International Journal of Current Research and Review
ISSN: 2231-2196 (Print)ISSN: 0975-5241 (Online)
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IJCRR - Vol 11 Issue 08, April, 2019

Pages: 13-16

Date of Publication: 30-Apr-2019


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In vivo study of Depigmentation Using Tyrosine Ammonia Lyase from Trigonella Foenum-graecum on Zebra Fish Embryos

Author: Kavitha G. Singh; Umme Umaima S.; Khushbu Jangid

Category: Healthcare

Abstract:Aim: To extract Tyrosine ammonia Lyase(TAL) from Trigonella foenum-graecum and utilize it for the treatment of depigmentation by carrying out in-vivo analyses on Zebra fish embryos
Tyrosine ammonia Lyase is an enzyme that is involved in deamination of L-tyrosine to P-coumaric acid. The enzyme TAL was extracted from Trigonella foenum-graecum. This enzyme has been targeted to be used in depigmentation of the skin in the current study using zebra fish as a model organism. This enzyme participates in depigmentation by inhibiting the production of melanin pigment, where in conversion of L-tyrosine in the melamine synthesizing pathway has been prevented by deaminating L-tyrosine to P-coumaric acid. The methodology of separation of proteins using acetone has been implemented in the extraction of the enzyme. Standardization of enzyme was accomplished to detect the presence of the enzyme. The formation of P-coumaric acid indicated the presence of the enzyme that was detected utilizing UV-Visible spectrophotometer at 380nm. In-vivo experimental study was carried on the zebra fish embryos for detecting the presence of the enzyme that was confirmed by observing the depigmented patterns in the developed embryos that were been treated with TAL.

Keywords: L-tyrosine ammonia Lyase, Depigmentation, P-coumaric acid, Trigonella foenum-graceum, Zebra fish

Full Text:

INTRODUCTION

Depigmentation is a process where the synthesis of melanin pigment is inhibited resulting in de-coloration of skin. The melanocytes losses their ability to synthesize melanin pigment by inhibiting the further conversion of L-tyrosine to participate in the melanin synthesizing pathway, by deaminating L-tyrosine to P-coumaric acid.

The purpose of the current study is to detect the presence of the enzyme tyrosine ammonia Lyase by extracting it from the leaves of Trigonella foenum-graecum, and there by utilizing it to cause depigmentation. The color of the body is induced by melanin pigment that is produced by melanocytes which are present in the lower layer of the epidermis. Tyrosine ammonia Lyase is an enzyme that deaminates L-tyrosine to P-coumaric acid, as a result of which the conversion of L-tyrosine  to further products in the pathway is eliminated and the melanin pigment from the melanocytes is not synthesized.

Trigonella foenum-graecum also known as fenugreek plant, belong to the family of Fabaceae, Papillionaceae subfamily. It is an efficient aromatic herb found mostly in Asia and Europe and also in North African areas. It is an ancient plant that is used in treatment of various types of diseases, among which diabetes mellitus is one of them. The leaves of the plant are used as spice in many dishes to improve the taste; the leaves are also known as methi leaves.

 

Zebra fish is a fresh water  living organism  found usually in  Himalayas and some parts of west Bengal. It belongs to minnow family, It is the most frequently used model organism in most of the research work carried out through conducting in-vivo experiments. The zebra fish genome is fully sequenced and shows 80% of genomic similarities with that of human genomic sequence and hence has been considered to be most frequently used model organism all over the globe to carry out medicinal research work. The best examples of it are conditions like Parkinson’s disorder, alziemer, and atherosclerosis. The embryonic developmental stages have been well studied and it rapidly develops. The embryos occur to be transparent, with natural pigmentation. It also has the capability to undergo external fertilization.

MATERIALS

Reagents: Chilled acetone, Chilled distilled water, Tris HCL, HCL (1N), NaOH (1N), L – tyrosine, Methanol.

Instruments: Chilled grinder, Cooling centrifuge, UV spectrophotometer, Computer, Incubator,

Refrigerator,  pH Meter.

Other Requirements: Glass wares, Mortar & pestle, Centrifuge tubes, Muslin cloth, Fresh leaves of Trigonella foenum-graecum.

METHODOLOGY

Preparation of Tyrosine Ammonia Lyase:

Trigonella foenum-graecum leaves were collected from north areas of Bangalore. Leaves were washed and air dried to remove water content. 26.6g of leaves were weighed and grinded with 100ml of distilled water using a mixer grinder. The prepared mixture was filtered using muslin cloth. The filtrate was measured; its volume was found to be 110ml that was refrigerated in freezer for 24 hours. Then to the filtrate equal amount of chilled acetone was added and kept undisturbed in freezer at -20 degree C for 24 hours. After precipitation two layers were formed, upper layer were discarded and the precipitate containing the protein was collected. Then the precipitate was centrifuged at 5000 rpm for 5 minutes at low temperature in cooling centrifuge. The pallet was collected, washed using acetone and the mixture was then poured into Petri plate for acetone to dry.

