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Biotarnsformation of Terpenes Using Medicinal Labiates and Fusion Effect on the Products

Thesis Info

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Author

Hammad Afzal Kayani

Program

PhD

Institute

University of Karachi

City

Karachi

Province

Sindh

Country

Pakistan

Thesis Completing Year

2015

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/13468/1/Hammad_Afzal_Kayani_Biotechnology_2015_HSR_UoK_Karachi_02.09.2016.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676725646452

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The thesis was divided into parts; first part consisted of plant cell and tissue culture studies, while second part constituted biotransformation studies using Labiates cell culture and genetic modification effect on biotransformed products. PART A In first part, in vitro protocol for direct regeneration of Lamiaceae plants were established for biotransformation and other modification studies. For this, three Labiates (Ocimum basilicum, Ocimum sanctum and Agastache anisata) were studied. In O. basilicum, the best response for direct plant regeneration was observed in Murashige and Skoog (MS) culture medium containing IAA 0.025 mg/L + BAP 0.1 mg/L, producing 16.4 shoots having a shoot length of 5.33 cm, and 6.86 roots having a root length of 5.36 cm. The plants were best acclimated in sand: farm yard manure (50:50 w/w) and sand (100 %). In O. sanctum, direct plant regeneration was observed in MS medium containing IAA 0.025 mg/L + BAP 0.1 mg/L, producing 12.4 shoots with a shoot length of 5.94 cm, and inducing 15.0 roots having a root length of 6.05 cm. The plants were best acclimated in sand: farm yard manure (50:50) and sand (100 %). Till date, this is the first report of in vitro propagation of O. basilicum using nodal segment as explants. In Agastache anisata, plant regeneration was observed in MS medium containing IAA 0.05 mg/L + BAP 0.1 mg/L, producing 7.93 shoots with a shoot length of 6.0 cm, and inducing 5.96 roots having a root length of 4.01 cm. The plants were best acclimated in sand: farm yard manure (50:50) and sand (100 %). Till date, this is the first report of in vitro propagation of A. anisata using tissue culture technology. The plant was tested for its bioactivity, and it exhibited potent cytotoxic activity. In order to establish and optimize callus induction protocol, required for the biotransformational studies, different combinations of plant growth regulators (PGR) with MS medium were tested. Out of 60 different combinations, best combination for O. sanctum was DN6 (MS + NAA 2 mg/L + 2,4- D 0.25 mg/L) that produced friable callus. When this callus was transferred into the liquid medium, it started dispersing easily making good suspension culture for biotransformation studies. In O. basilicum, yellow friable granular callus was induced in DN9 (MS + NAA 2 mg/L + 2,4-D 0.5 mg/L), and this was further used for the establishment of cell suspension culture for the biotransformational studies. PART B For the biotransformational studies, six substrates were used in which five were terpenes and one steroid. In total, fifteen compounds were isolated for the first time via this route (plants), but twelve (2, 3, 6, 8-11, 13, 16, 18, 19 and 21) are known and three are new (4, 5 and 14) metabolites. Known compounds were mostly synthetically produced compounds or/and some of them bioconverted from fungus but not through plant cell suspension cultures. These compounds have not been acquired this way by any plant cell culture. In order to seek different derivatives of a given compound; various cell suspension cultures were used against several kind of compounds to get more valuable metabolites. In this connection, podocarpic acid (1) was subjected to biotransformation process employing cell suspension culture of Ocimum species. Both cell culture (Ocimum) and substrate (1) were used for the first with each other. Biotransformation of podocarpic acid (1) by cell suspension culture of Ocimum yielded compounds Methyl podocarpate (2), Podocarpic acid acetate (3), 6α, 7α-epoxy podocarpic acid (4), 6β, 7β-epoxy podocarpic acid (5) and 7-keto podocarpic acid (6). Metabolites 4 and 5 were found to be new metabolites, as deduced on the basis of spectroscopic techniques. Ethynodiol diacetate (7), a steroidal compound has not previously been used for the biotransformation employing the cell suspension culture of Ocimum basilicum. Biotransformation of ethynodiol diacetate (6) by culture of Ocimum basilicum yielded compounds 8-11. 