Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan
By Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD PhD Thesis In Pharmacy COMSATS Institute of Information Technology Abbottabad - Pakistan
Fall, 2015
COMSATS Institute of Information Technology
Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan
A Thesis Presented to
COMSATS Institute of Information Technology, Abbottabad
In partial fulfillment
of the requirements for the degree of
PhD (Pharmacy)
By
Muhammad Hassham Hassan Bin Asad
CIIT/FA11-R60-003/ATD
Fall, 2015
ii Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan
A Post Graduate Thesis submitted to the Department of Pharmacy as partial fulfillment of the requirement for the award of Degree of PhD in Pharmacy.
Name Registration Number
Muhammad Hassham Hassan CIIT/FA11-R60-003/ATD Bin Asad
Supervisor
Dr. Izhar Hussain Professor Department of Pharmacy Abbottabad Campus. COMSATS Institute of Information Technology (CIIT) Abbottabad Campus. December, 2015
iii Final Approval
This thesis titled Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan
By Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD Has been approved
For the COMSATS Institute of Information Technology, Abbottabad
External Examiner: ______Dr………… ………
External Examiner: ______Dr………… ………
Supervisor: ______Prof. Dr. Izhar Hussain Department of Pharmacy, Abbottabad
HoD/ Chairperson: ______Prof. Dr. Nisar-ur-Rehman Department of Pharmacy, Abbottabad
Dean, Faculty of Sciences: ______Prof. Dr. Arshad Saleem Bhatt
iv Declaration
I, Muhammad Hassham Hassan Bin Asad, CIIT/FA11-R60-003/ATD hereby declare that I have produced the work presented in this thesis, during the scheduled period of study. I also declare that I have not taken any material from any source except referred to wherever due, and that the amount of plagiarism is within acceptable range. If a violation of rules on research has occurred in this thesis, I shall be liable to punishable action under the plagiarism rules of the HEC.
Signature of the student:
Date: ______Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD
v Certificate
It is certified that Muhammad Hassham Hassan Bin Asad, CIIT/FA11-R60-003/ATD has carried out all the work related to this thesis under my supervision at the Department of Pharmacy, COMSATS Institute of Informational Technology, Abbottabad and the work fulfills the requirements for award of PhD degree.
Date: ______
Supervisor:
______Prof. Dr. Izhar Hussain Department of Pharmacy CIIT, Abbottabad
Head of Department:
______Prof. Dr. Nisar-ur-Rehman Department of Pharmacy CIIT, Abbottabad
vi
DEDICATION
This thesis is dedicated to my parents
Mrs. Tasneem Habib & Mr. Allah Dad Asad Awan
Who realized me the power of knowledge
vii ACKNOWLEDGEMENTS
This thesis would not have been concluded without inspiring and untiring efforts of many individuals who contributed and extended their guidelines in completion of this work. First and foremost I would like to express my deep gratitude to my PhD supervisor, Prof. Dr. Izhar Hussain, Department of Pharmacy, COMSATS Institute of Information Technology (CIIT), Abbottabd, Pakistan for his constant motivation, illumination of ideas and valuable advices throughout the research work. I also indebted to Dr. Ghulam Murtaza, Assistant Professor, Department of Pharmacy, CIIT, Abbottabad for his care, collaboration and sincere efforts. I owe a great debt of appreciation to the Dr. Durr-e-Sabih, Director, Multan Institute of Nuclear Medicine and radiotherapy (MINAR), Nishtar Hospital Multan, Pakistan for his sympathetic and scholarly approach. Moreover I would like to thanks to the Dr. Khan Muhammad Sajid (Deputy Chief Scientist), Dr. Muhammad Saqib Khan (Senior Radiologist), Israr Ahmad (Radio Pharmacist), Rubada Mehmood (Clinical Biochemist) and other lab members for all the assistance they have done during my stay at MINAR, Nishtar Hospital Multan, Pakistan. During my stay at Copenhagen I highly acknowledge my supervisors Dr. Anna Katherine Jager (Head, Natural Product Research, University of Copenhagen, Denmark) and Dr. Dan Streak (Professor of NMR, University of Copenhagen, Denmark) for their useful guidelines, sympathetic approach and enlightening ideas throughout my research. At the end I would like to thanks from core of heart to my mentor at National University of Singapore (NUS) Prof. Dr. R. M. Kini (Department of Biological Sciences, National University of Singapore, Singapore) for his educated, valuable and critical guidelines towards completion of my snake venom Ph.D research project.
Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD
viii ABSTRACT Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan
Background Snake bite envenomation is one of the vivid examples of neglected occupational hazards that accounts for tens of thousands of deaths all over the world. One of such instance is Naja naja karachisis bite, a nightmare for the inhibitants of Southern Punjab (Paksitan), often endup with countless deaths and sequela. To address this problem present study was designed to highlight scientific grounds for Naja naja karachisis envenomation and to rationalize folklore claimed Pakistani medicinal plants as a first aid treatment before proper hospitalization.
Methods Proteomic characterization of Naja naja karchiensis venom was carried out with electrophoresis (SDS-PAGE) and HPLC (SEC & RP-HPLC) coupled LC-MS/MS whereas inorganic constituents were quantified with ICP-OES technique. Bio distribution and kinetic profile of venom was monitored with short lived radiotracer (99mTc) via direct radio isotopic binding technique. Lethal biological effects of crude venom were examined in terms of its LD50, hemolytic and anticoagulant behavior while toxic biochemical parameters (in vivo), towards liver (AST & ALT), heart (CK-MB & LDH) and kidneys (urea & creatinine) damage were investigated by following the recommendations of DGKC and IFCC methods. Venom was analyzed for different enzymatic activities
(PLA2, ALPase, 5ʹ-ND, hyaluronidase and protease) by adopting conventional biochemical assays (in vitro). Twenty eight medicinal plants of Pakistan were extracted with methanol by simple maceration process and thereafter used to reverse deleterious actions of cobra venom. RP-HPLC coupled bioassay guided fractionation technique was used to characterize bioactive constituent/metabolite(s), responsible for anti-PLA2 activity in Bauhinia variegata L extract.
ix Results SDS-PAGE indicated Naja naja karachiensis venom as a concoction of proteins, which ranges in molecular weight from 6 KDa to 200 KDa. Proteinous bands ranges from 50 KDa to 90 KDa were found homologous, however, after venom reduction the bands (15 KDa to 36 KDa) were not in homology (rather appreared at 9 KDa to 12 KDa) suggesting protein complex(s) in this venom. Total protein content was found to be 188 ± 0.011 µg per 200 µg of dry weight, which constitutes overall 94% proteineous stuff in cobra venom. LC-MS/MS analysis of protein component further identified and sequenced
3FTXs (58%), PLA2 (19%), SVMPs (5%), LAAO (5%), helvepryn (3%), vespryn (2%), CVF (2%), 5ʹ-ND (2%), vNGF (2%) and kunitz type serine protease inhibitor (2%). Among 3FTXs the identified components included CTXs (32%), WNTX (24%), LNTX (24%), SNTX (8%), MTLP (8%) and post synaptic-NTX (4%). MTLP-3 was found unique among 3FTXs due to 78% homology in amino acids sequence with novel haditoxin. Majority of the RP-HPLC fractions were heterogeneous while some were homogeneous. ICP-OES analysis revealed that other than protenous stuff metallic (95%) and non-metallic constituents were also present in cobra venom. Among metallic ions sodium (4519 ± 2 µg/g, 30%), potassium (2013 ± 5.5 µg/g, 13%), zinc (3473 ± 28 µg/g, 23%), magnesium (3047 ± 31 µg/g, 20%), calcium (1442 ± 19 µg/g, 9%), manganese (6.5 ± 0.65 µg/g, 0.05%) and copper (0.6 ± 0.09, 0.003%) were quantified, however, phosphorus (718 ± 8.5 µg/g, 5%) was the only detectable non-metallic component in this venom. This is the first report about divalent copper ion that has not been previously detected in other elapid venom. Venom from Naja naja karachiensis was labeled (97.7%) successfully for the first time with technetium-99m. Its labeling yield was higher than some previously reported toxins. Venom was found stable in vivo (96%) and in vitro (saline 94.3% & serum 94.1%) more 99m than one t1⁄2 of Tc (4 h). Venom (0.5mCi, 0.25 mg) was evenly distributed (R/L ratio=1) in the middle compartment and completely excreted from blood pool within 24 h of injection in rabbits. Intravenous dose of venom was distributed in all parts of the rabbit such as, stomach (0.05% ID/g), brain (0.14% ID/g), skeleton muscles (0.3% ID/g), intestines (0.35% ID/g), skin (0.45% ID/g), blood (0.56% ID/g), heart (0.8% ID/g), bones (1.38% ID/g), liver (4.3% ID/g), lungs (14.4% ID/g), urinary bladder (23.7% ID/g) and
x kidneys (53.7% ID/g) in ascending manner. Moreover, single photon emission computed tomographic (SPECT) images supported biodistribution in different tissues of rabbit. Kidneys were the main organ for excretion whereas lungs and liver were found the possible sites for metabolism of venom. Lethal dose of Naja naja karachiensis venom for
50% population (LD50) in mice was found to be 2.0 µg/g (2.0 mg/kg) intraperitonially. Cobra venom (100µg/ml) was found to destabilize human red blood corpuscles membranes which ended up in hemolysis. Among various evaluated medicinal plants extract only Cedrus deodara (Roxb. ex D. Don) G. Don was found to pose highest (72%, P˃0.5) protection even better than reference standard antisera (56%). Majority of the medicinal plants extract (69%) were not proved to abort venom induced hemolysis. Stenolobium stans (L) D. Don (TT= 29 ± 0.5 sec, PT = 16±0.5 sec, aPTT = 36 ± 0.5 sec) and Enicostemma hyssopifolium (Willd.) Verdoorn (TT = 19±0.5 sec, PT = 22 ± 0.5 sec, aPTT = 36 ± 1 sec) were noticed the most effective (70% to 92%) to combat venom induced anticoagulation. In vivo studies revealed that Naja naja karachiensis venom is deadly toxic for liver, heart and kidneys in dose dependent manner. At the dose of 0.4 mg/kg surrogate markers for renal damage were moderately released. Similarly, at the dose of 0.8 mg/kg (~envenomation dose in human), venom was proved extremely toxic in terms of significant release of biochemical parameters for renal and hepatic damage. Stenolobium stans (L) D. Don was the only useful medicinal plant extract to neutralize elevated serum levels of GOT (70.5 ± 3.5 U/L), GPT (46.5 ± 6.5 U/L), creatinine (1.1 ± 0.06 mg/ml), urea (25.5 ± 6.5 mg/ml), LDH (787 ± 28 U/L) and CK-MB (13 ± 1.7 U/L) efficiently (p˃0.5) in comparison with reference standard antisera (LDH = 763 ± 6 U/L, CK-MB = 9 ± 0.85 U/L, GOT = 69.5 ± 18.5 U/L, GPT = 52.5 ± 3.5 U/L, creatinine = 1.0 ± 0.02 mg/ml & urea = 31.5 ± 0.5 mg/ml).
Toxic enzymatic activity of PLA2 was accessed via acidimetric, turbidimetric and anticoagulant biochemical procedures. Medicinal plants extract (0.1 to 0.6 mg) were proved helpful to neutralize PLA2 hydrolytic action (50% to 100%). However, Bauhinia variegate L., Stenolobium stans (L) D. Don, Citrus limon (L.). Burm. f., Enicostemma hyssopifolium (Willd.) Verdoorn, Ocimum synctum L and Psoralea corylifolia L extracts were observed entirely useful (100%) at minute concentration as shown by reference
xi standard. Bauhinia variegate L was the only shortlisted (p˃0.01) extract which showed maximum (67%) anti-PLA2 effect in comparison with reference standard EDTA (76%).
Anticoagulant role of PLA2 was proved at 0.005 mg of cobra venom which prolonged coagulation time (100 sec to 125 sec) in the assay mixture. To neutralize anticoagulant effect Citrullus colocynthis (L.) Schrad (p˃0.5), Stenolobium stans (L) D. Don (p˃0.5) and Rubia cordifolia L (p˃0.5) were found beneficial however, Bauhinia variegate L extract was proved 76% helpful in comparison with standard antidote. Naja naja karachisis was also found abundant in ALPase enzymatic activity in dose dependent manner. Medicinal plants extract (0.625µg/ml to 2.5µg/ml) were found 80% to 93% effective to minimize cobra venom (0.4 mg) induced ALPase toxicity. Among all evaluated extracts Sapindus mukorossi Gaertn was observed to pose maximum (93%, 2.5 µg/ml, p˃0.5) protection against ALPase activity as observed with reference standard anti-venom. Among twenty eight medicinal plants extract Bauhinia variegata L and Citrus limon (L.) Burm. f were found extremely useful (94%, 0.16 mg) and effective (p˃0.5) to minimize snake venom (0.01 mg) induced 5ʹ-ND activity as compared with reference antidote (94%, 0.08 mg). Moreover, hyaluronidase activity was also recorded in Naja naja karachiensis venom. Cobra venom at the dose of 10 mg/ml was proved to diminish turbidity of reaction mixture and considered 100% activity. Rutintrihydrate (reference hyaluronidase inhibitor) was proved 99% effective to neutralize hyaluronidase activity while Trichodesma indicum (L.) Sm extract was declared the best anti-hyaluronidase (97%, p˃0.5) when compared with reference standard. PLA2 activity. RP-HPLC chromatogram revealed that tannins chromophores (15-102 µg/mg of tannic acid equivalents) of different polarity (visualized as large hump) were present in Bauhinia variegata L extract. Tannins are poly-phenolic chromophores and form insoluble compounds with snake venom protein(s) thus impart anti-PLA2 effect.
Conclusion
Pakistani cobra venom was found unique (due to novel PLA2) which prolongs PT and TT tests which is not possible for other snake venoms. Existence of copper ion (to activate/deactivate enzymes) is another salient feature of this venom and still unreported
xii from any other elapids venom. Snake venom was found to impart little hemolysis, coagulopathies and severe damage to the heart, liver and kidneys. Once injected it is distributed to the all parts of the body and cleared from blood pool within 24 h of envenomation (metabolized in liver and lungs & excreted through kidneys). To neutralize snake venom toxicity shortlisted plants extract should be used, i.e., Bauhinia variegate L
(anti-PLA2&anti-5ʹ-ND), Enicostemma hyssopifolium (Willd.) Verdoorn (reverse anticoagulant), Sapindus mukorossi Gaertn (anti-ALPase), Trichodesma indicum(L.) Sm (anti-hyaluroniadse), Citrus limon (L.) Burm. f (anti-5ʹ-ND), Cedrus deodara (Roxb. ex D. Don) G. Don (anti-hemolytic) and Stenolobium stans (L) D. Don (reverse anticoagulant and to minimize severe damage to the liver, heart and kidneys). Anti-PLA2 potential of Bauhinia variegata L extract was due to the presence of tannins which precipitated snake venom protein(s) thus could be applied in the form of paste to the infected area before proper hospitalization.
xiii List of Publications from this Research Work
Published
Asad, M.H.H.B., Iqbal M., Ikram M.R., Khawaja N.R., Muneer S, Shabbir, M.Z., Khan, M.Q., Muratza, G. & Hussain, I. (2016). 5ʹ-nucleotidases enzymatic assay for Naja naja karachiensis venom: their toxicities and remedial approach by natural inhibitors (medicinal plants). Acta. Pol. Pharm. 73, 667-673.
Asad, M.H.H.B., Durr-e-Sabih, Ahmad, I., Choudary, B.A., Murtaza, G. & Hussain, I. (2015). Bio distribution and kinetic studies of technetium labeled Naja naja karachiensis venom via gamma scintigraphic and SPECT images. Pak. J. Pharm. Sci. 28, 1233-1238.
Asad, M.H.H.B., Murtaza, G., Ubaid, M., Durr-e-Sabih, Sajjad, A., Mehmood, R., Mehmood, Q., Ansari, M.M., Karim, S., Mehmood, Z. & Hussain, I. (2014). Naja naja karachiensis envenomation: Biochemical parameters for cardiac, liver and renal damage along with their neutralization by medicinal plants. Biomed. Res. Int. 970540, 1-13.
Asad, M.H.H.B., Durr-e-Sabih, Choudary, B.A., Asad, A.F., Murtaza, G. & Hussain, I.
(2014). Compensatory effects of medicinal plants of Pakistan upon prolongation of coagulation assays induced by Naja naja karachiensis bite. Cur. Sci. 106, 870- 873.
Asad, M.H.H.B., Durr-e-Sabih, Yaqab, T., Murtaza, G., Hussain, M.S., Hussain, M.S.,
Nasir, M.T., Azhar, S., Khan, S.A. & Hussain, I. (2014). Phospholipases A2: Enzymatic assay for snake venom (Naja naja karachiensis) with their neutralization by medicinal plants of Pakistan. Acta. Pol. Pharm. 71, 625-630.
Asad, M.H.H.B., Durr-e-Sabih, Chaudhory, B.A., Ahmad, I., Hussain, M.S., Izhar, N.,
Akmal, N., Shahzad, A.H. & Hussain, I. (2014). Antihemolytic property of local medicinal plants upon Pakistani cobra venom induced hemolysis. The J. Anim. and Plant. Sci. 24, 1701-1708.
xiv Asad, M.H.H.B., Murtaza, G. & Hussain, I. (2014). Enzymatic assay for alkaline phosphatase present in snake venom Naja naja karachiensis with its neutralization by medicinal plants. Pak. J. Zool. 46, 1775-1781.
Asad, M.H.H.B., Razi, M.T., Durr-e-Sabih, Najam-us-Saqib, Q., Nasim, S.J., Murtaza, G. & Hussain, I. (2013). Anti-venom potential of Pakistani medicinal plants: inhibition of anticoagulation activity of Naja naja karachiensis toxin. Curr. Sci. 105, 1419-1424.
Asad, M.H.H.B., Murtaza, G., Siraj, S., Khan, S.A., Azhar, S., Hussain, M.S., Ismail, T., Hussain, M.S. & Hussain, I. (2011). Enlisting the scientifically unnoticed medicinal plants of Pakistan as a source of novel therapeutic agents showing anti- venom activity. Afr. J. Pharm. Pharacol. 5, 2292-2305.
Submitted
Asad et al., (2016). ICP-OES analysis of Najanajakarachiensis venom: Inorganic (metallic & non-metallic) elements for turning on and off various enzymatic activities. Curr. Sci.
Asad et al., (2016). Hyaluronidase and phospholipase inhibitory activity of plants used in Pakistan in traditional treatment of Naja naja karachiensis snake bites. J. Ethnopharmacol.
Asad et al., (2016). Proteomic characterization of Naja naja karachiensis venom: A neglected subspecies of Pakistani Naja naja. Toxicon.