The acetone powder obtained was weighed. 1.0g of the powder was weighed and homogenized with 20ml of 1mM Tris HCl (pH=8.2) utilizing a pestle and mortar. The mixture obtained was centrifuged at 5000 rpm for 5 minutes. The crude enzyme extract was obtained in a supernatant form which was used for further analysis. (Umme Umaima et al., 2016) (Sarina P. Khabade, 2014)

Enzyme Assay of Tyrosine Ammonia Lyase:

Reaction mixture tube was prepared with 1ml of enzyme source, 0.2ml of 1mM L-Tyrosine (substrate) and 0.8 ml of 0.1M Tris HCl buffer (pH=8.9). A control tube was also prepared using 1ml of distilled water instead of 1ml of enzyme extract. Both the tubes were incubated in incubator at 37°C for 30 minutes. After incubation reaction is terminated by the addition of 0.5 ml of 1N HCl. Tyrosine Ammonium Lyase enzyme deaminates L-Tyrosine and produce p-coumaric acid with the release of ammonia. Then it was quantitatively measured by using UV – Visible spectrophotometer at 380nm. (Umme Umaima et al., 2016)

Experiment on Zebra Fish (In-Vivo) To reach depigmentation:

Zebra fishes were maintained in lab condition and allowed to mate in a breeding tank. Embryos of Zebra fish were collected  after 20 hours of fertilization before the pigmentation was produced and were transferred in a Petri plate containing embryo water. Two Petri plates containing zebra fish embryos were taken and marked as control and test. Control plate with the zebra fish embryos was left as such without the addition of  enzyme Tyrosine ammonia Lyase, where as test Petri plate with zebra fish embryos was treated with  1 ml of enzyme extract of Tyrosine Ammonia Lyase. Both the plates containing zebra fish embryos were kept at optimum temperature and observed under microscope after 24 hours of incubation. After observation the embryo water in the petri plate was cleaned and the plates were again added with enzyme Tyrosine Ammonia Lyase. Then the petri plates containing zebra fish embryos were further incubated at optimal temperature and were observed after 24 hours for better result. (Umme Umaima et al., 2016) (Sarina P. Khabade, 2014)

RESULT

Presence of enzyme Tyrosine Ammonia Lyase was confirmed in Trigonella foenum-graecum. Enzyme was assayed as per the standard conditions. UV-Visible spectrophotometer was used to check the activity of enzyme at 380nm. When the in-vivo experiment was performed on the zebra fish embryos, showed a gradual decrease in the pigmentation of the test samples induced with particular concentration of enzyme when compare to the control.

Discussion

The test sample (Trigonella foenum-graecum) in comparison   with that of control indicated  the presence of

 Enzyme tyrosine Ammonia Lyase  in a rich amount as a result of conversion of L-Tyrosine to p-coumaric acid by the action of enzyme(TAL). The presence of enzyme in a higher concentration   in the sample   enables the

use of the sample  to carry out the  depigmentation  experiment through carrying out  in-vivo study on zebra fish embryos. The clear indication of depigmentation is obtained through observing the above figure 2 and 3.       

The  above  figure2 and  figure3 that represent the embryos in the control sample ( figure 2) that is left untreated  with  enzyme Tyrosine Ammonia Lyase and  the embryos in the test sample ( figure 3) treated with the enzyme  Tyrosine Ammonia Lyase. The absence of   pigmented stripes in the test sample embryos  (figure 3)  indicates the identical effect  of the enzyme obtained through the sample Trigonellafoenum- graecum  which resulted in Depigmentation.

Conclusion                         

Leaves of Trigonella foenum-graecum are found to contain numerous useful and multi nutritive values, hence is used as herb in many Ayurveda medicines in case of treatment of many diseases, one of them is diabetes mellitus. Depigmentation is characterized as condition where in skin becomes white, as result of inhibition of melanin pigment that is responsible for darkening of skin color. There are many therapeutics and sunscreens creams being used in obtaining the depigmented state, which are useful only for a short period, moreover there are many side effects that get induced affecting the skin and sometimes other parts of the body. As a result of this consequences Trigonella foenum-graecum containing multi nutritive value, medicinal properties along with its zero side effect inducing ability, has been implemented to be utilized inducing depigmentation in the current study and as well as can be utilized in the treatment of many other diseased condition.

References:

1.  P Khosla, DD Gupta, RK Nagpal.  Effect of Trigonella foenum graecum ( Fenugreek) on blood giucose normal and diabetic rats. Indian journal of physiology and pharmacology 39, 173-173, 1995.