17α-Ethynyl-17β- acetoxyestr-4-en-3-one (8), 17α-Ethynyl-17β-hydroxyestr-4-en-3-one (9), 17α-Ethynyl-3β-hydroxy- 17β-acetoxyestr-4-ene (10) and 17α-Ethynyl-5α,17β-dihydroxyestr-3-ene (11). When totarol (12) was incubated with the cell suspension culture of O. sanctum for 20 days, two metabolites totarol acetate (13) and 6-dehydrototarol (14) were obtained. The metabolite 14 was found to be new as deduced on the basis of spectroscopic techniques. This compound (12) has not been used before for the biotransformation using the plant cultures. It was observed that when artesunate (15) was incubated with O. sanctum cell suspension culture for 10 days, it produced metabolite artemisinin (16). When Sclareol (17, diterpene) was incubated with rapidly growing cell suspension cultures of O. sanctum, it afforded two known compounds 3-keto sclareol (18) and 3β, 3-hyrdroxy sclareol (19), but this is a new route for these biotransformed products. When humulene (sesquiterpene) was incubated with rapidly growing cell suspension cultures of O. basilicum, it afforded a known compound 2,6-diepoxy humulene (22), but this is a new route for this biotransformed product. Bioassay studies were also performed for transformed products obtained through biotransformation. The activities employed were anticancer, antioxidant and enzyme inhibition studies. Transformed compounds did not exhibit any remarkable activity as compared to substrates. To evaluate the diversity of plant cell cultures for biotranformation studies, protoplast isolation and fusion method was tested. For protoplast isolation, in vitro developed leaves and callus of O. sanctum and O. basilicum were used as source tissue. The crucial factors were yield (per ml) and viability of protoplasts for successful protoplast isolation. So, at the optimized enzyme concentration of 0.5 % each of Cellulase R-10 and Macerozyme R-10, the 12 hour incubation is sufficient for optimum protoplast yield (5 x 105 cells/ml) and viability (85%). These isolated protoplasts were then cultured on BH3 + KL medium containing 2,4-D (0.5 mg/L) and NAA (2 mg/L). The further subculturing initially on solid MS medium supplemented with 2,4-D with Kinetin for four weeks, and then BAP 0.1 mg/L and IAA 0.05 mg/L for four more weeks resulted in plantlet formation in O. sanctum only. O. basilicum protoplast did not regenerate into plantlet. The fusion of Ocimum spp. was also performed using PEG and electrofusion method. In electrofusion, the protoplasts of Ocimums did fuse to become a putative hybrid, but they did not survive in culture medium after two weeks of culture. As for PEG fusion method, protoplast did not successfully hybridize and eventually died. The hairy root culture was then established to evaluate their potential for biotransformation and comparison studies were conducted. Hairy root culture was developed by co-cultivating Agastache anisata leaf and stem with root inducing bacterium, Agrobacterium rhizogenes. The induced hairy roots and normal roots were cultured in liquid medium and compound (17) was incubated that yielded biotransformed product (19). However, hairy root culture produce high yield (almost twice) and utilized the substrate (17) completely in 20 days as compared to normal root suspension culture. Finally, tomato (Lycopersicon esculantum) transgenic (expressing Hepatitis B surface antigen) and non-transgenic callus, and suspension culture were employed for biotransformation for comparison studies. Both cultures transformed the substrate (17) yielding metabolite 19. There was no significant difference in biotransformation ability.
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گیا سینے وچ بھانبڑ بل