xv TABLE OF CONTENTS 1 Introduction……………………………………………………………… 1 1.1 Asiatic Cobras (Classification & Distribution)…………………… 2 1.2 Black Pakistan Cobra (Naja naja karachiensis)………………… 4 1.3 Snake Bite and Naja naja karachiensis Envenomation…………… 4 1.4 Composition of Snake Venom…………………………………….. 5
1.4.1 Phospholipase A2…………………………………………. 6 1.4.2 Protease…………………………………………………… 8 1.4.3 Phosphatase (Alkaline Phosphatase)……………………… 9 1.4.4 5ʹ-Nucleotidase……………………………………………. 10 1.4.5 Hyaluronidase…………………………………………… 11 1.5 Surrogate Markers for Cobra Venom Toxicity Determination…… 14 1.6 Proteomics as a Tool to Study Snake Venom Components……… 14 1.7 Radio Labeling Technique to Observe Bio-distribution of Venom. 15 1.7.1 Technetium: A Radionuclide & its Generation…………… 15 1.7.2 General Properties of Technetium……………………… 16 1.7.3 Labeling of Compounds (Snake Venom Proteins) with 99mTc...... 17
1.8 Antidote(s) to Neutralize Snake Venom Toxicity………………… 19 1.8.1 Immunoglobulin’s (Equines Anti-sera) Therapy…………. 19 1.8.2 Medicinal Plants: Cheap Alternate Source to Treat Snake Venom Toxicity…………………………………………... 19
1.8.2.1 Albizia lebbeck (L.) Benth……………………… 20 1.8.2.2 Allium cepa L…………………………………… 21 1.8.2.3 Allium sativum L……………………………… 22 1.8.2.4 Althaea officinalis L…………………………… 24 1.8.2.5 Bauhinia variegate L…………………………… 24
xvi 1.8.2.6 Brassica nigra (L.) W. D. J. Koch…………...... 26 1.8.2.7 Calotropis procera (Aiton) W. T. Aiton……….. 27 1.8.2.8 Cedrus deodara (Roxb. ex D. Don) G. Don…… 28 1.8.2.9 Citrullus colocynthis (L.) Schrad……………….. 30 1.8.2.10 Citrus limon (L). Burm. f……………………….. 31
1.8.2.11 Cuminum cyminum L…………………………… 32 1.8.2.12 Enicostema hyssopifolium (Willd.) I. Verd…….. 33 1.8.2.13 Fagonia cretica L………………………………. 34 1.8.2.14 Leucas capitata Desf……………………………. 35 1.8.2.15 Matthiola incana (L.) W. T. Aiton……………... 36 1.8.2.16 Momordica charantia L………………………… 37 1.8.2.17 Nerium indicum Mill……………………………. 37 1.8.2.18 Ocimum synctum L……………………………... 39 1.8.2.19 Pinus roxburghii Sarg…………………………... 40 1.8.2.20 Pistacia integerrima J. L. Stewart……………… 41 1.8.2.21 Psoralea corylifolia L…………………………... 42 1.8.2.22 Rhazya stricta Decne…………………………… 44 1.8.2.23 Rubia cordifolia L………………………………. 45 1.8.2.24 Sapindus mukorossi Gaertn…………………… 46 1.8.2.25 Stenolobium stans (L.) Seem…………………… 47 1.8.2.26 Terminalia arjuna (Roxb. ex DC.) Wight & Arn. 48
1.8.2.27 Trichodesma indicum (L.) Sm………………… 49 1.8.2.28 Zingiber officinale Roscoe……………………… 50 1.9 Hypothesis of this Study…………………………………...... 51 1.10 Aims & Objectives of this study…………………………………... 51 2 Materials & Methods……………………………………………………… 52 2.1 Equipments…………………………………………………………. 53 2.2 Experimental Animals………………………………………………. 54
xvii 2.3 Medicinal Plants……………………………………………………. 54 2.4 Collection of Snakes (Naja naja karachiensis)……………………. 56 2.5 Chemical Agents…………………………………………………….. 56 2.6 Extraction of Plants Material………………………………………... 59 2.7 Milking of Naja naja karachiensis Venom…………………………. 59 2.8 Proteomic Analysis of Pakistani Cobra (Naja naja karachiensis) Venom………………………………………………………………. 60 2.8.1 Sodium Dodecylsulpahte Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Crude Cobra Venom (Naja naja karchiensis)…………………………………… 60
2.8.1.1 SDS-PAGE Protocol………………………… 60 2.8.1.2 Staining with Coomassie Brilliant Blue R 250 60 Dye…………………………………………….. 2.8.1.3 Silver Staining Technique……………………... 60 2.9 Chromatographic Separation of Naja naja karachiensis 64 Venom…………………………………………………………….. 2.9.1 Size Exclusion Chromatographic (SEC) Analysis………... 64 2.9.2 Reverse Phase HPLC Analysis…………………………… 64 2.9.3 Protein (Trypsin) Digestion of Crude Venom Sample and its Fractions……………………………………………...... 65 2.9.4 Mass Spectrometric (LC-MS/MS) Analysis of Tryptic Digested Venom Sample(s)……………………………...... 65 2.10 Protein Estimation by Bicinchoninic Acid (BCA) for Naja naja krachiensis Venom………………………………………………... 66 2.10.1 Protein Estimation by Bicinchoninic Acid (BCA)………. 66 2.11 Elemental Analysis for Naja naja karachiensis Venom via Inductive Couple Plasma-Optical Emission Spectroscopy (ICP-OES) Technique…………………………………………………... 67
xviii 2.11.1 Multi-Elemental Analysis for Naja naja karachiensis Venom…………………………………………………... 67 2.11.1.1 Closed Pressurized Digestion of Samples…… 67 2.11.1.2 ICP-OES Analysis…………………………… 67 2.12 Biodistribution & Kinectic Study of Naja naja karachiensis Venom via Radio Tracer (99mTc) Binding Technique…………… 68 2.12.1 Tracer (99mTc) Binding Protocol for Cobra Venom and its Percentage Yield…………………………………….. 68 2.12.2 Stability Study for 99mTc Labeled Cobra Venom………. 68 2.12.3 Biological Activity of Radio Labeled and Unlabeled Crude Cobra Venom……………………………………. 69 2.12.4 Blood Kinetic Study of Technetium Labeled Cobra Venom…………………………………………………... 69 2.12.5 Bio distribution Study of Technetium Labeled Cobra Venom…………………………………………………... 69 2.12.6 Gamma Scintigraphy & Single Photon Emission Computed Tomography (SPECT) of Technetium Labeled Pakistani Cobra Venom……………………….. 70 2.13 Toxic Biological/Biochemical Activities of Naja naja karachiensis Venom & their Neutralization with Medicinal Plants of Pakistan………………………………………………………… 70 2.13.1 Lethal Dose of Naja Naja Karachiensis Venom for 50%
Population (LD50)………………………………………. 70 2.13.2 Hemolytic Activity……………………………………... 71 2.13.3 Anticoagulant Activity…………………………………. 71 2.13.3.1 Preparation of Platelet Poor Plasma (PPP)… 72 2.13.3.2 Prothrombin Time (PT) Coagulation Assay.. 72 2.13.3.3 Activated Partial Thromboplastin Time 72
xix (aPTT) Coagulation Assay…………………. 2.13.3.4 Thrombin time (TT) Coagulation Assay…… 72 2.13.4 Study of Surrogate Markers (Biochemical Parameters) for Liver, Heart and Kidney Damage…………………... 73 2.13.4.1 Laboratory Animals Preparation…………... 73 2.13.4.2 In vivo Anti Snake Venom Potential of Medicinal Plants Extract…………………… 73 2.13.4.3 Estimation of Biochemical Parameters: GPT/Alanine Aminotransferase (ALAT) & GOT/Aspartate Aminotransferase (ASAT) to Access Hepatic Damage………………… 74 2.13.4.3.1 Assay Procedure……………… 74 2.13.4.4 Estimation of Biochemical Parameters: Creatine Kinase (CK-MB) & Lactate Dehydrogenase (LDH) to Access Cardiac Damage…………………………………….. 74 2.13.4.4.1 Assay Procedure……………… 74 2.13.4.5 Estimation of Biochemical Parameters: Urea & Creatinine to Access Renal Damage……. 75 2.13.4.5.1 Assay Procedure……………… 75 2.14 Various Enzymatic Assays for Naja naja karachiensis Venom and their Neutralization by Medicinal Plants of Pakistan……………... 79 2.14.1 Enzymatic Assays for Snake Venom Phospholipase…...... 79 2.14.1.1 Assay I (Acidimetric Assay)………………...... 79 2.14.1.2 Assay II (Turbidimetric Assay)……………...... 79 2.14.1.3 Assay III (Coagulation Assay)………………... 80 2.14.2 Enzymatic Assay for Snake Venom Alkaline Phosphatase 81 2.14.3 Enzymatic Assay for Snake Venom 5ʹ-Nucleotidase……. 81
xx 2.14.4 Enzymatic Assay for Snake Venom Hyaluronidase……... 82 2.14.5 Enzymatic Assay for Snake Venom Protease……………. 83 2.15 Identification of Specific Enzyme(s) Inhibitor(s)…………………. 83
2.15.1 Potential Anti-PLA2 Plant Extract……………………….. 83 2.15.1.1 High Resolution (HPLC) Microplate Based
PLA2 Bioassay Guided Fractionation………… 83
2.15.1.2 IC50 Determination (Dose Response Curves of
Effective Anti-PLA2 Inhibitors)………………. 84 3 Results……………………………………………………………………. 85 3.1 Proteomic Strategy for Analysis of Naja naja karachiensis Venom 86 3.1.1 Proteomic Strategy for Analysis of Naja naja karachiensis Venom……………………………………... 86 3.1.2 Electrophoresis Pattern / (SDS-PAGE) Profile………….. 87 3.1.3 Chromatographic Separation of Snake Venom Components……………………………………………… 87 3.1.4 Analysis of Tryptic Digested Venom Samples via LC- MS/MS…………………………………………………… 87 3.1.5 Protein Concentration of Naja naja karchiensis Venom… 88 3.2 Elemental Analysis of Naja naja karachiensis Venom…………… 114 3.2.1 Quantitative Estimation of Inorganic Constituents………. 114 3.3 Bio-distribution and Kinetic Study of Naja naja karachiensis Venom via Radio Tracer (99mTc) Binding Technique……………... 118 3.3.1 Optimization of Radio Labeling Procedure and its Percentage Yield…………………………………………. 118 3.3.2 Stability Profile of Technetium Labeled Naja naja karachiensis Venom……………………………………… 118 3.3.3 Technetium Labeled Cobra Venom and its Toxic (Hemolytic) Potential…………………………………….. 118
xxi 3.3.4 Blood Kinetics Profile of Technetium Labeled Naja naja karachiensis Venom……………………………………… 118 3.3.5 Bio-distribution of Technetium Labeled Naja naja karachiensis Venom……………………………………… 119 3.3.6 Gamma Scintigraphy and SPECT Images……………….. 119 3.4 Toxic Biological/Biochemical Activities of Naja Naja Karachiensis Venom and their Neutralization with Medicinal Plants of Pakistan………………………………………………….. 130 3.4.1 Lethal Toxic Dose of Naja Naja Karachiensis Venom for
50% Population (LD50)…………………………………… 130 3.4.2 Hemolytic Potential of Naja naja karachiensis Venom and its Neutralization with Medicinal Plants…………………. 131 3.4.3 Anticoagulant Activity of Naja naja karachiensis Venom and its Neutralization with Medicinal Plants……………... 136 3.4.4 Biochemical Parameters for Heart, Liver and Kidney Damage and their Neutralization by Medicinal Plants…… 143 3.5 Different Enzymatic Assays for Naja naja karachiensis Venom with their Neutralization by Medicinal Plants…………………….. 151 3.5.1 Enzymatic Assays for Phospholipase Enzyme…………… 151
3.5.1.1 Acidimetric Phospholipase (PLA2) Enzymatic Assay and its Neutralization…………………... 151
3.5.1.2 Turbidimetric Phospholipase (PLA2) Enzymatic Assay and its Neutralization………. 155 3.5.1.3 Anticoagulant Phospholipase (PLA) Enzymatic Assay and its Neutralization…………………... 160 3.5.2 Enzymatic Assay for Alkaline Phosphatase (ALPase) and its Neutralization…………………………………………. 164 3.5.3 Enzymatic Assay for 5ʹ-Nucleotidase and its
xxii Neutralization…………………………………………….. 168 3.5.4 Enzymatic Assay for protease……………………………. 173 3.5.5 Enzymatic Assay for Hyaluronidase and its Neutralization 174 3.6 Identification of Specific Enzyme(s) Inhibitor(s)…………………. 179
3.6.1 Anti-PLA2 Activity of the Most Efficient Plant Extract…. 179 3.6.2 RP-HPLC Profile of Bauhinia variegata L Extract……… 179 3.6.3 Screening of Fractionated Extract of Bauhinia variegata L……………………………………... 179 3.6.4 Dose Response Relationship of Bauhinia variegata L…… 180 4 Discussion………………………………………………………………… 185 4.1 Proteomic Characterization of Naja naja karachiensis Venom…… 186 4.2 Elemental Analysis of Naja naja karachiensis Venom…………… 189 4.3 Biodistribution and Kinetic Study of Naja naja karachiensis Venom via Radio Tracer (99mTc) Binding Technique……………... 193 4.4 Toxic Biological/Biochemical Activities of Naja Naja Karachiensis Venom and their Neutralization with Medicinal Plants of Pakistan………………………………………………….. 194
4.4.1 LD50 Dose of Naja naja karachiensis Venom……………. 194 4.4.2 Hemolytic Potential of Naja Naja Karachiensis Venom and its Neutralization with Medicinal Plants……………... 195 4.4.3 Anticoagulant Activity of Naja naja karachiensis Venom and its Neutralization with Medicinal Plants……………... 197 4.4.4 Biochemical Parameters for Heart, Liver and Kidney Damage and their Neutralization by Medicinal Plants…… 198 4.5 Various Enzymatic Assays for Naja naja karachiensis Venom and their Neutralization by Medicinal Plants of Pakistan……………… 200 4.5.1 Enzymatic assays for phospholipase enzyme and its Neutralization…………………………………………….. 200
xxiii 4.5.2 Enzymatic Assay for Alkaline Phosphatase (ALPase) and its Neutralization………………………………………….. 201 4.5.3 Enzymatic Assay for 5ʹ-Nucleotidase and its Neutralization…………………………………………….. 202 4.5.4 Enzymatic Assay for Hyaluroniadse and its Neutralization……………………………………………….. 203 4.6 Identification of Specific Enzyme(s) Inhibitor(s) from Bauhinia variegata L Extract against Turbidimetrimic Phospholipase Activity……………………………………………. 205 5 Conclusion………………………………………………………………... 207 6 Future perspective ………………………………………………………. 210 7 References………………………………………………………………... 211
xxiv LIST OF FIGURES
Fig 1.1 Apparatus of a Poisonous Snake………………………………………… 3 Fig 1.2 Naja naja karachiensis / Black Pakistan Cobra…………………………… 4
Fig 1.3 Represents Hydrolysis of Phospholipids by the Action of PLA2………….. 7 Fig 1.4 General Representation of Mechanism of Protease Enzyme to Hydrolyze Protein Molecule…………………………………………………………... 8 Fig 1.5 Possible Mechanism for Induction of Alkaline Phosphatase Toxicities Post Naja naja karachiensis Envenomation……………………………….. 12 Fig 1.6 Possible Deleterious Mechanism of Action of 5ʹ-Nucleotidase Enzyme Post Naja naja karachiensis Envenomation……………………………….. 13 Fig 1.7 Describes the Structure of Hyaluronan and Site of Action of Hyaluronidase Enzyme……………………………………………………. 13 Fig 1.8 Represents Typical Generator System (Left) & a 99Mo-99mTc Generator (Right)……………………………………………………………………... 16 Fig 1.9 Represents Chemical Basis for Binding of 99mTc with Naja naja karachiensis Venom……………………………………………………….. 18 Fig 1.10 Describes the Contour of Albizia lebbeck (L.) Benth with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen……….. 21 Fig 1.11 Describes the Contour of Allium cepa L with Specific Part (Bulbs) Collected as Anti-Venom Along with Voucher Specimen………………... 22 Fig 1.12 Describes the Contour of Allium sativum L with Specific Part (Bulbs) Collected as Anti-Venom Along with Voucher Specimen……………… 23 Fig 1.13 Describes the Contour of Althaea officinalis L with Specific Part (Roots) Collected as Anti-Venom Along with Voucher Specimen………………... 25 Fig 1.14 Describes the Contour of Bauhinia variegate L with Specific Part (Roots) Collected as Anti-Venom Along with Voucher Specimen………………... 26 Fig 1.15 Describes the Contour of Brassica nigra (L.) W. D. J. Koch with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen…... 27 Fig 1.16 Describes the Contour of Calotropis procera (Aiton) W. T. Aiton with Specific Part (Flowers) Collected as Anti-Venom Along with Voucher
xxv Specimen…………………………………………………………………... 28 Fig 1.17 Describes the Contour of Cedrus deodara (Roxb. ex D. Don) G. Don Specific Part (Bark) Collected as Anti-Venom Along with Voucher Specimen…………………………………………………………………... 29 Fig 1.18 Describes the Contour of Citrullus colocynthis (L.) Schrad with Specific Part (Fruits) Collected as Anti-Venom Along with Voucher Specimen…………………………………………………………………... 30 Fig 1.19 Describes the Contour of Citrus limon (L). Burm. f with Specific Part (Fruits) Collected as Anti-Venom Along with Voucher Specimen……….. 31 Fig 1.20 Describes the Contour of Cuminum cyminum L with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen………………... 32 Fig 1.21 Describes the Contour of Enicostema hyssopifolium (Willd.) I. Verd Plant Collected as Anti-Venom Along with Voucher Specimen………………... 33 Fig 1.22 Describes the Contour of Fagonia cretica L with Specific Part (Leaves and Twigs) Collected as Anti-Venom Along with Voucher Specimen…… 34 Fig 1.23 Describes the Contour of Leucas capitata Desf Plant Collected as Anti- Venom Along with Voucher Specimen…………………………………… 35 Fig 1.24 Describes the Contour of Matthiola incana (L.) W. T. Aiton with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen…... 36 Fig 1.25 Describes the Contour of Momordica charantia L with Specific Part (Fruits) Collected as Anti-Venom Along with Voucher Specimen…………………………………………………………………... 38 Fig 1.26 Describes the Contour of Nerium indicum Mill with Specific Part (Leaves and Roots) Collected as Anti-Venom Along with Voucher Specimen……. 39 Fig 1.27 Describes the Contour of Ocimum synctum L Plant as Anti-Venom Along with Voucher Specimen…………………………………………………… 40 Fig 1.28 Describes the Contour of Pinus roxburghii Sarg with Specific Part (Oleoresins) Collected as Anti-Venom Along with Voucher Specimen… 41 Fig 1.29 Describes the Contour of Pistacia integerrima J. L. Stewart with Specific Part (Galls) Collected as Anti-Venom Along with Voucher Specimen…… 42 Fig 1.30 Describes the Contour of Psoralea corylifolia L with Specific Part (Seeds)
xxvi Collected as Anti-Venom Along with Voucher Specimen………………... 43 Fig 1.31 Describes the Voucher Specimen of Leaves of Rhazya stricta Dcne Plant Collected as Anti-Venom………………………………………………….. 44 Fig 1.32 Describes the Contour of Rubia cordifolia L with Specific Part (Stems) Collected as Anti-Venom Along with Voucher Specimen………………... 45 Fig 1.33 Describes the Part (Fruits) of Sapindus mukorossi Gaertn Used as Anti- Venom in Voucher Specimen Deposited at the Herbarium……………….. 46 Fig 1.34 Describes the Contour of Stenolobium stans (L.) Seem with Specific Part (Roots) Collected as Anti-Venom Along with Voucher Specimen……….. 47 Fig 1.35 Describes the Contour of Terminalia arjuna (Roxb. ex DC.) Wight & Arn with Specific Part (Bark) Collected as Anti-Venom Along with Voucher Specimen………………………………………………………………….. 48 Fig 1.36 Describes the Contour of Trichodesma indicum (L.) Sm Plant Collected as Anti-Venom Along with Voucher Specimen……………………………… 49 Fig 1.37 Describes the Contour of Zingiber officinale Roscoe with Specific Part (Rhizomes) Collected as Anti-Venom Along with Voucher Specimen…… 50 Fig 3.38 Schematic Overview of Combined Proteomic Strategy about Naja naja karachiensis Venom……………………………………………………….. 86 Fig 3.39 SDS-PAGE (4%-20% resolving gel) Electrophoresis Profile (Reduced & Non-reduced) of Naja naja karachiensis Venom with Coomassie Brilliant Blue R250 (a) and Silver Stain (b). Mobility of the Protein Ladder Markers is Shown on the Right (a) and in the Middle (b)…………………. 89 Fig 3.40 Gel Filtration Chromatographic Separation of Crude Naja naja karachiensis Venom on SuperdexTM 200, HiloadTM 16/60, Preparative Grade Column using 50mM Tris-HCl buffer pH 7.4……………………… 90 st Fig 3.41 RP-HPLC Profile of 1 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A° Pore Size) and ÄKTA Purifier LC Unicorn System……………………………………………….. 91 nd Fig 3.42 RP-HPLC Profile of 2 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A° Pore Size) and ÄKTA Purifier LC Unicorn System……………………………………………….. 92
xxvii rd Fig 3.43 RP-HPLC Profile of 3 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A° Pore Size) and ÄKTA purifier LC Unicorn System……………………………………………….. 93 th Fig 3.44 RP-HPLC Profile of 4 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A Pore Size) and ÄKTA Purifier LC Unicorn System……………………………………………….. 94 th Fig 3.45 RP-HPLC Profile of 5 Gel Chromatographic Peak using Jupiter C18 Column (4.6 × 250 mm, 5 µm Particle Size, 300 A Pore Size) and ÄKTA purifier LC Unicorn System……………………………………………….. 95 th Fig 3.46 RP-HPLC Profile of 6 Gel Chromatographic Peak using Jupiter C18 Column (4.6 × 250 mm, 5 µm Particle Size, 300 A Pore Size) and ÄKTA Purifier LC Unicorn System……………………………………………….. 96 Fig 3.47 Sequence Alignment of Unique 3FTX (MTLP-3) Detected in Naja naja karachiensis Venom with Recently Discovered Novel Haditoxin (Ophiophagus hannah)…………………………………………………….. 111 Fig 3.48 The Summarized Proteome Picture of Naja naja karachiensis Venom…… 112 Fig 3.49 Standard Curve for Bovine Serum Albumin (BSA) at its Various Concentrations for Determination of Protein Components in Naja naja karachiensis Venom……………………………………………………….. 113 Fig 3.50 Relative Abundance of Elementals (Metal & Non metal) Found in Naja naja karachiensis Crude Venom…………………………………………... 117 Fig 3.51 Blood Kinetics/Clearance Profile of 99mTc Labeled Naja naja karachiensis Venom Post Intravenous Injection in Rabbits……………………………... 125 Fig 3.52 Biodistribution Profile of 99mTc Labeled Naja naja karachiensis Venom after Intravenous Injection in Healthy Male Rabbits…………………….. 126 Fig 3.53 Whole Body Gamma Scintigraphic (2h Blood Flow Study) Scans after Intravenous Injection of 99mTc Labeled Cobra Venom in Rabbit…………. 127 Fig 3.54 Distribution Profile of Technetium Labeled Naja naja karachiensis Venom in Middle Compartment of Rabbits In Terms of R/L Ratio………. 128 Fig 3.55 Acquired Single Photon Emission Computed tomography (SPECT) Images of Naja naja karachiensis Envenomed Rabbit at 360°……………. 129
xxviii Fig 3.56 LD50 Determination of Naja naja karachiensis venom…………………… 130 Fig 3.57 Comparison of Short Listed Medicinal Plants Having Potentials to Neutralize Naja naja karachiensis Venom (100 µg/ml) Induced Hemolysis in Comparison with Reference Standard (Anti-Sera)…………. 134 Fig 3.58 Percentage Distribution of Plants for their Potential to Neutralize Hemolytic Effect of Naja naja karachiensis Venom……………………… 135 Fig 3.59 Schematic Overview of Different Stages of Blood Coagulation Pathway (I, II & III) and its Possible Hindrance by Three Ways (a, b & c) after Snake (Naja naja karachiensis) Bite Envenomation……………………… 142 Fig 3.60 Represents Absorbance of the Turbidity at Various Venom Concentrations along with Incubation Time………………………………. 157 Fig 3.61 Mechanism of Action of Secondary Metabolites (Plants Extract) to Neutralize Anticoagulant Effect of Phospholipase Enzyme Present in Naja naja karachiensis Venom………………………………………………….. 163 Fig 3.62 Standard Curve for p-Nitrophenol In Terms of Absorbance at Different Concentrations……………………………………………………………... 165 Fig 3.63 Standard Curve for Inorganic Phosphate In Terms of Absorbance at Various Concentrations……………………………………………………. 169 Fig 3.64 Comparison of Two Valuable Medicinal Plants Extract with Reference Standard Antidote in Neutralization of 5ʹ-Nucleotidase Activity Posed by Naja naja karchiensis (0.01mg) Venom…………………………………... 172 Fig 3.65 Screening for Protease Enzymatic Activity in Naja naja karachiensis Venom……………………………………………………………………... 173 Fig 3.66 Optimization of Hyaluroniadse Assay: Effect of Incubation Time at 37 °C on the Reaction Mixture Containing Hyaluroniadase Enzyme at Different Concentrations of Naja naja karachiensis Venom………………………... 175 Fig 3.67 Michaleous Menten Kinetics Data Obtained in Optimization of Hyaluroniadse Assay for Naja naja karachiensis venom…………………. 176 Fig 3.68 RP-HPLC Chromatogram of Ethanolic Bauhinia variegata L Extract (280 nm) with Detail Change in Percentage Gradient of Buffer B……………... 180 Fig 3.69 General representation of RP-HPLC Chromatogram of
xxix Bauhinia variegata L Extract……………………………………………… 181 Fig 3.70 High Resolution Biochromatogram of Bauhinia variegata L Extract…….. 182 Fig 3.71 Dose Response Curve for Bauhinia variegata L in the Turbidimetric
PLA2 Assay………………………………………………………………… 183
Fig 3.72 Dose Response Curve for EDTA in the Turbidimetric PLA2 Assay………. 184
xxx LIST OF TABLES
Table 2.1 List of Equipment Used for Experimental Work………………………… 53
Table 2.2 List of Medicinal Plants of Pakistan having Ethnobotanical Evidences as 54 Anti-Snake Venom Collected for this Work……………………………...
Table 2.3 Chemicals Used for Various Experimental Work………………………... 56
Table 2.4 Composition of Reducing Sample Loading Buffer (4X)………………… 61
Table 2.5 Composition of Non Reducing Sample Loading Buffer (4X)………...... 61
Table 2.6 Composition of Running Buffer for SDS PAGE………………………… 62
Table 2.7 Composition of Coomassie Brilliant Blue R 250 Staining Solution……... 62
Table 2.8 Composition of Destaining Solution……………………………………... 62
Table 2.9 Recipe for Preparation of Various Solutions Used for Silver Staining… 63
Table 2.10 Composition of Mono Reagent (By Mixing 4R1 & 1R2) Used for ALAT
Estimation……………………………………………………………… 76
Table 2.11 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for ASAT
Estimation………………………………………………………………... 76
Table 2.12 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for LDH
Estimation………………………………………………………………... 77
Table 2.13 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for CK-
MB Estimation…………………………………………………………… 77
Table 2.14 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for Urea
Estimation………………………………………………………………... 78
Table 2.15 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for
Creatinine Estimation…………………………………………………….. 78
xxxi Table 3.16 Assignment of Naja naja karachiensis Crude Venom Proteins to Various
Families after Digestion of Peptides via Trypsin by LC-MS/MS Analysis 97 Table 3.17 Assignment of RP-HPLC Separated Fractions of Acidic Naja naja karachiensis Proteins to Various Families after Digestion of Peptides by 101 Trypsin via LC-MS/MS Analysis………………………………………...
Table 3.18 Quantitative Estimation of Different Elements (Metal & Non metal) Found in Naja naja karachiensis Venom (100 mg) via ICP-OES 114 Analysis…………………………………………………………………...
Table 3.19 LOD for Different Elements Detected in Naja naja karachiensis Venom. 115
Table 3.20 Accuracy and Other Parameters for the Concentration of Elements in Certified Reference Material (NIST 1515) Apple Leaf Measured After 116 Closed Pressurized Digestion……………………………………………..
Table 3.21 Effect of Various Concentrations of Stannous Chloride Dihydrate in Optimization of Radio Labeling Procedure (Binding of 99mTc with Naja 120 naja karachiensis Venom) In Terms of Labeling Percentage at pH 7……
Table 3.22 Effect of Different pH for Optimization of Radio Labeling Procedure In
Terms of Labeling Percentage at 100 µg SnCl2.2H2O…………………… 122 Table 3.23 Stability Profile of Technetium Labeled Naja naja karachiensis Venom Complex via Both In vivo and In vitro Experimentation………………… 124 Table 3.24 Comparison of Toxic (Hemolytic) Potential of 99mTc Labeled and Unlabeled Cobra Venom…………………………………………………. 124 Table 3.25 Aptitudes of Different Agents Towards Induction of Hemolysis In Terms of Hemoglobin Release Measured at 540 nm…………………….. 132 Table 3.26 Effect of Various Antidotes (Medicinal Plants Extract and Standard Antidote) Evaluated to Halt Venom (100 µg/ml) Induced Hemolysis…... 132
xxxii Table 3.27 Anticoagulant Effect (Delay in PT) of Naja naja karachiensis Venom and its Neutralization by Various Antidotes……………………………... 137 Table 3.28 Anticoagulant Effect (Delay in aPTT) of Naja naja karachiensis Venom and its Neutralization by Various Antidotes……………………………... 139 Table 3.29 Anticoagulant Effect (Delay in TT) of Naja naja karachiensis Venom and its Neutralization by Various Antidotes……………………………... 140 Table 3.30 Naja naja karachiensis Envenomation: Various Biochemical Parameters for Cardiac, Liver and Kidney Damage in Experimental Rabbits……….. 144 Table 3.31 A List of Medicinal Plants Extract (100 mg/kg) used to Minimize Cardiotoxicity Posed by Naja naja karachiensis Venom (800 µg/kg) in 145 Rabbits……………………………………………………………………
Table 3.32 A List of Medicinal Plants Extract (100 mg/kg) used to Minimize Hepatotoxicity Posed by Naja naja karachiensis Venom (800 µg/kg) in 147 Rabbits……………………………………………………………………
Table 3.33 A List of Medicinal Plants Extract (100 mg/kg) used to Minimize Nephrotoxicity Posed by Naja naja karachiensis Venom (800 µg/kg) in
Rabbits…………………………………………………………………… 149
Table 3.34 Dose Dependent Effect of Cobra Venom for the Release of Free Fatty Acids In Terms of Variation in pH………………………………………. 152 Table 3.35 List of Evaluated Medicinal Plants Extract Having Anti-Phospholipase
A2 Property In Terms of Increase in pH…………………………………. 153 Table 3.36 Data Points Acquired to Optimize Various Cobra Venom Concentrations
at Different Incubation Time for Validation of PLA2……………………. 156
Table 3.37 Anti-Phospholipase A2 Potential of Various Antidotes in Neutralization of Decrease in Turbidity Posed by Naja naja karachiensis Venom……... 158
xxxiii Table 3.38 Reverse Anticoagulant Potential of Pakistani Medicinal Plants Extract Posed by Weak PLA Enzymes Abundant in Naja naja karachiensis 161 Venom…………………………………………………………………….
Table 3.39 Effect of Various Concentration of Naja naja karchiensis Venom Abundant in Alkaline Phosphatase (ALPase) Enzyme in the Release of 166 p-Nitrophenol……………………………………………………………..
Table 3.40 Medicinal Plants of Pakistan Having Anti-Alkaline Phosphatase (Anti- ALPase) Activity Posed by Naja naja karachiensis (0.4 mg/0.1ml) 166 Venom…………………………………………………………………….
