PhD Thesis

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Chemical and Biological Investigation on Zanthoxylum rhetsa, Micromelum minutum and their Endophyte Isolates
(©University of Dhaka, 2022-02-01) ZOHORA, FATEMA-TUZ-
Zanthoxylum rhetsa (Roxb.) DC and Micromelum minutum (G. Forst.) Wight & Arn belonging to the family Rutaceae have been investigated in this research to isolate secondary metabolites and evaluate bioactivities. In addition, five endophytic fungi were isolated from Z. rhetsa and identified at the genus level. Phytochemical investigation of the two plants yielded twenty four compounds which were identified by extensive NMR (1H NMR, 13C NMR, DEPT-135, 1H-1H COSY, HSQC, HMBC and NOSEY) experiments and ESI mass spectrometry. The root bark of Zanthoxylum rhetsa afforded twenty one chemical compounds viz. 2,11-didemethoxy-vepridimerine A, N-methylatanine, 3-dimethylallyl-4,8-dimethoxy-1-methyl-2-quinolone, zanthodioline, 8-methoxy-N-methylflindersine, skimmianine, canthin-6-one, arnottianamide, 8-O-demethylchelerythrine, chelerythrine, 7-methoxynitidine, oxynitidine, xanthyletin, pluviatilol, (+)-piperitol-γ-γ-dimethylallylether, lupeol, β-sitosterol, stigmasterol, methyl oleate, methyl stearate and stearic acid. Among them 2,11-didemethoxy-vepridimerine A is a new dimeric prenylated quinolone alkaloid. 7-methoxynitidine also appeared as a new natural compound containing a benzophenanthredine nucleus, however, it was synthesized previously. N-methylatanine and 3-dimethylallyl-4,8-dimethoxy-1-methyl-2-quinolone are 2-quinolone alkaloids and isolated for the first time from the genus Zanthoxylum. The pyranoquinoline alkaloid, zanthodioline, two benzophenanthridine alkaloids, 8-O-demethylchelerythrine and oxynitidine and the lignan, pluviatilol are reported for the first time from Z. rhetsa. Phytochemical investigations of the methanolic extract of the whole plant of Micromelum minutum provided two coumarins namely micromelin and murrangatin as well as one sterol, stigmast-4-en-3-one, the latter being reported for the first time from M. minutum. To evaluate bioactivities, different fractions of the crude methanol extracts of the two plants, obtained by partitioning, and two pure compounds, isolated in the present study, were screened for antioxidant, cytotoxicity, thrombolytic and antimicrobial activity. In vitro antioxidant activity was assessed by DPPH radical scavenging method using butylated hydroxytoluene as the standard. Among the fractions tested, the aqueous and chloroform soluble fractions of Z. rhetsa exhibited significant antioxidant activities with IC50 values of 55.25±2.78 and 96.00±4.13 μg/ml respectively as compared to the standard 29.23±5.38 μg/ml. Among the different fractions of M. minutum tested, pet ether and chloroform soluble fractions showed prominent antioxidant activities with IC50 value of 49.46 and 67.53 μg/ml compared to the standard 31.02 μg/ml. 8-methoxy-N-methylflindersine showed good antioxidant activity with IC50 value of 71.18±1.74 μg/ml. In the brine shrimp lethality bioassay, the aqueous fraction of Z. rhetsa showed very promising cytotoxicity with LC50 value of 0.46 μg/ml as compared to the standard vincristine sulphate (LC50 0.43 μg/ml). The pet ether and chloroform fractions of M. minutum showed good brine shrimp larvicidal activity with LC50 values of 1.15 and 1.50 μg/ml respectively as compared to vincristine sulphate (LC50 0.27 μg/ml). In the thrombolytic assay, the aqueous fraction of Z. rhetsa again showed the highest activity when compared to other fractions tested with clot lysis 50.5%±3.3% and the standard streptokinase showed 70.6%±7.9% lysis. Zanthodioline and 8-methoxy-N-methylflindersine showed mild thrombolytic activities of 32.0%±9.0% and 17.5%±1.6%. In vitro antimicrobial activity was determined against six gram-positive and nine gram-negative bacteria by the standardized disc diffusion method using kanamycin as the reference drug. Carbon tetrachloride, crude methanolic extract and aqueous fraction of Z. rhetsa demonstrated the highest antimicrobial activity against Vibrio cholera, Bacillus subtilis and Klebsiella pneumonia respectively with zone of inhibition of 18.67±3.21, 14.33±1.53 and 14.0±3.61mm as compared to the standard 24.67±0.58, 19.33±2.08 and 25.33±1.53 mm. 8-methoxy-N-methylflindersine showed poor antimicrobial activity against Bacillus subtilis, Sarcina lutea, Staphylococcus aureus. Pet ether soluble fraction of M. minutum showed good antimicrobial activity against Staphylococcus aureus and Escherichia coli with zone of inhibition of 19.0 mm and 17.0 mm as compared to the standard kanamycin 22.0 mm and 24.0 mm respectively. A total of five endophytic fungi were isolated from the plant Z. rhetsa and were identified morphologically up to the genus level on the basis of macroscopic and microscopic characteristics as Colletotricum sp., Fusarium sp., Colletotricum sp., Clonostachys sp. and Mucor sp. Among of the fungal extract, the extract of Clonostachys sp. showed good brineshrimp larvicidal activity with LC50 value of 0.94 μg/ml as compared to the standard vincristine sulphate (LC50 0.48 μg/ml). Finally, the molecular docking and molecular dynamics (MD) simulations was performed on thirteen compounds isolated in this study to determine their binding affinities against the main protease (Mpro) of SARS-CoV-2 by using AutoDock Vina and the YASARA software package respectively. Among the pure compounds, 2,11-didemethoxy-vepridimerine A, N-methylatanine, 3-dimethylallyl-4,8-dimethoxy-1-methyl-2-quinolone, showed the binding affinity of -8.5, -6.3 and -6.1 Kcal/mol respectively as compared to the standard remdesivir, which showed the binding affinity of -7.8 Kcal/mol.
Chemical and Biological Studies of Some Litsea Species Available in Bangladesh
(©University of Dhaka, 2023-02-23) Bulbul, Israt Jahan
In order to discover new phytochemicals as well as searching for evidence based information of traditional therapeutic uses of medicinal plants, four species of the genus Litsea belonging to the Lauraceae family were selected in this study. The selected plants are Litsea glutinosa Lour., L. monopetala Roxb., L. deccanensis Gamble. and L. lancifolia Hook. f. Two compounds were revealed from L. glutinosa and they are 4΄-O-methyl-(2 ̋,4 ̋,-di- E-p-coumaroyl)-afzelin (LGC-26, 95) and quercetin-3-O-(2 ̋,,4 ̋,-di-E-p-coumaroyl)-α- L-rhamnopyranoside [or, 5΄-hydroxyl-(2 ̋,4 ̋-di-E-p-coumaroyl) afzelin] (LGC-45-3, 96). Both of the compounds were reported for the first time from Litsea species. Five compounds were isolated and purified from the leaf extract of L. monopetala and characterized as vomifoliol (LML 363-1, 97), α-amyrin (LML 309, 98), β-amyrin (LML 301, 99), (E)-6,7,8,9,10,11-hexahydro-8,17:10,16-di(metheno)dibenzo- [h,l][1]oxa[5] azacyclotridecine-1,4-diol (LML 339-1, 100) and (Z)-1,2,3,4,5,6- hexahydro-8,11-etheno-2,13:4,12di(metheno)benzo[h][1]-oxa[5]aza-cyclopentadecine (LML 339-2, 101) by 1H and 13C NMR, COSY, HSQC, HMBC spectral data analysis. All these five compounds are reported for the first from L. monopetala, while compounds 101 and 101 appear to be new compounds. The obtained 1H NMR spectral data and the comparison with the reference value helped us to characterize lupeol (LDC-10-3, 102) and a mixture (4:1 ratio) of β-sitosterol and stigmasterol (LDC-10-2, 55 & 56) from L. deccanensis and β-sitosterol (LLC-10-1, 55) from L.lancifolia. Lupeol has been isolated from L. deccanensis for the first time. 4ʹ -O-methyl (2 ̋,4 ̋-di-E-p-coumaroyl) afzelin (95) Quercetin3-O-(2 ̋,4 ̋-di-E-p-coumaroyl)-α-Lrhamnopyranoside [or 5΄-hydroxyl-(2 ̋,4 ̋-di-E-p-coumaroyl) afzelin] (96) The crude extracts of L. glutinosa, L. monopetala, L. deccanensis and L. lancifolia were evaluated for biological activities through in-vitro and in-vivo screenings. For antidiarrheal activity test, 100, 200 and 400 mg/kg bw methanol extract of L. glutinosa (MELG), L. monopetala (MELM), L. deccanensis (MELD) and L. lancifolia (MELL) were administered in two animal models where 0.5 ml castor oil was used for diarrhea induction and 3 mg/kg bw loperamide was used as standard drug. In all the groups treated with MELG, MELM, MELD and MELL extracts wet feces number, total number of feces and total weight of the foecal output were decreased significantly (p<0.05) with rising of doses. The maximum peristaltic inhibition was observed 32.36%, 22.52%, 26.26% and 33.22% for 400 mg/kg by MELG, MELM, MELD and β- amyrin (99) (E)-6,7,8,9,10,11-hexahydro- 8,17:10,16-di(metheno)dibenzo- [h,l][1]oxa[5]azacyclotridecine- 1,4-diol (100) (Z)-1,2,3,4,5,6-hexahydro-8,11-etheno- 2,13:4,12-di(metheno)benzo- [h][1]oxa[5]azacyclopentadecine (101) Stigmasterol (56) Lupeol (102) β-Sitosterol (55) Vomifoliol (97) α- amyrin (98) MELL extracts respectively. The peristaltic indices were 59.0%, 79.0%, 59.1% and 63.0% for 400 mg/kg of MELG, MELM, MELD and MELL respectively compared to the control (90.0%) and standard (66.7%) groups. For all the plant extracts the percentage inhibition of gastrointestinal motility and peristalsis index were comparable to the standard. In analgesic activity test, MELG, MELM, MELD and MELL on acetic acid-induced writhing in mice at two different doses (100 and 200 mg/kg bw) showed significant reduction of squirming (p<0.001, p<0.01 and p<0.05) in a dose dependent manner as paralleled to control. In the second animal model (Eddy’s hot plate method), pain was induced by heat and analgesia was assessed by counting the time required for the initiation of the reaction. Out of the four plants, all the plants at 100, 200 and 400 mg/kg doses, increased the latency time. The pain-relieving activity data (formalin method) are presented as licking and biting time in seconds at early- and late-phases of treatment with plant extracts. In both the early- and late-phase, reaction time for licking and biting hind paw were decreased with the increment of the doses (from 100 mg/kg bw to 200 mg/kg bw) but in the late phase (20-30 min) the reaction time was decreased significantly (p < 0.05) with the increment of doses for all the studied plant extracts as well as standard indomethacin at 10 mg/kg bw. The effects of MELG, MELM, MELD and MELL on blood glucose level in streptozocin (STZ) induced diabetic rats were found to drop the blood glucose level (BGL) significantly (p<0.05) after 7th days of treatment with the plant extracts at 300 and 500 mg/kg/day doses. Percentage inhibition of blood glucose level for MELG, MELM, MELD and MELL were comparable with that of standard metformin and they are 66.69%, 57.06%, 68.16% and 69.33% respectively at 500 mg/kg/day dose as compared to the untreated diabetic control group. Hole cross test was performed to investigate the possible neuropharmacological effects (CNS stimulant or depressant) of MELG, MELM, MELD and MELL and all the extracts at two different doses (300 and 500 mg/kg bw) found to reduce locomotion in the test animals and to decrease the passing number through the hole in between the hole cross chamber by the animals in a dose dependent manner. Three fractions of four different species