Institute

The University of Agriculture

City

Peshawar

Province

KPK

Country

Pakistan

Thesis Completing Year

2017

Thesis Completion Status

Completed

Subject

Horticulture

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10515/1/Naveed_Ahmad_Horticulture_HSR_2017_UA_Peshawar_07.03.2018.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676724892107

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Stevia rebaudiana Bertoni is an important plant known for antidiabetic steviol glycosides and several other bioactive compounds. Owing to the importance of Stevia plant and demand of natural secondary metabolites, the current research was conducted at Plant Tissue Culture Lab., Departmnet of Plant Breeding and Genetics, The University of Agriculture Peshawar, during the year of 2014-15. The objectives of the study were to optimize/evaluate the effect of sucrose (05, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l-1), pH (5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0) and inoculum sizes (0.5, 1.0, 1.5 and 2.0 g) on biomass yield and valuable secondary metabolites accumulation in callus, cell and adventitious root cultures. Preliminary experiments were also carried out to optimize the effect of various monochromatic spectral lights (white, blue, green, yellow and red) on callus culture growth and secondary metabolites production. To check the effect of sucrose, media pH and inoculum sizes on cultures productivity, research work was conducted in Complete Randomized Design (CRD). Each culture was planned for a period of 30 days and 3 days intervals were kept to develop growth curves. The control light (16/8 hr) resulted in the maximum callogenic response (92.73%) along with the accumulation of maximum biomass (5.78 g l-1) during prolong log phase at 18th day of culture. Blue light was found the most effective for the production of total phenolics content (TPC), total flavonoids content (TFC) along with total antioxidant capicity (TAC) (102.32 μg/g-DW) (22.07 μg/g-DW) and (11.63 μg/g DW), respectively. On the other hand, reducing power assay (RPA; 0.71 Fe (II) g -1 DW) and DPPH-radical scavenging activity (DRSA; 80%) were considerably enhanced as a result of green and red lights, respectively. Sucrose concentrations also affected the biomass accumulation, growth kinetics and secondary metabolites production. The lag phase of 9 days was followed by log phase till 27th day of culture was recorded in callus cultures on 05, 10, 15 and 20 g l-1 sucrose. The rest of the cultures did not show a lag phase. All cultures, except a few displayed nonviability after 27 days of inoculation. The cultures initiated in media having 20, 25 and 30 g l-1 sucrose were found in stationary phase after 27 days of log phase. Similarly, cell cultures grown on 05-30 g l-1 sucrose concentrations displayed relatively shorter lag phase of 3 days as compared to 12 days lag phase in cultures with 35-50 g l-1 sucrose concentrations. The Lag phase in each cell culture was preceded by log phase till 18th day. Most cultures showed a stationery phases with or without decline phases. However, growth curve of adventitious root cultures was characterized with direct log phase till 18th day. The lag phase of 15 days was observed in cultures developed in media having 5 and 10 g l-1 sucrose, followed by very short log phase. The fresh and dry biomass of callus, cell suspension and adventitious root cultures was significantly affected by sucrose concentrations. The highest fresh and dry biomass (142.38 g l-1and 11.71 g l-1) in callus culture was with 40 g l-1 and 50 g l-1 sucrose, respectively. However, the maximum total phenolics content (TPC; 124.20 mg/g-DW), total flavonoids content (TFC; 49.36 mg/g-DW), rebaudioside contents (6.56 mg/g-DW) and antioxidant activity (92.82 %) in callus cultures was observed at sucrose concentration of 30 g l-1. By contrast, the highest stevioside (42.34 mg/g-DW) and rebaudioside (22.67 mg/g-DW) contents were recorded in callus culture grown at 15 and 20 g l-1 sucrose, respectively. In cell suspension culture, media having 20 g l-1 sucrose resulted in the maximum fresh (97.71 g l-1) and dry (8.57 g l-1) but the highest TPC (139.20 mg/g-DW) and TFC (41.46 mg/g-DW) was at 40 g l-1 sucrose. The highest antioxidant activity (83.87%) was observed at 30 g l-1 sucrose. While the stevioside content (40.32 mg/g-DW) was the maximum on media supplemented with 10 g l-1 sucrose, the highest rebaudioside (27.64 mg/g-DW) and dulcoside (6.43 mg/g-DW) contents were observed with 20 g l-1 sucrose. In case of adventitious root culture, the maximum fresh (175.43 g l-1) and dry (11.14 g l-1) biomass was accumulated in cultures having 50 g l-1 sucrose but the highest TPC (155.00 mg/g-DW) and TFC (94.78 mg/g-DW) were recorded with 30 g l-1 sucrose. While the highest antioxidant activity (94.43 %) was recorded in