Table 3.41 Effect of Various Concentration of Naja naja karachiensis Venom Abundant in 5ʹ-Nucleotidase Enzyme in the Release of Inorganic 170 Phosphate…………………………………………………………………
Table 3.42 List of Medicinal Plants of Pakistan having Anti-5ʹ-Nucleotidase Activity Posed by Naja naja karachiensis (0.01mg/0.1 ml) Venom…….. 170 Table 3.43 Medicinal Plants of Pakistan (0.1mg, 10 mg/ml) having Anti- hyaluronidase Enzymatic Activity Posed by Naja naja karachiensis 177 (0.1mg, 10 mg/ml) Venom……………………………......
xxxiv LIST OF ABBREVIATIONS
Single/three letters abbreviations of an aminoacids were followed as per recommendations of IUPAC-IUBMB Joint Commission on Biochemical Nomenclature. Rest of all abbreviations used in this thesis is described below. ADP Adenosine diphosphate AMP Adenosine monophosphate ATP Adenosine triphosphate α Alpha α-EPTX Alpha - elapitoxin ALAT Alanine aminotransferase +1 NH4 Ammonium ion A° Armstrong ALPase Alkaline phosphatase ASAT Aspartate aminotransferase aPTT Activated partial thromboplastin time β Beta Bq Becquerel BCA Bicinchoninic acid -1 HCO3 Bicarbonate ion BBB Blood brain barrier BUN Blood urea nitrogen B Boron BSA Bovine serum albumin Ca calcium °C Celsius cm Centimeter CRM Certified reference material ClustalW Clustal Omega CVF Cobra venom factor CID Collision induced dissociation cDNA Complementary DNA CK-MB Creatine kinase Co Cobalt Cu Copper Ci Curie CRISP Cysteine rich secretory protein CTX Cytotoxin dGDP Deoxy guanosine diphosphate DTT Dithiothreitol DMSO Dimethyl sulfoxide ESI-MS Electrospray ionization – mass spectrometer EDTA Ethylenediaminetetraacetic acid ECM Extra cellular matrix
xxxv FMN Flavin mononucleotide GGT Ɣ-glutamyl transferase GPCR G-protein coupled receptor G6P-DH Glucose-6-phosphate dehydrogenase GLDH Glutamate-dehydrogenase gm Gram IC50 Half Inhibitory concentration HMP Hemorrhagic metalloproteinase HK Hexokinase h Hour HRBCs Human red blood corpuscles HCl Hydrochloric acid H2O2 Hydrogen peroxide ICP-OES Inductive couple plasma-optical emission spectroscopy ID/g Injected dose per gram IFCC International Federation of Clinical Chemistry and Laboratory medicine i.c.v Intracerebroventricular i.m Intramuscular i.p Intraperitoneal i.v Intravenous Fe Iron K Kelvin Kg Kilogram KDa Kilo dalton LAAO L-amino acid oxidase LDH Lactate dehydrogenase LD50 Lethal dose that kills 50% of population L / l Liter LC Liquid chromatography LOD Limit of detection LNTX Long chain neurotoxin mPa Magapascal MDH Malate dehydrogenase MgSO4 Magnesium sulphate Mg Magnesium MMP Mammalian metalloproteinase Mn Manganese m/z Mass over charge ratio m Meter µl Microliter µg Microgram ml Milliliter mg Milligram mg/dl Milligram per deciliter mM Milli molar µmol Micromole
xxxvi min Minute M Molar concentration mol Mole 99Mo Molybdenum-99 MTLP Muscarinic-toxin like protein mAChRs Muscarinic acetyl choline receptors N Normal concentration NCBI National Center for Biotechnology Information nm Nanometer NIST National Institute of Standards and Technology NGF Nerve growth factor NADH Nicotinamide adenine dinucleotide (reduced form) nAChRs Nicotinic acetyl choline receptors HNO3 Nitric acid nr Non-redundant 5ʹ-ND 5ʹ-nucleotidase ppb Parts per billion p-NP p-nitrophenyl P Phosphorus % ID/g Percentage radioactivity per gram TcO4 Pertechnetate PPP Platelet poor plasma PRP Platelet rich plasma PDE Phosphodiesterase PLA Phospholipase PL Phospholipids PAGE Polyacrylamide gel electrophoresis psi Pounds per square inch PT Prothrombin time r Correlation coefficient Mr Relative molecular mass RP-HPLC Reverse phase high pressure liquid chromatography rpm Revolution per minute sec Second SNTX Short chain neurotoxin SPECT Single photon emission computed tomography SEC Size exclusion chromatography SVMP Snake venom metalloproteinase NaCl Sodium chloride SDS Sodium dodecylsulpahte NaOH Sodium hydroxide 99m Tc-NaTcO4 Sodium pertechnetate
SnCl2.2H2O Stannous chloride dihydrate s.c Subcutaneous
H2SO4 Sulphuric acid Ta Tantalum
xxxvii 99mTc Technetium 99m 3FTX Three finger toxin TT Thrombin time TFA Trifluoroacetic acid U/L Unit per liter vNGF Venom nerve growth factor V Volt W Watt H2O Water WNTX Weak neurotoxin ZMP Zinc depended metalloproteinase Zn Zinc Zr Zirconium
xxxviii
Chapter 1
Introduction
1 1.1Asiatic Cobras (Classification & Distribution)
Fright from snakes is as primordial as human history because they were being considered imperative by all civilization. Some people considered them sacred and worshipped while others abhorred them due to their threat to the human life. Certainly deadly venomous snakes are actual reason for their notoriety abundantly found from 4000 m high altitudes to the 100 m deep seas between 50°N and 50°S in western hemisphere and as 65°N and 50°S in the eastern hemisphere (Warrell, 2010). However interestingly few places have been found totally devoid of venomous snakes. Among these Antarctic, islands of Madagascar, Altantic & Caribbean, New Caledonia & New Zeeland, Hawaii & other pacific islands are included. It has been estimated out of 3000 snake species more than three hundreds are deadly venomous. Later on these species were grouped into Colubridae, Viperidae, Elapidae, Hydrophidae and Atractaspididae families. They belonged to the phylum Chordata, subphylum Vertebrata, class Reptilia, order Squamata and suborder Serpentes (Matsui et al., 2000; Jethanand, 2006).
Indeed snakes are considered very hazardous owing to their venom primarily secreted for capturing of a prey or as a defensive weapon. Fangs are designed to metered discharge of venom but it largely depends on the size of the prey(s). Interestingly few snakes (spiting cobras) have intrinsic ability to spit their venom up to a distance of three meters in to the eyes of the victim(s). Fags are therefore modified into a discharge orifice to perform this action (Fig 1.1). Venom is an extremely helpful secretion of venomous snakes not only used for killing but also useful in the digestion of prey (McCue, 2005; Jethanand, 2006).
Initially Wuster has been tried to categorize variety of cobras’ (Elapides: genus Naja) abundantly found in Asia and called them Naja naja species complex. Recent literature revealed that there are ten full species of Asiatic Naja. Among these N. atra (Chinese cobra), N. kaouthia (Monocellate cobra), N. naja (Indian spectacled cobra), N. oxiana (Central Asian cobra), N. philippinensis (Philippine cobra), N. sagittifera (Andaman cobra), N. samarensis (Visayan cobra), N. siamensis (Indochinese spitting cobra), N. sputatrix (southern Indonesian spitting cobra), N. sumatrana (Equatorial spitting cobra) are included. N. naja (Indian spectacled cobra) are found by different names in literature
2 according to geographical distribution. These are N. n. naja (Indian cobra), N. n. oxiana (pattern less species from northern India), N. n. indusi (NW India and in northern Pakistan), N. n. karachiensis (pattern less black southern Pakistan cobra), N. n. polyocellata (Sri Lanka) and N. n. caeca, (pattern less species reported from northern India) (Wuster, 1996). Interestingly, some other venomous snakes are also considered in Elapids. They are not belonging to genus Naja (true cobras) rather the word cobra in its common name. One of such example is king cobra (Ophiophagus hannah) abundantly reported from Southeast Asian countries (Jethanand, 2006).
Fig 1.1 Apparatus of a Poisonous Snake (adopted from snake copyright 1982. Marcel Decker Inc).
3 1.2 Black Pakistan Cobra (Naja naja karachiensis)
Like other countries of the world Pakistan is a hub of diverse variety of snakes. Venomous snakes’ inhabitant here primarily belongs to Elapidae, Viperidae and Hydrophidae families. Indeed eighty different species of snakes are quite abundant in Pakistan however fifteen of them are extremely toxic. Among them Elapids represented five different species while vipers’ categorized into seven distinct species/subspecies however rest of other is considered as sea snakes (Feroze et al., 2010). Genus Naja (Elapids) is represented by two species in Pakistan; Naja naja naja (Indian spectacled cobra) and Naja naja oxiana (central/brown cobra). They are quite common and constitute the most noxious members of genus Naja however lethality of Naja naja karachiensis (subspecies of genus Naja) can’t be overlooked. They are abundantly found in Cholistan desert and reported as a nightmare for the inhabitants of southern Punjab province of Pakistan as shown in fig 1.2 (Razi et al., 2011).
Fig 1.2 Naja naja karachiensis / Black Pakistan Cobra (adopted from Asad et al.,
2014(e)).
1.3 Snake Bite and Naja naja karachiensis Envenomation
Snake bite envenomation is a global occupational hazard that accounts for tens of thousands of deaths every year. It is the most neglected tropical and subtropical disorder annually affects 2.5 millions of people end up with 100 000 of deaths (Asad et al.,
2014(b)). According to a global survey report victims of snake bite belonging to the South
4 Asian countries are significantly higher than several regions of the world. It was noticed that snake bite incidence/100,000 ranges from 7.84 to 29.94 that is fairly elevated when compared with Europe, Australasia, Caribbean, North Africa/Middle East, Latin America (Southern), Sub-Saharan Africa (Southern) and even Central & East Asian countries (Kasturiratne et al., 2008). So far the highest deaths annually have been reported for Asia (15,385–57,636) as compared to Australasia (229–520), Europe (48–128), Latin America (647–3,459), North Africa/Middle East (43–78), North America (5–7) and Sub-Saharan Africa (3,529–32,117) regions. Moreover rural populations were found to carry largest burden of it due to the lack of proper health facilities along with ignorance about snake bite control program (Kasturiratne et al., 2008). Pakistan is one of the south Asian countries where numerous cases of snake bite have been reported previously particularly from Southern Punjab. Naja naja karachiensis is one the deadly venomous snake repeatedly documented as a consequence of 20,000 deaths annually in Pakistan (Asad et al., 2014(e)). Victims who survive from snake bite have to face diverse complications like edema, necrosis, severe pain, nausea, vomiting, respiratory muscle paralysis, headache, hypotension, cardiac arrhythmias (cardiac arrest), coagulopathies (prolonged PT, aPTT and TT), wounds bleeding, hematuria, proteinuria, mucus discharge, bleeding gums, high serum urea & creatinine, altered consciousness along with numerous physical handicaps and sequela (Asad et al., 2014(d); Razi et al., 2011).
1.4 Composition of Snake Venom
For therapeutic and scientific purposes snakes venom act as a gold mine saturated with biologically active prototypes compounds. Generally components of snake’s venoms have been classified into haemotoxins (degrade endothelium basement membrane lead to hemorrhage), myotoxins (damage muscle fibers to cause permanent tissue loss/amputation), cardiotoxins (produce cystolic heart arrest with cytotoxicity) and neurotoxins (antgonoize nicotinic (Ach) receptors with blocking of K+ currents and neurotransmitter) (Jethanand, 2006). Indeed it is a complex concoction of enzymatic and non enzymatic superfamilies. Being Elapides Cobra venom possesses 25%-70% of
5 enzymatic proteins (Hider et al., 1991). Among them Phospholipases, L-amino oxidases, phosphodiesterases, 5ˈ-nucleotidases, alkaline phosphatases, acetylcholinesterases, ribonucleases, serine proteases, metalloproteinases and cobra venom factors are included. Non enzymatic proteins consist of serine protease inhibitors (SPIs), three finger toxins (3FTXs), disintegrins, helvepryns, C-type lectin proteins (CLPs), sarafotoxins, waprins, vespryns and nerve growth factors. Apart of it carbohydrates, metal ions (Mg++, Ca++, Zn++ and Cu++), lipids and biological amines are also found in snakes’ venom. Concentration of enzymes in venom is hard to measure therefore biological activities are the only way to confirm their actions via various substrates. Biological activities mainly concerned with enzymatic proteins rather than non enzymatic part (Tu, 1977; Hider et al., 1991; Roy, 2011; Jethanand, 2006). In the following paragraphs, the significance of five enzymes will be addressed which have been focused in this project. These are phospholipase A2, protease, alkaline phosphatase, 5ˈ-nucleotidase and hyaluronidase.
1.4.1 Phospholipase A2
Phospholipase A2 (PLA2) is one of the superfamilies of snake venom enzymes having molecular mass ranging from 10 KDa to 15 KDa. They are of several types however possessing two properties in common: 1) they don’t cross blood brain barrier (BBB) therefore consider harmless for central nervous system and 2) they are mainly attributed for necrosis. Due to their difference in action at the site they are classified into phospholipase A1, phospholipase A2, phospholipase C and D. PLA1 hydrolyses 1-acyl group while PLA2 hydrolyses 2-acyl group with central part (Fig 1.3). Phosphodiester linkages are usually hydrolyzed by phospholipase C and D. (Asad et al., 2014(b); Montecucco et al., 2008).
PLA2 hydrolyses phospholipids (PLs) of the membrane and releases lysophospholipids and other fatty acids e.g., arcidonic acid. Lysophospholipids destroy red blood cells (RBCs) indirectly and directly degrade PLs within their membranes thus results in anemia and hypovolemic shock (Lee, 1979; Chippaux, 2006). Arachidonic acid is a precursor of various inflammatory mediators like prostaglandins and histamine which are attributed for vasodilatation and redness in victims. Apart of it prostaglandins is also
6 reported to intensify the responses of pain mediators (mainly bradykinin) and to accelerate the actions of thromboxane which causes acute inflammation (Hutt and Houghton, 1998; Sanchez and Rodriguez-Acosta, 2008). Anticoagulant role of venom
PLA2 is quite prominent as reported previously by several researchers. Basic PLA2 are stronger anticoagulant in action than neutral PLA2. However former produces anticoagulant effect by inhibition of both extrinsic tenase and prothrombinase complex while later produces this effect only by interruption at extrinsic tenase complex pathway.
Basic PLA2 binds to the activated factor X therefore inhibits further activation of factor V in blood coagulation pathway (Kerns et al., 1999). Anticoagulation is also attributed to less cellular release of cAMP (secondary messenger) which is responsible for transmission of hormonal signaling and facilitates the platelet aggregation. Deleterious actions of PLA2 are hemorrhage, coagulopathies, myotoxicity, oedema, necrosis and malfunctioning of the liver and kidneys of the victims. Generally most of the cases related to elapids (cobras) bite are responsible for death of the victims (Asad et al.,
2014(d); Chippaux, 2006; Chippaux, 1998; Montecucco et al., 2008).
Fig 1.3 Represents Hydrolysis of Phospholipids by the Action of Phospholipases A2.
++ PLA2 is reported as Ca dependent enzyme and usually produces its effect via enzyme- ++ Ca -substrate formation. However, monomers and dimmers of PLA2 are quite recognized which influence the catalytic action of phospholipases (Evans et al., 1980;
Iwanaga and Suzuki, 1979). Most of the snake venoms contain large number of PLA2 isoenzymes which clarify their difference in pharmacological and enzymatic actions. Up till now lot of PLA2 enzymes have been studied not only for toxicity determination and but also to identify mammalian protein targets (which bind to these enzymes) and for
7 ligands identification (Jethanand, 2006). It is therefore quite interesting and scope of the thesis to determine its role in Naja naja karachiensis envenomation.
1.4.2 Protease
Snake venom proteases are attributed to hydrolyze peptide bonds within the protein molecules. Generally they are classified into endopeptidases (hydrolyze peptide bonds within protein molecule) and exopeptidases (hydrolyze protein from C and N termini) as shown in fig 1.4. Broadly speaking they can be categorized into metallo, serine, cysteine, aspartic acid and threonine proteases. On the basis of structural domain snake venom metalloproteinases (SVMPs) are of four types: 1) P-I with only metalloproteinase domain; 2) P-II with metalloproteinases and disintegrin domains; 3) P-III with metalloproteinases, cysteine and disintegrin domains; 4) P-IV with all P-III domains and additionally lectin like peptide attached to the polypeptide chain of metalloproteinases by a disulphide linkages. Due to these differences SVMPs are found with molecular weight ranges from 22 to 100 KDa (Priolo et al., 2000; Jethanand, 2006).
Fig 1.4 General Representation of Mechanism of Protease Enzyme to Hydrolyze Protein Molecule.
Serine and metalloproteinase both are considered the most toxic enzymes in snake’s venom. Serine proteases are associated with normal physiological functions of human body. They are structurally related to the thrombin (however don’t inhibit by heparin) and acting as competitive agent for hydrolysis of fibrinogen. They are involved in blood coagulation, provoke immune responses and to assist in digestion. Immune responses are due to complement system and lead to the production of histamine and bradykinin therefore induces acute inflammation. Activation of cellular immune response further
8 stimulates the release of cytokine and tissue necrosis factor facilitating acute inflammation (Hedstrom, 2002; Chippaux, 2006; Rojnuckarin et al., 2006). Mtalloproteases are Zn++ dependent enzymes however Ca++ is also required for their structural stability. Furthermore, strong acidic condition (pH ˂ 3) will lead to cessation of their enzymatic activity (Chippaux, 1998). Metalloproteases destroy basement membrane of the endothelium lead to the extravasation of blood and usually appears as rashes and edema. Damage to the blood vessel later on promotes blood coagulation cascade pathway. Generally degradation of extracellular matrix (ECM) by proteases end up with interruption of several vital processes like cell differentiation and proliferation, their migration as well as cell-cell interaction and their death. It is well known accepted that damaging effects on ECM further provoke tissue anoxia and gangrene (Girish and Kemparaju, 2011; Chippaux, 2006; Elkington et al., 2005). Literature review revealed that various metalloproteinases have been isolated from various snakes’ venom. They are employed previously as a powerful tool to understand several complicated processes such as: 1) a useful candidate to treat vascular thrombotic diseases e.g., mutalysin from Lachesis muta muta (bushmaster snake) venom; 2) a tool for research into anti venom therapy e.g., jararhagin from Bothrops jaracara venom; 3) a tool to study exogenous mammalian metalloproteinases (MMPs) e.g., graminelysin from Trimeresurus gramineus venom (Jethanand, 2006).
1.4.3 Phosphatase (Alkaline Phosphatase)
Among variety of hydrolytic enzymes phosphatases are ubiquitously present in approximately all sort of snake’s venom. They are apparent in two different enzymatic activities viz., acid phosphatase (E.C.3.1.3.1) and alkaline phosphatase (ALPase) (E.C.3.1.3.2). Former is active at pH 5 while later is active at pH 9.5. ALPase has been found more abundant in crotalid and elapid (cobras) venoms. They are metalloenzymes with molecular weight usually ˃90 KDa. Metal ion chelator (EDTA) is best known to inhibit their activities (Dhananjaya and D’Souza, 2010).
ALPase hydrolyzes phosphate esters (ribo and deoxy ribonucleotides) non-specifically and further provokes their deleterious effects. They usually hydrolyze 5’-phosphoribose
9 1-pyrophosphate, deoxy-dinucleotide phosphates, 5ʹAMP, 3ʹAMP, ribose 3-phosphate, ATP, 5ʹ-dAMP, FMN and dGDP. Acid phosphatase is different from ALPase and hydrolyses glycerophosphates, glucose -6- phosphate and glucose-1-phosphate
(Dhananjaya and D’Souza, 2010; Asad et al., 2014(a)). Initially they were engrossed less attention by toxinologists owing to their digestive and non toxic role. However recently there is renewed interest among scientists due to their endogenously release of multitoxin ‘purines’. Purines produce their effects via purinergic signaling in case of snake venom poisoning. Various snake venom enzymes like cardiotoxin, cytotoxin and phospholipases assist in cell necrosis and resulted in production of nucleic acids. Later on they are cleaved by ALPases along with nucleotidases led to the formation of multitoxin ‘adenosine’. ALPases also act on ATP in presence of phosphodiesterases (PDE) and resulted in generation of adenosine (Fig 1.5). Adenosine induces miscellaneous toxic effects like cardiac arrest, eczema, inflammation, hypotension, reverse platelet aggregation, unconsciousness and kidney damage via A1, A2a, A2b and A3 (adenosine) receptors in the victims (figure 1.5) (Asad et al., 2014(a)). As far as acid phosphatase is concerned; it is still believed as an allergen and induces the release of histamine (Asad et al., 2014(a)). Due to these severe lethal effects it is the scope of this thesis to study (characterize biologically) these enzymes in detail such that their deadly venomous role is clearly established.
1.4.4 5ʹ-Nucleotidase
One of the widely distributed enzymes among venomous snake taxa is 5ʹ-nucleotidase. They were abundantly reported from various venomous species belonging to the family; elapidae, cortalidae and viperidae. However venoms of cortalidae possessed significantly (2.3 fold) higher 5ʹ- nucleotidases activity as compared to elapidae and viperidae venoms. They were also reported from other animal tissues (bull seminal plasma & mammalian tissues) and the name 5ʹ-nucleotidases was given by Ries in 1934 (Bragdon and McManus, 1952; Dhananjaya et al., 2010). 5ʹ-nucleotidases enzymes are considered to hydrolyze 5ʹ-nucleotides and play a pivotal role in nucleotide metabolism however complete information about their biological activities is still unclear. They digest ribose- 5-phosphate and other phosphate esters at a slow and extremely slow rate respectively.
10 Boffa and Boffa (1974) for the first time reported their role as an anti-platelet aggregator. Since then they were believed to halt blood coagulation cascade pathway synergistically with the help of other toxins like disintegrins, ADPases and PLA2 (Dhananjaya et al., 2006; Koshland and Springhorn, 1956). Furthermore Dhananjaya et al. (2010) proved this enzyme to exist in multimeric forms (glycoprotein or polypeptide) by the use of 5ʹ– nucleotidase activity inhibitor; concanavalin (Con-A). Additionally their deleterious role in snake venom poisoning is considered due to the release of purines- a multitoxin; however, this idea is still not confirmed by pharmacological experimental efforts (Fig 1.6). This may be due to their less concentration in venoms (˂0.3%) and loss of many products during purification processes (Dhananjaya et al., 2010). On the basis of this information it is inevitable to explore the role of 5ʹ-nucleotidases in snake bite cases and to eradicate them for complete and effective treatment of snake bite.
1.4.5 Hyaluronidase
Venom hyaluronidase commonly known as ‘spreading factor’ is one of the key enzymes attributed for degradation of extracellular matrix in the victims. It causes local tissue damage (necrosis) and inflammation by cleavage of internal glycosidic bonds of mucopolysaccharide (hyaluronan) [D-glucoronic CID (1-β-3) N-acetyl-D-glucosamine (1- β-4)], which acts as a substrate (Kreil, 1995). Indeed hyaluronic acid absorbs water to maintain extracellular matrix lubricated hence imparts viscoelastic property (El-Safory et al., 2010). Snake venom intoxication (hyaluronidase) causes hyaluronic acid to breakdown into smaller tetrasaccharide fragments (chondroitin & chondroitin -4- and -6-sulfate) which is attributed for angiogenic, inflammatory and immunostimulating responses in the victims (Fig 1.7) (Girish et., 2004; Kemparaju and Girish, 2006; El-Safory et al., 2010). Hyaluronidase was reported previously in various snake’s venom like Agkistrodon contortix contortix, Agkistrodon acutus, Vipera russelli and Naja naja however, few studies have been conducted for their isolation and characterization. Recently it has been recognized that impact of hyaluroniadse can’t be overlooked. It is an attractive tool clinically due to substrate (hyaluronic acid) involvement in several vital processes like cancer progression, cell motility, fertilization, inflammation, wood healing and embryogenesis. Moreover, hyaluronidase administration is extremely helpful to prevent oedema (post organ
11 transplantation), tissue destruction (neonates) and in closure of infected wounds (Jethanand, 2006). It is therefore the need of the time to search various sources for hyaluronidases to characterization and discover their role(s) in several awful complications like cancer and snake bite research.
Fig 1.5 Possible Mechanism for Induction of Alkaline Phosphatase Toxicities Post Naja naja karachiensis Envenomation.
12
Fig 1.6 Possible Deleterious Mechanism of Action of 5ʹ-Nucleotidase Enzyme Post Naja naja karachiensis Envenomation.
Fig 1.7 Describes the Structure of Hyaluronan and Site of Action of Hyaluronidase Enzyme.
13 1.5 Surrogate Markers for Cobra Venom Toxicity Determination
Naja naja karachiensis envenomation hits more drastically to the liver, heart and kidneys in the victims (Asad et al., 2014d). These important organs play several tremendous roles to sustain the life of an individual. Liver acts as a power house and performs several vital functions in the human body such as degradation of fat, glycogen formation, production of certain amino acids and urea, detoxification as well as maintenance of blood glucose level. Alanine aminotransferases (ALT) and aspartic aminotransferases (AST) are the major hepatic enzymes attributed for transfer of α-amino groups to the keto group for the production of pyruvic and oxaloacetic acid respectively. ALT is a specific indicator of hepatic injury abundantly found in liver, heart, kidneys, pancreas and skeleton muscles whereas AST is more widely distributed in the body. Alkaline phosphates (AP) and Ɣ- glutamyl transferase (GGT) are indicators of liver malfunction and considered reliable indicators for jaundice, cholestasis and related dysfunctions (Franca et al., 2009). Myotoxic action of cobra venom could be easily recognized by the presence of certain cardiac injury markers. Among them creatine kinase (CK-MB), creatine phosphokinase and lactate dehydrogenase (LDH) are the most important surrogate markers mainly found in the serum of victims. Similarly damage to the renal system could be accessed promptly by measuring the serum levels for urea, creatinine and BUN (Franca et al., 2009; Asad et al., 2014d).
