Evaluation of Anti-Angiogenic Effect of Naturally Occurring Compound from Ficus.Bengalensis on Regeneration & Development of Zebra-Fish Fin

Table of contents

1. Introduction

ngiogenesis, the process that leads to the formation of new blood vessels or neovascularisation, which is highly important during development but is largely not observed in the adult, except physiological exceptions in which angiogenesis occurs under tight regulation found in the female reproductive system and during wound healing. (Malin Dollinger et al.) In pathological situations, however, angiogenesis may be turned on, which contribute to the onset and progression of most severe human pathologies characterized by high mortality, including cancer, diabetes, obesity and retinopathies. Thus, angiogenesis is one of the largest and fastest evolving areas of research today, the knowledge of the molecular mechanisms that regulate neovascularisation continues to emerge, and there is increasing hope for the new discoveries that will lead to newer therapies targeting angiogenesis. (Uday P Kundap et al 2013) Angiogenesis is the physiological process through which new blood vessels develop from pre-existing vessels. This is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. Anti-angiogenesis is a form of targeted therapy that uses drugs or other substances to stop tumors from making new blood vessels. Without a blood supply, tumors can't grow. Anti-angiogenesis research began more than 35 years ago with the work of the late Judah Folkman, MD. (Bagchi. D et al 2004, Han-Chung Wul et al) Anthocyanin are the flavonoid compounds that produce plant colours ranging from orange and red to various shades of blue and purple. Anthocyanin are members of the flavonoid group of phytochemicals, which is a group predominant in teas, honey, wines, fruits, vegetables, nuts, olive oil, cocoa and cereals. The flavonoids are thought to be perhaps the most important single group of phenolic in food. (Gael McGill et al.) Adult zebrafish have a remarkable regenerative capability. Many tissues which may not be regenerated in mammals are quickly regenerated in zebrafish. Among these are the heart, retina, maxillary barbell and fins importantly, as they regenerate, new blood and lymph vessels grow into the regenerating tissue -which enables studies on regenerative angiogenesis. One commonly used assay in the adult zebrafish, based on this principle is the regenerating tail fin. After amputation, the tail fin will re-grow and after approximately 1 month, the fin is back to its original size.

The zebrafish embryo has become an important vertebrate model for assessing drug effects. It is well suited for studies in genetics, embryology, development, and cell biology. (L. D. Jensen1, et al) Zebrafish embryos exhibit unique characteristics, including ease of maintenance and drug administration, short reproductive cycle, and transparency that permits visual assessment of developing cells and organs. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for largescale drug screening. The main aim of this study is to evaluate Anthocyanin obtained from medicinal plant Ficus.bengalensis for Anti-angiogenesis & to standardize a method for the study of angiogenesis. A variety of animal models have been described to provide more quantitative analysis of in vivo angiogenesis and to characterize pro-and anti Angiogenic molecules. However, it is still necessary to establish a quantitative, reproducible and specific.

2. a) Collection of plant

The dried stem bark of Plant Ficus.bengalensis Linn. Was collected from Uran region of Navi-Mumbai Maharashtra India & were authenticated from Agarkar Research Institute, G. G. Agarkar Road, Pune, Sample deposited on 13/9/2012 & voucher number allotted is S/B-110.

3. b) Extraction of Anthocyanin

This unit describes methods for extraction, isolation, and purification of anthocyanin pigments from plant tissues. The choice of extraction method should maximize pigment recovery with a minimal amount of adjuncts and minimal degradation or alteration of the natural state.

Basic Protocol 1 describes the extraction of Anthocyanin with acetone and their partition with chloroform. Basic Protocol 2 describes a simple, fast, and effective method for purification of Anthocyanin from polyphenol compounds, sugars, and organic acids using solid-phase adsorption. This produces a uniform composite sample with a high surface area, which allows for efficient pigment extraction. (Oszmianski and Lee, 1990, Sheikh Anis, et al. 2012) Basic Protocol -1

