Pharmaceutical Sciences and Natural Products - Master Dissertation

Permanent URI for this collectionhttps://kr.cup.edu.in/handle/32116/53

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Anticancer activity of cassia fistula linn through invitro and insilicoapproach
(Central University of Punjab, 2014) Sharma, Akanksha; Jaitak, Vikas
Cassia fistula L. (Leguminoseae) is a plant species called "Aragvadha" that means "disease killer". C. fistula consists of plethora of medicinal properties. A detailed discussion is depicted of the therapeutic potential and chemical composition of C. fistula that is responsible for its highly important medicinal properties. C. fistula contains many chemical components like anthraquinones, anthocyanindins, proanthocyanidins, flavanoids, polyphenols, alkaloids, saponins, coumarins, tannins, etc. These constituents are reported to possess various biological properties such as antioxidant, antimicrobial, antidiabetic, antitumor, antimelasmic activities etc. In the current dissertation work we limited our study to cancer; specifically, Lung cancer (LC) and Breast cancer (BC) which are spread all over the world to a threatening level. The present study is an effort to contribute to such a multi-targeting moiety from the plant C. fistula, on the basis of in vitro and simulated analysis. To explore the anticancer activity of C. fistula; in vitro (cytotoxicity studies) on different extracts and in silico studies using MMGBSA (Molecular Mechanics - the Generalized Born model and Solvent Accessibility) on the earlier reported molecules was performed; to study protein - ligand binding interactions on four different targets. The different extracts (Petroleum v ether (SVA-1) (for defatting), Ethyl acetate (SVA-2), Methanol (SVA-3), Hydro- methanolic (SVA-4) Extracts) were prepared in order of their increasing polarity. In- vitro cytotoxicity studies done using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay on the extracts of plant and their IC50 values were calculated. It was performed to identify plant's cytotoxic activity. Targets studied via performing in silico protein-ligand binding interactions of majorly mutated yet interlinked pathways of tyrosine kinase and serine/ threonine kinase family; that are namely EGFR, PI3K, Akt and mTOR receptors on which reported molecules of plant were studied. In silico studies were performed using maestro 9.6 Schrodinger software. MMGBSA and ADMET analysis were performed to discover and understand protein - ligand interactions between the chosen ligands of Cassia fistula and selected kinase receptors. On conducting MMGBSA (Molecular Mechanics ' the Generalized Born model and Solvent Accessibility) studies 5,3,4-tri-hydroxy-6-methoxy-7-O-?-L- rhamnopyranosyl-(1?2)-O-?-D-galactopyranoside along with few other moieties illustrated fine binding interactions against standard inhibitors of all the protein targets. From in vitro experiment, it was found that Hydromethanolic extract (SVA-4) showed prominent cytotoxicity against MCF-7, A-549 and H-460 cell lines particularly near 50?g/ml of concentration. Results were found in comparable with the NCI criteria limits for IC50 values of extracts obtained in assay. Also extracts were stable in DMSO solvent even when kept at the ambient temperature for 30 days and gave consistent results against cancer cell lines. As per the results of the in silico studies, the 5,3,4-tri- hydroxy-6-methoxy-7-O-?-L-rhamnopyranosyl-(1?2)-O-?-D-galactopyranoside (dG Bind score -98.7866kcal/mol) for EGFR in comparison to gefitinib (dG Bind score - 86.5585kcal/mol); (dG Bind score -87.3524kcal/mol) for Akt as compared to AZD 5363 (dG Bind score -76.5959); (dG Bind score -87.1051kcal/mol) for PI3K as compared to wortmannin (dG Bind score -79.0654kcal/mol) and for mTOR it has shown the (dG Bind score of -81.964kcal/mol) against sirolimus (dG Bind score -192.354kcal/mol) is reported to have fair pharmacokinetic profile along with attractive binding interactions with EGFR, PI3K, Akt and mTOR receptors.