گیا سینے وچ بھانبڑ بل
تاں میں لکھی ایہہ غزل
جیوں کر تیل کڑاہی وچہ
دتا ہجر نے اینویں تل
ہنجواں نال پروئی جو
اگے قلم نہ سکدی چل
ہک واری تاں کول بلا
بیٹھے آن دوارا مل
تھکے کر فریاداں رو
تساڈے واسطے کوئی نہیں گل
تیرے ہتھاں دے وچہ جند
جیون ہے ہک جھٹ کہ پل
جیوندیاں تاں نہیں ملیا توں
ہُن تاں نال جنازے رَل

معاشرتی جرائم کے اسباب اور ان کا ممکنہ تدارک اسلامی تعلیمات کی روشنی میں

Today in the world, the human beings are crunch in crimes in developed society or in undeveloped society. As a result the whole world has become insecure and unsafe. A society and nation can perish and drowned due to its cruelty and crimes. The Muslim society is also being victims of such crimes. According to the scholars the basic reason of this issue is illiteracy and get for away from Islamic teachings while fearless from God and from the Day of Judgment, social injustice, instability, destabilization, disunity and dis-integrity, away from knowledge, negative role of media and impatience tolerance are also considered as major factors. The scholars and reformers are suggested to control the crimes through laydown of justice or establishment of Courts and spread of knowledge, bring awareness among the peoples, establishment of educational institutes in a society. In this article it has been point out the major causes of social crimes and their potential remedy in the light of Qur’an and Sunnah.

A Study on the Synthesis and Biological Evaluation of Some 3, 6-Disubstituted-1, 2, 4-Triazolo[3, 4-B]-1, 3, 4-Thiadiazoles and 2, 5-Disubstituted-1, 3, 4-Oxadiazoles

The 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and 1,3,4-oxadiazoles are prominent biological agents used as a cure against a number of human ailments. The present work deals with the synthesis of 3,6-disubstituted-1,2,4-triazolo[3,4-b]-1,3,4- thiadiazoles and 2,5-disubstituted-1,3,4-oxadiazoles bearing adamantyl and aryl moieties. The synthesis of 3,6-disubstituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles was carried out starting from aryl carboxylic acids. On the other hand, 2,5-disubstituted-1,3,4-oxadiazoles were synthesized using two distinct routes starting from either aryl carboxylic acids or aryl nitriles. The structures of the synthesized compounds were confirmed by IR, 1 H- and 13 C-NMR, EIMS and XRD analysis. The target compounds were screened for their antitumor, antiviral, antibacterial and antifungal potential. Among the tested compounds, 3-(2-methylphenyl)-6-adamantyl- 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole (4a) and 3-(2-fluorophenyl)-6-adamantyl-1,2,4- triazolo[3,4-b]-1,3,4-thiadiazole (4d) exhibited prominent antiproliferative activity against human CD4 + T-cells containing an integrated HTLV-1 genome (MT-4) with CC 50 of 45 μM and 47 μM, respectively. These compounds were also found to be active against human acute T-lymphoblastic leukemia (CCRF-CEM) cell lines with CC 50 of 40 μM each. 3-(3-Methylphenyl)-6-adamantylmethyl-1,2,4-triazolo[3,4-b]- 1,3,4-thiadiazole (4n) showed moderate inhibition of HIV-1 and HIV-2 with EC 50 of >10.90 μg/mL, but no selectivity could be witnessed. Similarly, 3-(3-methylphenyl)- 6-adamantylmethyl-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole (5n) was found to be potent exhibiting moderate inhibition of HIV-1 and HIV-2 with EC 50 of >10.86 μg/mL. Furthermore, oxadiazole (9a) and 2-adamantyl-5-(2-(2-methylphenyl)-1,3-thiazol-4-yl)-1,3,4- 2-adamantyl-5-(2-(2-bromophenyl)-1,3-thiazol-4-yl)-1,3,4- oxadiazole (9j) showed significant inhibition of HIV-1 and HIV-2 with EC 50 of >10.50 μg/mL and >10.90 μg/mL, respectively. However, antibacterial and antifungal activities of all the compounds were found to be non-significant at non-toxic concentrations.