of Litsea were studied for antimicrobial activity by disc diffusion method. The results of antimicrobial activity test of different fractions showed mild to moderate activity for L. monopetala, very good activity for L. lancifolia, moderate to good activity for L. glutinosa and mild to very good antimicrobial activity for L. deccanensis against the microorganisms selected for this study. The quantities of phenolic compounds were found in ethyl acetate fraction of L. glutinosa (103.04±0.06), followed by ethyl acetate fraction of L. lancifolia (79.94±0.07). Among the plants L. lancifolia and L. glutinosa have shown very good total phenolics compared to L. deccanensis and L. monopetala. In DPPH free radical scavenging activity test, the IC50 for pet-ether, chloroform and ethyl acetate fractions of L. deccanensis were 31.75 μg/ml, 24.62 μg/ml and 31.04 μg/ml, respectively. All the values are comparable with that of ascorbic acid (31.66 μg/ml). For L. lancifolia 63.97 μg/ml pet-ether, 65.91 μg/ml chloroform and 80.46 μg/ml ethyl acetate extractives were required for 50% scavenging of free radicals. The effective concentrations for pet-ether, chloroform and ethyl acetate fractions of L. glutinosa were measured as 25.19 μg/ml, 37.90 μg/ml and 67.41 μg/ml, respectively. The IC50 values were 31.94 μg/ml, 24.91 μg/ml and 31.10 μg/ml for pet-ether, chloroform and ethyl acetate fractions of L. monopetala, respectively. From results, it may be proposed that three different extractives of L. deccanensis, L. lancifolia, L. glutinosa and L. monopetala were able to exhibit the free radical scavenging activity compared to ascorbic acid, a potent antioxidant compound. The docking simulation was conducted against aldose reductase (AKR1B1) protein model with the purified compound 95 and 96 by using Auto Dock Vina software. Compound 95 exposes the higher negative binding affinity (-9.8 kcal/mol) as compared to the compound 96 with binding affinity (-9.4 kcal/mol) for the interaction of the target protein aldose reductase (AKR1B1). Compound 95 exhibited strong connection with eleven hydrophobic bonds, hydrogen bonds and one other bond while compound 96 developed stable interactions by three hydrogen bonds, and eleven hydrophobic bonds. During investigating interaction pattern, binding affinity, and best binding poses of the compounds it can be proposed that both structures might be promising inhibitors against aldose reductase (AKR1B1) protein. Molecular docking analysis of isolated compounds 95 and 96 (−9.4, and −8.9 kcal/mol, respectively) against human pancreatic alpha amylase showed promising docking affinity. Compound 95 formed polar contacts with Tyr-151, Thr-163, Arg-195, Asp-197, His-201, and His-299 residues and compound 96 showed polar contacts with Gln-63, Arg-195, Asp-197, and His-299 residues. These findings suggest that these compounds are promising inhibitors of human pancreatic alpha amylase. The docking results of vomifoliol clearly indicate that it is a better candidate as an analgesic agent. Vomifoliol (97) is a potent binder (-4.9 kcal/mol) to COX-2 than indomethacin (-1.1 kcal/mol) indicating that it is supposed to have better analgesic action.
Chemical and biological studies of some medicinal plants used by the small ethnic community people of Chittagong hill tracts
(University of Dhaka, 2019-03-28) Ibrahim, Mohammed
This dissertation describes the isolation and characterization of compounds from the leaves of Murraya koenigii (L.) Spreng (Fam.- Rutaceae) and Baliospermum montanum Willd. (Fam.- Euphorbiaceae) as well as the isolation of constituents of Plumbago indica L. (Fam.-Plumbaginaceae) and Alpinia conchigera Griff.( Fam.- Zingiberaceae). In addition, the crude methanol extract of leaves of M. koenigii, B. montanum and Perilla ocymoides L. (Fam.- Lamiaceae) along with crude methanol extract and different partitionates, i.e. n-hexane and chloroform soluble fractions of P. indica and A. conchigera were subjected to screenings for different biological activities. A total of 30 compounds were isolated, 14 from M. koenigii, 8 from B. montanum, 5 from P. indica and 3 from A. conchigera by using repeated column chromatography, TLC, PTLC and HPLC techniques. The structures of the 16 purified compounds were solved by extensive analysis of their high resolution NMR spectroscopic data as well as by comparison with published values and co-TLC with authentic sample whenever possible. The structures of the remaining compounds could not be solved due to unavailability of spectral data. The petroleum ether soluble fraction of methanol extract of leaves of M. koenigii upon gel permeation chromatography over lipophilic Sephadex LH-20 provided two alkaloids (MK-2, MK-8), seven coumarins (MK-1, MK-4, MK-5, MK-6, MK-7, MK-11 and MK12) and one flavonoid (MK-9). Further purification of the petroleum ether soluble partitionate by HPLC afforded two additional coumarins (MK-3 and MK-10). The isolated compounds MK-1, MK-2, MK-3, MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, MK-10, MK-11 and MK-12 were characterized as murracarpin (1), arborinine (2), murpanidin (3), epoxyosthol (4), isomeranzin (5), meranzin hydrate (6), murrangatin (7), mahanimbine (8), 3,3′,4′,5′,5,6,7,8-octamethoxyflavone, (exoticin, 9), demethylisomeranzin (10), 8-acetyl-7-hydroxycoumarin (11) and 7-methoxy-8hydroxycoumarin (12), respectively.On the other hand, the dichloromethane soluble Kupchan partitionate of methanol extract of leaves of B. montanum upon gel permeation chromatography over Sephadex LH-20 yielded two flavonoids (BM-1 and BM-2) while the petroleum ether soluble fraction yielded another two flavonoids (BM-3 and BM-4), which were identified as syringetin (13), 5,6,4′,5′-tetramethoxy-7,3′-dihydroxy-8-methylflavone (14), 4′,5,7-trihydroxy-3′,5′dimethoxyflavone (tricin, 15) and 3,5,7,3′,4′,5′-hexahydroxyflavone (myricetin, 16), respectively. Among these, to the best of our knowledge, compounds 10 and 11 appear to be new, whereas compound 3 is the first report of its occurrence from M. koenigii. On the other hand, compound 14 appear to be new, whereas compounds 13, 15 and 16 are the first report of their occurrence from B. montanum. Acquisition of additional spectroscopic data, including MS is in process to confirm the structure of compounds 10, 11 and 14.
Chemical and pharmacological profiling of Jatropha pandurifolia and Syzygium reticulatum
(© University of Dhaka, 2025-02-19) JAHAN, NISRAT
Jatropha pandurifolia Andrews (family: Euphorbiaceae) and Syzygium reticulatum (Wight) Walp. (family: Myrtaceae) were studied for their secondary metabolites and their biological activities were evaluated both in vitro and in vivo. Through extensive NMR experiments—including ¹H NMR, ¹³C NMR, and ¹H-¹H COSY, HSQC and mass spectrometry, a total of fifteen compounds were identified from the plant extracts. Thirteen of these chemical compounds were isolated specifically from the stem bark of J. pandurifolia including 2-epi-jatrogrossidione, integerrimene, jatrophatrione, citlalitrione, dotriacontyl trans-ferulate, triacontyl trans-ferulate, octacosyl trans-ferulate, hexacosyl trans-ferulate, octacosyl cis-ferulate, β-sitosterol, stigmasterol, n-dotriacontanol, and 1,2-dioleoyl-3-palmitoyl-glycerol. Dotriacontyl trans-ferulate was isolated and characterized as a previously unreported molecule. Bis(2-ethylhexyl) phthalate and stigmast-4-en-3-one were isolated from the leaves of S. reticulatum, marking the first report of these compounds from this plant species. In vivo investigation was carried out to assess CNS depressant, antidiarrheal, and analgesic activities, while in vitro study was assessed for antioxidant, cytotoxic, and thrombolytic properties. Furthermore, in silico investigation of selected pure compounds were conducted using molecular docking and ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling. CNS depressant activity was carried out by open field and hole cross methods at 100 mg/kg, 200 mg/kg, and 300 mg/kg, where diazepam was used as standard. In open field and hole cross method, highest inhibition of movements were 87.37% (***P<0.001) and 84.78% (*P<0.05), respectively for methanolic extract of J. pandurifolia leaf at 300 mg/kg. Furthermore, at the same dose highest inhibition of locomotor activity were 73.68% (***P<0.001) and 80.43% (**P<0.01) for ethyl acetate extract of S. reticulatum leaf in aforementioned methods. Diazepam exhibited 91.58% and 95.65% movement inhibition, respectively. The antidiarrheal effect was performed using the castor oil induced diarrheal model at 200, 400, and 600 mg/kg, using loperamide as the standard. The frequency of diarrhea was reduced by 85.95% at 600 mg/kg of Jp-ML, where loperamide showed 98.57% gastric inhibition. At the same dose, Sr-EAL exhibited a delay in diarrheal onset of 87.53% where loperamide showed 94.84% gastric inhibition. The analgesic activity was assessed by acetic acid induced writhing ix method and formalin induced nociception test. Jp-ML showed the highest dosedependent inhibition of writhing by 66.67% (**P<0.01) at 300 mg/kg, whereas indomethacin showed 78.43% pain reduction. In the formalin test, pain inhibition were 63.92% (***P<0.001) in the early phase and 70.35% (***P<0.001) in the late phase at the same dose. On the other hand, Sr-EAL showed the highest inhibition of writhing by 70.59% (***P<0.001) at 300 mg/kg. Inhibition of paw licking were recorded by 54.55% (***P<0.001) and 70.52% (***P<0.001) in the early phase and late phase, respectively. Antioxidant activity was estimated using the DPPH free radical scavenging assay, with BHT as the standard. The lowest IC50 value were 8.11 μg/mL and 10.34 μg/mL for JP-ML and Sr-EAL, respectively. Pure compounds (JP- 10, JP-13, JP-25, and JP-460) exhibited promising antioxidant activity, with highest antioxidant activity of JP-25 (IC50 =13.10 μg/mL). Cytotoxicity was assessed using the brine shrimp lethality assay, with vincristine sulphate as the standard. The LC50 values were 1.60 μg/mL for Jp-EAL and 1.70 μg/mL for Sr-ML. Among the four pure compounds tested, JP-460 exhibited the strongest cytotoxicity, with LC50 value of 1.27 μg/mL. Cytotoxicity test was also carried out on the HeLa cell line both qualitatively and quantitatively. In qualitative test JP-13 and JP-460 showed a cell survival rate of over 95%, whereas JP-200 resulted in a cell survival rate of only 5%, indicating strong cytotoxicity. In quantitative MTT assay, JP-200 exhibited LD50 value of 272.45 μg/mL. Thrombolytic activity was evaluated using streptokinase as standard. The highest clot lysis activity were 63.32% and 67.58% for the Jp-ML and Sr-EAL, respectively. JP-460 demonstrated significant clot lysis activity, with 71.04% clot rupture. Finally, molecular docking studies revealed that compound JP-460 exhibited strong binding affinity (-9.4 kcal/mol) and binding efficacy (0.41 (kcal/mol per non-hydrogen atom) to the human GABA-A receptor. The ADMET assessment confirmed that the isolated compounds have preferable pharmacokinetic and safety profiles, supporting their potential for therapeutic application.