Antiproliferative activity of Asparagus racemosus extracts
(Central University of Punjab, 2018) Sharma, Ram; Jaitak, Vikas
Cancer is regarded as uncontrolled progression and spread of cells. Cancer is not a singular, specific disease but a group of variable tissue responses that result in uncontrolled cell growth. Healthy cells have a specific size, structure, function and growth rate that best serves the needs of the tissues they compose. Cancer is one of the leading of morbidity and mortality worldwide, with approximately 14 million new cases in 2017. Breast cancer (BC) is a disease where cells in the tissue of the breast cancer grow and divide without normal control. Estrogen receptor is a group of proteins (or a twelve helix protein) present inside the cells of the female reproductive tissues or located in the nucleus of cells. ER?, ER? and ER gamma have different responses and they are located in different tissues. Quinone forms of catechol estrogen binds to DNA and forms adduct. Semi Quinone intermediates are free radicals can bind with oxygen to producing superoxide radicals. Superoxide radicals can attack and alter the structure of DNA and causing Breast cancer. Various synthetic drugs (Tamoxifen & Raloxifene) are used for treatment of breast cancer, but numerous side effects like menopausal symptoms, vaginal dryness, low libido, mood swings and Nausea. The discovery of novel natural drugs is important for reduction of side-effects, high selectivity, low toxicity, and better killing of cancer cells. Phytoestrogens are one the best category of natural products used for treatment of breast cancer. Phytoestrogens have similar structure to the endogenous estrogen. Distance between the hydroxyl groups is 14.5 A0 is similar to estrogen. Asparagus racemosus contain large number of phytoestrogens. In this context, the aim of the present study was to explore the roots of Asparagus racemosus in the terms of its medicinal values for Breast cancer. Anticancer activity of different extracts were evaluated by performing In vitro study by using Breast cancer cell lines T-47 D. from the Preliminary phytochemical investigation of extracts demonstrated that methanolic extract and Aqueous methanolic extract contain large number of phytoestrogens. Aqueous methanolic extract and methanolic extract showed maximum IC50 value as compare to other extract. Isolation of molecules from methanol extract, total four molecules isolated from methanol extract and three molecules from aqueous methanol extract. Moreover, in silico study of reported phytoestrogen from Asparagus racemosus was also carried out using glide docking to investigate interaction pattern with estrogen receptor ? and estrogen receptor ?. The top docking score was obtained for Rutin (Estrogen receptor ?) and Quercetin (Estrogen receptor ?). Tamoxifen and raloxifene used as standard for estrogen receptor ? and oestradiol used as standard for estrogen receptor ?. From the ADME study demonstrated that maximum flavonoids has highest oral absorption as compare to other. The results showed that phytoestrogens are expected prospective candidate for regulatory tumor progression with a special emphasis in breast cancer progression.