2.   S Kaviarasan, GH Naik, R Gangabhagirathi, CV Anuradha, KI Priyadarsini.  In vitro studies on antiradical        and antioxidant activities of fenugreek (Trigonella foenum graecum) seeds. Food chemistry 103(1), 31-37, 2007.

3.   K Srinivasan.  A review of Health Beneficial Physiological  Effect: Fenugreek(Trigonella foenum graceum). Food review international 22(2), 203-224, 2006.

4.   Zia Tayyaba, S Nazrul Hasnain,  SK Hassan. Evaluation of oral hypoglycemic effect of Trigonella Foenum graceum(Fenugreek) in normal mice. Journal of ethnopharmacology 75(2-3), 191-195, 2001.

5.  Kowichi Jimbow, Hiroko Obata, Madhu A Pathak, Thomas B Fitzpatrick. Mechanism of De-pigmentation by hydroquinone. Journal of investigation Dermatology 62(4), 436-449, 1974         

 6.  Gail K Naughton, Diana Reggiardo, Jean-Claude Bystryn. Correlation between vitiligo antibodies and extent of  De- pigmentation in vitiligo. Journal of American Academy of Dermatology 15(5), 978-981, 1986

7.  TP Dooley. Topical skin  de- pigmentation agents: current products and discovery of novel  inhibitors of  melanogenesis, Journal of Dermatological Treatment 8(4), 275-283, 1997.

8.  K M AlGhamdi, A Kumar. De-pigmentation therapies for normal skin in vitiligo universalis. Journal of the European Academy of Dermatology and Venereology  25(7), 749-757, 2011.

9.  Jens Rosler, Florian  Krekel, Nikolaus Amrhein, Jurg Schmid.  Maize phenylalanine ammonia-lyase has tyrosine ammonia- lyase  activity . Plant physiology 113(1), 175-179, 1997.

10.  M Jason MacDonald, Godwin B D’Cunha. A modern view of  phenylalanine ammonia lyase. Biochemistry and Cell Biology 85(3), 273-282,  2007.

11. Robert Gerlai. Zebra fish: an uncharted behavior genetic model. Behaviour genetics 33(5), 461-468, 2003.

12.  Charles B Kimmel, Jill  Patterson, Richard O Kimmel. The development and behavioral Characteristics of the startle response of  Zebra fish. Developmental psychobiology 7(1),  47-60, 1974.

13.   Charles B Kimmel, William W Ballard, Seth R Kimmel, Bonnie Ullmann, Thomas F Schilling. Stages of embryonic development of the  Zebra fish. Developmental dynamics 20393), 253-310, 1995.

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15.  Graham J Lieschke, Peter D Currie. Animals models of human disease: zebra fish swim into view, Nature Reviews Genetics 8(5), 353,  2007.

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One article from every issue is selected for the ‘Best Article Award’. Authors of selected ‘Best Article’ are rewarded with a certificate. IJCRR Editorial Board members select one ‘Best Article’ from the published issue based on originality, novelty, social usefulness of the work. The corresponding author of selected ‘Best Article Award’ is communicated and information of award is displayed on IJCRR’s website. Drop a mail to editor@ijcrr.com for more details.