Cytotoxic and anti-microbial constituents from some Bangladeshi Bridelia and Erythrina species
(University of Dhaka, 2016-06-02) Anjum, Adeeba
This thesis describes the isolation and structure elucidation of secondary metabolites from four plants Bridelia verrucosa, B. stipularis, B. tomentosa (Family: Phyllanthaceae) and Erythrina fusca (Family: Fabaceae) as well as biological studies of the extractives from these plants along with E. variegata (Family: Fabaceae). A total of nineteen compounds were isolated of which three appear to be new. The structures of the isolated compounds were elucidated mainly by spectroscopic studies including high field NMR. The stem bark of B. verrucosa afforded eight compounds viz. glochidonol (BVSP-1, 46), brassicasterol (BVSP-2, 47), friedelin (BVSP-4, 48); 5β-24S-ethylcholestan-3β-ol (BVSP-6, 49); stigmasterol (BVSP-34, 50); 5,5'-dihydroxysesamine (BVBC-2, 51), 3-ketoleanane (53) and pinoresinol (BVS-65, 54). Except stigmasterol (50) and pinoresinol (54) this is the first report of the isolation of compounds 46-49, 53 and 54 from the genus Bridelia and 51 has been identified as a new natural product. Phytochemical investigations of methanolic extract of the stem bark of B. stipularis provided two triterpenes namely, glut-5(6)-en-3-one (BS-01, 55), glut-5(6)-en-3α-ol (BS-06, 56) and a cholestane type compound (22E)-7-hydroxy-28methylcholesta-4,22-dien-3-one (BS-11, 57). All these are the first report of their occurrence from B. stipularis while (22E)-7-hydroxy-28-methylcholesta-4,22-dien-3-one is a new compound. On the other hand, phytochemical investigation of methanol extract of the stem bark of B. tomentosa yielded friedoolean-5(6),14(15)-dien-3-one (BT-1, 58), β-taraxerol (BT6, 59), D -stigmasterone (BT-8, 60) and lupeol (BT-14, 61). This is the first report of their occurrence from this plant and friedoolean-5(6),14(15)-dien-3-one (58) is a novel compound. The carbon tetrachloride soluble fractions of a methanol extract of the stem bark of E. fusca provided three flavonoids namely, shinpterocarpin (EF-7, 62), lupinifolin (EF-8, 63) and 3,9dihydroxy-4-(3,3-dimethylallyl)[6aR,11aR]-pterocarpan (64) and a steroid, β-sitosterol (EF38, 65). All these are the first report except 65 from E. fusca. The crude extracts and organic soluble material of the investigated plants and some purified compounds were screened for their antimicrobial activity against a wide range of Grampositive and Gram-negative microorganisms and fungi by the standardized disc diffusion method. Kanamycin and Griseofulvin were used as reference drugs for the test.
Cytotoxic and antimicrobial constituents from some Bangladeshi Bridelia and Erythrina species
(© University of Dhaka, 2025-03-10) Anjum, Adeeba
Design, development and evaluation of formulations of poorly water soluble drugs intended for oral delivery
(©University of Dhaka, 2023-11-22) Rahman, Md. Rezowanur
Ledipasvir and Daclatasvir, belonging to the BCS class 2, and Velpatasvir, belonging to the BCS class 4, are directly acting anti-viral agents used to treat Hepatitis C virus infections. Owing to poor aqueous solubility and oral bioavailability, development of effective delivery system for these drugs has been enormously challenging. Moreover, suitable dosage forms for pediatric and geriatric patients and patients having difficulty in swallowing as well pose added burden. Therefore, the aim of the present study was to develop a nanosuspension, via solid dispersion technique, based liquid oral suspension using Quality by Design (QbD) approach. Primarily, the compatible polymers for Ledipasvir were screened using FTIR and DSC, and finally the polymers - Poloxamer 188, Poloxamer 407, HPC and HMPC were selected, considering their ability to convert the API into amorphous state in solid dispersions. Design of formulation and analysis with the DOptimal design using Design Expert ® software revealed that Poloxamer 188 and Poloxamer 407 in 0.3:0.7 ratio of Ledipasvir:Polymer produced the optimized nanosuspension formulations with a statistically significant mathematical model. Subsequently, the formulations were stabilized using suspension vehicle optimized via Box Behnken Design using the amount of xanthan gum (gm), avicel ® RC-591 (gm) and citric acid monohydrate (gm) as independent variables whereas viscosity (cp) and zeta potential (mv) as responses. The dissolution profiles revealed that the prepared suspensions of Ledipasvir had much faster dissolution than pure API, suspensions prepared with micronized and nonmicronized API, and the market products available as tablet dosage form. In-vivo simulation studies using PKSolver ® suggested that the absorption of drug from the formulated suspensions was comparable to that of market product up to single dose level (90mg) and superseded in triplicate dose level (270 mg). The formulated suspensions were found to be stable over three- and six-months periods, identified via accelerated stability studies. Interestingly, dissolution profile of the stabilized suspensions was found to be similar after six months. An RP-HPLC method to determine the assay content of Ledipasvir in the finished product has also been developed using 3 2 full factorial design with a Diphenyl column (250 mm X 4.6 mm, 5 µm), the detection wavelength of 330 nm and the injection volume of 20 µL. The optimized method consisted a mobile phase of buffer:acetonitrile at 48:52 ratio and flow rate of 1.7 ml/min. A simple and rapid UV method was developed simultaneously to analyze Ledipasvir and shown to be equivalent to the developed RP-HPLC method. To determine the content of the residual solvent in Ledipasvir solid dispersions, a GC method was developed using the same 3 full factorial design and fused silica GC capillary column (30-m x 0.32-mm x 1.8-µm), Nitrogen with 14.0 psi through head space as carrier gas. Validation of all the developed methods were carried out by following ICH Q2 (R1) guideline. The best results were found with Poloxamer 188 in the Ledipasvir study at a drug:polymer ratio of 0.7:1.3 in terms of in-vivo simulation. Therefore, both Daclatasvir and Velpatasvir were further studied to develop solid dispersion based nanosuspensions and finally a stabilized oral suspension using Poloxamer 188. In case of Daclatasvir, the drug failed to produce amorphous solid dispersion and hence, was not further evaluated for nanosuspension preparation. On contrary, Velpatasvir produced amorphous solid dispersion and thus, nanosuspension was prepared using the same approach applied for Ledipasvir. Afterward, the nanosuspension of Velpatasvir were stabilized using the same method used to stabilize Ledipasvir nanosuspension. The study of the dissolution profiles revealed that stabilized suspension of Velpatasvir had much faster dissolution than its market product available as tablet dosage form. Finally, in-vivo simulation study revealed that single dose of formulated suspension gave the comparable absorption profile to that of the market product.
Development of sustainable method for the Quality Control of traditional medicines on the basis of phytoequivalence and chemical fingerprinting
(©University of Dhaka, 2023-12-07) Koly, Sabiha Ferdowsy
Background: Herbal drugs are composed of single or several types of medicinal plants with additives. This type of preparation is gaining a wide range of popularity among a large a number of people throughout the world. With the increase of usage, it is necessary to maintain the quality of these drug preparations. Therefore, analytical approaches for their intended use in drug quality evaluation need to be validated. This study was planned to develop simple, rapid, selective, precise and economical method for the quality control of herbal preparations. Methods and Materials: Infrared (IR) spectroscopy was used to determine phytoequivalence among the samples. This work outlined a method for identifying herbal drugs on the basis of phytoequivalence. However, as this is a time-consuming and expensive process, the current analysis did not attempt to identify the specific chemical compounds found in the herbs. The marked bands served as a comparative tool for herbal plants and the medications made from them. The quantitative method of comparison was applied for the comparison of crude herbal standard with herbal sample containing formulations. Thin Layer Chromatography (TLC) was used as another analytical method for identification and showing phytoequivalence. It has mostly been used for the qualitative analysis of herbal medicines and to characterize and track the components visually or as an initial separation technique. Single or multiple herbs containing preparation can be analyzed using ultraviolet (UV) spectroscopy. In this technique, it involves the measurement of ultra violet radiation absorption by the substance dissolved in solution. Both qualitative and quantitative analysis can be done through this technique. In the present study, it was aimed at development and validation of UV-spectroscopic technique according to International Conference on Harmonization guidelines which is known as ICH (Q2) guidelines for the analysis of herbs containing polyherbal formulation. The method validation parameters like specificity, precision, accuracy, linearity, range, repeatability and robustness were studied according to ICH (Q2) guidelines. Results and Discussion: Amlaki (Phyllanthus emblica) showed the presence of prominent peak and maximum absorption at 303 nm. The detector response for the Amlaki was linear over the selected concentration range of 1 to 5 μg/mL with a correlation coefficient of 0.998. The absorbance values for intraday precision, found for 1 μg/mL, 3 μg/mL and 5 μg/mL were 0.0082, 0.234 and 0.396 having %RSD of 0.998%, 0.0080% and 0.0058%, respectively. The absorbance values for intermediate precision, found for 1 μg/mL, 3 μg/mL and 5 μg/mL were 0.0084, 0.234 and 0.398 having %RSD 0.6846%, 0.0137% and 0.0038%, respectively. The absorbance value for repeatability was 0.084 having %RSD 0.6901. The accuracy was between 99.348% and 101.478%. Robustness of the method was studied. The %RSD for analyst to analyst variation was 0.4851% and instrument to instrument variation was 0.9726%. The assay results of Amlaki were about 86.588%, 82.150% and 90.828% for three market preparations A, B and C, respectively, indicating insignificant interference from the other ingredients in the formulation. Black plum (Syzygium cumini) showed the presence of prominent peak and maximum absorption at 279 nm. The detector response for the S. cumini was linear over the selected concentration range of 0.1-0.5 μg/mL with a correlation coefficient of 0.9914. The absorbance values for intraday precision found for 0.1 μg/mL, 0.3 μg/mL and 0.5 μg/mL were 0.234, 0.432 and 0.735 having %RSD 0.9245%, 0.5751% and 0.4668%, respectively. The absorbance values for intermediate precision found for 0.1 μg/mL, 0.3 μg/mL and 0.5 μg/mL were 0.233, 0.432 and 0.736 having %RSD 0.8921%, 0.8346% and 0.3421%, respectively. The absorbance value for repeatability was 0.225 having %RSD 0.7950%. The accuracy was between 99.647% and 101.943%. Robustness of the method was studied. The %RSD for analyst to analyst variation was 0.8251% and instrument to instrument variation was 0.3609%. The assay results of S. cumini were about 83.152%, 86.821%, 90.082% and 80.579% for four market preparations A, B, C and D, respectively, indicating insignificant interference from the other ingredients in the formulation. During the method development phase, a number of solvents were used. Among them, methanol was selected in analysis of Amlaki and ethanol was selected in analysis of black plum. These solvents were selected as these solvents satisfied all the conditions relative to peak quality and non-interference at the specified wavelength. The wavelength of maximum absorption (λ max ) was found to be 303 nm and 279 nm in Amlaki and Black plum, respectively. As the values of %RSD is <2%, the method is validated according to ICH (Q2) guidelines. Conclusion: It can be capitulated that this method can be conveniently employed for routine quality control analysis of herbal drugs in bulk drug and other formulations.