Chemical Investigation and Antiproliferative screening of extracts from Stevia rebaudiana (Bertoni)
(Central University of Punjab, 2018) Saxena, Aditi; Jaitak, Vikas
Cancer is the uncontrolled development of abnormal cells in the body. It is considered as the leading public health problem in both developed and the developing countries. As no drug of cancer is establish to be completely efficient and safe as anticancer therapy and is responsible for the prolonged toxicity and also causes various side effects. Chemoprevention of cancer by natural products is beneficial, as these compounds have the nominal side effects and short of toxicity compared to the synthetic compounds. The phenomena of Carcinogenesis is very complex and includes so many signaling pathways. Thus Phytochemicals are measured as the right candidates for developing the anticancer drug. The study for developing more potent candidates which can obstruct or slow down the expansion of the cancer cells without c au s in g any side effects from these phytochemicals are still in progress and Many new phytochemicals and its derived analogs have been recognized as potential candidates for anticancer therapy among these one of the potent plant is Stevia rebaudiana. The leaves of Stevia rebaudiana tends to possess zero calories, and consists mainly of ent kaurene diterpene glycosides generally recognized as steviol glycosides. Stevioside is the main sweet component found in S. rebaudiana. studies suggest that the stevioside along with v other associated compounds including rebaudioside A, steviol, and isosteviol tends to have therapeutic benefits including anticancer activity. Taking in consideration the above mentioned factors we have investigated the Anticancer potential of extracts of S. rebaudiana. Four extracts was prepared using petroleum ether, chloroform, ethyl acetate and aqueous methanol. T47D cell line have been used to evaluate the anticancer potential using MTT assay. AD-2 that is chloroform extract showing IC50 value of 7.79μg/ml. Moreover IC50 value of AD-4 that is aqueous methanol was also comparatively better and found to be 9.53 μg/ml and AD-1 that is petroleum ether had shown IC50 value of 9.58μg/ml. Thus, various extracts have shown good Antiproliferative activity and S. rebaudiana can be further investigated for its anticancer potential. Furthermore docking study on estrogen receptor–alpha, androgen receptor and aromatase receptor discovered that the phytochemicals of the plant have good binding affinity towards all the three mentioned receptors and can be suitably customized to search its anticancer potential. Moreover unfavorable ADME profile can be overcome by structure modification. Thus on the basis of in-vitro and in-silico data we can conclude that S. rebaudiana extracts have promising anticancer potential. further isolation of compounds have been done successfully and total four compounds have been isolated. two compounds ASP-2 and ASP-4 have been successfully characterized and found to be Stevioside and Rebaudioside A respectively which are already known compounds.
In-vitro guided fractionation of crude root extracts of potentilla atrosanguinea lodd and in-silico of polyphenolic compounds
(Central University of Punjab, 2013) Gupta, Vinay Kumar; Jaitak, Vikas
Modern therapeutic system is a kind of inspiration from traditional plant based medicine used for various diseases and ailments. Advancement in drug discovery technology including computational drug design and bioassay guided fractionation emboldens the interest of Medicinal Chemists? concerning to lead identification from medicinal plants for complicated diseases, in the last few years. There are so many traditional plants which are used in the treatment of various diseases in different parts of our country but scientific information is missing for the same. Potentilla atrosanguinea is a native to the western Himalaya region has been used traditionally for the treatment of wound-healing, diarrhea, influenza and bleeding but there is not even a single published evidence about its activity accept antioxidant activity of aerial part. In this context, the aim of the present study was to explore the roots of P.atrosanguinea