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Best Article Award

A Study by Amr Y. Zakaria et al. entitled "Single Nucleotide Polymorphisms of ATP-Binding Cassette Gene(ABCC3 rs4793665) affect High Dose Methotrexate-Induced Nephrotoxicity in Children with Osteosarcoma" is awarded Best Article for Vol 13 issue 19
A Study by Kholis Ernawati et al. entitled "The Utilization of Mobile-Based Information Technology in the Management of Dengue Fever in the Community Year 2019-2020: Systematic Review" is awarded Best Article for Vol 13 issue 18
A Study by Bhat Asifa et al. entitled "Efficacy of Modified Carbapenem Inactivation Method for Carbapenemase Detection and Comparative Evaluation with Polymerase Chain Reaction for the Identification of Carbapenemase Producing Klebsiella pneumonia Isolates" is awarded Best Article for Vol 13 issue 17
A Study by Gupta R. et al. entitled "A Clinical Study of Paediatric Tracheostomy: Our Experience in a Tertiary Care Hospital in North India" is awarded Best Article for Vol 13 issue 16
A Study by Chandran Anand et al. entitled "A Prospective Study on Assessment of Quality of Life of Patients Receiving Sorafenib for Hepatocellular Carcinoma" is awarded Best article for Vol 13 issue 15
A Study by Rosa PS et al. entitled "Emotional State Due to the Covid – 19 Pandemic in People Residing in a Vulnerable Area in North Lima" is awarded Best Article for Vol 13 issue 14
A Study by Suvarna Sunder J et al. entitled "Endodontic Revascularization of Necrotic Permanent Anterior Tooth with Platelet Rich Fibrin, Platelet Rich Plasma, and Blood Clot - A Comparative Study" is awarded Best Article for Vol 13 issue 13
A Study by Mona Isam Eldin Osman et al. entitled "Psychological Impact and Risk Factors of Sexual Abuse on Sudanese Children in Khartoum State" is awarded Best Article for Vol 13 issue 12
A Study by Khaw Ming Sheng & Sathiapriya Ramiah entitled "Web Based Suicide Prevention Application for Patients Suffering from Depression" is awarded Best Article for Vol 13 issue 11
A Study by Purushottam S. G. et al. entitled "Development of Fenofibrate Solid Dispersions for the Plausible Aqueous Solubility Augmentation of this BCS Class-II Drug" is awarded Best article for Vol 13 issue 10
A Study by Kumar S. et al. entitled "A Study on Clinical Spectrum, Laboratory Profile, Complications and Outcome of Pediatric Scrub Typhus Patients Admitted to an Intensive Care Unit from a Tertiary Care Hospital from Eastern India" is awarded Best Article for Vol 13 issue 09
A Study by Mardhiah Kamaruddin et al. entitled "The Pattern of Creatinine Clearance in Gestational and Chronic Hypertension Women from the Third Trimester to 12 Weeks Postpartum" is awarded Best Article for Vol 13 issue 08
A Study by Sarmila G. B. et al. entitled "Study to Compare the Efficacy of Orally Administered Melatonin and Clonidine for Attenuation of Hemodynamic Response During Laryngoscopy and Endotracheal Intubation in Gastrointestinal Surgeries" is awarded Best Article for Vol 13 issue 07
A Study by M. Muthu Uma Maheswari et al. entitled "A Study on C-reactive Protein and Liver Function Tests in Laboratory RT-PCR Positive Covid-19 Patients in a Tertiary Care Centre – A Retrospective Study" is awarded Best Article of Vol 13 issue 06 Special issue Modern approaches for diagnosis of COVID-19 and current status of awareness
A Study by Gainneos PD et al. entitled "A Comparative Evaluation of the Levels of Salivary IgA in HIV Affected Children and the Children of the General Population within the Age Group of 9 – 12 Years – A Cross-Sectional Study" is awarded Best Article of Vol 13 issue 05 Special issue on Recent Advances in Dentistry for better Oral Health
A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" is awarded Best Article of Vol 13 issue 06
A Study by Chen YY and Ghazali SRB entitled "Lifetime Trauma, posttraumatic stress disorder Symptoms and Early Adolescence Risk Factors for Poor Physical Health Outcome Among Malaysian Adolescents" is awarded Best Article of Vol 13 issue 04 Special issue on Current Updates in Plant Biology to Medicine to Healthcare Awareness in Malaysia
A Study by Kumari PM et al. entitled "Study to Evaluate the Adverse Drug Reactions in a Tertiary Care Teaching Hospital in Tamilnadu - A Cross-Sectional Study" is awarded Best Article for Vol 13 issue 05
A Study by Anu et al. entitled "Effectiveness of Cytological Scoring Systems for Evaluation of Breast Lesion Cytology with its Histopathological Correlation" is awarded Best Article of Vol 13 issue 04
A Study by Sharipov R. Kh. et al. entitled "Interaction of Correction of Lipid Peroxidation Disorders with Oxibral" is awarded Best Article of Vol 13 issue 03
A Study by Tarek Elwakil et al. entitled "Led Light Photobiomodulation Effect on Wound Healing Combined with Phenytoin in Mice Model" is awarded Best Article of Vol 13 issue 02
A Study by Mohita Ray et al. entitled "Accuracy of Intra-Operative Frozen Section Consultation of Gastrointestinal Biopsy Samples in Correlation with the Final Histopathological Diagnosis" is awarded Best Article for Vol 13 issue 01
A Study by Badritdinova MN et al. entitled "Peculiarities of a Pain in Patients with Ischemic Heart Disease in the Presence of Individual Combines of the Metabolic Syndrome" is awarded Best Article for Vol 12 issue 24
A Study by Sindhu Priya E S et al. entitled "Neuroprotective activity of Pyrazolone Derivatives Against Paraquat-induced Oxidative Stress and Locomotor Impairment in Drosophila melanogaster" is awarded Best Article for Vol 12 issue 23
A Study by Habiba Suhail et al. entitled "Effect of Majoon Murmakki in Dysmenorrhoea (Usre Tams): A Standard Controlled Clinical Study" is awarded Best Article for Vol 12 issue 22
A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by situs slot and Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh et al. situs slot "Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma situs slot entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by situs slot et al entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09
Late to bed everyday? You may die early, get depression situs slot
Egg a day tied to lower risk of heart disease situs slot
88 Percent Of Delhi Population Has Vitamin D situs slot  Deficiency: ASSOCHAM Report

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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