1.6 Proteomics as a Tool to Study Snake Venom Components
Recent advances in the field of proteomic studies have opened a new arena for characterization and immunological profiling of toxins even of minute amount of proteins. Due to limitation in the database depository there is always a need in expansion of proteomics and genomics databases by identification and purification of novel peptides/proteins and toxins. The detailed knowledge of snake venom components not only helped the clinicians in prompt understanding of pathogenesis of envenomation but also provided reasonable guidelines in production of effective antidotes. Recent insight in proteomics has improved in-depth understanding of knowledge to discover new potent drugs and to develop latest biomedical tools (Yap et al., 2014; Malih et al., 2014). Snake
14 venom is an intricate concoction of various compounds. Molecular weight of a desired compound(s) can be acquired via liquid chromatography (LC) coupled with mass spectrometer (MS). Electron spray ionization- mass spectrometry (ESI-MS) adequately allows ionization of all well separated tryptic digestive peptides (macromolecules) at sufficient alternate current voltage and radio frequency. By comparing molecular weight of a desired compound(s) to various classes of proteins, we can guess the identity of a desired protein(s). It is therefore a valuable method to characterize unknown protein and have superseded over traditional gel electrophoresis (SDS-PAGE) technique(s). Automated Edman degradation technique allows us to determine exact sequence of a purified protein which is enough to determine primary structure and to calculate exact molecular mass (Jethanand, 2006). Present study encompasses classical biochemical techniques coupled with mass spectrometry to enlist various components of Naja naja karachiensis venom.
1.7 Radio Labeling Technique to Observe Biodistribution of Venom
1.7.1 Technetium: A Radionuclide & its Generation
In the past several years radionuclides have been used extensively in the field of nuclear medicine primarily for diagnosis and therapeutic purposes. They are produced in the cyclotron or reactor and categorized into short and long lived radionuclides. Due to ease in large doses of administration with minimal radiation dose along with excellent image quality, short lived radioisotopes are considered more significant than long lived radionuclides. These salient features have led to the development of radionuclide generators as a convenient source for their production (Colombetti, 1983; Saha, 1984). Typical generator consists of a glass column fitted with fritted disc at the bottom and parent nuclide is usually adsorbed on the material filled within the column. As a result of decay in parent nuclide daughter radionuclide grows until transient or secular equilibrium is reached within its several half lives and ultimately it decays as parent nuclide (same half life). Appropriate eluting solvent is required for elution of daughter activity in a carrier free state and it can be made repeatedly as daughter activity grows again within the column. Vial filled with required solvent (eluant) is usually inverted onto the needle
15 A. Vacuum in the evacuated vial on needle B draws the solvent through the column and results in the elution of daughter nuclide as presented by typical generator system in the figure given below (Saha, 1984). Moly or molybdenum-99 technetium-99 (99Mo-99mTc) generator (Fig 1.8) is one of the examples of frequently used generators to extract short 99m 99m life technetium-99m ( Tc) in the form of sodium pertechnetate (Na TcO4) with 0.9% NaCl solution. 99Mo has half life of 67 hours and decays by beta emission; 87% decay goes to metastable state (99mTc) and 13% to the ground state 99Tc (Baker, 1971; Tippetts and Kennedy, 1969; Saha, 1984).
Fig 1.8 Represents Typical Generator System (Left) & a 99Mo-99mTc Generator (Right) (Courtesy of E.R. Squibb & Sons, Inc.).
1.7.2 General Properties of Technetium
Indeed technetium (atomic number 43) is a transition metal belonging to the group VIIB in the periodic table of elements. It is eluted easily from 99Mo-99mTc generator (as described above) and readily available in sterile, carrier free and pyrogen free forms. Technetium has no stable isotope however 7+ and 4+ are the most common oxidation states. 99m Tc is the most stable in the form of pertechnetate ion at oxidation state of 7+. It has half life of 6 hours and decays to 99Tc by gamma transition of 140 keV which is ideal
16 for current generation of imaging devices in nuclear medicine practices. Chemistry of technetium is still hard to explore and poses an intriguing challenging task to the radio chemist/pharmacist due to its minute carrier free amount (~10-9) in various 99mTc labeled compounds. Chemical behavior of technetium has been studied enormously at molar level ranges from 10-4 to 10-5. However it is still considered hard to extrapolate these finding at micro or pico molar levels (Saha, 1984; Asad et al., 2015).
1.7.3 Labeling of Compounds (Snake Venom Proteins) with 99mTc
In the few decades the need of selected compounds labeled with radiolnucliodes has grown tremendously in different fields of science particularly medical science. Among these Ɣ-emitting radionuclides have much wider applications than β-emitting isotopes. Since several years tagging of snake venom protein(s) with the selected radionuclide has been introduced by incorporation of a foreign label via chelate formation. Technetium is a transition metal and has natural tendency to form a chelate with different chemicals groups such as –SH within the proteins. These groups donate lone pair of electrons to form coordinate covalent bonds with 99mTc (Asad et al., 2015; Shirmardi et al., 2010).
99m - Chemically pertechnetate ions ( TcO4 ) are nonreactive in nature and addition of a reducing agent is required to bring down oxidation state of 99mTc from 7+ to lower one (3+, 4+ or 5+) usually by the use of concentrated HCl, ferrous sulphate, dithionite, stannous chloride dihydrate, sodium borohydrate, ascorbic acid and ferric chloride. Routinely technetium-99m is reduced to 4+ state by Sn2+ salt in acidic condition (Saha, 1984). The chemical reactions occur in acidic condition by stannous chloride can be stated as follows:
3 Sn 2+ → 3 Sn 4+ + 6 e- (1-1)
99m - + - 99m 4+ 2 TcO4 + 16 H + 6 e → 2 Tc + 8 H2 O (1-2)
By combining the two equations, one has
99m - + 2+ 99m 4+ 4+ 2 TcO4 + 16 H + 3 Sn → 2 Tc + 3 Sn + 8 H2 O (1-3)
17 Interestingly, as the number of technetium atoms within eluate is very little (~10-9 M), therefore small amount of Sn2+ is sufficient for reduction of 99mTc. However enough quantity of Sn2+ should be added within reaction mixture to ensure complete reduction of technetium-99m (Fig 1.9). Hydrolysis of stannous chloride at pH 6-7 leads to the formation of insoluble colloids which compete with chelating agents in the labeling procedure. Care must be exercised by the addition of an acid along with enough quantity of chelating agents to circumvent the process of hydrolysis. Subsequent labeling procedure thin layer chromatographic techniques are recommended to estimate three
99m 99m - Tc species: (1) Free form that has not been reduced ( TcO4 ), (2) hydrolyzed fraction
99m that didn’t react with chelating agent ( TcO2) and (3) bound fraction of desired labeled compound. Free and hydrolyzed fractions of radioactivity are useless and must be removed or minimized to such an extent that they don’t interfere with diagnosis test in question (Asad et al., 2015; Saha, 1984).
Fig 1.9 Represents Chemical Basis for Binding of 99mTc with Naja naja karachiensis Venom (adopted from Asad et al., 2015).
18 1.8 Antidote(s) to Neutralize Snake Venom Toxicity
1.8.1 Immunoglobulin’s (Equines Anti-sera) Therapy
Although administration of equines immunoglobulin’s (anti-sera) is an accepted way to treat snake bite cases all over the world. However anaphylactic shock, pyrogenic reactions, late serum sickness and less protective coverage against venom induced necrosis and local tissue damage have shown some limitations of anti-sera. Moreover tedious production, limited supply and strict storage conditions further added in worries. To overcome these difficulties alternate appropriate treatment(s) is inevitable that should be available as a first aid treatment for victims during hospitalization (Razi et al., 2011).
1.8.2 Medicinal Plants: Cheap Alternate Source to Treat Snake Venom Toxicity
This world is gifted with a rich capital of medicinal plants and record of these is as aged as human society. Medicinal plants have been utilized to overcome all sorts of problems since time unmemorable in all known societies. According to the World Health Organization (WHO) 80% of the world’s population relay on medicinal plants for their health related problems and 11% drugs are exclusively from plant origin. They have been used for food, shelter, clothes and therapeutic purposes (Rates, 2001; Khan, 2006). According to OPS medicinal plant is any plant which is used to mitigate, cure or to treat an ailment via an alteration in the physiological or pathological processes hence act as a source of drug(s) or precursor(s) (Arias, 1999). Crude extracts, standard enriched fractions (phytopharmaceutical preparations) or natural compounds isolated from these sources have been proved scaffold for modern therapeutic sciences therefore enabled us to develop empirical system of medicines. Natural compounds can be source of leads for rational planning, design and biomimetic synthetic development of new drugs (Hamburger and Hostettmann, 1991). Now a day there has been an increasing tendency in alternative treatment by the use of natural products that derived particularly from plants origin. It is primarily due to abusiveness, severe adverse effects and incorrect/ineffective use of synthetic drugs. Secondly lot of people does not have access to conventional medicine beside that ecological and folklore survey of medicines propose
19 nontoxic behavior of natural products. Economic and social concepts of health services, market requirements of pharmaceuticals and research on medicinal plants have opened new avenues for development of new drugs (Elisabetsky, 1987; Calixto, 1996). In Pakistan medicinal plants are quite ubiquitous and have been used frequently to solve various health related problems particularly snake bite (as an antidote). Striking parallelism between potential of medicinal plants with chemical constituents conferred their characteristics as an antidote and explained their proposed mechanism of action (Dhananjaya et al., 2006; Mors et al., 2000). It is therefore interesting to rationalize scientifically folklore claims about Pakistani medicinal plants as antidote to snake bite (Asad et al., 2011). Below follows a description of the plants that were selected for this project.
1.8.2.1 Albizia lebbeck (L.) Benth.
Albizia lebbeck (L.) Benth widely distributed in Pakistan. It belongs to the family Fabaceae having compound leaves with glabrous shoots. Pods are pale to brown in color. Flowers are bisexual and appear after new leaves. It is hermaphroditic and its length is usually from 15-20 m with diameter of 50 cm (Asad et al., 2011). It is beneficial in various ailments for instance; barks are effective in toothache, gum infections and for opthalmia. In piles and diarrhea barks and seeds are very useful. Leaves are used in bronchial asthma and allergy. The root is used in hemicranias. Extract of Pods of this plant is used as hypoglycemic, anticancer, antimicrobial, antifertility and antiprotozoal. It possesses anticonvulsant, nootropic and anxiolytic activities. Apart of it all parts of this plant is recommended in snake bite (Saha and Ahmed, 2009; Mahmood et al, 2012; Pathak et al., 2010). In order to test anti venom potentials seeds were collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of May, from division Bahawalpur, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.381) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.10). It has variety of chemical constituents but few are very much important like albigenin (a triterpene from bean), albigenic acid (a triterpenoid sapogenin from bean) and albiziahexoside, saponin from bark (Pathak et al., 2010). Alkaloid, tannins, carbohydrate,
20 flavanoids, proteins, echinocystic acid and amino acids are major constituents of this plant (Asad et al., 2011).
Fig 1.10 Describes the Contour of Albizia lebbeck (L.) Benth with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.2 Allium cepa L.
Allium cepa L. (family Amaryllidaceae) is the most widely cultivated specie among the genus 3Allium. It is biennial condiment crop but can be triennial or a perennial. It has two phases viz, first phase from seed to bulb formation followed by reproductive phase, for production of seeds. Flowers of onion after pollination developed into seeds. Onions above ground leaves are tubular and green. But bulb of the onion is also considered as modified leaves. In Pakistan it is famous for all cuisine and culinary preparations to enliven them. Its importance in health care can’t be neglected since it has tremendous role in lowering blood pressure and cholesterol due to organosulfur compounds. It is effective in the prevention of artherosclerosis and coronary heart diseases (CHF) by inhibition of platelet aggregation to form clot. It is anti-rheumatic, diuretic, heart stimulant and good for eyes (Karimi et al., 2012; Shah et al., 2011). Paste of its bulb is recommended in snake bite (Makhija and Khamar, 2010). For anti venom activity bulbs were collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of April,
21 from district Bhakkar, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.42) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.11). Main active constituent is allyl propyl disulfide, APDS (Kumari et al., 1995). It is free of fat and cholesterol and its bulb abundant in proteins (1.2 grams), fiber (0.6 grams), carbohydrates (11 grams) and water contents (86.8 grams) per hundred grams of material. Vitamins like A (0.012 mg), C (11 mg), riboflavin (0.01 mg), niacin (0.2 mg), thiamine (0.08 mg) and minerals for instance, Ca (27 mg), K (157 mg), Fe (0.7 mg), P (39 mg) and Na (1.0 mg) are also reported in them per hundred grams of material (Anon, 1978).
Fig 1.11 Describes the Contour of Allium cepa L with Specific Part (Bulbs) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.3 Allium sativum L.
Allium sativum L belongs to the family Amaryllidaceae and perennial in nature. This plant can attain a height of two feet. The leaves of this plant are resembled with grass. They are long and narrow and six to twelve in number. Bulbs are composed of 4-20 bulblets (cloves). The flowers of this plant are whitish or umbel in color at the end of the stalk originating from bulb. It is hermaphrodite and propagated by cloves. Garlic has tremendous role in health care system. It has been proved useful to treat cardiovascular diseases, cancer, malaria, asthma, candidiasis, colds, and diabetes and to
22 improve immunity. It has been shown marvelous effects against Citrobacter, Enterobacter, Pseudomonas, Kilabsella and Bacillus anthrax. Antifungal, antiseptic, antihelmantic, antiviral and anti-inflammatory are the salient properties of Allium sativum bulb extract (Daka, 2011). Besides all these activities crushed bulbs are recommended ethobotanically in the treatment of snake bite (Ugulu, 2011). Therefore Bulbs were collected to test against snake venom under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of June, from district Bhakkar, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.46) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.12). Major constituent of this plant is allicin found in the cloves. However, thiosulfinates, anthocyanins, 2 mercapto-L-cysteins, polysaccharides, allinase, sativin I and II, quercetin, glycosides of kaempferol and scordinines A & B are other famous constituents reported in the literature (Rahman et al., 2012; www.garlic-source.com).
Fig 1.12 Describes the Contour of Allium sativum L with Specific Part (Bulbs) Collected as Anti-Venom Along with Voucher Specimen.
23 1.8.2.4 Althaea officinalis L.
Althaea officinalis L. belongs to the family Malvaceae. It is perennial herb and grows up to the length of 2.2 meters. It is cultivated for medicinal uses and likes to grow in marshy and tidal areas. It has pink color flowers with white thick roots. Its leaves are heart- shaped. It is propagated by seeds (Prajapati et al., 2010). It is very effective medicinally. It has soothing effects on mucous membranes. Roots are used to combat stomach acid, ulcers, gastritis, inflammation, abscesses and for boils. It is very useful in constipation, different intestinal ailments, ileitis, colitis, diverticulitis and in irritable bowel syndrome. Leaves have clinical significance in cystitis and to treat frequent urination. Flowers (as infusion) are extremely effective to soothe inflamed areas. It brings relief in cough, asthma, catarrh and pleurisy (Prajapati et al., 2010). Its roots are recommended ethnobotanically in the treatment of snake poisoning (Asad et al., 2011). In order to proof snake venom poisoning roots were collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of July, from the area of Rawalpindi, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.411) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.13). Important chemical constituents abundantly found in the roots are asparagines, sucrose, phenolic acids, fatty oil, butyric acid, phytosterin, flavonoids, pectin (11%), starch (37%) and 11% mucilage (Prajapati et al., 2010; Asad et al., 2011).
1.8.2. 5 Bauhinia variegate L.
Bauhinia variegate L. belongs to the family Fabaceae. It is a deciduous tree ubiquitous in Pakistan. Leaves of this plant are 10-15 cm in length and are bilobed. Flowers of this plant are large and white or purple in color having fragrance. Fruits (pods) of this tree are fifteen to thirty centimeter in length. They are flat, dehiscent and possess 10-15 seeds. Bark is cracked and grey whilst wood is hard and grayish brown in color with irregular darker patches. Bauhinia variegate is propagated by seeds (Shinwari et al., 2007; Prajapati et al., 2010).
24 From medical point of view, its bark is anthelmintic, tonic, astringent and alterative hence useful in skin disorders, scrofula and ulcers. Bark also possesses acrid, vulnerary, anti- inflammatory and styptic properties. Buds have significance in piles, diarrhea, dysentery and in worms. Roots are extremely useful in dyspepsia and snake bite treatment (Shinwari et al., 2007). Therefore, for testing folk claim scientifically as an antidote of snake poisoning, roots were collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of November, from district Haripur, KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.374) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.14). Different constituents were reported in literature. Important constituents are gums, tannins are found in bark and fatty oil from seeds. Apart of it β- sitosterol, lupeol, kaempferol-3-glucoside and 5, 7- dehydroxy, and 5,7- dimethoxyflavanone-4-0-a-L-rhamnopyranosyl-β-D-glucopyranosides (Shinwari et al., 2007; Prajapati et al., 2010).
Fig 1.13 Describes the Contour of Althaea officinalis L with Specific Part (Roots) Collected as Anti-Venom Along with Voucher Specimen.
25
Fig 1.14 Describes the Contour of Bauhinia variegate L with Specific Part (Roots) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.6 Brassica nigra (L.) W. D. J. Koch
Brassica nigra (L.) W. D. J. Koch belongs to the family Brassicaceae. It is also called black mustard in English and rai in Urdu languages. It is branched hairy annual herb having leaves in 10-20 cm. Flowers are bright yellow in color with four sepal and petal and 8-13 mm in diameter. Flowers bloom in the spring. Seeds are oblong in shape (Baquar, 1989). Seeds are the most imperative part of this plant and recommended in different complications as stimulant, vescicant, rubifacient and as an antidote in snake poisoning (Baquar, 1989). In order to rationalize its use as antidote seeds were collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of May, from district Mansehra, KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.302) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.15). Plant contains different chemical constituents like glucoside, sinigrin and essential oil (Baquar, 1989).
26
Fig 1.15 Describes the Contour of Brassica nigra (L.) W. D. J. Koch with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.7 Calotropis procera (Aiton) W. T. Aiton
Calotropis procera (Aiton) W. T. Aiton belongs to the family Apocynaceae. It is commonly found throughout in Pakistan. It is erected shrub with many branches ascending from the base. Plant is covered with white tomentum releasing milky juice. Leaves are 5.0-12.5 cm long with waxy bloom. Flowers are 2-2.5 cm broad with whitish and purple tips. Seeds are found with fluffy hairs for scattering (Shinwari et al., 2007; Baquar, 1989). Plant has been reported previously for its effectiveness in different complications. It is used as anthelminic, expectorant and alterative. Bark is used to cure dysentery. Bark is also famous as cholagogue, diaphoretic, emetic and diuretic. Flowers of this plant are used as digestive, stomachic, purgative and for remedy of guinea worms. Fresh leaves are used in inflammations while dried leaves are used for smoke to cure asthma and cough. Root is effective as purgative. Hairs from seed are used for stuffing pillows. Milky latex is antispasmodic, nervine tonic and acts as an antidote against scorpion and snake venom. (Shinwari et al., 2007; Baquar, 1989; Asad et al., 2011). For testing of anti snake venom potentials flowers and latex were collected under the supervision of renowned botanist
27 Prof. Dr. Altaf Ahmad Dasti in the month of June, from the area of district Haripur, KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.566) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya- University, Multan, Pakistan (Fig 1.16). Important chemical constituents are calotropin, calotropagenin, uscharin, calotoxin, calactin, sterol, resin, oil (in seeds) and tannins are found in leaves (Shinwari et al., 2007; Baquar, 1989).
Fig 1.16 Describes the Contour of Calotropis procera (Aiton) W. T. Aiton with Specific Part (Flowers) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.8 Cedrus deodara (Roxb. ex D. Don) G. Don
Cedrus deodara (Roxb. ex D. Don) G. Don (family, Pinaceae) is a tall evergreen conifer tree found abundantly in northern areas of Pakistan. Its height is normally up to eighty five meters. This plant has needle shaped, sharp, triquetrous pointed leaves with spreading horizontal branches. Both male and female cones are solitary but male cones are cylindrical at the end of branchlets. This plant has rough and furrowed bark. It has pale brown seeds but wings are longer than the seeds. It is propagated by seeds (Prajapati et al., 2010). It has significant role in treatment of various health problems. Its leaves are thermogenic, acrid and extremely useful in inflammatory and tubercular glands. Its oil is diaphoretic,
28 depurative as well as diuretic and helpful in syphilis, leprosy, wounds, ulcers, skin diseases and in fever. Wood of this plant is very effective in pulmonary and urinary problems. It is also recommended in the treatment of snake bit (Baquar, 1989). Therefore, bark of this plant was collected in order to confirm scientifically, ethnobotanical claim as antidote in snake bite. It was gathered under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of September, from the area of Nathia Gali, KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.25) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya- University, Multan, Pakistan (Fig 1.17). Various constituents had been reported in the literature. Among them essential oil, cholesterin, gum, ascorbic acid, himachalol, allohimachalol, himadarol, cantdarol, cedrinoside dihydrodehydrodiconiferyl alcohol, cedrin, isocentdarol, dihydromyricetin, dewarene, dewarol, dewardiol and taxifolin are very important (Prajapati et al., 2010; Baquar, 1989).
Fig 1.17 Describes the Contour of Cedrus deodara (Roxb. ex D. Don) G. Don Specific Part (Bark) Collected as Anti-Venom Along with Voucher Specimen.
29 1.8.2.9 Citrullus colocynthis (L.) Schrad
Citrullus colocynthis (L.) Schrad belongs to the family Cucurbitaceae. It is annual herb with angular branching stems. It has woolly tender shoots and leaves are deeply divided. Flowers are 2.5 cm in diameter and monoecious with yellow in color. Both male and female are solitary. Fruits are 7-9 cm in diameter. They are rounded with green and white striped. Seeds are 4-6 in cm with pale brown in color. Propagation takes place by seeds and vegetative methods (Prajapati et al., 2010; Baquar, 1989). It has also momentous effect in health care system. Roots are effective in jaundice, amenorrhoea, ascites, uteralgia, mammillitis, rheumaltalgia, visceromegaly, uropathy and ophthalmia. Fruits are very useful in splenomegaly, tubercular glands of neck, tumors, leucoderma, ulcers, asthma, bronchitis, elephantiasis, constipation, dyspepsia and urethrorrhea. Fruits and roots are used to combat snake poisoning (Baquar, 1989). Therefore, fruits were collected to confirm folk claim as anti-dote to snake bite. They were gathered under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of December, from division Bahawalpur, Punjab, Pakistan (Fig 1.18). Voucher specimen (as shown above) was deposited with number (STW.702) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan. Lot of chemical constituents has been reported for this plant. Among them citrulluin, citrulluene, citrulluic acid, dihydric alcohol, citrullol, p-hydroxybenzyl, methylether, bitter oil, citbittol, elaterin, hentriacontane, saponins, various alkaloid, glycosides and tannins are very prominent (Prajapati et al., 2010; Baquar, 1989).
Fig 1.18 Describes the Contour of Citrullus colocynthis (L.) Schrad with Specific Part (Fruits) Collected as Anti-Venom Along with Voucher Specimen.
30 1.8.2.10 Citrus limon (L). Burm. f.
Citrus limon (L). Burm. f. belongs to the family Rutaceae. It is shrub or small tree having leaves 7-15 cm long. Flowers are white but sometime tinged with red. Flowers are 1-2 cm in size. Fruit is yellow, ovoid and its pulp is stuffed with acid (Baquar, 1989). Fruit of this plant has essence medically. Ripe fruit juice is used in scurvy, dysentery, rheumatism and in diarrhea. Rind of fruit is stomachic and carminative. Its peel is used as cosmetics as hair rinse and mouth freshener. Root of this plant is anthelmintic (Baquar, 1989; Jarald and Jarald, 2006). Fruit is also used to cope with snake poison (Rita et al., 2011). In order to combat snake poisoning fruits were collected under the aegis of eminent botanist Prof. Dr. Altaf Ahmad Dasti in the month of November, from the area of Haripur, KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.XX) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.19). Essential oil of leaves have neral, geranial and limonene. Apart of it oil from peel contains d-x-pinene camphene, d-limonene, linalool, ichangin 4-β-glucopyranoside, nomilinic acid, 4-β-glucopyranoside (Baquar, 1989; Jarald and Jarald, 2006).
Fig 1.19 Describes the Contour of Citrus limon (L). Burm. f with Specific Part (Fruits) Collected as Anti-Venom Along with Voucher Specimen.
31 1.8.2.11 Cuminum cyminum L.
Cuminum cyminum L belongs to the family Apiaceae. It is fifteen to thirteen centimeter tall slender annual herb. Leaves of this herb are divided into filli form. Fruits are cylindrical with tips that are narrowed primary ridges filiform. They are five to six millimeters long. Propagation takes place by seeds (Prajapati et al., 2010; Baquar, 1989). Fruit of this plant is used as carminative, stimulant, astringent, stomachic, helpful in diarrhoea, dyspepsia and used in veterinary medicine. Oil of cumin is used in culinary preparations, confectionery, liqueurs, cordials, beverages and to flavor curries. Its seeds are recommended in snake bite (Baquar, 1989). That is why seeds were gathered under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of June, from Sargodha, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.516) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.20). As far as chemical substances are concerned, various important constituents are found in this plant. Cumin oil contains sminaldehyde, 1, 3-p-menthadien-7-al, 1,4-p-menthadien- 7-al. Other essential oils are also present in this plant (Prajapati et al., 2010; Baquar, 1989).
Fig 1.20 Describes the Contour of Cuminum cyminum L with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen.
32 1.8.2.12 Enicostema hyssopifolium (Willd.) I. Verd.
Enicostema hyssopifolium (Willd.) I. Verd. belongs to the family Gentianaceae. It is stiff glabrous herb and divided from base. It has quadrangular stems and it is sessile in nature. Leaves of this plant have three nerved and moreover linear to oblong in shape. This plant has small white flowers. Capsules are ellipsoids in shape with globose seeds (Daniel, 2006). This plant is very effective as carminative, hypoglycemic and restorative. Fresh plant is grinded and applied on the areas of snake bite (Daniel, 2006). Therefore to rationalize its use in snake bite whole plant was collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of August, from the area of Jhellum, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.553) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya- University, Multan, Pakistan (Fig 1.21). Various chemical constituents were documented. Among them betuline, swertioside, sylyswertin, swertiamarin, isoswertisin, enicoflavine, erythrocentaurine, apigenin, genkwanin, isovetexin, swertisin and saponarin are very abundant (Daniel, 2006).