in terms of its medicinal value for instances in-vitro photoprotective and antioxidant activity. The photoprotective activity was evaluated in the term of SPF (sun protection factor) by spectrophotometric method in the range of 290-320 nm (UVB region) whereas antioxidative activity was evaluated using a free radical scavenging assay (DPPH, superoxide anion scavenging and CUPRAC). Total phenolic contents of the extract/fractions were v determined by Folin Ciocalteu reagent. The ability of photo-protection of different fraction against UVB region followed the trend Pa-AcOEt > Pa- n-BuOH > Pa-H2O-MeOH > Pa-H2O. Ethyl acetate fraction of Potentilla atrosanguinea indicated the highest sun protection factor (SPF) (7.319 ' 0.353) at a concentration of 120 ?g/ml. IC50 values of aqueous methanolic (Pa-H2O-MeOH) and ethyl acetate fraction (Pa-AcOEt) for DPPH assay was comparable as that of rutin (80 ?g/ml). Superoxide anion scavenging activity of all fractions was found to be excellent than standard (IC50 150 ?g/ml). Calculated IC50 value for the aqueous-methanolic, ethyl acetate, n-butanol (Pa- n-BuOH) and aqueous fractions (Pa-H2O) were 60, 70, 90 and 140 ?g/ml respectively. In CUPRAC assay percentage reduction capacity of the aqueous methanolic crude extract was highest among all other fractions. Total phenol contents of aqueous methanol extract and ethyl acetate fraction were almost comparable and indicated high phenol content. Results indicated the importance of ethyl acetate extract of P. atrosanguinea as a photoprotective agent in sunscreen preparation in the pharmaceutical industry and natural antioxidants as well. Further isolation of molecules from ethyl acetate fraction was performed using column chromatography which led to the isolation of total seven molecules out of them two were characterized namely methyl pentatetraconta-30, 32, 34, 36, 38, 40, 42-heptaenoate (VVR-I) and pentadecyl butyrate (VVR-III). VVR-I is novel compound while VVR-II is already reported in literature. Moreover, in-silico study of already reported polyphenolic compounds which are considered to be anticancer agents were also carried out using Glide docking to investigate interaction pattern with MDR receptors (MRP1 and GSTP-1) involved in cancer chemotherapy. In-silico findings suggest that rutin may be used as dual modulator for MRP-1 and GSTP1-1 mediated multidrug resistance
Integrated in-vitro antioxidant and in-silico anti-apoptotic study of essential oil components of aconitum heterophyllum wall
(Central University of Punjab, 2013) Bhall, Yashika; Jaitak, Vikas
Aconitum heterophyllum Wall. is consumed for its promising medicinal properties in several parts of the world. Present study consists of hydrodistillation, antioxidant potential and in-silico antiapoptotic study of A. heterophyllum oil. Antioxidant activities were evaluated by in-vitro assays namely DPPH, Superoxide anion scavenging and CUPRAC. It was found that the anti-oxidative effect of A. heterophyllum oil was dose dependent up to 200 g/ml. For studying the apoptotic nature of the volatile constituents, in silico studies were carried out using BCL-2 anti-apoptotic receptors (BCL-2, BCL-XL, MCL-1). To understand the cascade of mechanisms leading to apoptosis, NF-?B was also considered. From the comparative study of the constituents with that of the standard inhibitor it has been observed that the constituents show favorable binding affinity for the receptors as in the case of BCL-2 receptor, ?--longipinene has a dock score of -4.26 kcal/mol as comparable to that of standard inhibitor ABT 263 (-4.67 kcal/mol); BCL-XL receptor, neryl acetate has a dock score of -4.05 kcal/mol as compared to ABT 737 (standard inhibitor) which was -9.47 kcal/mol. Best results were observed in the case of NF-?B with ?-fenchol, having the dock score of -4.36 kcal/ mol which shows higher binding affinity of the ?-fenchol molecule for the receptor site as compared to the selective inhibitors parthenolide whose dock score was -3.04 kcal/ mol. In summary, based on our in silico and in vitro results, it can be postulated that essential oil of A.heterophyllum could be used as functional antiapoptotic inhibitor and as natural antioxidant.