Investigation of Anticancer and Antibacterial Metabolites from Seaweeds of the Bay of Bengal and their Associated Endophytic Fungi
(© University of Dhaka, 2026-03-02) NOOR, SADIA
The relentless pursuit of novel anticancer and antibacterial agents has directed significant research toward marine ecosystems, with seaweeds and their endophytic fungi emerging as exceptionally promising reservoirs of bioactive metabolites. This research aimed to systematically investigate the bioactive potential of marine endophytic fungi derived from seaweeds of the Bay of Bengal, Bangladesh. Three seaweed species—Ulva sp. (Chlorophyta), Gracilaria sp. (Rhodophyta), and Sargassum sp. (Phaeophyta)—were collected, morphologically identified, and served as hosts for the isolation of endophytic fungi on potato dextrose agar (PDA) media. Twelve fungal strains were isolated and identified through a polyphasic approach combining macroscopic/microscopic morphological characterization and molecular phylogenetic analysis of ITS sequences. The fungal strains comprised Chaetomium globosum (UE-1, SE-1), Nigrospora magnoliae (UE-2), Curvularia sp. (UE- 3), Curvularia moringae (UE-4), Aspergillus terreus (UE-5, GE-2, SE-2), Collariella gracilis or C. virescens (UE-6), Aspergillus subversicolor (GE-1), Cladosporium halotolerans (GE-3), and Curvularia perotidis (SE-3). Critically, all twelve endophytic fungal strains were reported for the first time from the Bay of Bengal, Bangladesh, with Nigrospora magnoliae being reported as a fungal endophyte for only the second time worldwide. Preliminary chemical profiling of crude extracts via Thin Layer Chromatography (TLC) and Gas Chromatography-Mass Spectrometry (GC-MS) indicated a rich diversity of secondary metabolites, including steroids, terpenoids, flavonoids, and fatty acid derivatives. Bioassay-guided fractionation led to the isolation of twelve pure compounds using various chromatographic techniques, and their structures were elucidated through 1D and 2D NMR spectroscopy. From C. globosum, six compounds were isolated: the new natural compounds UC-71 [(E)-1-(2′,4′- dihydroxy-3′,5′-dimethylphenyl)-2-buten-1-one] and UC-58 [1-(3′,4′-dihydroxy-2′,5′- dimethylphenyl)-3-hydroxybutan-1-one], alongside known metabolites chaetoviridin E (UC-59), a mixture of chaetoviridin A epimers (UC-60), chaetoglobosin G (UC-62), and chaetoglobosin B (UC-63). Another six compounds were isolated and characterized for the first time from N. magnoliae: ergosta-4,6,8(14),22-tetraene-3-one (UN-172), sterigmatocystin (UN-171), dihydrosterigmatocystin (UN-176), 11-oxo-(9E)- octadecenoic acid (UN-233), ostopanic acid (UN-235), and cerevesterol (UN-411). Bioactivity screening revealed that the crude extracts possessed significant iv antimicrobial activity against Gram-positive (Staphylococcus aureus, Bacillus megaterium) and Gram-negative bacteria (Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa), and antifungal activity against Aspergillus niger and A. flavus in disc diffusion assays, outperforming their host seaweeds which showed no activity. Antioxidant activity, evaluated via DPPH radical scavenging, was particularly potent in extracts of A. terreus strains (IC50 value as low as 7.88 μg/mL). Brine shrimp lethality bioassay indicated strong cytotoxicity (LC50 value less than 30 μg/mL) for numerous extracts. The isolated pure compounds also exhibited notable bioactivities; for instance, UC-60 showed remarkable antifungal activity against Candida albicans (31.3 mm zone at 100 μg/disc), and several compounds demonstrated significant cytotoxicity in subsequent MTT assay on the HeLa cervical cancer cell line. The MTT assay revealed that compound UN-235 exhibited significant dose-dependent cytotoxicity against HeLa cells (IC50 value 243.04 μg/mL), demonstrating potency comparable to doxorubicin, thereby identifying it as a promising cytotoxic candidate worthy of further investigation. These comprehensive findings underscore the immense, largely untapped potential of endophytic fungi from Bangladeshi seaweeds as prolific sources of novel chemotherapeutic agents, with the isolation of new natural compounds and first-reported metabolites highlighting their critical value in anticancer and antibacterial drug discovery pipelines.
Phytochemical and Biological Studies on Ravenia spectabilis and Erythrina variegata
(University of Dhaka, 2021-02-28) Tabassum, Fatema
The methanolic extract of the leaves of Ravenia spectabilis Lindl. (Family: Rutaceae) and stem bark of Erythrina variegata L. (Family: Fabaceae) were investigated for the isolation of secondary metabolites and evaluation of bioactivities. A total of twenty one compounds were isolated from these two plants, among them six appeared to be new. The structures of the compounds were elucidated mainly by spectroscopic studies including 1H NMR, 13C NMR, HSQC, HMBC, 1H-1H COSY and NOSEY experiments and the molecular weights were determined by ESI mass spectrometry. Among the new compounds, four were alkaloids and two were very unusual C34 terpenoids. These are 3,5-diprenylindole, 3-prenyl-5-(2-keto-but-3-enyl)indole, 3-prenyl-indole-5-carbaldehyde, iso-oligophyline, ravespanol and ravespanone, all of which were isolated from Ravenia spectabilis. The known compounds isolated from this plant include ravenoline, γ-fagarine, arborinine, atanine, oligophyline, ravenine, methyl linoleate and β-sitosterol. Phytochemical investigation of Erythrina variegata afforded seven known compounds namely scandenone, alpinumisoflavone, lupeol, stigmast-4-en-3-one stigmasta-4,22-dien-3-one, stigmasterol and 3β,28-dihydroxyolean-12-ene. Different fractions of the crude methanolic extract of the investigated plants and some pure compounds, isolated in this study, were screened for their cytotoxic, antimicrobial, thrombolytic and antioxidant activities by standard methods. The new compounds 3,5-diprenylindole, 3-prenyl-5-(2-keto-but-3-enyl)indole and 3-prenyl-indole-5-carbaldehyde were investigated for cytotoxicity using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] colorimetric assay method. Among the three compounds, 3,5-diprenylindole was found to be most cytotoxic to human pancreatic adenocarcinoma cell lines with IC50 value of 9.5 ± 2.2 μM, moderately cytotoxic to human cervical and lung cancer cell lines with IC50 values of 11.3 ± 1.3 μM and 13.5 ± 1.66 μM respectively and weakly cytotoxic to non-tumour cell line (WI-38 ) with IC50 value of 68.5 ± 3.5 μM as compared to the standard (0.19 ± 0.12 to 6.3 ± 0.3 μM). The rest two compounds showed very poor cytotoxicity (IC50 >50 μM) against the four cell lines tested. In vitro antimicrobial activity was measured by disc diffusion method against ten gram positive and gram negative bacterial strains using kanamycin as the standard. Among the samples tested, the pet ether fraction of Ravenia spectabilis and the carbontetrachloride fraction of Erythrina variegata demonstrated the highest antimicrobial activity against Bacillus subtilis and Bacillus cereus respectively with zone of inhibition of 20.5 ± 0.74 mm and 19.5 ± 1.18 mm as compared to the standard (34.0 ± 0.50 mm and 24.30 ± 0.44 mm). Ravenoline isolated from R. spectabilis showed moderate inhibition against Vibrio cholerae (17.2 ± 0.41 mm). Mild to moderate thrombolytic activities were observed by arborinine and different fractions of the crude extract with clot lysis ranging from 30.43 ± 1.03 to 57.78 ± 0.24 % as compared to the standard streptokinase with clot lysis of 74.34 ± 0.73 % for Ravenia extract and 76.54 ± 0.90% for Erythrina extract. The antioxidant activity was evaluated by DPPH radical scavenging method using butylated hydroxytoluene as the standard. Among the crude extract tested, the chloroform and aqueous extract of E. variegata exhibited moderate antioxidant activities with IC50 values of 67.59 ± 1.87 μg/ml and 75.02 ± 2.62 μg/ml respectively as compared to the standard 23.09 ± 1.37 μg/ml. The pure compounds arborinine and ravenoline showed very poor antioxidant activity.
Phytochemical and pharmacological evaluation of Pongamia pinnata, Sesbania grandiflora and associated endophytic fungi
(©University of Dhaka, 2023-06-11) AFRIN, SEAGUFTA
Sesbania grandiflora (L.) Pers and Pongamia pinnata (L.) Pierre, belonging to the Fabaceae family, have been investigated for isolating new natural compounds with promising pharmacological activities. A total of tweenty five compounds have been isolated from these two plants and endophytic fungi associated with Sesbania grandiflora. The isolated compounds were identified by extensive analysis of high resolution NMR ( 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, HSQC and HMBC) experiments and the molecular weights were determined by ESI mass spectrometry. The stem bark of Sesbania grandiflora afforded fourteen natural compounds namely, Sesbagrandiflorain A, (-) maackiain, medicarpin, sativan, 4-hydroxy-2methoxybenzaldehyde, nobiletin, kaurenoic acid, betulinic acid, lupeol, β-amyrin, stigmast-4-en-3-one, stigmasta-4, 22-dien-3-one, stigmasterol and linoleic acid. Among them, eight compounds like (-) Maackiain, 4-hydroxy-2methoxybenzaldehyde, nobiletin, kaurenoic acid, β-amyrin, stigmast-4-en-3-one, stigmasta-4, 22-dien-3-one and linoleic acid were isolated for the first time from Sesbania grandiflora. Five flavonoids and lupeol were isolated from Pongamia pinnata and were identified as pongachromene, kanugin, demethoxykanugin, 5’methoxypongapine and karanjin. Seven different endophytic fungi were isolated and identified from the plant Sesbania grandiflora, such as Fusarium solani, Fusarium fujikuroi, Fusarium equisetti, Xylaria bambusicola, Cladosporium sp, Clonostachys rosea and Colletotrichum aeschynomenes. Two endophytic fungal extract were also investigated for chemical constituents and anhyrofusarubin, bostrycoidin and myristic acid were isolated from Fusarium solani and 3β, 5α, 9α-trihydroxy-ergosta-7,22diene-6-one, 3β,5α-dihydroxy-6β-acetoxy-ergosta-7,22-diene and 3β, 5α,dihydroxyergosta-7, 22-diene-6-one were isolated from Xylaria bambusicola. All these compounds were reported for the first time from the associated endophytic fungi of Sesbania grandiflora. Different solvent fractions of the crude extract and the pure compound Sesbagrandiflorain A, were subjected to several biological activities like antimicrobial, antioxidant, brine shrimp lethality and thrombolytic activities. Among the different fractions tested against five microorganisms, the ethylacetate soluble fraction showed the highest anti-bacterial and antifungal activity against Bacillus megaterium and Aspergillus niger with zone of inhibition of 17.67 ± 1.53 mm and 12.67 ± 0.58 mm compared to the standard, kanamycin (30.67 ± 1.15mm) and ketoconazole (28.00± 1.00 mm) respectively. The pure compound displayed mild activity against Bacillus megaterium (11.67 ± 0.58 mm). In vitro antioxidant activity was studied by DPPH radical scavenging method using butylated hydroxy anisole (BHA) as the standard. Among all the samples tested, the pure compound Sesbagrandiflorain A and crude methanolic extract showed very significant antioxidant activity with IC 50 value of 28.26 ± 0.35 μg/ml and 54.59 ± 0.09 μg/mL as compared to the standard BHA (9.21 ± 0.10 μg/ml). In brine shrimp lethality assay the ethylacetate soluble fraction and methanolic extract demonstrated significant cytotoxic activity with LC 50 of 0.68 ± 0.10 and 0.66 ± 0.04 μg/ml respectively compared to Tamoxifen (0.30 μg/ml). The dichloromethane soluble fraction exhibited promising thrombolytic activity with % of clot lysis of 54.29% as compared to the standard Streptokinase, 73.57%. Among the different extractives of P. pinnata, the methanolic extract showed highest anti-bacterial activity against Bacillus megaterium and antifungal activity against Aspergillus niger with zone of inhibition of 20.67 ±1.15 mm and 17.00 ±1.00 mm respectively compared to the standards Kanamycin (30.67 ± 1.15 mm) and Ketoconazole (28.00 ± 1.00 mm). The Highest antioxidant activity was demonstrated by pongachromene with IC 50 values of 19.69 µg/ml. (BHA, 8.70 ± 0.29 µg/ml). Very mild cytotoxic activities were found for different extracts of P. pinnata in the brine shrimp lethality assay. The isolated endophytic fungi from Sesbania grandiflora were evaluated for their antioxidant activity, cytotoxicity and antimicrobial activity. Among the seven samples tested, SGFRE-1 showed highest antibacterial activity with zone of inhibition of 22.67 mm against Bacillus megaterium as compared to the standard antibiotic kanamycin (30.67 mm) and highest antifungal activity with zone of inhibition of 19.33 mm each against A. niger and A. flavus as compared to the standard antifungal Ketoconazole (28.00 mm). Moderate antioxidant and cytotoxic activities were observed by SGFE-1 with IC 50 value of 54.06 ± 0.20 µg/ml and LC 50 value of 1.90 µg/ml as compared to the standards BHA (9.30 ± 0.06) and tamoxifen 0.34 µg/ml respectively.