Fig 1.21 Describes the Contour of Enicostema hyssopifolium (Willd.) I. Verd Plant Collected as Anti-Venom Along with Voucher Specimen.
33 1.8.2.13 Fagonia cretica L.
Fagonia cretica L (family: Zygophyllaceae) is spiny shrub and widespread in calcareous rocks in Pakistan. It has small leathery leaves which have 1-3 lobed. Flowers have pale rose or purple in color and small in size. It has five petals and ten stamens with five celled. In each cell two seeds are present (Razi et al., 2011; Baquar, 1989). This plant is extremely helpful as astringent, febrifuge, anticancer, anti-inflammatory, antiemetic, prophylactic against small-pox, antidiarrheal and antiasthmatic. It is often recommended to apply as paste on swelling areas of the neck and on tumors. Apart of it arial parts (twigs and leaves) are effective in snake bite (Razi et al., 2011). For rationalizing its use in snake poisoning leaves and twigs were collected under the aegis of well-known botanist Prof. Dr. Altaf Ahmad Dasti in the month of December, from the area of Lasbella, Baluchistan, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.433) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.22). Several chemical constituents have been reported in the literature like saponin glycosides, saponin-I & saponin-II, various proteins from aqueous extract, docosyl docosanoate from n-hexane extract, ursolic acid, pinitol and nahagenin are very important (Saeed et al., 1999).
Fig 1.22 Describes the Contour of Fagonia cretica L with Specific Part (Leaves and Twigs) Collected as Anti-Venom Along with Voucher Specimen.
34 1.8.2.14 Leucas capitata Desf.
Leucas capitata Desf belongs to the family Lamiaceae. It is an erect aromatic annual herb. Its length is 35 cm. This plant possesses leaves that are ovate and 5-10 centimeter in diameter. Flowers are in large terminal and round clusters, 2.5 to 5 cm in diameter. This plant has 1.8 cm calyx, four stamens (unequal pairs), two lipped corolla, four nutlets which are ovoid in nature (Shinwari et al., 2007). It has insecticidal, anthelmintic, diaphoretic and stimulant effects. Fresh juice of this plant when applied externally helpful in scabies. Syrup of flowers has momentous effect in cough and cold. This plant is also used as culinary herb and oil of its seeds is used for illumination. Whole plant is recommended to combat snake bite poisoning (Shinwari et al., 2007). For scientific confirmation as anti-dote in snake envenomation, plant was collected under the aegis of well-known botanist Prof. Dr. Altaf Ahmad Dasti in the month of October, from an area of Rawalpindi, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.615) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.23). Important chemical substances are essential oil and alkaloids found in them (Shinwari et al., 2007).
Fig 1.23 Describes the Contour of Leucas capitata Desf Plant Collected as Anti-Venom Along with Voucher Specimen
35 1.8.2.15 Matthiola incana (L.) W. T. Aiton
Matthiola incana (L.) W. T. Aiton belongs to the family Brassicaceae. This plant is annual or perennial herb with woody root. Its leaves are sinuate or entire. Flowers are fragrant and large. Sepals are erect while petals are like claw long and 1.5-2.5 centimeter long. Seeds are flattened having membranous wing which are very narrow (Baquar, 1989). Seeds are very important from health care point of view. They are diuretic, stimulant, aphrodisiac, bitter, expectorant, tonic and stomachic. Apart of it infusion is valuable in cancer and recommended in poisonous bites (Baquar, 1989). In order to confirm its use as folklore medicine as antidote seeds were collected under the aegis of well-known botanist Prof. Dr. Altaf Ahmad Dasti in the month of September, from an area of Rawalpindi, Punjab, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.322) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.24). Sulforaphene (Brinker and Gayland, 1993), oil rich in γ-linolenic acid (Mahgoub et al., 2011), chlorphyll a & b, carotenoids, N, P, K and Na ions are important constiyuents of this plant (Abd El Aziz et al., 2011).
Fig 1.24 Describes the Contour of Matthiola incana (L.) W. T. Aiton with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen.
36 1.8.2.16 Momordica charantia L
Momordica charantia L belongs to the family Cucurbitaceae. It is climbing herbaceous vine with undivided tendrils. Its leaves are alternate with 2.5-7.5 centimeter in diameter. Flowers are pale yellow to orange in color and are across in 2.5 centimeter. Both male and female flowers are found on the same plant. Male flowers are solitary on slender stalks with length of 5-10 cm. Bracts of male flowers are at or below the middle. Female flowers are also solitary on stalks whose bracts are to the base. Fruits are 2.5-12.5 centimeter in length and found with seeds (8 mm) in red pulp. It is propagated by seeds and vegetative methods (Prajapati et al., 2010; Baquar, 1989). Leaves are purpative and emetic and extremely effective in bilious affections. They are used to combat burning sole of foot. Its root is astringent and useful in haemorrhoids. Fruit and leaves are useful in jaundice, leprosy and piles. Both are anthelmintic and vermifuge. Fruit is stomachic and its juice is used to neutralize snake bite (Baquar, 1989). In order to test anti venom potentials fruits were collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of December, from division Abbottabad, KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.706) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.25). Plant contains mycose, steroidal glucoside, vicine, momordicines I and II, momorcharaside A,B, cucubitane triterpenoids, cycloeucalenol, stigmasterol, spinasterol, taraxerol, momordicosides, lophenol, thiocyanogen, diosgenin, 24- methylencycloartenol, squalene, phenyl propanoids, stigmastadien-3-beta-ol, glucoside and carotenoids (Prajapati et al., 2010).
1.8.2.17 Nerium indicum Mill.
Nerium indicum Mill belongs to the family Apocynaceae. It is evergreen glabrous shrub with milky juice. It has leaves that are narrow at both ends and arranged in three whorls. Leaves are linear lanceolate and 10 to 15 cm by 8 to 22 mm. This plant has white, red or pink flowers which are 3.5 cm across. Fruits (follicles) are connate from 12.5 to 20 cm by
37 8 mm. Seeds are oblong, densely villous and 5 mm in length. It is propagated by cutting (Baquar, 1989; Prajapati et al., 2010). Although this plant is poisonous, however roots are used externally in the form of paste to reduce ulcers and cancers on penile. Roots are acrid, astringent, thermogenic, bitter, stomachic, diuretic, febrifuge and aphrodisiac. Bark of root is helpful in ringworm. Leaves are used for scabies as repellent and in piles. Leaves juice is effective in ophthalmia with abundant tearing. Flowers are used to purify air. Its roots, leaves and flowers are used to combat snake envenomation (Asad et al., 2011; Baquar, 1989; Prajapati et al., 2010). To test this plant as anti-venom leaves and roots were collected under the supervision of expert botanist Prof. Dr. Altaf Ahmad Dasti in the month of October, from division Abbottabad (Haripur), KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.564) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.26). Important constituents are neriodorin, karabin, nerioderin and odorin (Baquar, 1989). Neriodorin and karabin were reported to have paralyzing effect on heart like digitalin while possess stimulating action on spinal card exactly like strychnine (Prajapati et al., 2010).
Fig 1.25 Describes the Contour of Momordica charantia L with Specific Part (Fruits) Collected as Anti-Venom Along with Voucher Specimen.
38
Fig 1.26 Describes the Contour of Nerium indicum Mill with Specific Part (Leaves and Roots) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.18 Ocimum synctum L.
Ocimum synctum L (family Lamiaceae) is medium sized perennial herb having height of 30-60 cm. Its leaves are simple, oblong, opposite and 2.5 to 6 cm in length. Leaves are narrow at the both ends. Flowers are whitish pink or purplish in color and very small in size. Fruits are nutlets nearly round, smooth and pale reddish brown in color. They are not mucilaginous when wetted. It is propagated by seeds and vegetative methods (Baquar, 1989; Prajapati et al., 2010). Leaves are utmost important. Their juice is used as antiperiodic, stimulant, expectorant, and diaphoretic. They are also helpful in catarrh, earache and bronchitis. Their infusion is used to treat gastric disorders. Roots are effective to cope with malaria. This plant has significance in cardiopathy, haemopathy, leucoderma, asthma, otalgia, hapatopathy, vomiting, lumbago, genito-urinary disorders and ringworms. Apart of it this plant is used to treat snake bite and mosquito bites (Baquar, 1989; Prajapati et al., 2010). For the sake of confirmation as an anti-dote for snake poisoning whole plant was collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of September, from a garden in Islamabad, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.626) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.27).
39 Major chemical constituents in leaves are essential oil containing 3.2% carvacrol, 71.3% eugenol, 1.7% caryophyllene and 20.4% methyl eugenol. Others are linalool, methyl chavicol cineole and eugenol methol ether (Baquar, 1989; Prajapati et al., 2010).
Fig 1.27 Describes the Contour of Ocimum synctum L Plant as Anti-Venom Along with Voucher Specimen.
1.8.2.19 Pinus roxburghii Sarg.
Pinus roxburghii Sarg (family: Pinaceae) is a tall coniferous tree having thick and rough bark of 2-5 cm. Its leaves are 20-30 cm long and present in bundles of three. Male cones are found having length of 13 mm while ripe female cones are of 10-20 cm. Its seeds are winged whilst wings are membranous and 2-3 times longer than the seeds. It is propagated by seeds and vegetative methods (Baquar, 1989; Prajapati et al., 2010). Its wood is sweet, emollient, haemostatic, acrid, thermogenic, antiseptic, stimulant, deodorant, liver tonic, digestive, anthelmintic, diuretic, rubefacient and diaphoretic. It is very effective in burning of body, fainting, cough and ulceration. Its resin when used internally will act as stomachic and for gonorrhea. For suppuration it is applied externally to abcesses or buboes in the form of plasters. Oleoresin is bitter, acrid, expectorant, anti- inflammatory, purgative, anodyne and demulcent. It has shown significant role in mitigation of asthma, hepatopathy, otalgia, bronchitis, splenopathy, scabies, urethrorrhea, epilepsy, tuberculous glands, lumbago and haemorrhoids (Prajapati et al., 2010). Wood
40 and oleoresin is also recommended in scorpion and snake bite (Baquar, 1989). For the sake of scientific proof as an anti-dote for snake envenomation oleoresin of this plant was collected under the supervision of botanist Prof. Dr. Altaf Ahmad Dasti in the month of June, from the hills of Murree, Punjab, Pakistan (Fig 1.28). Voucher specimen (as shown above) was deposited with number (STW.26) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan. Important chemical constituents are β-carene, α-pinene, β-longifolene, β-pinene, longifolene, longicyclene and careen (Prajapati et al., 2010).
Fig 1.28 Describes the Contour of Pinus roxburghii Sarg with Specific Part (Oleoresins) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.20 Pistacia integerrima J. L. Stewart
Pistacia integerrima J. L. Stewart (family: Anacardiaceae) is a medium sized deciduous tree having gray or blackish bark with a height of approximately 18 m. This plant has pinnately compound leaves with a length of 15-22.5 cm. Flowers are small and reddish in lateral panicles. Male flowers (in compact panicles) are found with a length of 5-15 cm while female flowers have laxer panicles of 15-25 cm length. Petals are not found in flowers. Fruits are globose drupes and on ripening grey in color. Young galls are coriaceous but later on become hard. Galls are horn shaped, hollow, twisted and greenish brown or pinkish in color. They may be straight or curved. Galls are generated on
41 branches of tree by the insect Dasia aedifactor. It is propagated by seeds and vegetative methods (Baquar, 1989; Prajapati et al., 2010). From therapeutic point of view galls are effective in pthisis, asthma, and dysentery. They are expectorant and tonic. Galls are used to neutralize snake as well as scorpion poison (Baquar, 1989). For the sake of scientific illustration as anti-snake venom galls of this plant were collected under the supervision of renowned botanist Prof. Dr. Altaf Ahmad Dasti in the month of May, from Murree hills, in Pakistan. Voucher specimen (as shown above) was deposited with number (STW.458) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.29). As far as chemical constituents are concerned galls contain 1.3% essential oil having A- pinene, camphene, d-limonene, cineole, A-terpineol, aromadendren and caprylic acid. Apart of it two crystalline acids are also reported (Baquar, 1989).
Fig 1.29 Describes the Contour of Pistacia integerrima J. L. Stewart with Specific Part (Galls) Collected as Anti-Venom Along with Voucher Specimen.
1.8.2.21 Psoralea corylifolia L. Psoralea corylifolia L belongs to the family Fabaceae. It is an annual, mediun sized erect herb maximally attain the height of 90 cm. Its leaves are rounded oval having length of 6- 9 cm whilst width is 5-7 cm. They are covered with black spots on both sides (above and below). It has yellowish or whitish flowers (5 mm) tipped with purple color. Flower is
42 found with 10 stamens and sessile ovary. Its fruit is dry, short, black, glabrous and an oval pod wrapped by calyx. It is propagated by seeds and vegetative methods (Baquar, 1989; Prajapati et al., 2010). This plant has significant role in relief of diarrhea, enuresis, micturition (frequent), impotence, lumbago, seminal emissions, weakness of legs, cough, tinea capitis, psoriasis and vitiligo. Fruits (seeds) are laxative, diaphoretic, aphrodisiac and tremendous useful in leucoderma, leprosy and inflammatory conditions (Prajapati et al., 2010). Seeds are extremely effective to neutralize snake and scorpion poison (Baquar, 1989). For confirmation as anti-snake venom seeds were gathered under the supervision of famous botanist Prof. Dr. Altaf Ahmad Dasti in the month of May, from Peshawar KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.418) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya- University, Multan, Pakistan (Fig 1.30). Lot of chemical constituents was reported in literature previously. Among them seeds possess corylifolean, corylifolin, corylifolinin, raffinose, psoralidin, psoralen, isopsoralidin, isopsoralen, bavachin, isobavachin, bakuchiol, bavachinin, 7-O-methylbavachin, 4-O-methylbavachalcone, neobavaisoflavone, isobavachalcone, bavachromene, corylidin, traincontane, β-sitosterol- D-glucoside, bavachalcone, neobavachalcone, isobavachalcone, isoneobavachalcone, bakuchalcone, stigmasterol, psoralidin-2,3-oxide diacetate. Limonene, β- caryophylenoxide, α-elemene, linalool, 4-terpineol, angelicin, geranylacetate, bakuchiol and psoralene are important chemical substances extracted from seed oil (Prajapati et al., 2010).
Fig 1.30 Describes the Contour of Psoralea corylifolia L with Specific Part (Seeds) Collected as Anti-Venom Along with Voucher Specimen.
43 1.8.2.22 Rhazya stricta Decne.
Rhazya stricta Decne belongs to the family Apocynaceae. It is glabrous shrub having length of approximately 90 cm. It’s spirally set of leaves are 7-10 cm in length and 1-2 cm in width. Yellowish green leaves are narrow at both ends and lanceolate or oblanceolate in nature. Faintly fragrant flowers are 3 mm across. Its seeds are oblong (8 mm in length), compressed, angular and winged at the ends (Baquar, 1989). Its fruit and leaves are recommended to treat eruptions and boils. Leaves are more important and are significantly used in skin eruptions, fever, throat infections and general debility. Apart of it leaves are widely used to treat cancer and to neutralize snake poison (Asad et al., 2011; Baquar, 1989). For testing of anti-snake venom activity leaves were collected under the supervision of renewed botanist Prof. Dr. Altaf Ahmad Dasti in the month of September, from Lakki Marwat KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.565) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.31). Essential chemical constituents are triterpenes (Mg quinate, β-sitosterol & urosoic acid), glycosides (3-7-rhamnoside, isorhamnetin-3-7-rhamnoside & roblnin), alkaloid (sewarine), flavonoids (rhazianosides A&B), among enzymes: NADPH dependent tetrahydroaistonine and strictosidine synthase are important (Asad et al., 2011; Baquar, 1989).
Fig 1.31 Describes the Voucher Specimen of Leaves of Rhazya stricta Dcne Plant Collected as Anti-Venom.
44 1.8.2.23 Rubia cordifolia L.
Rubia cordifolia L belongs to the family Rubiaceae. It is climbing perennial herb with stout 4- angled stems and long reddish cylindrical, flexuose roots. Leaves are variable in a whorl from 2-8, often 4 in number. It has darkly red, whitish or greenish yellow flowers whilst fruits are 5 mm in diameter which are round in shape, blackish upon ripening and may be 1 or 2 celled. There are two seeds which are also small in size. It is propagated by seeds and vegetative methods (Baquar, 1989; Prajapati et al., 2010). Root is tonic, astringent, alterative, bitter, thermogenic, digestive, constipating, anti- inflammatory, antiseptic, carminative, vulnerary, depurative, anthelmintic, antidysenteric, diuretic, emmenagogue, ophthalmic, febrifuge, galacto-purifier and rejuvenating (Prajapati et al., 2010). Stem is used to neutralize snake and scorpion poisoning (Baquar, 1989). For confirmation as anti-snake venom stems were gathered under the supervision of famous botanist Prof. Dr. Altaf Ahmad Dasti in the month of July, from Murree, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.689) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya- University, Multan, Pakistan (Fig 1.32). Roots possess coloring matter which is a mixture of purpurin & munjistin. Apart of it other chemical substances are pseudopurpurin and xnthopurpurin. Alizarin and munjistin with their glycosides are also found in this plant (Baquar, 1989; Prajapati et al., 2010).
Fig 1.32 Describes the Contour of Rubia cordifolia L with Specific Part (Stems) Collected as Anti-Venom Along with Voucher Specimen.
45 1.8.2.24 Sapindus mukorossi Gaertn
Sapindus mukorossi Gaertn (family: Sapindaceae) is a large deciduous tree having height of approximately 20 meters with grayish smooth bark. Its leaves are alternate and 30-50 cm in length. Flowers are white or greenish white and 5 mm across. Fruits are solitary, 2- 2.5 cm in diameter. They are globose, fleshy drupes (indehiscent), single seeded and black in color. On drying its pulp become wrinkled rind. They are propagated by seeds (Baquar, 1989; Prajapati et al., 2010). Its bark and roots are demulcent and expectorant. Roots are effective in hysteria, epilepsy as well as in hemicranias. Its fruit are bitter, thermogenic, acrid, astringent, expectorant, abortifacient and emetic. They are used to combat severity of salivation, chlorosis, asthma, diarrhea, lumbago, gastralgia (due to dyspepsia) and verminosis (Prajapati et al., 2010). It is used as fish poison (Baquar, 1989). Apart of above mentioned uses fruits are recommended ethnobotanically as anti-venom (Parganiha et al., 2012). Therefore, for scientific confirmation of anti-snake venom fruits were purchased under the supervision of famous botanist Prof. Dr. Altaf Ahmad Dasti from local market (Golden Pansar store, Niswari Bazaar) in Rawalpindi. Voucher specimen (as shown above) was deposited with number (STW.463) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.33). Essential chemical substances found in the nuts are kaempferol, β-sitosterol and quercetin. Among saponins: sapindoside A, sapindoside B and saponin emarginatoside (from fruits) is reported previously (Prajapati et al., 2010).
Fig 1.33 Describes the Part (Fruits) of Sapindus mukorossi Gaertn Used as Anti-Venom in Voucher Specimen Deposited at the Herbarium.
46 1.8.2.25 Stenolobium stans (L.) Seem.
Stenolobium stans (L.) Seem belongs to the family Bignoniaceae. It is a small tree or somewhat large shrub. Leaves of this plant are long and pinnate having length of 17.5-25 cm. Its flowers are yellow in color and 2.5 cm across. Flowers have 15 cm long calyx and corolla of 4-5 cm length (Baquar, 1989). Roots of this plant are extensively used to neutralize rat bite. They are also effective to combat snake and scorpion poison (Baquar, 1989). In order to proof as anti-snake venom roots were collected under the supervision of famous botanist Prof. Dr. Altaf Ahmad Dasti in the month of November from Haripur, KPK, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.669) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.34). Plant contains some important constituent’s viz., phenolic acids, β-setosterol, triterpenoids-ursolic acid, α-amarine, oleanic acid, indole metabolizing enzymes, indole- oxygenase, zeaxanthin, lutein-zeaxanthin and β-carotene (Kandakatla et al., 2010).
Fig 1.34 Describes the Contour of Stenolobium stans (L.) Seem with Specific Part (Roots) Collected as Anti-Venom Along with Voucher Specimen.
47 1.8.2.26 Terminalia arjuna (Roxb. ex DC.) Wight & Arn.
Terminalia arjuna (Roxb. ex DC.) Wight & Arn (family: Combretaceae) is a large evergreen tree. Its bark is flesh colored inside while greenish grey in color from outside. Leaves are simple, oblong, subopposite, crenulate. They are pale green from above side and pale brown in color below side. Yellowish white flowers are 5-7.5 cm in length. Fruits are oblong or ovoid found with hard angles or wings. It is propagated by seeds (Prajapati et al., 2010). This plant has significant essence from health point of view. Its fruit is deobstruent and tonic while juice of leaves is used in earache. Bark of this plant is sweet, cooling, astringent, acrid, demulcent, styptic, antidysenteric, expectorant, urinary astringent, lithontriptic, aphrodisiac and demulcent (Prajapati et al., 2010). Apart of above said uses, its bark is used as an antidote to different poisons (Baquar, 1989). To test this plant as anti-venom bark was collected under the supervision of expert botanist Prof. Dr. Altaf Ahmad Dasti in the month of August, from Islamabad, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.502) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.35). Its bark contains lot of chemical substances like arjunetin, arjunine, essential oil, calcium salts, aluminium and magnesium salts, coloring materials, reducing sugars, pyrocatachol (tannin) and a laactone. Among others tomentosic acid, ellagic acid, arjunolic acid and β- sitosterol are very abundant (Prajapati et al., 2010).
Fig 1.35 Describes the Contour of Terminalia arjuna (Roxb. ex DC.) Wight & Arn with Specific Part (Bark) Collected as Anti-Venom Along with Voucher Specimen.
48 1.8.2.27 Trichodesma indicum (L.) Sm.
Trichodesma indicum (L.) Sm belongs to the family Boraginaceae. It is annual hispid herb with a maximum height of 45 cm. Leaves are sessile, ovate, simple, opposite, lanceolate and obtuse. Their length is 2.5-10 cm. Flowers are pale blue but change to pink or white on drooping axillary stalks. Fruits of this plant is pyramidal 4-ribbed nutlets, smooth, rugose, pitted on inner surface and oblong with rounded rounds. It is propagated by seeds (Prajapati et al., 2010; Baquar, 1989). This plant is emollient, alexeteric, bitter, acrid, anodyne, anti-inflammatory, carminative, diuretic, constipating, ophthalmic, pectoral and febrifuge. Roots are used to reduce swelling on joints and dysentery of children. It is also helpful in leprosy, skin diseases, strangury, dysmenorrhoea, fever and sores. Apart of it roots and leaves are effective to neutralize snake poison (Baquar, 1989). To test this plant as anti-venom whole plant was collected under the supervision of expert botanist Prof. Dr. Altaf Ahmad Dasti in the month of September, from Sind province, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.604) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.36). Important chemical constituents from leaves are hexacosane, ethyl hexacosanoate and 21, 24-hexacosadienoic acid ethyl esters. Seed oil possesses oleic, linoleic, palmatic, stearic and linolenic acid (Dachani et al., 2012).
Fig 1.36 Describes the Contour of Trichodesma indicum (L.) Sm Plant Collected as Anti- Venom Along with Voucher Specimen.
49 1.8.2.28 Zingiber officinale Roscoe
Zingiber officinale Roscoe belongs to the family Zingiberaceae. It is large biennial rhizomatous herb. Its leaves are sessile, glabrous and 30 cm by 5-7.5 cm oblong- lanceolate. Flowers of this plant are 2.5-3.8 cm in length. They are oblong having dark green or purplish black in color. Fruits are oblong capsules. Rhizomes are branched, annualated, laterally flattened and white to yellowish brown in color. Its surface is smooth while fibers of vascular bundles come out from the cut ends. It is propagated by rhizomes (Prajapati et al., 2010; Baquar, 1989). Rhizome is very imperative from medical point of view. It is condiment, stimulant, carminative, flavorful and sialagogue. It is effective to cure dyspepsia, rheumatism, vomiting, colic, chest problems, diarrhea, dysentery, dropsy, nausea, tympanites, pulmonary and catarrhal complications. It is used with honey to reduce the severity of asthma as well as tonic (Baquar, 1989). It is also recommended to neutralize snake venom (Duke and Ayensu, 1985). In order to test this plant as anti-venom rhizome was collected under the supervision of expert botanist Prof. Dr. Altaf Ahmad Dasti in the month of September, from Lahore, Pakistan. Voucher specimen (as shown above) was deposited with number (STW.66) at the herbarium of Department of Pure and Applied Biology, Bahauddin- Zakariya-University, Multan, Pakistan (Fig 1.37). Important chemical constituents are camphene, cineol, singiberine, shagaol, potassium oxalte, citral borneol, gingerol, β-phellandrene, α-curcumene, α-bergamotene, β-D-curcumene and gamma-bisabolene (Prajapati et al., 2010; Baquar, 1989).
Fig 1.37 Describes the Contour of Zingiber officinale Roscoe with Specific Part (Rhizomes) Collected as Anti-Venom Along with Voucher Specimen.
50 1.9 Hypothesis of this Study Snake bite envenomation is one of the vivid examples of neglected occupational hazards that accounts for tens of thousands of deaths all over the world. One of such instance is Naja naja karachisis bite, a nightmare for the inhibitants of Southern Punjab (Paksitan), often endup with countless deaths and sequela (Asad et al., 2016). To address this problem present study was designed to highlight scientific grounds for Naja naja karachisis envenomation and to rationalize folklore claimed Pakistani medicinal plants as a first aid treatment before proper hospitalization. In order to prove the present hypothesis aims and objectives were defined to sort out scientifically hidden cause responsible for numerious detahs in southern Punjab province of Pakistan.