culture, established in media augmented with 20 g l-1 sucrose. The stevioside (73.97 mg/g-DW) and rebaudioside (24.57 mg/g-DW) content were the highest in media containing 10 g l-1 sucrose. By contrast, the dulcoside content (12.24 mg/g-DW) was the maximum at 40 g l-1 sucrose. It is suggested that sucrose concentration modulates biomass and metabolites of interest in callus, cell suspension and adventitious root cultures of Stevia rebaudiana. The media pH significantly influenced fresh and dry biomass of callus, cell suspension and adventitious root cultures. The biomass accumulation revealed a short lag phase of 3 days in callus cultures on media pH 4.0, 5.9 and 6.0; and cell suspension culture on pH 5.6 and 5.7; while the adventitious roots culture expressed a lag phase of 3 days on media pH 5.5-6.0. The log phase was followed by decline phases in callus and cell suspension cultures. However, root growth was not restricted in cultures having pH 5.2 and 5.4 even after 27 days of the culture. pH 5.6 was optimized for the maximum fresh (130.57 g l-1) and dry biomass (12.10 g l-1) of callus cultures. However, the highest TPC (43.38 mg/g-DW), TFC (37.55 mg/g-DW) and antioxidant activities (87.68 %) in callus cultures were recorded on media pH 5.6. Media pH 5.6 was also found optimum for the maximum stevioside (62.20 mg/g-DW) and rebaudioside (22.79 mg/g-DW), while 5.1 for dulcoside (5.92 mg/g-DW) production in callus cultures. Similarly, the maximum fresh and dry biomass (85.81 g l-1; 8.84 g l-1) of cell suspensions were observed on media pH 5.6 and 5.5, respectively. The highest TPC (72.13 mg/g-DW), TFC (57.32 mg/g-DW), DRSA (93.99%), rebaudioside (7.01 mg/g-DW) and dulcoside (4.72 mg/g-DW) contents were observed in cell suspension cultures established in media having pH 5.8. However, stevioside contents were induced to their maximal level (41.47 mg/g-DW) at pH level 5.2 in cell culture. In contrast, the highest fresh (112.86 g l-1) and dry (8.29 g l-1) biomass were accumulated in adventitious root culture on media pH 6.0. The maximum TPC (70.06 mg/g-DW), TFC (50.19 mg/g-DW), DRSA (92.67 %) and dulcoside contents (2.57 mg/g-DW) in adventitious root cultures were recorded on media pH 5.8. However, the highest stevioside (79.48 mg/g-DW) and rebaudioside (13.10 mg/g-DW) contents in adventitious root cultures were accumulated at 5.1 media pH. Various inoculum sizes also had significant influence on growth kinetics, biomass and secondary metabolites production in callus, cell suspension and adventitious root cultures. Relatively short lag phase of 3 days in callus cultures, while elongated lag phases from day 3rd to 12th day of the cultures developed from inoculum sizes (0.5-2.0g) was recorded. However, adventitious root cultures did not display lag phases. An increase in biomass with elongated log phases from day 3rd to 27th day of the culture was observed in callus cultures. Among all inoculum sizes, 2.0 g started sudden increased in biomass accumulation up to 15 days and increments in growth was further continued till 27th day of culture. Log phase was followed by sudden decline phase without having any stationery phase in all cultures. Similarly, the highest fresh and dry biomass (112.29 g l-1; 7.71 g l-1) in callus cultures was observed at 2.0 g inoculum. However, cultures developed from smaller inoculum (0.5 g) resulted the maximum TPC (28.54 mg/g-DW), TFC (24.78 mg/g-DW), DRSA (77.57 %), stevioside (43.89 mg/g-DW) and rebaudioside (36.54 mg/g-DW) contents in callus cultures, while the highest dulcoside contents (2.57 mg/g-DW) were observed at 1.0 g inoculum. On the other hand, Cell suspension also accumulated the maximum fresh (102.71 g l-1) and dry biomass (5.38 g l-1) at 1.5 g inoculum. Moreover, the highest TPC (45.36 mg/g-DW), TFC (36.50 mg/g-DW), stevioside (59.89 mg/g-DW), rebaudioside (24.41 mg/g-DW) and dulcoside (1.85 mg/g-DW) contents were found in cell cultures established from 0.5 g inoculum. However, the maximum DRSA (78.30%) was found in cell cultures having initial inoculum size 2.0 g. Similarly, adventitious root cultures accumulated the maximum fresh biomass (106.86 g l-1), dry biomass (5.05 g l-1) and dulcoside contents (0.71 mg/g-DW) at 1.5 g inoculum. Inoculum size 2.0 g was optimized for the maximum TPC (41.46 mg/g-DW), TFC (33.44 mg/g-DW) and DRSA (98.82 %). However, stevioside (64.75 mg/g-DW) and rebaudioside (29.67 mg/g-DW) contents were significantly increased to their maximal level using initial inoculum size (1.0 g). Herein, we concluded that the utilization of various colored spectral lights, concentrations, pH levels and inoculum sizes are promising strategies for enhanced biomass yield and secondary metabolites production in callus, cell suspension and adventitious root cultures of Stevia rebaudiana." xml:lang="en_US
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یونس رضوی