Acetone Extraction & Chloroform Partition of Anthocyanin: In this method, acetone extracts the Anthocyanin from the plant material and chloroforms partitioning further isolates and partially purifies the pigments. The addition of chloroform results in phase separation between the aqueous portion (which contains the anthocyanin, phenolics, sugars, organic acids, and other water-soluble compounds) and the bulk phase (which contains the immiscible organic solvents, lipids, carotenoids, chlorophyll pigments, and other nonpolar compounds). This method has the advantage of producing an extract with no lipophilic contaminants. The absence of a concentration step minimizes the risk of acid-dependent pigment degradation. (Oszmianski and Lee, 1990) Materials: Powdered plant material,Frozen Acetone 70% (v/v) aqueous acetone or aqueous acidified acetone: 70% aqueous acetone with 0.01% HCl, Chloroform, Acidified water: 0.01% (v/v) HCl in deionized, distilled water,Waring Blender with stainless steel container (Waring) or general-purpose homogenizer, Whatman no. 1 filter paper, Buchner funnel, Separatory funnel, 500-ml boiling flask Rotary evaporator with vacuum pump or water aspirator, 40°C Basic protocol -2 Anthocyanin Purification: Purification of anthocyanin-containing extracts is often necessary, as the solvent systems commonly used for extraction are not specific for anthocyanin. Considerable amounts of accompanying materials may be extracted and concentrated in the coloured extracts, which can influence the stability and/or analysis of these pigments (Jackman and Smith, 1996). Anthocyanin purification using solid-phase extraction (Figure) permits the removal of several interfering compounds present in the crude extracts. Mini-columns containing silica gel 60 chains bonded on silica retain hydrophobic organic compounds (e.g., anthocyanin, phenolics), while allowing matrix interferences such as sugars and acids to pass through to waste. Washing the retained pigments with ethyl acetate will further remove phenolic compounds other than anthocyanin. ( Regenerated Zebrafish fin was observed under the microscope after 7-8 days of regeneration, fish from control group shows the normal & complete growth of blood vessels in fin. Vehicle control group also shows the complete & normal growth of blood capillary bloodvessels in regenerated fin. Unique observation was noticed in standard drug group, the growth of capillary blood vessels in fin was stunted, the growth of blood vessels was blocked in standard drug paclitaxal (0.5ppm conc). Fishes in the test drug shows the simillar features as standard drug group. The growth of the blood vessels was also blocked by the test compound. Adult zebrafish have a remarkable regenerative capability. Many tissues which may not be regenerated in mammals are quickly regenerated in zebrafish. Among these are the heart, retina, maxillary barbell and fins importantly, as they regenerate, new blood and lymph vessels grow into the regenerating tissue -which enables studies on regenerative angiogenesis. One commonly used assay in the adult zebrafish, based on this principle is the regenerating tail fin. After amputation, the tail fin will re-grow and after approximately 1 month, the fin is back to its original size. Angiogenesis process was activated in zebrafish by cutting there fin. Test compound (1mg/300ml of water) was added in the water.

4. Material & Methods

5. Discussion

standard drug group, the growth of capillary blood vessels in fin was stunted, and the growth of blood vessels was blocked in standard drug paclitaxel (0.5ppm conc). Fishes in the test drug (3.3ppm) shows the similar features as standard drug group. The growth of the blood vessels was also blocked by the test compound which shows 78.21% inhibition of angiogenesis.

Design and development of small molecule therapeutics to inhibit angiogenesis has gained considerable importance in anti-angiogenesis research. We demonstrate that the zebrafish is a viable model for screening small molecules that can inhibit angiogenesis. The mechanism of Anthocyanin action is not yet known, but hypotheses include decreased levels of tumor necrosis factor alpha (TNF-a)-induced VEGF expression), inhibition of H2O2-and as well as through the inhibition of VEGF and VEGF receptor expressionThus, our study suggests that even mill molar concentration of test drug (Anthocyanin) could be an effective drug for in vivo inhibition of angiogenesis and thus might gain significance in future therapeutics.