2.0 Aims & Objectives of this Study
Aims and objectives of the present research were to
Identify various components (proteinous & non-proteinous) of Naja naja karachiensis venom. Identify structurally, therapeutically or functionally active novel toxic constituent(s) present in this venom (if any). Carry out biodistribution and kinetics studies of Naja naja karchiensis venom (post envenomation) with radio ligand binding technique. Study various biological and biochemical toxicity parameters for this venom. Evaluate different enzymatic (phospholipase, alkaline phosphatase, 5ʹ- nucleotidase, hyaluronidase and proteolytic) activities present in Naja naja karchiensis venom. Validate scientifically ethnobotanical claims about twenty eight medicinal plants of Pakistan to neutralize toxic biological, biochemical and enzymatic activities present in this venom. Study potent medicinal plant extract with detailed liquid chromatography coupled
bioassy procedures to find out anti-PLA2 agent(s) (if any).
51
Chapter 2 Material&Methods
52 This section comprised of information about various equipments, medicinal plants, laboratory animals, chemical agents along with adopted procedures for different experimental purposes.
2.1 Equipment
Below follows a description about equipments (Table 2.1) that were used for this work.
Table 2.1 List of Equipment Used for Experimental Work.
Equipment Company Purpose Closed pressurized microwave UltrWave system, Milestone Sample digestion system Srl, Sorisole, Italy Electrophoresis assembly (Model: Bio-Rad, USA Separation of venom ready gel cell; serial:108BR components 03116) ELx808 micro plate reader Biotek Measure absorbance Gamma radiation counter Cap-Ria 16 by MedWOW Radio labeling yield Gamma camera Siemens, Digitrac 75 by Scanning/ images Dotmed HPLC (AKTAPURIFIER) Ambersham Pharmacia Biotech To purify components Heater/heating block Multi-Block by Fisher To heat/warm ICP-OES system Optima 5300 DV, Elemental analysis PerkinElmer, USA Lyophilizer Hetosica To obtain dry powder Mass spectrometer PerkinElmer Life Sciences, To determine USA molecular mass 99mMo/99mTc generator PINSTECH, Islamabad, Radioactive source Pakistan Rota vapor with water bath Buchi Rotavapor R-114 & Evaporation of solvent Buchi water bath B-480 under reduced pressure Selectra Junior Vital Scientific Netherland Bioassays SPD121P Savant speed-vac Thermo Scientific Evaporation
53 Spectrophotometer (UV-Visible) UV-1280 by Shimadzu Absorption
2.2 Experimental Animals
Male adult rabbits (1±0.5 kg) were purchased from local market of Multan, Pakistan while male Swiss albino mice were supplied by University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan. Animals were kept in animal house of Multan Institute of Nuclear Medicine and Radiotherapy (MINAR), Nishtar Hospital, Multan, Pakistan after getting permission from local ethical committee (Ref. letter no: Administration /432/27/04/13/MINAR/Multan).
2.3 Medicinal Plants
Medicinal plants of Pakistan for this project were selected on the basis of folklore evidences as anti-snake venom. They were collected from different locations in Pakistan (Table 2.2) expect Sapindus mukorossi Gaertn that was purchased from Niswari Market, Rawalpindi, Pakistan.
Table 2.2 List of Medicinal Plants of Pakistan having Ethnobotanical Evidences as Anti- Snake Venom Collected for this Work.
Medicinal plants of Family Location Part Voucher Pakistan collected # Albizia lebbeck (L.) Benth Fabaceae Bahawalpur Seeds STW.381 Allium cepa L Amaryllidaceae Bhakkar Bulb STW.42 Allium sativum L Amaryllidaceae Bhakkar Bulb STW.46 Althaea officinalis L Malvaceae Rawalpindi Roots STW.411 Bauhinia variegata L Fabaceae Haripur Roots STW.374 Brassica nigra (L.) W. D. J. Brassicaceae Manshera Seeds STW.302 Koch Calotropis procera (Aiton) Apocynaceae Haripur Exudates & STW.566a W. T. Aiton Flowers STW.566b Cedrus deodara (Roxb. ex Pinaceae Nathia Gali Bark STW.25
54 D. Don) G. Don Citrus limon (L). Burm. f Rutaceae Haripur Fruit STW.XX Citrullus colocynthis (L.) Cucurbitaceae Bahawalpur Fruits STW.702 Schrad Cuminum cyminum L Apiaceae Sargodha Seeds STW.516 Enicostemma hyssopifolium Gentianaceae Jhelum Whole plant STW.553 (Willd.) I. Verd. Fagonia cretica L Zygophyllaceae Lasbella Leaves STW.433 Leucas capitata Desf Lamiaceae Rawalpindi Whole plant STW.615 Matthiloa incana (L.) W. T. Brassicaceae Rawalpindi Seeds STW.322 Aiton Momordica charantia L Cucurbitaceae Abbottabad Fruit STW.706 Nerium indicum Mill Apocynaceae Haripur Roots & STW.564 leaves Ocimum sanctum L Lamiaceae Islamabad Whole plant STW.626 Pinus roxburghii Sarg Pinaceae Murree Oleoresin STW.26 Pistacia integerrima J. L. Anacardiaceae Murree Galls STW.458 Stewart Psoralea corylifolia L Fabaceae Peshawar Seeds STW.418 Rhazya stricta Dcne Apocynaceae Lakki Leaves STW.565 Marwat Rubia cordifolia L Rubiaceae Murree Stems STW.689 Sapindus mukorossi Gaertn Sapindaceae Local Fruits STW.463 market Stenolobium stans (L.) Seem Bignoniaceae Haripur Roots STW.669 Terminalia arjuna (Roxb. ex Combretaceae Islamabad Bark STW.502 DC.) Wight & Arn Trichodesma indicum (L.) Boraginaceae Sind/Karachi Whole plant STW.604 Sm Zingiber officinale Roscoe Zingiberaceae Lahore Rhizome STW.66
55 2.4 Collection of Snakes (Naja naja karachiensis)
Pattern less black Pakistani cobras (Naja naja karachiensis) were collected from Cholistan desert in the southern Punjab province of Pakistan. After collection they were dully identified by zoologist.
2.5 Chemical Agents
Below follows a description about chemical agents (Table 2.3) that were used for this work.
Table 2.3 Chemicals Used for Various Experimental Work
Chemicals Supplier Purpose Acetic acid (glacial) Sigma-Aldrich Buffer Acetonitrile (LC-MS grade) Thermo scientific Solvent for LC/MS Adenosine monophosphate Sigma-Aldrich Substrate monohydrate Ammonium bicarbonate Sigma-Aldrich Buffer Anti-sera (Immunoglobulin’s) Bharat Serum and Anti-dote for snake Vaccines India venom aPTT reagent Weiner lab Argentina Coagulation test β-mercaptoethanol Sigma Reducing agent Bromophenol blue Sigma-Aldrich Dye Calcium chloride dihydrate Sigma Calcium source Casein CALBIOCHEM Substrate Cetyl trimethyl ammonium ABCR GmbH & Co, To form aggregates bromide (CTAB) Karlsruhe Coomassie Brillantblau R250 Merck Dye Disodium hydrogen phosphate Sigma Buffer (anhydrous) Dithiothreitol (DTT) Thermo Scientific To stabilize enzyme and sulfhydryl groups Dimethyl sulfoxide (DMSO) Sigma Solvent
56 Disodium hydrogen phosphate Sigma-Aldrich Buffer Egg yolks from chicken Sigma Substrate Ethanol Kemetyl Solvent Ethylenediaminetetraacetic acid Sigma Standard protease (EDTA) inhibitor/Anticoagulant Formic acid (analytical) Fluka Improve peak shape/ provide proton for LC- MS/MS Folin-Ciocalteu’s phenol reagent Merck Chromophor developer Glycerol 1st BASE Protein stabilizer/increase density of sample Glycine Bio-Rad Electrophoresis Hyaluronic acid sodium salt Sigma-Aldrich Substrate Hydrochloric acid (HCl) Schedeloco To acidify Hydrogen peroxide Sigma-Aldrich For digestion of sample for ICP-OES Iodoacetamide (IAA) Sigma Alkylating agent/peptide mapping Kits for GPT, GOT and urea Innoline, Merck For bioassays Kit for LDH & creatinine DiaSys Diagnostic For bioassay Systems, Germany Kits for CK-MB Minias Globe Diagnostics, For bioassay Italy Magnesium sulfate heptahydrate Sigma-Aldrich Buffer Methanol Sigma-Aldrich Solvent Milli-Q water Sartorius Stedium Biotech Solvent Micro BCA protein kit Pierce Protein concentration estimation Mini-protean precast Gels Bio-Rad Gels for electrophoresis NIST 1515, apple leaf National Institute Certified reference
57 of Standards and material (CRM) Technology, Gaithersburg, MD, USA Nitric acid Sigma-Aldrich Digestion of sample for ICP-OES PageRuler plus Thermo scientific Prestained protein ladder p-nitrophenyl phosphate Sigma-Aldrich Substrate p-nitrophenol Sigma-Aldrich End product of p- nitrophenyl phosphate ProteaseMAX Promega Trypsin enhancer/surfactant PT reagent (Soluplastin) Weiner lab Argentina Coagulation test Reference antidote Bharat serums and Standard anti-venom (immunoglobulins) vaccines, India Rutin trihydrate Sigma-Aldrich Standard cobra hyaluronidase inhibitor Silver staining kit Pierce Thermo Scientific Detection of protein(s) Stannous chloride dihydrate Sigma-Aldrich Reducing agent in acidic condition Sodium dihydrogen phosphate Merck Used to Prepare Buffer dihydrate Sodium dodecyl sulfate (SDS) Sinopharm Chemical Denaturing agent Reagent Sodium acetate Sigma Used to Prepare Buffer Sodium chloride Sigma Used to Prepare Buffer Sodium hydroxide Fluka Used to Prepare Buffer Standard solutions (P/N 4400- CPI, Amsterdam, External calibrators for 132565 and P/N 4400-ICP- Netherland ICP-OES MSCS) Trypsin Gold (MS grade) Promega Trypsin enzyme
58 Trifluoroacetic acid (TFA) Iris Biotech, Germany Ion pairing reagent Tris base Sigma Buffer Trichloroacetic acid (TCA) Sigma-Aldrich Stopping reagent TT reagent Human Wiesbaden, Coagulation test Germany
2.6 Extraction of Plants Material
After collection various plants material were properly washed and shade dried. They were chopped and grinded to obtain bulk powder and further subjected to extraction process after passing through sieve no. 22. One kilogram of each plant material was soaked in 5L of methanol in extraction bottles. Homogenates were shaken occasionally and kept at 25 ± 3 °C for four weeks. Filtrate was obtained by the use of ordinary and Whatman filter paper no. 41. Methanol was evaporated at 25 ± 3 °C in a water bath and finally extracts were weighed and preserved for further experimentation (Asad et al.,
2014(e)). Moreover, milky exudates of Calotropis procera (Aiton) W. T. Aiton was lyophilized to obtain dry powder.
2.7 Milking of Naja naja karachiensis Venom
Non-captive Pakistani black cobra snakes were selected to squeeze their venom at ambient temperature in a low light atmosphere. Snake venom was collected in container filled with deionized water (0.1L) by pressing the glands below their eyes and further subjected to centrifugation (10,000 rpm for 60 min). Subsequently it was freeze dried and stored in a light resistance container in a refrigerator. Before use it was reconstituted in terms of its dry weight (Asad et al., 2014(e)).
59 2.8 Proteomic Analysis of Pakistani Cobra Venom (Naja naja karachiensis)
2.8.1 Sodium Dodecylsulpahte Polyacrylamide Gel Electrophoresis (SDS- PAGE) of Crude Cobra Venom (Naja naja krachiensis)
2.8.1.1 SDS-PAGE Protocol
SDS-PAGE was performed in a mini-protean precast gradient (4%-20%) resolving gel system. Venom (36µg, 40mg/ml) was mixed separately in 4X (reduced & non-reduced) sample loading buffers (Table 2.4 & 2.5) to observe its component(s) in both conditions. Samples were heated for 10 minutes at 95 °C and subsequently quick spinned for 10 seconds at maximum rpm (14680). Samples (12 µl) were applied to the gel troughs (30 µl capacity) and slab was run at 200 V for 40 min after filling the electrophoresis tank with running buffer (Table 2.6). Current was turned off immediately when dye front was reached to the lower end of the gel. Gel was removed from the plates and stained by two different dyes individually to analyze various components of Pakistani cobra venom adequately. Prestained protein ladder (PageRuler Plus) was applied and run simultaneously with the evaluated samples to estimate the molecular weight of individual protein (Laemmli, 1970; Jethanand, 2006; Khan, 2008).
2.8.1.2 Staining with Coomassie Brilliant Blue R 250 Dye
Gel was soaked in Coomassie Brilliant Blue R 250 dye solution (Table 2.7) for a period of 30 min on a rocker. Subsequently gel was destained with destaining solution as mentioned in table 2.8 (Jethanand, 2006; Badhe et al., 2006).
2.8.1.3 Silver Staining Technique
Gel was stained according to the instruction provided by Thermo ScientificTM Pierce Silver Stain Kit (Product No. 24612) and furthermore various reagents (fixing, ethanolic, sensitizer, staining, developer and stopping solutions) were prepared (Table 2.9). Gel was washed with Milli-Q water for 5 seconds and repeated this process. After this gel was fixed in fixing solution (100 ml) for 15 min and repeated. Now the gel was washed with
60 ethanolic solution (100 ml) for 5 min and repeated. It was again soaked in Milli-Q water for 5 min. Subsequently gel was sensitized in sensitizer solution for 01 min and repeated this process with Milli-Q water. Gel was incubated in staining solution for 30 min and subsequently washed with Milli-Q water for exactly 20 seconds. After that gel was soaked in developer solution till the desired bands appeared (within 2-3 min). After appearance of desired intensity band(s) gel was immediately soaked in stopping solution and incubated for 10 min (Badhe et al., 2006; www.thermoscientific.com/pierce).
Table 2.4 Composition of Reducing Sample Loading Buffer (4X)
Chemicals mixed Quantity Tris-base (1M, pH 6.8) 6 ml SDS 2.4 gm Glycerol 12 ml EDTA (0.5M, pH 8) 3 ml Bromophenol blue 24 mg β-mercaptoethanol 1.2 ml Milli-Q water 7.8 ml
Table 2.5 Composition of Non Reducing Sample Loading Buffer (4X) Chemicals mixed Quantity Tris-base (1M, pH 6.8) 6 ml SDS 2.4 gm Glycerol 12 ml EDTA (0.5M, pH 8) 3 ml Bromophenol blue 24 mg Milli-Q water 9 ml
61
Table 2.6 Composition of Running Buffer for SDS PAGE Chemicals mixed Quantity/l Glycine 14.375 gm Tris base 3 gm SDS 01 gm
Table 2.7 Composition of Coomassie Brilliant Blue R 250 Staining Solution Chemicals mixed Quantity Methanol 25 ml Milli-Q water 20 ml Glacial acetic acid 01 gm Coomassie Brilliant Blue R 250 150 mg
Table 2.8 Composition of Destaining Solution Chemicals mixed Quantity Methanol 250 ml Glacial acetic acid 70 ml Distilled water 680 ml
62
Table 2.9 Recipe for Preparation of Various Solutions Used for Silver Staining Reagent/ Fixing 10% Sensitizer Staining Developer Stopping chemical solution ethanol solution solution solution solution Milli-Q water 120 ml 180 ml 50 ml - - 190 ml Glacial acetic 20 ml - - - - 10 ml acid Ethanol 60 ml 20 ml - - - - Sensitizer - - 0.1 ml - - - Enhancer - - - 0.5 ml 0.5 ml - Silver stain - - - 25 ml - - Developer - - - - 25 ml - Total quantity 200 ml 200 ml 50.1 ml 25.5 ml 25.5 ml 200 ml
63 2.9 Chromatographic Separation of Naja naja karachiensis Venom
2.9.1 Size Exclusion Chromatographic (SEC) Analysis
Naja naja karachiensis (100 mg) venom was dissolved in 1ml MilliQ water. Subsequent sonication and filtration (0.45 µm) venom was fractionated by gel filtration column (SuperdexTM 200, HiloadTM 16/60, prep grade, 120 ml column volume) previously equilibrated (2 column volume) with 50mM Tris-HCl buffer (pH 7.4). Elution was performed with the same buffer at the flow rate of 1ml/min having maximum pressure (01 mPa) using an ÄKTA purifier LC system (GE Healthcare Life Sciences, Piscataway, NJ, USA). Elution was observed at 280 nm and each fraction (1 ml) was collected in 1.5 ml (12 mm) tube via fraction collector (frac-900). All the data was acquired by Unicorn (5.31version) software. Similar fractions were pooled together and lyophilized and stored for further experimentation (Malih et al., 2014).
2.9.2 Reverse Phase HPLC Analysis
Acidified gel filtration fractions were further separated on reverse phase Jupiter C18 analytical (4.6 × 250 mm, 5 µm particle size, 300 A pore size, 4.155 ml column volume) and semi-preparative (10 × 250 mm, 5 µm particle size, 300 A° pore size, 19.635 ml column volume) columns using an ÄKTA purifier LC system (GE Healthcare Life Sciences, Piscataway, NJ, USA). Elution buffer A was 0.1%TFA in MilliQ water while elution buffer B was 0.1% TFA in 60% acetonitrile. First four gel chromatographic peaks (~10 mg) were separated by semi-preparative column (flow rate, 4ml/min) however peak 5 and 6 (~1mg) was separated by analytical column (flow rate, 1ml/min). For gel chromatographic peaks 1 to 4 gradient was 0% B for 25 min, followed by 0-30% B for 30 min. Subsequently gradient was 30-70% for 170 min and finally it was 70-100% B for 40 min. For gel chromatographic peak 5 & 6 gradient was 0% B for first 12 min followed by 0-30% B for 16 min. Later on gradient was 30-70% B for 105 min and finally it was 70- 100% B for 16 min. Protein concentration was monitored at 280 nm and each fraction was collected automatically via fraction collectors (frac-950 & 900). Similar fractions were pooled together and further lyophilized. All the data was processed by the use of HPLC Unicorn (5.31version) software (Malih et al., 2014).
64 2.9.3 Protein (Trypsin) Digestion of Crude Venom Sample and its Fractions
For trypsin digestion of proteins in solution, crude venom and its fractions (~ 0.2 mg,
50µl) were mixed with protease max (1%, 2 µl), 50 mM NH4HCO3 buffer (42 µl) and 0.5M dithiothreitol (DTT) (1 µl). Reaction mixture was subjected to incubation at 56 °C for 20 min. After cooling at ambient temperature proteins were alkylated with iodoacetamide (IAA) (0.55M, 2.7 µl) for 15 min in the dark atmosphere. Subsequently protease max (1%, 1 µl) and trypsin (1µg/µl, 1.8 µl) solutions were added and reaction mixture was incubated at 37 °C for the period of 3h. After quick spin (14680 rpm, 10 sec) trifluoroacetic acid (0.5 µl) was added to halt the process of digestion. Later on sample(s) was subjected to centrifugation (10 min, 14680 rpm) and supernatant was analyzed by LC-MS/MS (ESI-MS) for complete separation and identification of peptide(s) to confirm the presence of proteins (www. promega.com).
2.9.4 Mass Spectrometric (LC-MS/MS) Analysis of Tryptic Digested Venom Sample(s)
Trypsin digested samples were loaded on C18 Hypersil Gold (50 × 2.1 mm, particle size 1.9 µm) column for further separation of peptides. Buffer A was 0.1% formic acid in MilliQ water while buffer B was 0.1% formic acid in pure acetonitrile. Gradient was 0% B for first 6 min followed by 0-40% B for 38 min. Subsequently gradient was 40-80% B for 56 min and maintained at 80%B for 63 min. Later on gradient was 0%B for 66 min and maintained at 0%B for 78min. Fractionated venom components were injected into LCQ FLEET mass spectrometer via electrospray ionization and fragmented by collision induced dissociation (CID) with ion trap analyzer. ESI-MS was set at positive ion mode and scan range was adjusted between 500-2500 m/z. Nitrogen gas was used as curtain (flow rate of 0.6 l/min) and nebulizer gas with a pressure of 100 psi. Capillary voltage was set at 2000V while capillary temperature and orifice potential were 350°C and 80V respectively. Mass spectrometry data was analyzed by Proteome discoverer (1.3.0.339) and proteins were identified with the aid of database search program SEQUEST. For SEQUEST search, the parameters were: Lepidosauria port database & O. hannah transcriptomic (cDNA) database (NCBI nr database); missed cleavages: 2; precursor
65 mass tolerance: 1 Da; dynamic side chain modification: carbamidomethylation and oxidation; enzyme: trypsin. Complete sequence analysis was done by the use of BLAST program against non redundant NCBI proteins database (www.ncbi.nlm.nih.gov) and proteomics ExPASy tools (www.expasy.ch). Multiple sequences were aligned by online Clustal Omega (ClustalW) server (www.ebi.ac.uk). Best possible alignments were acquired manually and protein identity was confirmed on the basis of significant hit as high protein score with coverage (Tayo et al., 2010; Yap et al., 2014; Malih et al., 2014).
2.10 Protein Estimation by Bicinchoninic Acid (BCA) for Naja naja krachiensis Venom
2.10.1 Protein Estimation by Bicinchoninic Acid (BCA)
Protein content of the required samples was determined via Thermo ScientificTM Micro BCA Protein Assay Kit (Product No. 23235). Standard curve for bovine serum albumin was prepared between BSA standard and its various concentration at 0.5-200µg/ml. Working reagent (WR) was prepared by mixing 25 parts of Micro BCA Reagent MA and 24 parts with Reagent MB with 01 part with Reagent MC. Sample replicates in thrice (150 µl) were mixed with equal volume of WR and incubated at 37 °C for two hours in 96 well plate. Subsequently plate was cooled at room temperature and absorbance was measured at 562 nm via Tecan infinite M 200pro (Magellan 7 software) microplate reader after subtracting the average value of absorbance blank. Subsequently standard curve was used to determine protein concentration for Naja naja karachiensis sample with a final concentration of 0.2 mg/0.15 ml (Malih et al., 2014; Smith et al., 1985; Wiechelman et al., 1988; Kessler and Fanestil 1986; Brown et al., 1989). The results were expressed as mean ± SD by using Microsoft Excel 2007 and performed three times for the sake of confirmation.
66 2.11 Elemental Analysis for Naja naja karachiensis Venom via Inductive Couple Plasma-Optical Emission Spectroscopy (ICP-OES) Technique
2.11.1 Multi-Elemental Analysis for Naja naja karachiensis Venom
2.11.1.1 Closed Pressurized Digestion of Samples
Closed microwave pressurizng system (UltraWave system, Milestone Srl, Sorisole, Italy) was selected for digestion of evaluated samples. Closed system possessed few advantages: in terms of less risk of contamination without loss of volatile analytes. Furthermore elevated boiling point of an acid (via nitrogen gas, 40 bars pressure) ensured rapid and more through matrix digestion. Digestion vial (Capitol vial, Fulton Ville, NY, USA) was used for measuring freez dried (100 mg) material and afterthat digestion media was added. A mixture of hydrogen peroxide (30%, 350 µl) and nitric acid (70%, 750 µl) was used for digestion of samples for 10 min at 240 °C (1500W microwave power) after initial 15 min of ramping. Furthermore, samples were cooled to 80 °C before releasing the pressure. At the end Milli-Q water was used to dilute (15 ml) the samples to ensure nictric acid 3.5% in concentration (Hansen et al., 2013).
2.11.1.2 ICP-OES Analysis
Elemental analysis for cobra venom was performed by ICP-OES (Optima 5300 DV, PerkinElmer, USA) equipped with cyclonic spray chamber, Meinhard nebulizer and auto sampler with automatic direct injection system. Following parameters were set for ICP- OES instrument: sample flow, 1.5 ml/min; nebulizer flow, 0.65 l/min; plasma flow, 15 l/min; auxiliary flow, 0.2 l/min; radio frequency power, 1400 W. Selected wavelengths were interference free and used in radial or axial mode. Software Winlab32 (version 3.1.0.0107, PerkinElmer) was used to process the acquired data. External calibration was done with inorganic standards (P/N4400-132565 A&B, P/N 4400-ICP-MSCS by CPI International, Amsterdam, Netherlands) and certified reference material (CRM), apple leaves (NIST 1515 from US Department of Commerce, National Institute of Standards and Technology, Gaithersburg, MD, USA) was used for analytical accuracy. Limit of
67 detection was three times SD of at least seven blank samples and all the data were acquired above the limit of detection. Furthermore, data were not acceptable if the accuracy of the elements was ˂ 90% of reference standard (Laursen et al., 2011; Hansen et al., 2009). All experiments were performed at least three times and numerical data was expressed as mean of individual results.
2.12 Biodistribution & Kinectic Study of Naja naja karachiensis Venom via Radio Tracer (99mTc) Binding Technique
2.12.1 Tracer (99mTc) Binding Protocol for Cobra Venom and its Percentage Yield
Technetium (99mTc) was eluted freshly with saline by Moly (99Mo/99mTc) generator in the 99m form of sodium pertechnetate ( Tc-NaTcO4). Various concentrations of stannous chloride dihydrate (50, 100, 150 and 200 µg) at different pH (5, 6 and 7) were evaluated for proper optimization and to get maximum percentage labeling yield. Fixed volume (125 µl) of cobra venom (250µg, 2mg/ml) solution was added to the 50µl acidified (3N HCl) solution of stannous chloride dihydrate. Subsequently radioactivity (18.5 M Bq or 0.5 mCi) was incorporated followed by 10 min of incubation. Percentage labeling was determined by spotting small aliquot (2µl) of tagged venom at the end of a strip (1.5 cm × 10 cm) of Whatman paper no 1. Chromatogram was developed with acetone present in a small vial fitted with screw cap. Subsequently chromatographic strip was dried and divided into 10 segments. Radioactivity for each segment was evaluated via well type NaI (TI) gamma counter (Cap-Ria 16 gamma counter) and percentage labeling was calculated (Asad et al., 2015; Priyadarshani et al., 2010; Sajid and Mahmood, 2012; Yonamine et al., 2005; Saha, 1984).