یونس رضوی(۱۹۲۷ء) سیالکوٹ میں پیدا ہوئے۔ آپ نے ۱۹۴۹ء میں محکمہ انکم ٹیکس میں ملازمت حاصل کی۔ ۱۹۵۴ء میں فلم انڈسٹری لاہور سے رابطہ قائم کیا۔ آپ نے غزل ،نظم اور دیگر اصناف میں طبع آزمائی کی لیکن فلم انڈسٹری سے منسلک ہونے کی وجہ سے آپ کی زیادہ توجہ گیت نگاری کی طرف تھی۔(۷۵۵) ان کا شعری کلام ملک کے مختلف رسائل و جرائد میں چھپتا رہا۔ ایک شعری مجموعہ ’’میرے آنسو میرے گیت‘‘ ،زمزمہ پرنٹنگ پریس سیالکوٹ سے ۱۹۷۶ء میں شائع ہوا۔

یونس نے اردو شاعری میں کوئی نئی اور انوکھی راہیں دریافت نہیں کیں ۔بلکہ وہ اپنی شاعری میں روایت پسندنظر آتے ہیں۔ ان کی شاعری غم و اندوہ کی شاعری ہے۔ مگر ان کے ہر شعر کے پردے میں ایک ایسی چھپی ہوئی مضبوط انا کا وجود ملتا ہے۔ جو حوادث کی ستمرانیوں سے کبھی زخمی نہیں ہوتی۔ اور زندگی کا ہر آنے والا زخم انھیں پہلے سے کہیں زیادہ حوصلہ مند اوربا وقار بنا دیتا ہے۔

یونس رضوی کا نمونہ کلام ملاحظہ ہو:

شبِ سیاہ مکمل شبِ سیاہ نہ تھی

 

تمہاری زلف کا سایہ بھی اس میں ڈالا گیا

 

بساطِ عشق کی بازی تمام ہار گئے

 

مذاقِ عشق ہمارا بلند و بالا گیا

(۷۵۶)

 

غم زمانے کا متاع جسم و جاں تک آگیا

 

آگ کا شعلہ لپک کر آشیاں تک آگیا

 

کٹ تو جائیں گے شب و روز فراق ان کے بغیر

 

دکھ یہی ہے کہ رونق شام و سحر جاتی رہی

(۷۵۷)

 

گردش دوراں کے ہاتھوں اس قدر مجبور ہوں

 

زندگی کی ہر مسرت سے میں کوسوں دور ہوں

(۷۵۸)