Quality Assessment of Some Commonly Used Enantiomeric Drugs in Bangladesh
(University of Dhaka, 2019-10-03) Rahman, Asma
This thesis describes rapid, accurate, precise, less time consuming analytical methods with excellent resolution for separation and determination of chiral drugs. By performing significant number of trial and error methods using a large number of polar and non-polar solvent mixtures as mobile phase, new and suitable chiral HPLC methods were developed for the separation of common enantiomeric drugs such as omeprazole, esomeprazole, rabeprazole, pantoprazole, salbutamol, levosalbutamol, ibuprofen, dexibuprofen, ofloxacin, levofloxacin, citalopram, Scitalopram, carvedilol and propranolol which are commonly prescribed in pharmaceutical formulations in Bangladesh. The chiral separation was achieved on different chiral columns, such as, Chiralcel OD-H, Chiralpak AGP, Chiralcel IC, Lux cellulose-3 and Chiral CD-PH (250 x 4.6 mm, 5 µm particle size, Daicel Chemical Industries Ltd., Tokyo, Japan) for various chiral drugs. This thesis presents suitable chiral HPLC methods for the respective drugs which have been validated according to the guidelines of the United States of Pharmacopeia (USP) and International Conference on Harmonization (ICH). For all enantiomeric drugs, the calibration curve showed good linearity with coefficient of determination (r ) values of ≤0.995. The percentage recovery for all drugs was found to be within the limit (97%-103%) and also the percentage of relative standard deviation (%RSD) of repeatability and intermediate precision was within the acceptable limit (%RSD ≤2). It showed that all the proposed methods met the system suitability criteria with resolution (> 2) though it is very difficult to separate enantiomers having similar physical and chemical properties. The limit of detection (LOD) and the limit of quantitation (LOQ) were also evaluated. Others parameters such as, capacity factor and selectivity were also determined for each method. Finally, all methods have been applied for quantitative determination of enantiomers of the respective drugs and calculated percentage purity of enantiomeric drugs. 2 The current research presents the enantiomeric quantitation and purity profiles of the following enantiomeric formulations of different manufacturers of Bangladesh: I. Omeprazole of twenty-six pharmaceutical companies and S-omeprazole (esomeprazole) of twenty-six companies, II. Rabeprazole of sixteen companies, III. Pantoprazole of twenty companies, IV. Salbutamol of ten companies and levosalbutamol of twelve companies, V. Ibuprofen of twelve companies and dexibuprofen of twelve companies, VI. Citalopram of two companies and S-citalopram of nine companies, VII. Ofloxacin of three companies and levofloxacin of nine companies, VIII. Carvedilol of five companies, and IX. Propranolol of six companies. Thus, all the proposed chiral HPLC methods can be used for routine analysis of the respective drugs for the enantiomeric determination in bulk as well as in pharmaceutical formulations of Bangladesh by simultaneous quantification of (S)- and (R) enantiomers.
Studies of Drug-drug and Drug-metal Interactions of Some Selected Antidiabetic, Antihypertensive and Lipid Lowering Drugs
(©University of Dhaka, 2022-04-13) Akter, Fahima
Drug-drug and drug-metal interactions are important area of research in drug discovery as well as pharmacodynamic actions of the drugs. Drug-drug and drug-metal complexation may introduce new molecules having new and/or better therapeutic activities in the body. In this research work, various types of complexes from antidiabetic, antihypertensive and lipid lowering drugs were investigated while interacting with each other and with the metal ions like chromium(III), lead(II), zinc(II), iron(II) and copper(II). Different types of physicochemical properties and in vivo as well as in vitro pharmacological effects of the complexes were also studied. Drug-drug interactions of three antidiabetic agents (metformin, dapagliflozin and vildagliptin) with an antihypertensive drug (olmesartan medoxomil) were performed by co-evaporated dispersion method and three complexes were synthesized as olmesartanmetformin (OM), olmesartan-dapagliflozin (OD) and olmesartan-vildagliptin (OV). Three complexes of a lipid lowering drug (rosuvastatin) with an antidiabetic drug (vildagliptin), as well as an antihypertensive drug (perindopril) were synthesized viz. perindopril-vildagliptin (PV), perindopril-rosuvastatin (PR) and rosuvastatinvildagliptin (RV). Eleven drug metal complexes were also synthesized viz.Cr-metformin, Cr-glimepiride, Cr-vildagliptin, Cr-dapagliflozin, Pb-metformin, Pb-glimepiride, Pb-vildagliptin, Pbdapagliflozin, Zn-atorvastatin, Cu-atorvastatin and Fe-atorvastatin. The co-evaporated dispersion method was used for the synthesis of both drug-drug and drug-metal complexes. TLC, HPLC, FT-IR, UV, DSC and NMR studies confirmed the synthesis of the complexes. The melting points, DSC and TGA analyses demonstrated the thermal stability as well as thermochemical properties of the synthesized complexes. The thermodynamic parameters of the interactions of BSA (bovine serum albumin) with these pure drugs and synthesized complexes were observed using fluorescence quenching method. For TLC studies of the precursor drugs and complexes, the Rf values were found to be different from each other. Although, the NMR spectra of the pure drugs (metformin, dapagliflozin, vildagliptin, glimepiride, olmesartan medoxomil, perindopril, rosuvastatin, atorvastatin) and synthesized complexes (OM, OD, OV, Cr-metformin, Crglimepiride, Cr-vildagliptin, Cr-dapagliflozin, Zn-atorvastatin, Cu-atorvastatin and Featorvastatin) were recorded. No attempt was taken for in-depth analysis of the NMR spectral data. The main objective of acquiring the 1H NMR spectra of the above mentioned drugs and complexes was to see the differences in the spectral patterns between the parent drug(s) and the corresponding synthesized complex(es). Careful analysis of the 1H NMR spectra demonstrated that differences could be seen between the spectra of the parent drug and synthesized complex. This was only to show that complexes were formed which was further supported by TLC, TGA, DSC and FTIR analyses. Melting points were found to be 221-225 oC, 80-85 oC, 150-154 oC, 175-180 oC, 126-130 oC, 156-160 oC and 163-167 oC for metformin, dapagliflozin, vildagliptin, olmesartan medoxomil, perindopril, rosuvastatin and atorvastatin, respectively. The synthesized complexes exhibited melting points at 68-72 oC, 80-85 oC, 100-105 oC, 55- 60 oC, 110-115 oC, 115-118 oC, 102-107.6 oC, 106.5-111 oC and 105.6-110 oC for OM, OD, OV, PR, RV, PV, Zn-atorvastatin, Cu-atorvastatin and Fe-atorvastatin, respectively which were also different from the precursor drugs and the complexes. The DSC thermograms of metformin, dapagliflozin, vildagliptin, olmesartan medoxomil, OD, OV, OM, Cr-metformin, Cr-glimepiride, Cr-vildagliptin, Cr-dapagliflozin, Pbmetformin, Pb-glimepiride, Pb-vildagliptin and Pb-dapagliflozin revealed the melting endotherms which were found to be different from each other. The Rt of HPLC chromatograms were found to not be identical of some parent drugs and drug complexes in the same analytical conditions. The mechanism of interactions of olmesartan, dapagliflozin, vildagliptin, metformin, and OD, OV and OM complexes with BSA were studied and found as dynamic quenches because the values of Ksv were increased with increasing temperature. The thermodynamic factors were determined from the linear plot of Van’t Hoff which indicated the spontaneous (negative value of ΔG) interaction where hydrophobic interaction was the major contributing force (positive values of ΔH and ΔS) except for olmesartan. For olmesartan-BSA, it was found that ΔH ˂0 ˂ΔS which indicated that the interaction was electrostatic force driven. The binding constants and number of binding sites were also calculated and found that one mole of the reactant (drug) and/or complex interacted with one mole of BSA. On the other hand, the interaction mechanisms of rosuvastatin, perindopril, vildagliptin, RV, PV and PR with BSA were also studied and found that perindopril, vildagliptin, PV and PR showed dynamic quenches as the values of Ksv increased along with increasing temperature. But the rosuvastatin-BSA and RV-BSA systems were developed by static quenching where, Ksv values decreased with increasing temperature. The interactions of perindopril, vildagliptin, PR, PV with BSA were mediated by enthalpy driven hydrophobic bonding (negative value of ΔG). But the rosuvastatin-BSA and RV-BSA systems were driven by the Vander Waal’s forces and H-bonds (negative value of ΔH along with ΔS˂0). The binding constants and number of binding sites were also analyzed and found that one mole of the reactant (drug) and/or complex interacted with one mole of BSA. In vivo exploration of anti-diabetic activity was done on alloxan induced mice. The study revealed that after 14 days of treatment the antidiabetic drugs e.g. metformin, dapagliflozin and vildagliptin reduced the blood sugar by 39.70%, 56.73% and 51.22%, respectively while the newly synthesized complexes e.g. OM, OD and OV reduced the blood sugar by 42.95%, 50.50% and 48.66%, respectively. Hence only OM demonstrated synergistic effect as it reduced the blood sugar level more than that exhibited by metformin. Other complexes OD and OV did not show better effect than the parent drugs dapagliflozin and vildagliptin, respectively. The OM can be demonstrated as safe because the complex revealed no damage to hepatic and nephrotic tissues. But the other complexes OD and OV produced moderate to severe dysplasia in kidney and liver tissues after 14 days of treatment. All the three complexes elevated levels of serum creatinine and uric acid than that for metformin, dapagliflozin and vildagliptin itself. The levels of serum creatinine for control, metformin, dapagliflozin, vildagliptin were found as 3.8 mg/dL, 3.38 mg/dL, 3.42 mg/dL and 3.60 mg/dL, respectively but for OM, OD and OV the concentrations were found as 4.09 mg/dL, 4.56 mg/dL and 5.95 mg/dL, respectively. Uric acid levels for control, metformin, dapagliflozin, vildagliptin, OM, OD and OV were found as 17.59 mg/dL, 10.06 mg/dL, 11.37mg/dL, 16.84 mg/dL, 12.75 mg/dL, 15.64 mg/dL and 17.81 mg/dL, respectively. The serum SGPT level for control, metformin, dapagliflozin, vildagliptin, complex OM, OD and OV treated mice were calculated as 23.85 U/L, 20.28 U/L, 21.02 U/L, 21.17 U/L, 17.35 U/L, 20.15 U/L and 27.78 U/L, respectively. Serum SGOT level in mice after treatment with drugs and drug complexes were found 21.23 U/L, 18.42 U/L, 17.24 U/L, 17.70 U/L, 15.54 U/L, 18.91 U/L and 25.67 U/L, respectively for