2.12.2 Stability Study for 99mTc Labeled Cobra Venom
99mTc labeled Naja naja karachiensis venom was evaluated for its stability by both in vitro and in vivo experiments. For in vitro determination 50 µl of labeled venom was incubated individually with 1000 µl of human serum and saline at 37 °C. Later on percentage labeling was determined by spotting 10µl of mixture on thin layer
68 chromatographic strip (1.5 cm × 10 cm) for a period of 4h (with 1h time lapse). After development of chromatogram percentage labeling yield was calculated. For in vivo experiments fixed volume (300 µl) of radio labeled venom (250 µg) was injected to the healthy male rabbits and blood serum was analyzed for its stability profile (Asad et al.,
2015(a); Shirmardi, 2010; Priyadarshani et al., 2010).
2.12.3 Biological Activity of Radio Labeled and Unlabeled Crude Cobra Venom
Radio activity didn’t alter any toxic potential(s) of Pakistani crude cobra venom was confirmed by hemolytic activity by the method reported by Asad et al. (2014)e. Healthy volunteer(s) blood (Rhesus positive) was collected to obtain packed cells volume. Equal volume (1000 µl) of radio labeled and non labeled venom (125, 250 & 500 µg) was incubated with 1% human red blood corpuscles (HRBCs) and phosphate buffer (0.15 M, pH 7.4) at 37 ºC for 30 min. Subsequently mixture was subjected to centrifugation at 1000 rpm for 3 min. Absorbance of supernatant was measured at 540 nm while ultra pure water and 0.9% NaCl solution was used as positive and negative control respectively. Hemolytic activity was accessed by the formula: Hemolytic activity = Absorbance of sample / Absorbance of control × 100.
2.12.4 Blood Kinetic Study of Technetium Labeled Cobra Venom
Radio labeled Naja naja karachiensis venom (250 µg with 18.5 M Bq / 0.5 mCi activities) was injected intravenously to the rabbits for determination of blood clearance. Blood was collected at specific intervals up to 24 h and monitored for radioactivity (Asad et al., 2015; Priyadarshani et al., 2010).
2.12.5 Biodistribution Study of Technetium Labeled Cobra Venom
Technetium labeled venom (250µg/0.3ml) was injected in rabbits through dorsal ear vein to study its bio distribution. For ex vivo studies animals were scarified exactly after 3h of envenomation. Different parts of rabbits (brain, blood, bones, heart, lungs, kidneys, liver, skin, stomach, intestines, skeleton muscles and urinary bladder) were separated, weighed and radio activity was counted with the help of gamma counter. Activity was expressed
69 as percentage of venom injected per gram of specified tissue (% ID/g). Radioactive decay corrections were applied by decay constant equation (1× e-λ t) (Asad et al., 2015; Yonamine et al., 2005).
2.12.6 Gamma Scintigraphy & Single Photon Emission Computed Tomography (SPECT) of Technetium Labeled Pakistani Cobra Venom
Animals were anesthetized initially with pre-anesthetic drug (buprenorphine) at the dose of 0.05 mg/kg. Subsequently anesthesia was induced by intravenous injection of propofol (8 mg/kg) and maintained at the dose of 0.6 mg/kg/min. Post venom injection (250µg/0.3ml) rabbits were subjected to acquire gamma scintigraphic images (blood pool & whole body images) for 2 h followed by SPECT images at 360º for transverse, coronal and sagittal sections via Siemens, Digitrac 75 gamma cameras (Asad et al., 2015; Priyadarshani et al., 2010; Martín-Cancho et al., 2006).
2.13 Toxic Biological/Biochemical Activities of Naja naja karachiensis Venom & their Neutralization with Medicinal Plants of Pakistan
2.13.1 Lethal Toxic Dose of Naja Naja Karachiensis Venom for 50%
Population (LD50)
Lethal dose for 50% population (LD50) of Naja naja karachiensis venom was determined by following the method of Meier and Theakston (1986) and necessary guidelines were considered as suggested by Aird and Kaiser (1985).
Briefly, three doses (D) of crude cobra venom (5, 10 and 25 µg/g, 0.2 ml) were prepared in saline and injected intraperitoneally (i.p) in male Swiss albino mice. Two mice were selected for each dose and time between injection and death was recorded in minutes. For each dose of a venom average survival time (T) was calculated. A plot was generated between D (ordinate) and D/T (abscissa) and LD50 value was obtained where regression line intersected the ordinate scale. According to the method LD50 is the least amount
70 venom attributed for 50% death in laboratory animals in an unlimited period of time (Roy, 2011; Meier and Theakston, 1986; Aird and Kaiser, 1985).
2.13.2 Hemolytic Activity
Hemolytic potential of Naja naja karachiensis venom was accessed by following the method reported by Asad et al. (2014) e. Moreover folklore claim about local Pakistani medicinal plants as anti-hemolytic was also evaluated.
Human volunteer (Rhesus positive) blood was collected and put in a vial containing heparin as anti-coagulant. Blood was subjected to centrifugation (3000 rpm) after washing three times with saline packed cells volume was separated. Human red blood corpuscles (HRBCs) (1% v/v, 1ml), phosphate buffer (0.15 M, pH 7.4, 1ml) and reconstituted snake venom solution (1ml, 0.1 mg/ml) was mixed together and incubated for 30 min at 37 °C. Later on mixture was subjected to centrifugation (1000 rpm) for 3 min and supernatant was measured at 540 nm. Anti-hemolytic property of 26 medicinal plants extract was evaluated by incubating equal volume of venom (0.1 mg/ml) with plants extract (20-320 µg/ ml) at 37 °C for 30 min. All plants extract were prepared in saline. Hyposaline (0.25% NaCl) was served as positive control while 0.9% NaCl solution was used as negative control (Asad et al., 2014(e)). All results were expressed as mean ± SD by using Microsoft Excel 2007. Moreover student t-test was applied to compare the results with standard antidote.
Percentage hemolysis and protection was calculated by the formula mentioned below
Hemolytic percentage = Absorbance of sample / Absorbance of control × 100 = Y
Protection percentage = 100 – Y = Z
2.13.3 Anticoagulant Activity
To evaluate anticoagulant activity of Naja naja karachiensis venom general purpose coagulation tests (prothrombin time, activated partial thromboplastin time and thrombin time) were performed on platelet poor plasma (PPP) as reported by Asad et al. (2014)c.
71 2.13.3.1 Preparation of Platelet Poor Plasma (PPP)
Blood was obtained from healthy volunteers in a tube containing k3-EDTA as an anticoagulant. Platelet rich plasma (PRP) was obtained by subjecting the blood to centrifugation at 200 g for 15 min. Later on PRP was again subjected to centrifugation for
20 min at 2000 g to obtain PPP (Asad et al., 2014(c)).
2.13.3.2 Prothrombin Time (PT) Coagulation Assay
Snake venom (200µg/ml) was mixed with PPP (100 µl) at 37ºC for 3 minutes in a water bath. Stop watch was started when this solution was transferred in a tube containing 200 µl of reconstituted PT reagent (Soluplastin by Weiner lab Argentina). Tube was moved back and forward (two times per second) and time was recorded in seconds until clot was appeared. Furthermore, to nullify coagulopathy (delay in PT) snake venom was incubated with various concentrations (5-640 µg/ml) of medicinal plants extract and clotting time was measured (Asad et al., 2012; Asad et al., 2014(c); Franc et al., 2003).
2.13.3.3 Activated Partial Thromboplastin Time (aPTT) Coagulation Assay
Snake venom (200 µg /ml) was mixed with 100 µl PPP. Subsequently equal volume of aPTT reagent (APPTest, Weiner lab Argentina). The mixture was incubated for 3 minutes at 37 ºC in a water bath. Calcium chloride (100 µl, 0.025 mol/l) solution which was already maintained at 37 ºC was added to the above mixture. Stop watch was started and tube was moved back and forth in a water bath for initial 25 sec. After this tube was removed from water bath and clotting time was recorded in seconds. To neutralize delay in aPTT snake venom was pre-incubated with various plants extract (5-640 µg/ml) and clotting time was determined (Asad et al., 2012; Asad et al., 2014(c); Franc et al., 2003).
2.13.3.4 Thrombin time (TT) Coagulation Assay
PPP (200 µl) and snake venom (200 µg /ml) was maintained at 37 ºC for 3 min. Timer was started after the addition of 100 µl thrombin time reagent (Human Wiesbaden, Germany) until clot was appeared. To inhibit delay in TT snake venom was pre-incubated with various plants extract in a concentration range of (5-640 µg/ml) and ultimately clotting time was recorded (Asad et al., 2012; Asad et al., 2014(c); Franc et al., 2003).
72 All numerical data related to PT, aPTT and TT was expressed as mean ± SD by using Microsoft Excel 2007. Moreover 95% confidence interval of mean of standard antidote was calculated to compare the efficacy of medicinal plants extract.
2.13.4 Study of Surrogate Markers (Biochemical Parameters) for Liver, Heart and Kidney Damage
In this section biochemical parameters for heart, liver and kidneys damage were evaluated in rabbits after snake venom envenomation. Snake venom toxicities were evaluated at the dose of (~700 µg/kg) that is attributed to severe envenomation in human (Audebert et al., 1994).
2.13.4.1 Laboratory Animals Preparation
Growing male healthy rabbits (1±0.5 kg) were selected for toxicity determination. Rabbits were acclimatized by providing their standards for food, water and light for a week in laboratory cages. Subsequently they were divided into 5 different groups. Group I: reserved for baseline measurements; Group II: reserved for 1st doses of venom; Group III: reserved for 2nd doses of venom; Group IV: reserved for negative control; Group V: reserved to evaluate the extract of medicinal plants. Group V was redivided into 29 sub groups (V/1-V/29) such that each plant extract was assigned to a single subgroup (Asad et al., 2014(d)).
2.13.4.2 In vivo Anti Snake Venom Potential of Medicinal Plants Extract
Naja naja karachiensis venom was injected subcutaneously for its toxicity determination to the heart, liver and kidneys at two different doses (400 µg/kg & 800 µg/kg). Before injection rabbits were anaesthetized with ketamine (50 mg/kg) and saline was used as control. To evaluate anti-venom potential plants extract (100 mg/kg) were incubated with fixed (800 µg/kg) amount of venom at 37 °C for half an hour. After 3 h of injection blood was collected from marginal ear artery with hypodermic syringe. Blood serum was separated by following the conventional method for its extraction. Later on blood serum was subjected to various biochemical assays for toxicity determination with the help of
73 their respective kits. Reference standard (anti-sera) was used to compare neutralizing tendency of various medicinal plants extract (Asad et al., 2014(d)).
2.13.4.3 Estimation of Biochemical Parameters: GPT/Alanine Aminotransferase (ALAT) & GOT/Aspartate Aminotransferase (ASAT) to Access Hepatic Damage
2.13.4.3.1 Assay Procedure
ALAT and ASAT activities were determined by Selectra Junior (Vital Scientific B.V, Netherlands) with the help of their respective kits (Innoline, Merck). Serum sample (50µl) was mixed with 500 µl of ALAT and ASAT mono reagent at 37 °C. After 1 min of mixing absorbance at (340nm) was measured for 3 min at 1 min interval. Change in absorbance per minute was determined and activity was calculated by the formula given below. Mono reagent was prepared by mixing of two solutions R1 (4 parts) with R2 (1 part). Complete detail about composition of mono reagents for ALAT and ASAT is mentioned in table 2.10 and 2.11 respectively (Thomas, 1998; Henderson and Moss, 2001).
ALAT / ASAT [U/l] = ∆A/min sample × factor (1746)
All results related to ASAT/ALAT were expressed as mean ± SEM by using Microsoft Excel 2007. Additionally student t-test was applied to compare the results with standard inhibitor.
2.13.4.4 Estimation of Biochemical Parameters: Creatine Kinase (CK-MB) & Lactate Dehydrogenase (LDH) to Access Cardiac Damage
2.13.4.4.1 Assay Procedure LDH enzymatic activity was evaluated by Selectra Junior (Vital Scientific B.V, Netherlands) with the help of LDH kit (DiaSys Diagnostic Systems, Germany). Rabbit serum (20 µl) was mixed with 1000 µl freshly prepared LDH mono reagent (Table 2.12 and 2.13) at 37 °C while distilled water was used for blank measurement. Absorbance at 340 nm was measured exactly after 1 min of mixing and continued to measure up to 4 min at 1 min interval. Change in absorbance / min was determined and LDH activity
74 (U/l) was calculated as: LDH (U/l) = (∆A/min sample - ∆A/min blank) × factor 8095 (Schumann et al., 2002).
CK-MB activity was determined via UV spectrophotometer with the help of CK-MB kit (Minias Globe Diagnostics, Italy). Serum sample (40 µl) was mixed with 1000 µl CK- MB mono reagent at 37°C for 5 min while distilled water was used for blank measurement. Absorbance at 340 nm was measured and subsequently for each and every min absorbance was recorded up to 3 min. Change in absorbance was measured and CK- MB activity was calculated as: CK-MB (U/l) = (∆A/min sample - ∆A/min blank) × factor 8254 (Lang and Würzburg, 1982; Gerhardt and Waldenstrom, 1976; Stein, 1985).
All results related to LDH and CK-MB was expressed as mean ± SEM by using Microsoft Excel 2007. Additionally student t-test was applied to compare the results with standard inhibitor.
2.13.4.5 Estimation of Biochemical Parameters: Urea & Creatinine to Access Renal Damage
2.13.4.5.1 Assay Procedure
Urea concentration was measured by Selectra Junior (Vital Scientific B.V, Netherlands) with the help of urea kit (Innoline, Merck). 10 µg serum sample or standard (50 mg/dl) was mixed with 1000 µl of mono reagent (Table 2.14 and 2.15) while distilled water was used as blank. After 01 min of incubation at 37 °C absorbance (A1) was measured at 340 nm. Exactly after 1 min again absorbance (A2) was observed (Fawcwtt and Scott, 1960; Newman and Price, 2001; Talke and Schubert, 1965). Change in absorbance was recorded and urea concentration was calculated by the formula given below:
∆A = [(A1 - A2) sample or standard] - [(A1 - A2) blank]
Urea (mg/dl) = ∆A sample / ∆A standard × concentration of standard (50 mg/dl)
Serum estimation for creatinine was performed by Selectra Junior (Vital Scientific B.V, Netherlands) with the help of creatinine kit (DiaSys Diagnostic Systems, Germany). Mono reagent for creatinine (1000 µl) was mixed at 37 °C with 50 µl serum or standard
75 (2 mg/dl) sample and distilled water was used as blank. After 1min of mixing absorbance was measured (A1) at 505 nm and absorbance (A2) was read after further 2 min (Thomas, 1998; Mazzachi et al., 2000; Newman and Price, 1999). After determining change in absorbance creatinine concentration was calculated by the formula given below:
∆A = [(A1 - A2) sample or standard] - [(A1 - A2) blank]
Creatinine (mg/dl) = ∆A sample / ∆A standard × concentration of standard (2 mg/dl)
All results related to urea and creatinine was expressed as mean ± SEM by using Microsoft Excel 2007. Additionally student t-test was applied to compare the results with standard inhibitor.
Table 2.10 Composition of Mono Reagent (By Mixing 4R1 & 1R2) Used for ALAT Estimation. Reagent name Chemicals Quantity
Reagent R1 Tris buffer pH 110 mmol/l 7.5 L-Alanine 550 mmol/l
Reagent R2 LDH 1200 U/l α-ketoglutarate 16 mmol/l NADH 0.20 mmol/l
Table 2.11 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for ASAT Estimation. Reagent name Chemicals Quantity
Reagent R1 Tris buffer pH 7.8 110 mmol/l L-Aspartate 320 mmol/l MDH 800 U/l LDH 1200 U/l
Reagent R2 2-Oxaglutarate 65 mmol/l NADH 1 mmol/l
76
Table 2.12 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for LDH Estimation. Reagent name Chemicals Quantity
Reagent R1 N-Methyl-D-Glucamine (pH 325 mmol/l 9.4) L-Lactate 25 mmol/l + Reagent R2 NAD 10 mmol/l
Table 2.13 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for CK-MB Estimation. Reagent name Chemicals Quantity
Reagent R1 Imidazol buffer, pH 6.7 100 mmol/l N-acetylcysteine 20 mmol/l Magnesium acetate 10 mmol/l Glucose 20 mmol/l NADP 2.5 mmol/l HK ≥4 KU/l
Reagent R2 Creatine phosphate 30 mmol/l AMP 5 mmol/l ADP 2 mmol/l Diadenosine pentaphosphate 10 µmol/l G6P-DH ≥1.5 KU/l CK-M human antibody Quantity sufficient
77
Table 2.14 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for Urea Estimation. Reagent name Chemicals Quantity
Reagent R1 Tris, pH 7.8 150 mmol/l 2-Ketoglutarate 8.75 mmol/l ADP 0.75 mmol/l Urease ≥7.5 kU/l Glutamate-dehydrogenase ≥1.25 kU/l Sodium azide ≤0.95 g/l
Reagent R2 NADH 1.32 mmol/l Sodium azide ≤0.95 g/l
Table 2.15 Composition of Mono Reagent (Mixture of 4R1 & 1R2) Used for Creatinine Estimation. Reagent name Chemicals Quantity
Reagent R1 Sodium hydroxide 0.2 mol/l
Reagent R2 Picric acid 20 mmol/l
78
2.14 Various Enzymatic Assays for Naja naja karachiensis Venom and their Neutralization by Medicinal Plants of Pakistan
2.14.1 Enzymatic Assays for Snake Venom Phospholipase Enzyme
Phospholipase enzymatic activity was accessed by three different procedures (biochemical assays).
2.14.1.1 Assay I (Acidimetric Assay)
Acidimetric phospholipase (PLA2) enzymatic assay was performed by following the method reported by Asad et al. (2014) b.
Assay was performed on egg yolk suspension which was prepared by mixing sodium deoxycholate (8.1 mM), calcium chloride (18 mM) and egg yolk in equal amount. Subsequently mixture was subjected to stirring (10 min) to get homogenous suspension and pH was adjusted at 8 with 1M NaOH. Various concentration of cobra venom (0.1- 8 mg/0.1 ml) was added to the suspension (15 ml) to start the process of hydrolysis while 0.9% NaCl was served as control. After 2 min interval a decline in pH of the suspension was noted and decrease in 1.0 pH unit is equivalent to 133 µmol of free fatty acids released. Later on PLA2 activity at different doses of cobra venom were calculated in terms of units/mg of lyophilized venom. To evaluate anti-venom (anti-PLA2) potential medicinal plants extract (0.1-0.6 mg/ml) were incubated with fixed amount of venom (0.1 mg) and their efficacy was recorded in terms of percentage inhibition. Standard anti-dote (anti-sera) was used to compare neutralizing efficiency of various medicinal plants extract and the results were expressed as mean ± SEM by using Microsoft Excel 2007.
(Asad et al., 2014(b)).
2.14.1.2 Assay II (Turbidimetric Assay)
Phospholipase enzymatic activity was performed turbidimetrically as reported previously by Molander et al. (2014).
79 Egg yolk mixture (1.1%) was prepared by mixing calcium chloride (0.01M, 0.1ml), phosphate buffer saline (0.1M, 26ml, pH 8.1) and egg yolk powder (0.3g). Snake venom (10µl, 20 mg/ml) was incubated with 200µl of suspension while saline was served as control. Decrease in turbidity of suspension is due to PLA2 enzyme and was measured at 5 min interval in a micro plate reader for 20 min at 630nm. To determine the anti-venom activity, medicinal plants extract (20µl, 10mg/ml) were pre-incubated with fixed amount of venom (200 µg) at 37 ºC for 15 min.
PLA2 activity was represented in percentage and considered cent percent in the absence of any anti-venom. Specific metalloenzyme inhibitor EDTA (0.01M) was used to compare neutralizing potential of different medicinal plants extract (Chakrabarty et al., 2000; Molander et al., 2014). Moreover results were expressed as mean ± SD by using Microsoft Excel 2007. Additionally student t-test was applied to compare the results with standard inhibitor.
2.14.1.3 Assay III (Coagulation Assay)
To evaluate anticoagulant (weak or strong) potential of phospholipase (PLA) enzyme hen’s egg yolk model was selected as adopted by Asad et al. (2013).
Hen’s egg yolk suspension was prepared by mixing following chemical substances: Tris- HCl buffer (2 ml, 50 mM, pH 7.5); isosaline (0.56 ml); calcium chloride (1%, 4.44 ml); EDTA (0.5%, 1.49 ml); sodium chloride (2%, 2.51 ml); egg yolk (9 ml). Snake venom (200 µl, 25 µg/ml) was added to the fixed volume of suspension (2 ml) and further incubated at 37 °C for 60 min while saline was used as control. Later on mixture was shifted to the boiling water bath and clotting time was recorded. To estimate anti-PLA (anti-coagulant) activity different plants extract (5 µg, 25 µg/ml) were pre-incubated with fixed amount of venom (5 µg) at 37 °C for 30 min and clotting time was recorded. Standard antidote (anti-sera) was used to compare neutralizing potential of various plants extract and results were expressed in percentage (Asad et al., 2013). Moreover all numerical values were expressed as mean ± SD by using Microsoft Excel 2007.
80 2.14.2 Enzymatic Assay for Snake Venom Alkaline Phosphatase
Enzymatic activity for alkaline phosphatase (ALPase) present in Naja naja karachiensis venom was performed by adopting the method reported by Asad et al. (2014)a.
An ALPase activity was measured by its hydrolytic action on p-nitrophenyl phosphate led to release in p-nitrophenol which confers yellow coloration with NaOH and absorb maximally at 400 nm. Reaction mixture was prepared by adding p-nitrophenyl phosphate
(0.5ml, 0.01M), glycine buffer (0.5 ml, 0.5M) and MgSO4 (0.3 ml, 0.01M) together. After addition of cobra venom (0.2-0.8 mg/0.1 ml) mixture was incubated at 37 °C for 30 min. Reaction was stopped with the addition of NaOH (2ml, 0.2 M) and further kept at room temperature for 20 min. Additionally venom (0.4 mg) was evaluated by heating it at 100 °C for 10 min and after cooling at room temperature for 45 min. Standard curve for known p-nitrophenol concentration was constructed and ALPase activity was expressed in U/mg of lyophilized venom (Chakrabarty et al., 2000; Asad et al., 2014(a)).
To test anti-venom (anti-ALPase) activity various anti-dotes (plants extract & reference standard anti-sera) were pre-incubated with 0.4 mg of cobra venom within concentration range of 0.312 to 5 µg/ml at 37 °C for 30 min. Additionally specific metalloenzyme inhibitor EDTA (2 mM) was also evaluated for inhibitory effect on ALPase activity
(Chakrabarty et al., 2000; Asad et al., 2014(a)). All the numerical values were expressed as mean ± SEM by using Microsoft Excel 2007. Additionally student t-test was applied to compare the results with standard antidote.
2.14.3 Enzymatic Assay for Snake Venom 5ʹ-Nucleotidase
Enzymatic assay for 5ʹ-nucleotidase cobra venom was performed using adenosine monophosphate (5ʹ-AMP) as a substrate by following the method adopted by Tan et al. (2011) (Asad et al., 2016).
Reaction mixture was prepared by mixing glycine buffer (0.5 ml, 0.2M, pH 8.5), MgSO4 (0.1 ml, 0.1M) and 5ʹ-AMP (0.5 ml, 0.02 M) together. One hundred microliters of cobra venom (5-40 µg) was added to the reaction mixture and incubated for 10 min at 37 °C to liberate inorganic phosphates. Enzymatic reaction was stopped with addition of 10%
81 trichloroacetic acid (1.5 ml) and phosphate contents were determined with freshly prepared ascorbic acid (ascorbate-molybate color) reagent. Ascorbic acid reagent was prepared by mixing one volume of 6N H2SO4, 2.5% ammonium molybdate and 10% ascorbic acid with twice the volume of distilled water. Reaction mixture was kept at 25 °C for 30 min and absorbance at 820 nm was measured after subtracting the value of sample blank (distilled water).
To estimate anti-venom potential different antidotes (plants extract & reference standard anti-sera) were pre-incubated with 0.01 mg of venom within concentration range of 0.010- 0.640 mg/0.1ml for 15 min at 37 °C. Standard curve for various concentrations of inorganic phosphate (µM) was constructed and enzymatic activity of was expressed in Unit/ml (Tan et al., 2011; Dhananjaya et al., 2006; Ushanandini et al., 2006; Asad et al., 2016; Chen et al., 1956). Results were expressed as mean ± SEM by using Microsoft Excel 2007. Additionally student t-test was applied to compare the results with standard inhibitor.
2.14.4 Enzymatic Assay for Snake Venom Hyaluronidase
Enzymatic assay for hyaluronidase present in cobra venom was performed turbidimetrically by following the method described by Molander et al. (2014) with few modifications.
Acetate buffer (0.2M, pH 5, 0.07ml) containing sodium chloride (0.15M) was mixed with cobra venom (0.1mg, 0.01ml) and DMSO (3%, 0.01ml) and further subjected to incubation at 37°C for 15 min. Subsequently, optimum quantity of hyaluronic acid (Km= 19 µg, 0.01ml) was added to the reaction mixture and futher incubated for 10 min. Enzymatic activity was ceased with the addition of 2.5% CTAB and absorbance was measured at 400 nm within 10 min of incubation. Hyaluronidase activity was considered 100% in the absence of an antidote.