Phytochemical investigation and biological evaluation of secondary metabolites from asparagus racemosus l through in-vitro and in-silico approach
(Central University of Punjab, 2013) Singla, Ramit; Jaitak, Vikas
Nature has been a source of medicinal products for millennia, and with many useful drugs developed from different natural resources, with majority of drugs are from plant origin. Asparagus racemosus belonging to family liliaceae, is one such important medicinal plant. This plant species is used traditionally in India and other parts of the world in epilepsy, Vaat disorders, brain tonic, hypertension, hepatoprotection, immunostimulant and antiabortifacient. In this context, the aim of the present study was to explore the roots of A.racemosus in terms of its medicinal values for instances antimutagenic, and advanced glycation end-product inhibitor. Antimutagenic activity of different extracts were evaluated using Ames test. A. racemosus methanolic extract (RME) and aqueous extract (RAE) have been found to have effective in the inhibition mutation induced by NPD and sodium azide. Among the two extracts, RAE and RME showed maximum inhibition of 49.2%, and 40.63% in Co-incubation mode respectively. The inhibition of BSA-glucose for the determination of antiglycation activity showed that the inhibition varied significantly among different extracts of A. racemosus. The highest inhibition measured by BSA-glucose was observed for (Ethyl acetate extract) REE (IC50 37.56 ± 1.65 ?g/mL) followed by (methanolic extract) RME (IC50 51.32 ± 1.48 ?g/mL). Isolation of molecules from methanol extract led to the characterisation of one molecule v namely nyasol out of total seven isolated molecules. The molecular docking study of isolated molecule Nyasol displayed strong binding affinity with estrogen receptor ? and estrogen receptor ?, indicating that Nyasol is beneficial in hormone responsive breast cancer. Moreover, in-silico study of already reported phytoestrogens from A.racemosus was also carried out using Glide docking to investigate interaction pattern with Human placental estrone sulphatases (1P49), human 17?-hydroxysteroid-dehydrogenase type 1 (1FDS), human glucose 6-phosphate dehydrogenase (2BH9) and tubulin protein receptors. The top docking score was obtained for rutin (estrogen receptor ?), 3,6,4'-trimethoxy-7-O-?-D-glucopyranosyl [1?4]-O-?-D-xylopyranoside glucopyranpsyl (HSP90), 8-Methoxy-5,6,4-trihydroxyisoflavone-7-O-?-D-glucopyranoside (human placental estrone sulphatase), Shatavarin X (17?-hydroxydehydrogenase`), Racemoside A (Glucose-6-phosphate dehydrogenase), Immunoside (Colchicine binding site of tubulin). The results indicated that phytoestrogens are likely potential candidate for controlling tumor progression with a special emphasis in breast cancer progression.
Phytochemical investigation in vitro anti-mutagenic activity of potentilla fulgens lodd and silico study of flavonoids with CDK-2,CDK-6 receptors
(Central University of Punjab, 2013) Monga, Prakriti; Jaitak, Vikas
Plants have been used for thousand years in the treatment of various diseases. Plant secondary metabolites have proved to be an excellent source of new medicinal compounds. They offer protection against variety of chronic diseases including diabetes, cardiovascular diseases, obesity and cancer. Mutation is an important factor that is linked to carcinogenesis. It has been found that occurrence of cancer can be reduced by decreasing the incidence or rate of mutation. Plants are promising source of antimutagens agents which are present in them as secondary metabolites such as flavonoids, alkaloids, terpenoids, glycosides etc. Potentilla fulgens is an important medicinal plant of higher Himalayas that is known globally for its therapeutic importance. A number of antioxidant constituents have been reported from the plant which mainly consists of polyphenolic compounds. It has been observed that diet rich in polyphenolic compounds such as flavonoids can reduce the risk of cancer. P.fulgens reported to have polyphenolic compounds such as flavonoids which are potent bioactive molecules that possess anticarcinogenic effects as they can interfere with initiation, development and progression of cancer by the modulation of cell cycle, apoptosis, and angiogenesis. Anti-mutagenic activity on different fractions of P.fulgens was carried out, which indicates that n-butanol and water fractions of the plant are strongly anti-mutagenic. Inhibitory activity for n-butanol fraction was 60.4% and 35.4% in co-incubation and pre-incubation respectively in TA 98 tester strains while 55.6% and 62.0% inhibition was observed in TA100 tester strains respectively for co and pre-incubation. For water fraction 56.6% and 60.7% inhibitory activity in co-incubation and pre-incubation mode of treatment respectively against TA98 strain and 34.5% and 50.6% inhibition in TA100 strain for co-incubation and pre-incubation treatment respectively. Results specify the importance of P.fulgens as a new source of anti-mutagenic agents. Isolation of molecules from ethyl acetate and n-butanol v fractions led to the characterisation of one molecule namely catechin out of total six isolated molecules. In-silico study of various reported flavonoids were performed on CDK-2, CDK-6 as these receptors are linked to cell cycle and mutation in cell cycle may lead to cancer. In-silico study indicates that natural as well as synthetic flavonoid molecules can be considered as a treatment for cancer by inhibiting CDK-2 and CDK-6 receptors.