control, metformin, dapagliflozin, vildagliptin, OM, OD and OV. Serum SGPT and SGOT levels were elevated by the treatment with OV. But OM treatment revealed reduced serum SGPT and SGOT levels than by only metformin treatment (20.28 U/L to 17.35 U/L and 18.42 U/L to 15.54 U/L, respectively). Considering all the issues the complex OM can be demonstrated as safe and promising ligand and can be suggested for further extensive studies to evaluate as therapeutic agent. In case of antidiabetic activity of four Cr-complexes viz. Cr-metformin, Cr-glimepiride, Cr-vildagliptin and Cr-dapagliflozin, they improved glucose metabolism in alloxan induced hyperglycemic mice. The treatment with the Cr-complexes significantly reduced the blood glucose level than that of the positive control group mice. Among the four Cr-drug complexes, Cr-dapagliflozin complex reduced blood glucose level significantly and it was found to be 64.20% more effective than the standard dapagliflozin. The result was followed by Cr-glimepiride by 26.72% blood glucose reduction, Cr-metformin by 23.35% reduction and Cr-vildagliptin by 7.61% reduction than the standard glimepiride, metformin and vildagliptin, respectively. But Crvildagliptin and Cr-dapagliflozin showed moderate dysplasia in hepatic tissues after 14 days of treatment. So, whether Cr-complexes can offer long-term health benefits or not is still unknown as extensive toxicological data could not be established yet. In case of Pb-antidiabetic drug complexes, they did not show significant positive effect to reduce the blood glucose level. After 14 days of treatment with metformin, glimepiride, vildagliptin, dapagliflozin, it was found that the average glucose levels of mice decreased from 31.54, 30.24, 31.50 and 30.37 to 19.02, 17.20, 19.70 and 17.60 mmol/L, respectively in mice whereas the complexes Pb-metformin, Pb-glimepiride, Pb-vildagliptin and Pb-dapagliflozin did not reduce blood glucose level considerably and blood sugar levels were found as 25.82, 29.23, 25.32 and 29.32 mmol/L, respectively. Moreover, the Pb-complexes increased serum creatinine and serum uric acid levels of mice as well as produced necrosis of the hepatic and nephrotic tissues which suggested cellular damage in liver and kidney of mice. After treatment with metformin, glimepiride, vildagliptin, dapagliflozin, the serum creatinine levels of mice were found to be 3.38, 3.96, 3.60 and 3.42 mg/dL, respectively whereas for Pbmetformin, Pb-glimepiride, Pb-vildagliptin and Pb-dapagliflozin the creatinine levels were increased to 4.57, 5.36, 5.21 and 5.24 mg/dL, respectively. The levels of uric acid of the experimental mice were elevated into 53.13 from 42.91, 57.40 from 44.83, 49.36 from 40.21 and 53.32 from 41.49 mg/dL for Pb-metformin, Pb-glimepiride, Pb vildagliptin and Pb-dapagliflozin, respectively than that of metformin, glimepiride, vildagliptin and dapagliflozin. In vivo evaluation of lipid lowering activity was done on high fat diet fed rabbits and from the experiment, it was found that all the synthesized complexes viz. complex PR, PV and RV reduced cholesterol, triglycerides, low density lipoprotein (LDL) cholesterol, very low density lipoprotein (VLDL) cholesterol, non high density lipoprotein (HDL) cholesterol levels but enhanced the high density lipoprotein (HDL) cholesterol level. The complex PR decreased the levels of serum cholesterol, triglycerides, low density lipoprotein (LDL) cholesterol, very low density lipoprotein (VLDL) cholesterol to 139.92±8.23 mg/dL, 210.1±38.34 mg/dL, 81.0±10.12 mg/dL, 30.0±2.12 mg/dL and 112.0±8.79 mg/dL, respectively while the reference drug rosuvastatin lowered the levels at 144.8±9.12 mg/dL, 280.13±40.25 mg/dL, 87.0±12.10 mg/dL, 37.0±3.23 mg/dL and 114.0±9.23 mg/dL, respectively. The complex RV also decreased cholesterol, triglycerides, low density lipoprotein (LDL) cholesterol, very low density lipoprotein (VLDL) cholesterol 172.35±10.12 mg/dL, 152.01±41.45 mg/dL, 111.0±10.11 mg/dL, 27.0±3.32 mg/dL and 137±8.32 mg/dL, respectively than the reference drug rosuvastatin did. The promising complex PV reduced the LDL and VLDL to 69.0±10.65 mg/dL and 26.0±4.13 mg/dL, respectively. But rosuvastatin as well as the three newly formed complexes PR, RV, PV increased HDL cholesterol levels to 30.34±2.01 mg/dL, 28.33±2.5 mg/dL, 34.9±2.7 mg/dL and 49.44±2.3 mg/dL, respectively than that of the control group of rabbits (12.48±2.3 mg/dL). In support of lipid lowering activity the antioxidant, thrombolytic and membrane stabilizing activities of the complexes PR, PV and RV were also evaluated in vitro. Three complexes showed better thrombolytic activity than rosuvastatin. Among the complexes, the RV demonstrated the highest thrombolytic activity (29.52±0.09%) whereas PR, PV and rosuvastatin showed 26.39±0.06%, 20.97% and 20.96±0.09% activities, respectively. The synthesized complexes PR, PV and RV displayed better antioxidant activity than the lipid lowering drug rosuvastatin. For free radical scavenging activity, the highest IC50 was produced by PV (67.71 μg/mL) among all the samples followed by RV (56.83 μg/mL), PR (54.79 μg/mL) and rosuvastatin (131.6 μg/mL). Three new complexes also were investigated for membrane stabilizing activity and showed significant effect. The rosuvastatin inhibited 39.92% hemolysis of RBCs followed by RV (35.18 %), PR (26.55 %) and PV (19.43 %) in the condition of induced by hypotonic solution.
Studies of stress degradation and impurity profiles of some 5-ht3 antagonists
(University of Dhaka, 2019-10-06) Hossain, Md. Mokaram
Three issues of fundamental importance in drug therapy are safety, efficacy and stability. This thesis paper has reports the scientific data of five 5-HT3 antagonists in the field of stress degradation, degradation kinetics and impurity profiles. The stress conditions are aqueous, acid, base, oxidation and photodegradation. Ramosetron HCl, ondansetron HCl, granisetron HCl, tropisetron HCl and palonosetron HCl are the five 5-HT3 antagonists that were selected for the studies. Extensive information derived from studies of stress degradation, degradation kinetics and impurity profiling expanded the scientific thought further to ensure the achievement of intended quality of drug substances and drug products available in the market. Stress degradation screening of 5-HT3 antagonists in aqueous conditions, different strengths of acid-base conditions, oxidative condition and photo degradation at different time point and temperature revealed the pH dependent stability, oxidative and photo sensitivity. From stressed degradation conditions, it was evident that ramosetron HCl and palonosetron HCl are more stable in aqueous conditions. However, ondansetron HCl, granisetron HCl and tropisetron HCl produced degradants in aqueous conditions. On the other hand, four antagonists except ondansetron HCl produced degradants and growing impurities in acid stressed conditions. Each 5-HT3 antagonist out of five was evident to produce degradants and growing impurities. Degradation kinetic studies were conducted for four 5-HT3 antagonists, ramosetron HCl, ondansetron HCl, granisetron HCl, and tropisetron HCl, to observe the effect of temperature and also to calculate activation energy (Ea). Degradation kinetics of ramosetron HCl was studied in 0.1N NaOH and ondansetron HCl, granisetron HCl, and tropisetron HCl in 2.0N NaOH at 60°C, 70°C and 80°C at different time interval. The calculated activation energy (Ea) was found as 10.05 kcalmol -1 , 7.57 kcalmol -1 , 16.98 kcalmol -1 , and 16.86 kcalmol -1 for ramosetron HCl, ondansetron HCl, granisetron HCl and tropisetron HCl, respectively. Degradants and growing impurities were reported after evaluation of HPLC and MS data taking consideration of the relative retention time (RRT), mass spectroscopy (MS) intensity and molecular weights of each 5-HT3 antagonist generated. In acidic conditions, ramosetron HCl produced some potent degradants with relative retention time (RRT) of 0.79, 0.89, 2.18, 2.33 and 3.50. In basic conditions, ramosetron hydrochloride also produceed potent degradants with relative retention time (RRT) of 0.38, 0.51, 0.56, 0.64, 0.67 and 0.70. Potent degradants of ramosetron HCl with relative retention time (RRT) of 0.65, 0.76, 0.94 and molecular weight of 113.9, 141.1 and 361.2 Da were evident in oxidative conditions. Ondansetron HCl increased the content of two growing impurities with relative retention time (RRT) of 0.51, 0.65 and molecular weight of 82.1 and 211.26 Da. Growing impurities of ondansetron HCl with relative retention time (RRT) of 0.44, 0.47, 0.49 and molecular weight of 604.77, 211.26 and 279.34 Da were evident in oxidative conditions. On the other hand, potent degradants of ondansetron HCl with relative retention time (RRT) of 0.51, 0.56, 0.65, and 1.39 was also observed in oxidative conditions. Ondansetron hydrochloride is light sensitive and increased the concentration of a growing impurity with relative retention time (RRT) of 0.25 and molecular weight of 256.2 Da when exposed to 3.6 million lux fluorescence light and 600 watts hour/m 2 UV light. Granisetron hydrochloride was observed to produce one degradant with relative retention time (RRT) of 0.85 in aqueous condition. In acidic conditions, granisetron HCl produced some potent degradants with relative retention time (RRT) of 0.24, 0.30, 0.58, and 1.22. In basic conditions, granisetron hydrochloride was seen to increase the concentration of a growing impurity with molecular weight of 335.1 Da. It was found to be stable in 10.0% hydrogen peroxide. It showed no photosensitivity. Tropisetron hydrochloride produced two degradants with relative retention time (RRT) of 0.35 and 0.40 in aqueous condition. In acidic conditions, tropisetron HCl produced some potent degradants with relative retention time (RRT) of 0.17, 0.21, 0.24, 0.40 and 0.59. In basic conditions, two growing impurities with relative retention time (RRT) of 0.51, 0.65 and molecular weight of 82.1 and 211.26 Da were evident for tropisetron HCl. Potent degradants of tropisetron HCl with relative retention time (RRT) of 0.23, 0.24, 0.38 and 0.43 were produced in oxidative conditions. In acidic conditions, palonosetron HCl produced a potent degradant with relative retention time (RRT) of 0.13. In basic conditions, palonosetron HCl also produced a potent degradant with relative retention time (RRT) of 0.13. One known growing impurity with molecular weight of 310.4 Da and three unknown potent degradats with molecular weight of 314.4, 328.4 and 344.4 Da were evident for palonosetron HCl in oxidative conditions. Therefore it is clearly evident that more attention should be given during formulation development, process validation and stability testing to minimize or control these growing impurities and potential degradants.
