THERAPEUTIC POTENTIAL OF SOME HERBS AND STRUCTURE OF BERBERRUBINE-DNA COMPLEX

Abstract

Over the last two decades many strategies have been developed to design drugs to control diseases like cancer and diabetes mellitus. Advancement of in our understanding of these diseases at the molecular level has provided us with new effective armors to diagnose, combat and control the spread of these diseases. In this regard the understanding of the interaction of small molecules with macromolecules like protein or DNA has opened the doors for rational drug design against cancer and type 1 and 2 diabetes mellitus. Although it is type 2 diabetes which is more prevalent and the main driver of the diabetes epidemic in India, it is noted that prevalence of type 1 diabetes in India is also on the rise. In spite of the introduction of hypoglycemic agents, diabetes and the related complications continue to be a major medical problem. Several bioactive molecules have been studied and employed for their antidiabetic activity but have shown toxic effects which cannot be bypassed. Recently the role of herbal remedies is in focus due to the side effects associated with oral hypoglycemic agents and insulin. More than 400 plants with glucose-lowering effects are known in India. Some of them have been tested and the active components are being isolated. In this context similar cytotoxicity of synthetic drugs are observed for antineoplastic agents prevalent in cancer treatment. Thus there is continuing search for new antidiabetic and anticancer drugs of herbal origin. A substantial body of research has been directed towards screening of drugs acting as stimulator of insulin (insulinotropic agent) or drugs which regenerate the injured pancreatic beta cells. At the same time, focus is on for developing and improvising the existing methodology to screen potent anticancer drugs from herbal origin. The present research work is directed towards screening components which shows insulinotropic properties. As well as screen phytochemical enriched fractions which targets topoisomerases and bind to specific nucleic acid sequences of oncogenes. Therefore, this work will allow us to screen both antidiabetic and antitumor agents. To achieve the goal of screening drugs from herbal origin; we have used both in vitro and in vivo method to screen active insulinotropic agents phytochemical from Cinnamomum zeylanicum and antitumor components through topoisomerase I and II inhibition assay and restriction inhibition assay using specific restriction endonucleases. In order to gain understanding of the mode of action berberrubine-a protoberberine analogue which is known to have antitumor potential, we present here the structure- I i 1 conformation of berberrubine d-(CCAATTGG)2 complex based on ID, 2D H and P NMR spectroscopy measurements followed by restrained Molecular Dynamics simulations based on observed NOEs. The present work is a significant step in screening and understanding the molecular basis of action of the active phytochemical rich fractions and berberrubine. The PhD thesis work has been reported in the form of 6 chapters. Chapter 1 contains introduction of the subject, a comprehensive review of the literature and scope of thesis. Chapter 2 deals with materials, methods, results and discussion of the antidiabetic study of Cinnamon zeylanicum. The methodology employed for preparation of methanol and aqueous extract, selection of animals used for in vivo studies, induction of type 1 diabetes mellitus in male wistar rats, estimation of blood glucose level of extract treated of both normal and induced type 1 fasted rats, estimation of OGTT (oral glucose tolerance test) of both normal and induced type 1 diabetic fasted rats, estimation of lipid, glycogen, SGOT, SGPT, alkaline phosphatase, urea, creatinine and insulin level of both normal and treated rats is satisfied. Preparation of different phytochemical rich fractions from the aqueous extract of Cinnamon zeylanicum, estimation of in vitro release of insulin from normal and treated rat pancreas and also methodology employed for semiquantitative reverse transcriptase-PCR, histopathological studies, toxicity study and statistical analysis is also given. The present study showed better hypoglycemic and antihyperglycemic property of 200 mg/kg body weight aqueous extract than methanol extract of cinnamomum zeylanicum in normal and streptozotocin induced type 1 diabetic mellitus (T1DM) animals. In case of treated animals, the hepatic glycogen increased by 63.27 %and serum SGOT, SGPT, ALKP, creatinine and urea levels were reduced by 68.04, 64.42 , 47.05, 52.63 and 62.03 % respectively, after 15 days of oral administration of the extract. While small change towards normalcy was observed in the lipid profile of experimental animals. In order to understand the molecular mechanism of action, the expression profile of enzymes involved in glucose homeostasis was analyzed. The role of saponins as an antidiabetic and insulinotropic agent from Cinnamomum zeylanicum was identified. The results indicate the favorable effect of saponin rich fractions of the aqueous extract of cinnamomum zeylanicum in bringing down the severity ofT1DM. Chapter 3 deals with materials, methods, results and discussion of the qualitative and quantitative study of drug-DNA interaction by inhibition of restriction endonucleases and its application in screening DNA interacting components from herbal plants. This includes methodology for transformation ofbacterial cells, plasmid isolation, quantification ofDNA using UV visible spectrophotometer, DNA fragment isolation from pBCKS+ plasmid DNA for restriction inhibition assay, transformation and isolation and linearization of pQE32 plasmid DNA, preparation of phytochemical rich fractions from aqueous extract of Cinnamomum zeylanicum, binding