Phytochemical investigation of natural sweetener from stevia rebaudiana (Bartoni)
(Central University of Punjab, 2014) Mayank; Jaitak, Vikas
S. rebaudiana is an important plant because of the high concentration of steviol glycosides (SGs). Sugars and artificial sweeteners, which are used in general as well as in pharmaceutical field have shown multiple toxic effects. On the other hand SGs have shown sweetness profile many fold compared to sucrose along with many health benefits.The substitution of SGs as sweeteners seems to be a reasonable solution towards the safety issues. The antidiabetic activity of S. rebaudiana extract as well as SGs is well documented in literature. The association of diabetes with cancer is also well known factor. Taking in consideration the above mentioned factors we have investigated the anticancer potential of extracts of S. rebaudiana. Extracts was prepared using petrolieum ether, ethyl acetate, methanol, aqueous methanol and water. Three cell lines (A-549, H-460 and MCF-7) have been used to evaluate the anticancer potential using MTT assay. In case of A-549, MVE-5 showing IC50 value of 10 ?g/ml. Moreover, IC50 values of MVE-2 was also comparatively better and found to be less than 50 ?g/ml and MVE-4 had shown IC50 value of 90 ?g/ml. In case of H- 460 reasonably better IC50 have been observed for MVE-4 and MVE-5 which is 88 ?g/ml and 92 ?g/ml, respectively. In H-460 reasonably better IC50 have been observed for MVE-4 and MVE-5 which is 88 ?g/ml and 92 ?g/ml, respectively. But in case of MCF-7 breast cancer cell line MVE-1 and MVE-2 have shown IC50 value of 90 ?g/ml and 53 ?g/ml, respectively. Thus, various extracts have shown good antiproliferative activity and S. rebaudiana can be further investigated for its anticancer potential. Furthermore docking study on EGFR, v PI3K and mTOR receptor revealed that SGs have good binding affinity towards all the three mentioned receptors and can be suitably modified to explore its anticancer potential. Moreover unfavourable ADME profile can be overcome by structure modification. Thus on the basis of in-vitro and in-silico data we can conclude that S. rebaudiana extracts have promising anticancer potential. Further isolation of compounds have also been done successfully and total seven compounds have been isolated. Two compounds MVR-1 and MVR-5 have been successfully characterized and found to be quercetin and stevioside respectively which are already known compounds. Many reported SGs have shown poorer taste quality. Moreover taste quality of all SGs are different from one another. Thus docking studies were performed on SGs by constructing homology models of T1R2 and T1R3 subunits of human sweet taste receptors to explore the sweetness mechanism. Ramachandran plot, PROCHECK results and ERRAT overall quality factor indicated the acceptable quality of models. The binding pattern indicated that Asn 44, Ans 52, Ala 345, Pro 343, Ile 352, Gly 346, Gly 47, Ala 354, Ser 336, Thr 326 and Ser 329 are the main interacting amino acids residues of T1R2 and Arg 56, Glu105, Asp 215, Asp 216, Glu 148, Asp 258, Lys 255, Ser 104,Glu 217, Leu 51, Arg 52 of T1R3 respectively. Amino acids interact with SGs mainly by forming hydrogen bonds with hydroxyl group of glucose moieties. Maximum binding affinity has been obtained with SGs having total four glucose molecules attached with it and increase or decrease in glucose molecules reduced the binding afinityThere is significant variation in docked poses of all SGs. Taking in consideration the diverse binding patterns of various SGs as well as their structural features, we have proposed the mechanism of sweetness in the form of multiple point stimulation model. The present study will be helpful to know the proper mechanism of sweetness as well as binding patterns of SGs to sweet taste receptor. It will further helpful in understanding the difference in taste quality and will be used in improving the taste of SGs using semisynthetic approaches.