Study on Health and Drug Policies of Bangladesh to Ensure Health for All
(©University of Dhaka, 2022-04-13) Rahman, Md. Aknur
Background National Health and Drug policy are the standard guidelines to ensure the healthy life of the citizens of a country. According to World Health Organization, “Health is the physical, social and mental wellbeing and not merely absence of any disease”. Health is one of the basic needs of people in Bangladesh and improvement of nutritional and public health status is a Constitutional commitment of the Government of Bangladesh according to the Constitution, Article 15 and 18, respectively. The present study is focusing on the critical analysis of National Health Policy (NHP), perceptions of the service providers and service receivers regarding NHP, and Primary Health Care (PHC) in Netrokona Sadar Upazila - A Case Study on Expanded Program on Immunization (EPI), National Drug Policy (NDP), availability, affordability and price variation of essential antibiotics in Bangladesh. All issues, in the purview of NHP and NDP, have been analyzed to have an endeavor in contributing to ensure health for all. The objectives of the study were-(i) to check the consciousness of people in health sector about NHP and NDP, analyze the national budget for research and education on health and drugs, contribute on improvement of the NHP and NDP by taking some valuable suggestions from the respondents, find out the drawbacks that are in the policies and find out the strategies to deal with them; (ii) to find the areas of cooperation between Government Organizations (GOs) and Non-Government Organizations (NGOs) in the arena of PHC focusing the EPI and find the weaknesses in providing the PHC services; (iii) to measure prices of essential antibiotics for treating prevalent conditions in Bangladesh, assess the affordability of standard treatment regimens using these medicines, compare the prices of medicines found in the country with international reference prices and compare the prices of medicines of different years with statistical significance testing and (iv) to inform the policy makers to address those issues. Methods The study was conducted as survey as well as case study. Both primary and secondary sources of data were used in the study. NHP, NDP, EPI and availability, affordability and price variation of essential antibiotics of Bangladesh were analyzed. A total of 1100 respondents were selected for the study. The data were collected by random sampling method. Results NHP was consulted with the doctors, health personnel and researchers and important feedbacks were obtained. Majority of the respondents informed that they were acquainted with NHP and put some suggestions regarding the appropriateness of NHP. Role of public servant was addressed by the respondents. Some areas were identified which were not properly addressed in NHP such as management of diseases caused by Zika virus, Nipah virus, Dengue and Chikungunya, non-communicable disease, epidemic disease control. Some suggestions were also given by the respondents. Those were - need revision in NHP; update of NHP at a regular basis; involve civil society, and public representatives; ensure more punishment in case of false drug manufacture. The study showed that most of the service receivers did not know about NHP. It was observed that the percentage of people who knew about NHP was increasing with respect to the educational level. If the people are well informed about NHP, they can get better health service. The respondents put some important comments- the NHP does not ensure enough number of doctors, pharmacists (virtually absent / non-existent), nurses, and other stuffs; some medicine especially antibiotics should be prescribed by the doctors only; the NHP cannot resolve the communication gap between the service providers and the service receivers; does not ensure enough necessary supplies; does not ensure skilled doctors, pharmacists and the health workers in primary and secondary level hospitals; does not utilize the referral system properly etc. In case of service providers, acquaintance with NHP was not satisfactory. In fact, the scenario for service providers was worse than service receivers. In both the cases, it did not cross 50% and most of the service providers were not aware about the policy and as a result, they could not provide better treatment to the patients. It was evident from the study that diseases like child tuberculosis, diphtheria, whooping cough, tetanus, hepatitis B virus, Hib V, polio and measles can be prevented by vaccines through EPI as part of the endeavors of primary health care services of the government. In the journey of primary health care services through EPI, collaboration of GOs and NGOs works effectively to a great extent. The study also found some areas to be improved such as more coordination and endorsement of contributions of the players in the smooth running of the EPI. Maximum respondents (99%) opined that right use of drug practice can prevent abuse of antibiotics. Awareness and training needs were soughed in this respect. It was found that some of the respondents (22%) did not know about essential drug program. It was found that only 26 (5%) respondents out of 500 knew the exact number of essential drugs in Bangladesh. In surveyed pharmacies, the essential medicines were less available in general medicine stores as compared to the Model pharmacies. A comparative study on price variation across years had shown a insignificant increase in price. The median prices of surveyed medicines were obtained, and a gross comparison was done that indicated percent increase or decrease in price. In the studied programs, the median price ratios of surveyed medicines varied from 0.36% - 2.56% and 0.33% - 2.39% in year 2015 and 2019, respectively. While noting the WHO target that consumer should pay no more than four times the IRPs, we observed that medicine prices were lower in Bangladesh compared to IRPs. The study confirmed that the essential antibiotics were affordable in Bangladesh. Conclusion Based on our findings, it can be inferred that the idea of ensuring health for all is not a very difficult task. The major plus point has been the positive gesture of all the related stakeholders of health and medication. Contribution and cooperation of all - politicians, GOs, NGOs, civil society etc. to the NHP and NDP can make them fullest and confident to ensure health for all in Bangladesh.
The in vitro and in vivo Pharmaceutical equivalence and stability studies of some antihypertensive drugs manufactured in Bangladesh in rat model
(©University of Dhaka, 2023-02-09) Begum, Rehana
Background Bangladesh is a densely populated country. To meet the healthcare needs of this huge population, huge amounts of medicines are required. Again, the first objective of National Drug Policy 2005 was to ensure that common people of Bangladesh should have easy access to effective, safe and good quality drug products at affordable prices. As hypertension is a very common disorder in Bangladesh, many pharmaceutical companies are now producing antihypertensive drugs from each class. But most of the companies do not conduct bioequivalence studies and for clinical trial and bioequivalence studies, even now we depend on another country like Malaysia, India and. No data are available in regard to pharmaceutical equivalence and bioequivalence studies of antihypertensive drugs manufactured in Bangladesh. The present study is carried out to perform in vitro and in vivo pharmaceutical equivalence and stability studies in comparison with reference innovator brands of some antihypertensive drugs manufactured in Bangladesh to compare the quality, efficacy and safety of these drug products by taking reference innovator brands as standard brands. This study will also help the physicians to choice a suitable brand which is easily available, have standards of quality, efficacy and safety. Methods In vitro pharmaceutical equivalence of some antihypertensive drugs was determined by comparing general quality assessment parameters such as weight variation, hardness, % friability, disintegration time, dissolution time and the amount of active substance between test brands and their respective reference innovator brands. Times required for 50% dissolution (T50%) and 90% dissolution (T90%) were also compared between test brands and their respective reference innovator brands. Mean of % dissolution versus time graph and statistical difference factor (f1) and similarity factor (f2) were also compared using dissolution profiles of test brands and their respective reference innovator brands. In vivo pharmaceutical equivalence of some antihypertensive drugs was done by plotting plasma concentration- time curves of test brands with their respective reference innovator brands after administration of drug in rat models. Stability testing was compared between test brands and their respective reference innovator brands under stress conditions in acidic and basic conditions at different temperatures (290C, 600C and 700C). Results Experimental three brands of tablet atenolol 50 mg were randomly designated as AA, AB, AC and reference innovator brand as ARI. Eight brands of tablet Carvedilol 6.25 mg were randomly designated as CA, CB, CC, CD, CE, CF, CG, CH and reference innovator brand as CRI. Ten brands of tablet Losartan potassium 50 mg were randomly designated as LA, LB, LC, LD, LE, LF, LG, LH, LI, LJ and reference innovator brand as LRI. Four brands of tablet ramipril 5 mg were randomly designated as RA, RB, RC, RD and reference innovator brand as RRI. All test brands including their respective reference innovator brands passed the general quality assessment parameters such as weight variation, hardness, % friability, disintegration time, dissolution and % potency. Still significant variations were observed in disintegration time of test brands of tablet losartan potassium and tablet ramipril with their respective reference innovator brands. A correlation was observed between disintegration time and the rate of dissolution in this study. All test brands including their respective innovator brands were found within % weight variation test acceptance limit. Test brands of atenolol showed weight variation percentage limit between - 2.39% and + 2.77%, whereas reference innovator brand showed weight variation percentage limit between - 1.84% and + 1.40%. Test brands of carvedilol showed weight variation percentage limit between - 2.72% and + 5.87%, whereas reference innovator brand showed weight variation percentage limit between - 1.08% and + 0.92%. Test brands of losartan potassium showed weight variation percentage limit between - 4.66% and + 4.08%, whereas reference innovator brand showed weight variation percentage limit between -2.60% and +2.14%. Test brands of ramipril showed weight variation percentage limit between -2.00% and + 2.85%, whereas reference innovator brand showed weight variation percentage limit between -1.85% and +1.49%. All test brands of tablet atenolol, tablet carvedilol, tablet losartan potassium and tablet ramipril including their respective reference innovator brands were found satisfactory for hardness testing. Test brands of atenolol showed lowest hardness value hardness between 4.55 kg and 6.13 kg, whereas reference innovator brand showed hardness 5.32 kg. Hardness of test brands of carvedilol were found between 3.88 kg and 7.68 kg, whereas 6.26 kg was found for reference innovator brand. Hardness of test brands of losartan potassium were found between 6.28 kg and 9.96 kg, whereas 6.89 kg was found for reference innovator brand. Test brands of ramipril showed hardness between 7.51 and 13.19, whereas reference innovator brand showed 7.16 kg All test brands of tablet atenolol, tablet carvedilol, tablet losartan potassium and tablet ramipril including their respective reference innovator brands met the acceptance criteria for % friability test. They had % friability values less than 1%. All test brands of tablet atenolol, tablet carvedilol, losartan potassium and tablet ramipril including their respective reference innovator brands met the acceptance criteria for disintegration time. No major variations were found in disintegration time of different test brands of atenolol. They were found to disintegrate between 0.43 and 1.36 minutes, whereas reference innovator brand disintegrated in 1.44 minutes. No momentous variations were found in disintegration time of test brands of carvedilol. They disintegrated between 0.39 and 5.33 minutes, whereas innovator brand disintegrated in 0.78 minutes. Test brands with higher disintegration time were CE, CH and CG. Significant variations were found in disintegration time of test brands of losartan potassium. They were found to disintegrate between 6.52 and 15.22 minutes, whereas reference innovator brand disintegrated in 7.19 minutes. Test brands with higher disintegration time were LA, LC, LF, LH and LI, having values ˃10 minutes. Test brands of ramipril showed significant variations in disintegration time. All test brands of ramipril disintegrated between 0.71 and 10.90 minutes, whereas innovator brand disintegrated in 1.09 minutes. Test brands with higher disintegration time were RA, RB having values ˃5 minutes and RC ˃10 minutes. All test brands of tablet atenolol, tablet carvedilol, tablet losartan potassium and tablet ramipril including their respective reference