studies, screening for the presence of EcoRl and Hindlll recognition sequence in the mRNA, STS and ESTs of breast oncogene and agarose gel electrophoresis. The present study showed the use of the restriction inhibition assay, using Hindlll, EcoRl and EcoRW restriction enzymes, to evaluate the binding specificity of DNA with anticancer drugs (mitoxantrone, berberine and palmitine). The inhibition of endonuclease Hindlll at 220 uM concentration was observed with mitoxantrone giving a direct evidence of the co-existence of concentration and sequence specificity for drug-DNA interaction. The present study showed that a simple and rapid method can be used to screen, plant extracts and the active phytochemicals involved in binding with restriction sequences of EcoRl and Hindlll in a relatively qualitative and quantitative manner. The results show that among; Fraction I- anthraglycosides, bitter principles, flavonoids and arbutin, Fraction II-saponins, Fraction III- Cardiac glycosides and Fraction IVterpenes, coumarins, phenol carboxylic acids, valepotriates. The Fraction II, saponins rich fraction from Cinnamomum zeylanicum shows inhibition at minimum concentration 15 pg/ml and 2.1 ug/ml for EcoRl and Hindlll restriction endonucleases, respectively. Secondly, the EcoRl and Hindlll restriction sites were found repeatedly in the cDNA and ESTs of BRCA2 early onset oncogene. These observations project the possible use of the screened phytochemical as an anticancer agent, targeting the expression of an oncogene (BRCA2). Through this study we postulate for the first time the use of restriction inhibition assay as a rapid and simple method to screen possible anticancer phytochemical from herbal plants. Chapter 4 deals with materials, methods, results and discussion of anti topoisomerases activity of Picrorrhiza kurroa. This includes the methodology employed for preparation of the Picrorrhiza kurroa extract, inhibition of catalytic activity of human, topoisomerase I (relaxation assay), stabilization of DNA-enzyme covalent complex studies, preincubation studies, human topoisomerase II a inhibition assay and agarose gel electrophoresis. The present study describes the inhibitory effect of the aqueous extract of Picrorrhiza kurroa on human topoisomerases by measuring the relaxation of superhelical plasmid pBR322 DNA. The aqueous extract inhibited topoisomerase I and II a in a concentration-dependent manner (Inhibitory concentration (IC) ~ 25 and 50 pg, respectively). From the stabilization studies of topoisomerase I-DNA complex and preincubation studies of topoisomerase I and II a with the extract, we conclude that the possible mechanism of inhibition is both; 1) stabilization of covalent complex of topoisomerase I-DNA complex and 2) direct inhibition of the enzyme topoisomerases. These findings might explain the antineoplastic activity of Picrorrhiza kurroa and encourage new studies to elucidate the usefulness of the extract as a potent antineoplastic agent. Chapter 5 deals with the materials, methods, result and discussion of the NMR studies of interaction of berberrubine with DNA. The detailed structural analysis is carried out using Nuclear Magnetic Resonance ID NMR, 2D Nuclear Overhauser Enhancement Spectroscopy (NOESY) and 31P - 3IP NOESY exchange techniques. The following experiments were performed on the berberrubine-DNA complex - 'H and 31P NMR titration studies at various drug (D)/DNA duplex (N) ratios up to 1.0 at 278, 283 K and 298 K in 90% H20 and 10% D20, temperature dependence of31P and 'HNMR ofthe berberrubine-DNA complex having D/N = 1.0 in the range of278 - 313 K; 2D 31P - 31P exchange spectra ofdrug-DNA complex by phase sensitive NOESY using mixing time of200 ms at 278 K, 283 Kand 298 Kfor D/N =1.0; 2D NOESY 'H - *H at D/N = 1.0 using mixing time xm = 200 ms at 278 K, 283 K, 298 K in 90 % H20 and 10 % D20. The 31P NMR shows a maximum downfield shift of 0.29 ppm for the A3pA4 resonance contrary to that observed for intercalating drugs e.g., Adriamycin or daunomycin) which show downfield shift upto 1.6 ppm at the interaction site due to changes in the phosphodiester angle of the DNA. The 2 D proton spectra of the 1:1 berberrubine-d-(CCAATTGG)2 complex at 298 K shows no significant shift in DNA base, sugar or the NH protons. The drug protons H 19, 20 and H 24 showed upfield shift of 0.24 ppm and 0.18 ppm, respectively and H 14 showed an upfield shift of 0.14 ppm upon binding to the DNA. The inter-drug cross peaks: H 10-H 5, H 10-H 14, H 10-H 24, H 10-H 31, 32, H 28-H 24 indicates that two drug molecules are stacked over each other in a possible antiparallel orientation in the complex. Drug protons H 10, H 16, 17 and H 31, 32 gave strong NOEs with C2NH2b, C2NH2nb, A4H2, T5H2' and T5H2", respectively. The observed 19 inter-molecular NOEs connectivities of the drug with the sugar HI', H272" and NH2nb/b, CH3 and the A3/A4H2 region of the DNA supports the minor groove mode of binding of berberrubine in the complex. The restrained molecular dynamics approach using INSIGHT II and DISCOVER showed that the two drug molecules with their aromatic region stacked in an antiparallel pattern lies at the upper minor groove region (5'-CAATT-3') of the DNA. The interaction of the drug at this site of the DNA was stabilized by van der Waals, electrostatic and hydrogen bonding forces. The specific drug-DNA structure thus obtained spells out exact contacts the drug makes with DNA, which is important for formation of ternary complex with enzyme and hence the molecular basis of topoisomerase II poisoning. Chapter 6 summarizes the result obtained and their implication in understanding the therapeutic potential of Picrorrhiza kurroa and Cinnamon zeylanicum against cancer and streptozotocin induced type 1 diabetes mellitus as well as role of berberrubine as topoisomerase II poison.