Figure 1. Figure 1 :Figure 2 :
12Figure 1 : Anthocyanin Purification Materials: Methanol, Acidified water: 0.01% (v/v) HCl in deionized, distilled water, Aqueous anthocyanin extract (see Basic Protocol 1), Ethyl acetate, Silica gel 60, Acidified methanol: 0.01% (v/v) HCl in methanol, 50-to 100-ml boiling flask, Rotary evaporator with vacuum pump or water aspirator, 40°C, Freeze-resistant container (optional).c) AnimalsAnimals were obtained from the Animal House of H(S)NCB's facility for Breeding & Experimentation, Dr. L.H. Hiranandani college of Pharmacy, opposite to Ulhasnagar station, CHM Campus, Ulhasnagar-03, maintained in an animal holding room (Protocol Registration no. 879/ac/05/CPCSEA). The experimental protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Efforts were made to minimize animal suffering and to reduce the number of animals used.The experiments were performed after the approval by the Institutional Animal Ethics Committee (IAEC) and were carried out in accordance with the current guidelines for the care of laboratory animals. Efforts were made to minimize animal suffering and to reduce the number of animals used.Zebrafish: Danio rerio (wild type) were obtained from local suppliers and maintained at 280C on a 14 hours light/10 hour dark cycle in 40 L glass tanks with four females and eight males in separate tanks. PH of breeding water was maintain at 6.6 -6.8, temperature of 78 -82 0 F. Embryos were collected by natural spawning with 2:1 male to female ratio and staged according toKimmel et al. Embryo stage is denoted as hours postfertilization (hpf). (Amy L Rubinstein.2003, Christian Lawrence. 2000).
Figure 2. FigureFigure 3 Figure 4 :
34Figure : 3 a
Figure 3. Figure 5 :Figure 6 :
56Figure 5 : Regenerated fin after 7-8days
Figure 4.
a) Anti-Angiogenic effects on zebrafish (Danio rerio) fin regeneration assay
Figure 5.
Comparison of test drug & Standard Drug effect was done with Control & vehicle control for regeneration of tail fin as a parameter for studying regenerative antiangiogenesis. Control group was kept to know the normal growth of fin. (Amy L Rubinstein, 2003, Westerfield M 2000) The test (Anthocyanin extract) & Standard (Paclitaxel) compound were evaluated for Anti-Angiogenic activity after 7 days of amputation. Standard drug showed 83.57± 12.43*** Test drug showed 78.21 ± 4.57*** angiogenesis inhibition, control group 12.09 ± 4.04, Vehicle control 20.67± 6.24 of angiogenesis inhibition which is significant according to statistical analysis (one-way annova). Regenerated Zebrafish fin was observed under the microscope after 7-8 days of regeneration, fish from control group shows the normal & complete growth of blood vessels in fin. Vehicle control group also shows the complete & normal growth of capillary blood vessels in regenerated fin. Unique observation was noticed in Volume XV Issue 1 Version I © 2015 Global Journals Inc. (US)
Figure 6. (
SimonaLucioli 2012, L. D. Jensen1,et al) Dr Radhikanand Dhavale, PhD in Spiritual Literature, Siddhayog Ashram, Pune, Mrs Rachana D Sarawade, HOD Prof. Department of Pharmacology, Dr L. H. Hiranandani College of Pharmacy, Ulhasnagar, Dr. Sadhana Sathaye, Department of Pharmacology, Institute of Chemical Technology, Matunga, Dr Ketan Patel, Post-doctoral fellow, Department of Pharmaceutics, Florida A&M University, 021, Dyson pharmacy, Tallahasse, FL USA, Pankaj Jain, Devang. S for their valuable guidance and support
Figure 7.
Figure 8.
Figure 9. Table 2 :
2
Mean+ S. E : *= P<0.05; **= P<0.01; ***=P<0.0001
1