innovator brands met the acceptance limit for % of dissolution. Test brands of tablet atenolol including their respective reference innovator brand attained more than 90% of dissolution within 30 minutes. Test brands of tablet carvedilol including their respective reference innovator brand achieved more than 90% of dissolution except brand CH which got more than 80% within 30 minutes. Test brands of tablet losartan potassium LA, LC, LE, LF, LG, LH and reference innovator brand LRI did more than 90% of dissolution except brands LB, LD, LI and LJ which got more than 80% within 30 minutes. Test brands of tablet ramipril including their respective reference innovator brand attained about 100% of dissolution within 30 minutes. All test brands of tablet atenolol, tablet carvedilol, tablet losartan potassium and tablet ramipril including their respective reference innovator brands met the acceptance limit for assay content. They had % potency between 99% and 103%. The mean % of drug dissolved of tablets of different test brands were compared with that of their respective innovator brands graphically by plotting the mean % of drug dissolved against time. All test brands including reference innovator of tablet atenolol released more than 80% of drug within 10 minutes. Except test brands CB, CC, CG, CH; all other brands including reference innovator brand of tablet carvedilol released more than 80% of drug within 20 minutes. Reference innovator brand and brands LC, LE, LF, and LG of tablet losartan potassium released more than 80% of drug in 20 minutes. Test brands LA, LB, LD, LH, LI, and LJ released more than 80% of drug in 30 minutes. Except brand RC; all test brands and reference innovator brand of tablet ramipril 5mg released more than 80% of drug in 10 minutes. The time required for 50% dissolution (T50%) and 90% dissolution (T90%) were determined. All test brands of tablet atenolol and also tablet ramipril including their reference innovator brands showed T50% values less than 10 minutes and T90% values less than 30 minutes. For tablet carvedilol; all test brands including reference innovator brand showed T50% values less than 10 minutes and T90% values less than 30 minutes except test brand CH. Test brand CH had T50% less than 10 minutes but T90% greater than 30 minutes. For tablet losartan potassium; test brands LA, LB, LD, LH, LI, LJ showed T50% values greater than 10 minutes whereas, other brands less than 10 minutes. Test brands LB, LD, LI showed T90% values greater than 30 minutes whereas, other brands less than 30 minutes. The mean percentage of drug dissolved of tablets of test brands and their respective reference innovator brands were used to calculate difference factor(f1) and similarity factor (f2) using the respective equations. All test brands of antihypertensive drugs showing f1 values less than 15 are acceptable in comparison with reference innovator brands. For test brands of tablet carvedilol CB and CH; f2 values were less than 50. For test brands of tablet losartan potassium LB, LD and LI; f2 values were less than 50. For test brand of tablet ramipril RC; f2 values were less than 50. Test brands with f2 values less 50 may not be equivalent to their respective reference innovator brands. In vitro dissolution profiles showed variations in availability of drug substances from test brands and reference innovators brands. All test brands of tablet atenolol; all test brands of tablet carvedilol except two brands CB & CH; all test brands of tablet losartan potassium except brands LB, LD & LI and all test brands of tablet ramipril except brand RC were observed to have T50% values less than 10 minutes, T90% values less than 30 minutes, f1 values less than 15 and f2 values more than 50. They appeared to have very good bioavailability. Test brands CB and RC showing f2 values less than 50 but T50% values less than 10 minutes, T90% values less than 30 minutes and f1 values less than 15 also seemed to have very good bioavailability. Test brands CH and LB, LD, LI having T50% values greater than 10 minutes, T90% values greater than 30 minutes and f2 values less than 50 were not equivalent to reference innovator brands in availability of drug substances. In vivo pharmaceutical equivalence study was done by plotting plasma concentration- time curves of test brands with their respective reference innovator brands after administration of drug products in rat models. The curves indicated that the tmax value for test brands and innovator brand of atenolol was 2.5 hrs and Cmax values for brands AA, AB, AC, ARI were 0.123, 0.128, 0.113, 0.129 μg/mL respectively. The tmax value for test brands and innovator brand of carvedilol was 1.5 hrs and Cmax values for brands CA, CB, CC, CD, CE, CF, CG, CH, CRI were 0.106, 0.106, 0.102, 0.103, 0.099, 0.096, 0.096, 0.098, 0.090, 0.106 μg/mL, respectively. The tmax value for test brands and innovator brand of losartan potassium was 1.5 hrs and Cmax values for brands LA, LB, LC, LD, LE, LF, LG, LH, LI, LJ, LRI were 0.122, 0.123, 0.126, 0.118, 0.122, 0.123, 0.118, 0.123, 0.117, 0.120, 0.124 μg/mL, respectively. The tmax value for test brands and innovator brand of ramipril was 2.5 hrs and Cmax values for brands RA, RB, RC, RD, RRI were 0.047, 0.061, 0.058, 0.053, 0.063 μg/mL, respectively. Comparing in vivo Cmax and tmax values of test brands with their respective innovators, all antihypertensive testing brands may be considered equivalent to their respective reference innovator brands. Stability studies of test brands of tablet atenolol, tablet carvedilol, tablet losartan potassium and tablet ramipril including their respective innovator brands were done by stress degradation in acidic and basic conditions at different temperatures (290C, 600C and 700C). Data showed no significant degradation of test brands and also their respective reference innovator brands. So, all antihypertensive test brands also may be considered equivalent to respective reference innovator brands regarding stability. In conclusion, this study indicated that except test brands CH, LB, LD and LI; all other test brands may be considered in vitro and in vivo pharmaceutically equivalent to their respective reference innovator brands and also equivalent in case of stability. These brands may be similar in quality, efficacy, safety and may be used interchangeably. But test brands CH, LB, LD and LI are not similar to their respective reference innovator brands and cannot be used interchangeably.
The potentiation and broadening the effect of low molecular hypoglycemic drugs through interaction with some CNS stimulant molecules
(© University of Dhaka, 2025-03-10) Mohiuddin, Mohammad
The potentiation and broadening the effect of low molecular hypoglycemic drugs through interaction with some CNS stimulant molecules
(University of Dhaka, 2015-03-18) Mohiuddin, Mohammad
The in-vitro study on the interaction of caffeine and theophylline with gliclazide, glipizide, glyburide and metformin has been carried out by IR spectroscopy, UV spectrophotometric, continuous variation, mole ratio, conductometric and Ardon’s spectrophotometric methods at room temperature and at different pHs to investigate the invitro complex formation and also to study the nature & strength of complexes which could be formed due to interaction of caffeine and theophylline with gliclazide, glipizide, glyburide and metformin. The results of the present study indicated that caffeine and theophylline form 1:1 complexes with gliclazide, glipizide, glyburide and metformin. Observations of infrared and ultraviolat spectral data have revealed the possibility of interaction of caffeine and theophylline with gliclazide, glipizide, glyburide and metformin. The spectra of target molecules alone and the 1:1 mixture of caffeine and theophylline each with gliclazide, glipizide, glyburide or metformin showed significant changes in their absorption intensities including some shifts in the absorption maxima. This may be due to interaction of caffeine and theophylline with the drugs that alter the absorption intensities. Study of continuous-variation, mole-ratio and conductometric methods has indicated initial complexation. Continuous-variation plots have conformed the formation of 1:1 complexes of caffeine with gliclazide, glipizide & metformin, 1:2 complexes of caffeine with glyburide, the formation of 2:1 complexes of theophylline with gliclazide & glipizide, 1:2 complexes of theophylline with glyburide and 1:1 complexes of theophylline with metformin. Mole-ratio plots conformed the formation of 1:1 complexes of caffeine with gliclazide, glipizide & metformin, 1:2 complexes of caffeine with glyburide, the formation of 1:2 complexes of theophylline with gliclazide & glipizide, 1:2 complexes of theophylline with glyburide & metformin.The conductometric method was used to further ascertain about the nature of interaction and stoichiometries. Conductometric titrations in demineralized water system at differe0nt pHs were carried out to find out the molar ratios at which complexation occurred. Conductrometric titrations have showed that 1:1 complexes are formed between caffeine and each of the interacting species. It has also showed that 1:1 complexes are formed between theophylline and each of the interacting species. The Ardon’s spectrophotometric method confirmed the formation of 1:1 molecular complexes and led to calculate the stability constants. It has been observed that the stability constants for caffeine-gliclazide system were higher than that of caffeine-glipizide, caffeine-glyburide, caffeine-metformin, theophylline-gliclazide, theophylline-glipizide, theophylline-glyburide and theophylline-metformin system in all pHs conditions. The in-vitro study of protein binding of gliclazide, glipizide, glyburide, metformin and their 1:1 mixtures with caffeine and theophylline have been conducted by equilibrium dialysis method performing measurement by direct spectrophotometric method at temperature 370 ± 0.50 C and at pH 7.4. The number of binding sites and affinity constants of gliclazide, glipizide, glyburide, metformin and their 1:1 mixtures with caffeine and theophylline have been calculated by scatchard method.Scatchard plots were prepared to reveal the number of binding sites and the affinity for protein binding. It has been found that both caffeine and theophylline cause lowering the affinity and percentage of binding of the drugs in the mixture to bovine serum albumin. Thus, the interaction of gliclazide, glipizide, glyburide and metformin with caffeine and theophylline can increase the free drug concentration of gliclazide, glipizide, glyburide and metformin in blood plasma. This may change the pharmacokinetic and pharmacodynamic properties of the drugs. An in-vivo study has been conducted in rats to observe the influence of caffeine and theophylline on plasma concentration of gliclazide, glipizide, glyburide and metformin. The plasma concentration of gliclazide, glipizide, glyburide and metformin were determined by UV spectrophotometric method after oral single administration of gliclazide, glipizide, glyburide, metformin alone and with caffeine & theophylline in rats. The in-vivo study for determination of plasma cocentration showed that concurrent administration of caffeine and theophylline with gliclazide and glyburide have not made noticeable changes in plasma concentration of gliclazide and glyburide. But administration of caffeine and theophylline with glipizide and metformin in rats has showed a significant change in plasma concentration of glipizide and metformin. So, a competitive inhibition of the binding to plasma protein by caffeine and theophylline increases the plasma concentration of glipizide and metformin.Thus any change in plasma concentration may affect the pharmacological effects of the drug. An in-vivo study has been conducted in rats to observe the effect of caffeine and theophylline on hypoglycemic activity of gliclazide, glipizide, glyburide and metformin. The blood sugar levels of animals were measured after administration of a drug alone and in combination. The blood sugar levels were estimated in two stages; firstly, after 2 weeks and secondly, after 4 weeks of the administration of drug. The results in this study have shown that caffeine and theophylline can enhance hypoglycemic effect of gliclazide, glipizide, glyburide and metformin in rats. It has also been found that the influence of caffeine on metformin is stronger than on gliclazide, glipizide and glyburide in respect of hypoglycemic activity.Thus, the hypoglycemic activities of gliclazide, glipizide, glyburide and metformin are potentiated and broadened by caffeine and theophylline concurrent application. The potentiation of the antidiabetic properties of gliclazide, glipizide, glyburide and metformin may be due to relaxation effect of caffeine and theophylline on smooth muscles of the rat as well as some sort of modification of the molecular conformations of the antidiabetic agents. Thus, the interaction of caffeine and theophylline with the potent antidiabetic drugs gliclazide, glipizide, glyburide and metformin hydrochloride may greatly influence the activity of these molecules. The data obtained from the present study would help to recommend that low molecular hypoglycemic drugs gliclazide, glipizide, glyburide as well as metformin may result into compatible combination therapies with CNS stimulant molecules caffeine and theophylline.