۷۵۶۔یونس رضوی ،’’ میرے گیت میر ے آنسو‘‘،ص: ۵۱

۷۵۷۔ایضاً،ص:۴۱،۴۲

۷۵۸۔ایضاً،ص:۴۸

الصراع الجيوسياسي الإقليمي وعلاقته بموارد الطاقة في الدول المطلة على الخليج العربي

تهدف هذه الدراسة الى التعريف بمحاور الصراع الجيوسياسي الإقليمي المتنافسة من اجل السيطرة على موارد الطاقة بغية الوقوف على مخططاتها وأهدافها الجيوستراتيجية التي تسلكها لتحقيق أهدافها الانية وتطلعاتها المستقبلية، ومن اجل تحقيق تلك الأهداف المرجوة فقد تم الاعتماد على المنهج التحليلي الذي يعتمد عليه في الدراسات الجغرافية السياسية والجيوبولتيك، وقد توصلت الدراسة الى جملة من النتائج ابرزها ان موارد الطاقة التي تزخر بها منطقة الخليج العربي من النفط والغاز الطبيعي جعلت منها ارضية خصبة للصراعات الإقليمية والدولية نظرا لتحرك محاور هذه الصراعات بقوة من اجل ايجاد موطئ قدم لها في هذه المنطقة الحيوية والظفر بهذه الموارد الطاقوية واحكام السيطرة عليها وضمان استمرارية تدفها وابعاد القوى الفاعلة الاخرى من الوصول اليها

Pharmacological Studies on Cardiovascular Effects of Selected Medicinal Plants: Activity-Guided Fractionation and Elucidation of Mechanism of Action