Appendix A

Appendix A.1 Acknowledgement

Appendix B

  1. Recent Potent Molecular Targets for Cancer Treatment -A Review. * Uday P Kundap , Rachanasarawade . AvailableOnlineatwww.ijpba.info International Journal of Pharmaceutical & Biological Archives 2013. 08 Jun 2013. 18 Sep 2013. 27 Sep 2013. 4 (4) p. . (Received)
  2. Zygogen LLC 520 Kell Hall 24 Peachtree Center Avenue Atlanta GA 30303 USA e-mail: [email protected] Current Opinion in Drug Discovery & Development, Amy L Rubinstein . 2003. 6 p. . (Zebrafish: From disease modeling to drug discovery)
  3. Development of a zebrafish 4-day embryolarval bioassay to assess toxicity of chemicals. B Fraysse , R Mons , J Garric . Ecotoxicol Environ Saf2006. 63 p. .
  4. Stages of embryonic development of the zebrafish. C B Kimmel , W W Ballard , S R Kimmel , B Ullmann , T F Schilling . DevDyn 1995. 203 p. .
  5. Atalay Anti-Angiogenic, Antioxidant, and Anti-carcinogenic Properties of a Novel Anthocyanin-Rich Berry Extract Formula. C D K Bagchi , M Sen , M Bagchi . Biochemistry (Moscow) 2004-01-01. Kluwer Academic Publishers-Plenum Publishers. 69 (1) p. .
  6. Brigham and Women's Hospital, Karp Family Research Laboratories. Christian Lawrence . One Blackfan Circle 16 January 2007. April 2007. April 2007. 25 p. . (The husbandry of zebrafish (Danio rerio): A review. received in revised form 24)
  7. Adenosinereceptor antagonism causes inhibition of Angiogenic activity of human ovariancancer cells. E Barcz , E Sommer , P Janik , L Marianowski , E Skopinska-Rózewska . Oncol Rep 2000. 7 (6) p. .
  8. Apoptosis in Tumorigenesis & Cancer therapy, Division of Pediatric Hematology/oncology, Dana Farbe Cancer Institute & Children Hospital. Gael Mcgill , & David , E Fisher . Harvard Medical School p. 2115.
  9. Drug Discovery and Evaluation Pharmacological Assays, H , Gerhard Vogel . Wolfgang H.VogelBernward A. Schölkens Jürgen Sandow Günter Müller Wolfgang F. Vogel Second Edition (ed.)
  10. Drug Discovery and Evaluation Pharmacological Assays, H , Gerhard Vogel . Wolfgang H.VogelBernward A. Schölkens Jürgen Sandow Günter Müller Wolfgang F. Vogel Second Edition (ed.)
  11. , Han-Chung Wul , De-Kaun Chang & Chia-Ting , Chia-Ting Huang . Targeted Therapy for Cancer, Institute of Cellular & Organismic Biology
  12. The Karolinska Institute, Stockholm, 2Institution of Medicine and Health. L D Jensen1 . Tumor and Cell Biology Linköping University, Linköping, SwedenNeeharika V. Vamsi KR. Reddy B (Animal Models of Angiogenesis and Lymphangiogenesis)
  13. A description of methods for isolating polyphenolics and Anthocyanin from grapes by solid-phase extraction, Lee Oszmianski . 1990. (See above)
  14. MD Everyone's guide to CANCER THERAPY, Malin Dollinger , MD , Ernest H Rosenbaum , Margaret Tempero , MD , Sean Mulvihill . Canadian Medical Association.
  15. Zebrafish genetics: the enigma of arrival. M C Fishman . ProcNatlAcadSci U S A 1999. 96 p. .
  16. The Zebrafish Book, Guide for the Laboratory Use of Zebrafish (Danio rerio), M Westerfield . 2000. University of Oregon Press. (4th Ed Eugene)
  17. , N John , Aelvini Abelson , Simon . METHODS IN ENZYMOLOGY March 2008. p. . Division of Biology California Institute of Technology Pasadena, California, Khandelwal KR, Practical pharmacognosy, Niraliprakashan
  18. Extraction, Isolation, and Purification of Anthocyanin. R E Wrolstad , G Skrede , P Lea , G Enersen . Contributed by Luis E. Rodriguez-Saona University of Maryland and Joint Institute for Food Safety and Applied Nutrition, (Washington, D.C
    )
  19. Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment. Sheikh Anis , Sameer Singh , ; Mandorianarendra , W L Seng , Kurt Eng , J Lee , P Mcgrath . 018-022. Institute of Pharmacy, ujjain, IJPRD Aug-2012. 2004. 4 (06) p. . (Angiogenesis)
  20. Centro di Ricerca per la Frutticoltura, CRA-Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Via di Fioranello, Simonalucioli . 2012. Roma, Italy. p. . (Anthocyanin: Mechanism of action and therapeutic efficacy)
  21. , Springer-Verlag . Berlin Heidelberg New York.
  22. , Springer-Verlag . Berlin Heidelberg New York.
Notes
1
© 2015 Global Journals Inc. (US)
Home 
Date: 2015-05-15