Cardiovascular diseases, especially the hypertension, have been a global threat for the last few decades because of non-curative effects of the available synthetic drugs and their high economic burden on the patients as their treatment requires lifelong use of drugs. Therefore, alternate effective and economical treatment options are urgently needed to be explored from all possible resources. Since ancient times plants have been utilized for treating various ailments and even some drugs have been derived from the traditionally used plants. This part of the world has been blessed with diverse medicinal flora that has been widely used on empirical folkloric basis. Due to these reasons, three medicinal plants claimed to be effective and curative for certain cardiovascular diseases were selected for the present investigations. The study consists of evaluation of antihypertensive, cardiac depressant and vasorelaxant effects of aqueous-methanolic (aq-meth) extracts and certain fractions of three indigenous medicinal plants viz. Sonchus asper, Linn. (aerial-parts), Pennisetum glaucum, Linn. (seeds) and Zanthoxylum armatum, DC. (fruits). The results obtained showed that aq-meth extracts of all three plants have significantly decreased the systolic blood pressure (SBP), mean arterial pressure (MAP) and diastolic blood pressure (DBP) and also reduced the heart rates of normotensive rats in the dose and time dependent manner. Further studies were carried out on the glucose and egg yolk fed hypertensive rats. The aq-meth extracts of all the three test plants were found to significantly prevent the diet-induced rise in blood pressure parameters as well as the heart rates of aq-meth extract treated rats. Acute toxicity study showed safety of the extracts as their LD50s in mice were quite high i.e., 3500 and 3000 mg /kg p.o., for the aq-meth extracts of S. asper and Z. armatum, respectively. While no mortality occurred with oral administration of P. glaucum extract upto 4000 mg/kg dose, so no LD50 was calculated for aq-meth extract of P. glaucum. In subchronic toxicity testing, non-significant changes were observed in liver ezymes and lipid profile parameters of treated rats. These data have clearly demonstrated that the test plants’ extracts do not possess significant amount of toxic substances. Therefore, investigations were further extended to determine their active fraction (s) and possible mechanism (s) of action. For this purpose, aq-meth extracts of S. asper (aerial-parts) and P. glaucum (seeds) were fractionated into ethyl acetate, butanolic and aqueous fractions while a polyphenolic compound, tambulin already reported to be present in Z. armatum (fruits) was isolated for further experiments. In isolated perfused rabbit hearts, the extracts, fractions and tambulin were found to significantly decrease the force of contraction, perfusion pressure and heart rate. Ethyl acetate fraction of S. asper and butanolic fraction of P. glaucum among their tested fractions showed more pronounced effects on the cardiac parameters. Then studies were conducted on porcine coronary artery rings and it was observed that aq-meth extracts, fractions and tambulin produced a dose dependent similar vasorelaxant effects in U46619 pre-contracted endothelium-intact as well as denuded artery rings. However, endothelium removal was not found to significantly affect their vasorelaxant potencies indicating the endothelium-independent effect. Interestingly, the effects of ethyl acetate fraction of S. asper and butanolic fraction of P. glaucum were more marked in this respect, too. Therefore, these fractions were selected for futher study in order to find possible mechanism (s) of their vasorelaxant effects. The tambuin (10 µM) produced 100% relaxation of intact and denuded coronary artery rings and was subjected to further series of experiments along with other potent fractions i.e., ethyl acetate fraction of S. asper and butanolic fraction of P. glaucum. It was observed that the response curves of the endothelium-dependent vasodilating agents including bradykinin and calcum ionophore were not shifted by the test fractions and tambulin showing that their relaxant effect was endothelium-independent and the effect was governed by the vascular smooth muscles. It was also found that ethyl acetate fraction of S. asper (0.01 mg/ml), butanolic fraction (0.03 mg/ml) of P. glaucum and tambulin (1µM) produced shifting of concentration response curves of the endothelium-independent vasodilators including, sodium nitroprusside (SNP), forskolin (FC) and isoproterenol. The experiments also showed that the test fractions and tambulin did not shift response curves produced by soluble guanyly cyclase (sGC) activators (YC-1 and BAY 41-2272) and K+ channel openers (levchromakalim and 1-EBIO). However, pretreatment with EAFS, BFPG and tambulin suppressed in dose dependent fashion the cumulative contractile responses of endothelium-denuded coronary artery rings induced by KCl, 5-HT, CaCl2 and U46619. Further, experiments were carried out to study the role of c-AMP and c-GMP on the vasorelaxant effect of the test fractions and tambulin. Specific inhibitors of PKA and PKG, namely H-89 and Rp-8-br-cyclic GMPS were used to inhibit the c-AMP and c-GMP elevation, respectively. The relaxations produced by the EAFSA and tambulin were observed to have significantly reduced in the coronary artery rings that were exposed to H-89 (10 µM) and Rp-8-br-cyclic GMPS (30 µM) whereas the relaxant effect of the BFPG was more significantly decreased by Rp-8-br-cyclic GMPS as compared to H-89. The qualitative phytochemical studies on the ethyl acetate fraction of Sonchus asper (aerialparts) and butanolic fraction of Pennisetum glaucum (seeds) showed that saponins, terpenoids, flavonoids, tannins and alkaloids are contained by them. HPLC studies of the test fractions showed the existance of quercetin, gallic acid, caffic acid, vanillic acid, and ferulic acid in both the test plants’ fractions where as chlorogenic acid, m-coumaric acid and synapic acid were detected in ethyl acetate fraction of S. asper while p-coumaric acid and ginamic acid were contained in butanolic fraction of the P. glaucum. It is conceivable, therefore, that the test medicinal plants, namely Sonchus asper (aerial-parts), Pennisetum glaucum (seeds) and Zanthoxylum armatum (fruits) empirically used in folkloric medicine to treat various cardiovascular diseases do contain compounds that are able to produce antihypertensive effects. Current findings have also suggested that the reported antihypertensive effect might have resulted from vasorelaxation produced by elevation of c-AMP and/or c-GMP levels in the vasculature. However, inhibition of calcium influx, prevention of calcium mobilization from sarcoplasmic reticulum into the cytoplasm, negative ionotropic and chronotropic effects on the heart and the prevention of the oxidative stress by test fractions could also be responsible for their antihypertensive property. Phytochemical analyses of these plants’ potent fractions have shown that no single chemical compound can be held responsible for the reported activities as several chemical entities have been detected in them. Furthermore, comprehensive chemical and pharmacological studies should be planned to isolate active principles, identify their chemical structure and elucidate exact mechanism (s) of the antihypertensive effect of tested plants. In the meantime, tambulin isolated from Zanthoxylum armatum (fruits) could be suggested as its major active antihypertensive principle. The results substantiate the antihypertensive activity of the indigenous medicinal plants and their fractions. May be here lies the role that the traditional system of medicine has to play in advancing