To evaluate antidotal property, medicinal plants extract (0.1mg) were pre-incubated with fixed amount of venom at 37ºC for 15 min. However, standard cobra venom hyaluronidase inhibitor, rutin trihydrate (2.5 mM) was used to compare neutralizing efficiency of various medicinal plants extract (Girish et al., 2009). All the results were
82 expressed as mean ± SD by using Microsoft Excel 2007. Furthermore student t-test was applied to compare the results with reference standard.
2.14.5 Enzymatic Assay for Snake Venom Protease
Proteolytic activity was performed the adopting the procedure described by Molander et al. (2014).
Naja naja karachiensis venom (0.3 mg) was incubated with casein (1%, pH 8.5) as substrate for the period of 50 min at room temperature in the triplicate wells of micro titer plate. The enzymatic activity was stopped with trichloroacetic acid (0.44 M, 0.09 ml) and mixture was left to stand for half an hour at 37°C. After centrifugation proteolytic activity of the supernatant was measured at 630 nm with the addition of Folin-ciocalteau (0.05 ml) and sodium carbonate (0.4M, 0.25 ml) reagents in 20 min. Furthermore results were expressed as mean ± SEM by using Microsoft Excel 2007.
2.15 Identification of Specific Enzyme(s) Inhibitor(s)
2.15. 1 Potential Anti-PLA2 Plant Extract
Selected plant extract was shown to have (˃90%) anti-PLA2 activity in at least one of the three given assays (section: 2.15.1.2) was further investigated in HPLC-UV coupled
(PLA2 turbidimetric) bioassay system.
2.15.1.1 High Resolution (HPLC) Microplate Based PLA2 Bioassay Guided Fractionation
Among various preliminary screened samples (section: 2.1.3.) the most active plant extract was further subjected to the HPLC analysis via Agilent 1200 series instrument (Santa Clara, CA) system. It was equipped with following accessories: quaternary pump (G1311A); thermostated column (G1316A); degasser (G1322); photodiode array detector (G1315C); high performance autosampler (G1367C); fraction collector (G1364C). HLPC system was controlled by Agilent ChemStation software (ver. B.03.02).
83 Gradient was changed from eluent A (95% water: 5% acetonitrile) towards eluent B
(95% acetonitrile: 5% water) with the addition of 0.1% formic acid. Reversed phase C18 Luna column was maintained at 40 C (Phenomenex, 150 × 4.6 mm, 3 µM, 100 ) and used to separate the component(s) of medicinal plant extract. The flow rate was maintained at 0.5 ml/min with the following elution pattern: 0 min, 5% B; 30 min, 95% B; 40 min, 95% B; 41 min, 5% B; along with 8 min of equilibrium. After a single injection of a sample (20 µL, 5 mg/ml) the eluate was fractionated from 2 min to 52 min by maintaining the column out flow towards automated fraction collector into 96-well micro plates (Sterilin, Aberbargoed Caerphilly, UK) having resolution of 8 points per min. The eluate was dried in the micro plate using a Savant SpeedVac concentrator (SPD 121P, Thermo Scientific) equipped with refrigerated vapor trap (RVT400) and OFP400 oil free pump. Rest of all the procedure was the same as described previously.
2.15.1.2 IC50 Determination (Dose Response Curves of Effective Anti-PLA2 Inhibitors)
IC50 curves were made for the best medicinal plant extract (anti-PLA2) and EDTA
(specific metalloenzyme PLA2 inhibitor) at various concentrations. All tests were made with triple determination. For IC50 determination maximum and minimum wells were used in each plate to ensure that data were comparable. Maximum wells were made without inhibitor while minimum wells were made without inhibitor and enzyme. IC50 curves were generated with the help of software Prism 5 (GraphPad, Inc., San Diego, CA 92130, USA).
For determination of IC50 about medicinal plant (PLA2 assay) following concentrations were used: 20 mg/ml, 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml, 0.62 mg/ml and 0.31 mg/ml.
For determination of IC50 about EDTA following concentrations were used: 0.15M, 0.1M, 0.05M, 0.01M, 0.001M and 0.0001M.
84
Chapter 3 Results
85 In this chapter results that were obtained from laboratory work will be presented.
3.1 Proteomic Characterization of Naja naja karachiensis Venom
3.1.1 Proteomic Strategy for Analysis of Naja naja karachiensis Venom
Recent advances in the field of proteomic have opened a new arena for identification and characterization of different venoms however complete panacea is still lacking. One technique may have useful edge for characterization of one proteome but may have disadvantageous for others attributed to the variation in sizes of peptides and proteins. As a resort two strategies were adopted: (1) SDS-PAGE as a rapid and cheap investigation method, and (2) using gel filtration and RP-HPLC for complete resolution of components together with LC-MS/MS (ESI-MS) for effective separation and identification of peptide sequences. Complete schematic overview about proteomic strategy of Naja naja karachiensis venom is shown in fig 3.38.
Fig 3.38 Schematic Overview of Combined Proteomic Strategy about Naja naja karachiensis Venom.
86 3.1.2 Electrophoresis Pattern / (SDS-PAGE) Profile
Various components for Naja naja karachiensis venom were analyzed and compared by SDS-PAGE (4-20% SDS-polyacrylamide gel) under reducing and non reducing conditions as shown in fig 3.39. Various protein bands (6 KDa to 200 KDa) were observed however surprisingly venom profiles under both conditions were not same. Venom protein bands from 15 KDa to 36 KDa were not found identical while they were similar between 50 KDa to 90 KDa under both conditions. Condensed area of bands (15 KDa to 36 KDa) was appeared at non reduced state while different bands were appeared in reduced form of venom between 9 KDa to 12 KDa. Protein bands greater than 95 KDa were appeared between 95 to 130 KDa after reduction with β-mercaptoethanol. Protein bands lesser than 10 KDa were found on Coomassie Brilliant Blue R250 (CBB) stained gel and bands ˃95 KDa were only appeared on silver stained gel however rest of all bands revealed the same.
3.1.3 Chromatographic Separation of Snake Venom Components
Snake venom was subjected to the gel chromatographic separation of components and yielded 6 different fractions namely peaks 1, peak 2, peak 3, peak 4, peak 5 and peak 6 as shown in figure 3.40. Moreover each fraction of gel filtration was further fractionated by RP- HPLC and yielded 10 fractions (1a-1j) from peak 1, 11 fractions (2a-2k) from peak 2, 12 fractions (3a-3l) from peak 3, 7 fractions (4a-4g) from peak 4, 8 fractions (5a-5h) from peak 5 and 5 fractions (6a-6e) from peak 6 (Fig 3.40 - 3.46).
3.1.4 Analysis of Tryptic Digested Venom Samples via LC-MS/MS
Proteomic (LC-MS/MS) analysis of (in solution) tryptic digested Naja naja karachiensis crude venom confirmed the presence of three finger toxin (3FTX), phospholipid A2
(PLA2), snake venom metalloproteinase (SVMP), helvepryn, cobra serum albumin, vespryn, L-amino acid oxidase (LAAO), cobra venom factor (CVF), phosphodiesterase, 5’-nucleotidase (5’-ND), venom nerve growth factor (vNGF) and kunitz type serine protease inhibitor. However subsequently SEC separated RP-HPLC fractions indicated relative abundance of various protein families. Overall 43 different proteins were
87 identified with their molecular weight, score and coverage matched from public available databases. They were assigned into 3FTXs (58%), PLA2 (19%), SVMPs (5%), LAAO (5%), helvepryn (3%), vespryn (2%), CVF (2%), 5’-ND (2%), vNGF (2%) and kunitz type serine protease inhibitor (2%). Cobra serum albumin and phosphodiesterase were not identified from RP-HPLC fractions. Among 3FTXs cytotoxins (CTXs) were abundant (32%) followed by weak (WNTX) and long (LNTX) neurotoxins (24%). Short neurotoxin (SNTX) and muscarinic-toxin like protein (MTLP) constituted 8% while post synaptic neurotoxin (post synaptic-NTX) was found the least (4%) among 3FTXs. MTLP-3 from Naja naja karachiensis was found unique among 3FTXs due to 78% homology in amino acids sequence when aligned with novel haditoxin (Fig 3.47).
It was observed that many of the RP-HPLC fractions were partially purified (heterogeneous) and required further separation. Some fractions were purified (homogeneous) while remaining couldn’t be identified and matched to known proteins from databases. Complete description about proteomic analysis / picture is mentioned in table 3.16 - 3.17 & fig 3.48.
3.1.5 Protein Concentration of Naja naja karchiensis Venom
Protein content in venom sample was determined by bicinchoninic acid method. Standard curve of bovine serum albumin (BSA) was obtained with positive correlation coefficient (r) equal to 0.999 while y = 0.014x + 0.000 as shown in fig 3.49. Total protein content was found to be 188 ± 0.011 µg of 200 µg of dry weight. It constitutes overall 94% protein components in Naja naja karachiensis venom.
88
Fig 3.39 SDS-PAGE (4%-20% resolving gel) Electrophoresis Profile (Reduced & Non- reduced) of Naja naja karachiensis Venom with Coomassie Brilliant Blue R250 (a) and Silver Stain (b). Mobility of the Protein Ladder Markers is Shown on the Right (a) and in the Middle (b).
89
Fig 3.40 Gel Filtration Chromatographic Separation of Crude Naja naja karachiensis Venom on SuperdexTM 200, HiloadTM 16/60, Preparative Grade Column using 50mM Tris-HCl buffer pH 7.4.
90
st Fig 3.41 RP-HPLC Profile of 1 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A° Pore Size) and ÄKTA Purifier LC Unicorn System.
91
nd Fig 3.42 RP-HPLC Profile of 2 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A° Pore Size) and ÄKTA Purifier LC Unicorn System.
92
rd Fig 3.43 RP-HPLC Profile of 3 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A° Pore Size) and ÄKTA purifier LC Unicorn System.
93
th Fig 3.44 RP-HPLC Profile of 4 Gel Chromatographic Peak using Jupiter C18 Column (10 × 250 mm, 5 µm Particle Size, 300 A Pore Size) and ÄKTA Purifier LC Unicorn System.
94
th Fig 3.45 RP-HPLC Profile of 5 Gel Chromatographic Peak using Jupiter C18 Column (4.6 × 250 mm, 5 µm Particle Size, 300 A Pore Size) and ÄKTA purifier LC Unicorn System.
95
th Fig 3.46 RP-HPLC Profile of 6 Gel Chromatographic Peak using Jupiter C18 Column (4.6 × 250 mm, 5 µm Particle Size, 300 A Pore Size) and ÄKTA Purifier LC Unicorn System.
96 Table 3.16 Assignment of Naja naja karachiensis Crude Venom Proteins to Various Families after Digestion of Peptides via Trypsin by LC-MS/MS Analysis. Protein Matched peptide sequence Accession Protein Sequence Molecular families No. score coverage weight (%) (kDa) Three finger GCIDVCPKSSLLVKYVCCNTDK 117682 71.35 83.33 6.6 toxins NLCYKMFMVAAPHVPVKR (CTX-II) (3FTXs) YVCCNTDKCN RGCIDVCPK KLVPLFSK LVPLFSK TGVDIQCCSTDDCDPFPTR 128932 50.71 90.14 7.8 RVDLGCAATCPTVR (LNTX-I) DCPNGHVCYTK TWCDGFCSIR CFITPDITSK NLCYKMFMVSTSTVPVKR 117664 49.33 60 6.7 YVCCSTDKCN (CTX-1) RGCIDVCPK GCIDVCPK TGVDIQCCSTDNCNPFPTR 128930 45.30 78.87 7.8 GKRVDLGCAATCPTVK (α- RVDLGCAATCPTVK EPTX/LNT DCPNGHVCYTK X) CFITPDITSK YRRGCAATCPEAKPR 136565 37.84 78.46 7.6 GCAATCPEAKPR (WNTX- 7) LTCLNCPEVYCR EIVQCCSTDK NLLGK FQICR ICFK GCIDVCPKSSLLVKYVCCNTDR 1345874 28.38 49.38 8.9 YVCCNTDRCS (CTX- 8) CNQLIPPFYK RGCIDVCPK GCIDVCPK
97 NLCYK SSLLVK LTCLICPEKYCNK 136564 24.59 69.23 7.6 EIVQCCSTDK (WNTX- 6) VHTCLNGEK GCADTCPVR LTCLICPEK ICFK LECHNQQSSQPPTTK 128973 15.79 68.85 6.9 VKPGVNLNCCR (SNTX-1) TCSGETNCYK GTIIER SIFGVTTEDCPDGQNLCFK 12230755 9.97 47.69 7.3 GCAATCPIAENR (MTLP-2)
MYMVSDK 298351640 9.51 63.33 3.4 LIPLAYK (CTX- 8) DLCYK GCTFTCPELRPTGIYVYCCRR 12230756 4.96 32.31 7.6 (MTLP-3) Phospholipase GDNNACAASVCDCDRLAAICFAGAP- 129514 65.11 96.64 13.3
A2 (PLA2) YNDNNYNID LAAICFAGAPYNDNNYNIDLK GDNNACAASVCDCDR SWWDFADYGCYCGR CCQVHDNCYNEAEK TYSYECSQGTLTCK GGSGTPVDDLDR NMIKCTVPSR ISGCWPYFK NLYQFK CCQVHDNCYDEAEK 2144440 58.69 54.62 13.3 TYSYECSQGTLTCK GGSGTPVDDLDR NMIQCTVPSR ISGCWPYFK NLYQFK LAAICFAGAPYNDNNYNIDLK 31615584 5.50 26.89 13.1
98 DFADYGCYCGR CCQTHDNCYDKAEK 55669539 21.06 11.29 14 WDFYRYSLR 239977492 4.74 7.38 14.3 Metalloprotei- HDCDLPELCTGQSAECPTDSLQR 32469675 52.88 25.19 44.5 nases CPTLTNQCIALLGPHFTVSPK (HMP) (SVMP) VYEMINAVNTKFRPLK TAPAFQFSSCSIR VYEMINAVNTK YIEFYVIVDNR CGDGMVCSK DYQEYLLR KRNDNAQLLTGIDFNGTPVGLAYIGS- 82223366 35.43 28.67 67.6 ICNPK (ZMP) AAKDDCDLPELCTGQSAECPTDVFQR STRMVAITMAHEMGHNLGMNHDK DPSYGMVEPGTKCGDGMVCSNR LQHEAQCDSEECCEK YIEFYMVVDNIMYR CPIMTNQCIALR CPIMTNQCIALR ATLDLFGEWR DSCFTLNQR CVMSTR LFCK ENDVKIPCAPEDIK 297593804 9.0 3.93 69.2 VTLDLFGEWR (ZMP)
ERPQCILNKPSR 294845712 6.52 4.45 68.2 FKGAETECR (ZMP) IPCAAK Helvepryns VLEGIQCGESIYMSSNAR 71041970 30.96 41.18 24.9 (CRISP) WANTCSLNHSPDNLR MEWYPEAASNAER TWTEIIHLWHDEYK RVSPTASNMLK VSPTASNMLK EIVDLHNSLR SNCPASCFCR
99 Cobra serum WECISNLGPDLSFVPPTFNPK 2134234 26.33 15.47 69.8 albumin SKPNISEEELAATILTFR MMPQAPTSFLIELTEK ELGDYFFTNEFLVK DSVLAQYIFELSR SPDLPPPSEEILK Vespryns ADVTFDSNTAFESLVVSPDKK 32363235 3.66 19.44 12 L-amino- TCADIVINDLSLIHDLPK 126035677 20.13 9.8 51.4 oxidases TFVTADYVIVCSTSR (LAAO) EGWYVNMGPMR SPLEECFQQNDYEEFLEIAR 347602328 6.15 21.05 11.2 Phosphodie- VRDVELLTGLDFYSALK Oh-132843 5.72 3.82 100.7 sterases SLVKPTSVPPSASDCLR Cobra -venom IEEQDGNDIYVMDVLEVIK 881915 19.26 3.7 184.4 -factors AVPFVIVPLEQGLHDVEIK (CVF) GDNLIQMPGAAMK VFSMDHNTSK GDNLIQMPGAAMK 228312101 8.59 5.58 69.5 ALYTLITPAVLR VFSMDHNTSK GDDVSHCRKENGAK 294845712 8.18 6.75 68.2 VYEMVNYLNTK FKGAETECR MVEPGTK 5’- YDAMALGNHEFDNGLNGLLDPLLK 537444870 14.73 16.72 62.9 nucleotidases VIKASGNPILLNKSIQEDPAVK (5’ND) FHECNLGNLICDAVVYNNLR VLLPSFLAAGGDGYYMLK VVSLNVLCTECR FHECNLGNLICDAVIYNNVR 547223115 12.04 14.68 55.5 LLLPSFLAGGGDGYYMLK DISEDQDVKAEVNK VVSLNVLCTECR EVVKFMNSLR Kunitz type - FIYGGCGGNANR 125050 18.61 57.89 6.4 serine RPGFCELPAAK protease- AHKPAFYYNK
100 inhibitor
Venom -nerve- GNTVTVMENVNLDNK 128164 22.69 32.76 13 growth factor ALTMEGNQASWR (vNGF) IDTACVCVITK All spectra acquired were searched against non- redundant NCBI database while protein families were classified on the basis protein scores.
Table 3.17 Assignment of RP-HPLC Separated Fractions of Acidic Naja naja karachiensis Proteins to Various Families after Digestion of Peptides by Trypsin via LC-MS/MS Analysis. RP- Protein(s) Matched peptide Accession Protein Sequence Molecular HPLC family sequence No score coverage weight fractions (%) (KDa) 1a &1b No significant ------hit
1c PLA2 TYSYECSQGTLTCKGDNNACAASVCDCDR 129514 47.88 96.64 13.3 GGSGTPVDDLDRCCQVHDNCYNEAEK LAAICFAGAPYNDNNYNIDLK SWWDFADYGCYCGR NMIKCTVPSR ISGCWPYFK 3FTX TSETTEICPDSWYFCYK 12230756 10.10 41.54 7.6 ISLADGNDVR (MTLP-3)
1d PLA2 TYSYECSQGTLTCKGDNNACAASVCDCDR 129514 86.98 96.64 13.3 GGSGTPVDDLDRCCQVHDNCYNEAEK LAAICFAGAPYNDNNYNIDLK NLYQFKNMIKCTVPSR SWWDFADYGCYCGR ISGCWPYFK GGSGTPVDDLDRCCQVHDNCYDEAEK 2144440 64.41 66.39 13.3 NLYQFKNMIQCTVPSR SWWNFADYGCYCGR TYSYECSQGTLTCK ISGCWPYFK NLYQFK LAAICFAGAPYNDNNYNIDLK 31615585 12.72 30.25 13.1 RSWRDFADYGCYCGR
101 1e PLA2 GGSGTPVDDLDRCCQVHDNCYNEAEK 129514 116.75 96.64 13.3 LAAICFAGAPYNDNNYNIDLK SWWDFADYGCYCGR GDNNACAASVCDCDR TYSYECSQGTLTCK NLYQFKNMIK ISGCWPYFK CTVPSR GGSGTPVDDLDRCCQVHDNCYDEAEK 67172 82.41 63.03 13.4 SWWDFADYGCYCGR NGNNACAAAVCDCDR TYSYECSQGTLTCK NLYQFK NMIQCTVPNRSWWHFADYGCYCGR 804794 35.89 58.22 16.2 LAAICFAGAPYNDNNYNIDLK CCQIHDNCYNEAEK TYSYECSQGTLTCK GGSGTPVDDLDR
1f PLA2 LAAICFAGAPYNDNNYNIDLK 129514 66.63 96.64 13.3 GDNNACAASVCDCDR SWWDFADYGCYCGR CCQVHDNCYNEAEK TYSYECSQGTLTCK GGSGTPVDDLDR NLYQFKNMIK ISGCWPYFK CTVPSR LAAICFAGAPYNDDNYNIDLK 48425762 57.20 45.38 13.2 GDNNACAASVCDCDR GGSGTPVDDLDR NLYQFK 3FTX TGVDIQCCSTDNCNPFPTR 128930 19.55 45.07 7.8 VDLGCAATCPTVK (α- EPTX/LNT X)
1g PLA2 LAAICFAGAPYNDNNYNIDLK 129514 61.39 96.64 13.3 GDNNACAASVCDCDR SWWDFADYGCYCGR CCQVHDNCYNEAEK TYSYECSQGTLTCK GGSGTPVDDLDR NLYQFKNMIK ISGCWPYFK CTVPSR SWWDFADYGCYCGR 67172 52.24 50.42 13.4
102 CCQVHDNCYDEAEK TYSYECSQGTLTCK GGSGTPVDDLDR NLYQFK vNGF GNTVTVMENVNLDNK 128164 22.69 32.76 13 ALTMEGNQASWR IDTACVCVITK 1h CVF VDMNPAGGMLVTPTIEIPAK 881915 49.20 15.90 184.4 IEEQDGNDIYVMDVLEVIK AVPFVIVPLEQGLHDVEIK CCEDVMHENPMGYTCEK QNQYVVVQVTGPQVR FFYIDGNENFHVSITAR LILNIPLNAQSLPITVR LNQDITVTASGDGK CAGETCSSLNHQER ACETNVDYVYK GIYTPGSPVLYR VNDDYLIWGSR YEVDNNMAQK VYSYYNLDEK INYENALLAR KCQEALNLK YVLPSFEVR YFTYLILNK IDVPLQIEK VPDTEIETK 1i LAAO HVVVVGAGMAGLSAAYVLAGAGHK 126035677 38.33 23.16 51.4 LNEFFQENENAWYYINNIR TCADIVINDLSLIHDLPK YPVKPSEEGK IYFEPPLPPK SDALFSYEK VTLLEASER EYLIK SPLEECFQQNDYEEFLEIAR 347602328 15.42 30.53 11.2 SDALFSYEK 1j CRISP LGPPCGDCPSACDNGLCTNPCTIYNK 71041970 78.63 58.37 24.9 VLEGIQCGESIYMSSNAR WANTCSLNHSPDNLR TWTEIIHLWHDEYK MEWYPEAASNAER QKEIVDLHNSLR RVSPTASNMLK NVDFNSESTR VSPTASNMLK
103 SNCPASCF
2a PLA2 LAAICFAGAPYNDNNYNIDLK 129514 11.47 58.82 13.3 SWWDFADYGCYCGR GDNNACAASVCDCDR CCQVHDNCYNEAEK NLYQFK 2b No significant ------hit 2c 3FTX GCTFTCPELRPTGIYVYCCR 12230756 21.57 86.15 7.6 TSETTEICPDSWYFCYK (MTLP-3) ISLADGNDVR TICYNHLTR SVMP HDCDLPELCTGQSAECPTDSLQR 32469675 50.33 29.93 44.5 CPTLTNQCIALLGPHFTVSPK NGHPCQNNQGYCYNGK TAPAFQFSSCSIR GQCVDVQTAY GDDGSFCR
2d PLA2 GGSGTPVDDLDRCCQVHDNCYNEAEK 129514 113.84 96.64 13.3 LAAICFAGAPYNDNNYNIDLK SWWDFADYGCYCGR GDNNACAASVCDCDR TYSYECSQGTLTCK NLYQFKNMIK ISGCWPYFK CTVPSR NMIQCTVPNRSWWHFADYGCYCGR 804794 32.61 58.22 16.2 LAAICFAGAPYNDNNYNIDLK CCQIHDNCYNEAEK TYSYECSQGTLTCK GGSGTPVDDLDR SVMP ICGVTDTTWESDEPIKK 82223366 17.83 8.0 67.6 LQHEAQCDSEECCEK CGDGMVCSNR IPCAAK
2e PLA2 GDNNACAASVCDCDRLAAICFAGAPY- 129514 152.06 96.64 13.3 NDNNY GGSGTPVDDLDRCCQVHDNCYNEAEK TYSYECSQGTLTCKGDNNACAASVCDC-DR CTVPSRSWWDFADYGCYCG NLYQFKNMIK ISGCWPYFK GGSGTPVDDLDRCCQVHDNCYDEAEK 2144440 94.34 57.98 13.3 SWWNFADYGCYCGR CCQVHDNCYDEAEK
104 TYSYECSQGTLTCK ISGCWPYFK NLYQFK GGSGTPVDDLDR 129403 27.07 31.09 13.5 NLYQFKNMIK ISGCRPYFK CTVPSR 2f 3FTX MFMVAAPHVPVKR 117682 13.08 70 6.6 YVCCNTDKCN (CTX-II) GCIDVCPK SSLLVK NLCYK YECCNTDRCN 117667 13.08 61.67 6.8 MYMVSNK (CTX-I) GCIDVCPK LIPLAYK NLCYK
PLA2 GGSGTPVDDLDRCCQVHDNCYNEAEKIS- 129514 78.93 96.94 13.3 GCWPYK LAAICFAGAPYNDNNYNIDLK GDNNACAASVCDCDR SWWDFADYGCYCGR TYSYECSQGTLTCK NLYQFKNMIK NMIKCTVPSR ISGCWPYFK CTVPSR
2g PLA2 LAAICFAGAPYNDNNYNIDLK 129514 69.76 96.64 13.3 GDNNACAASVCDCDR SWWDFADYGCYCGR CCQVHDNCYNEAEK TYSYECSQGTLTCK GGSGTPVDDLDR NLYQFKNMIK ISGCWPYFK CTVPSR vNGF EDHPVHNLGEHSVCDSVSAWVTK 128164 32.63 79.31 13 GIDSSHWNSYCTETDTFIK GNTVTVMENVNLDNK CKNPNPEPSGCR IDTACVCVITK 3FTX TGVDIQCCSTDDCDPFPTR 128932 13.37 60.56 7.8 RVDLGCAATCPTVR (LNTX) TWCDGFCSIR
2h PLA2 SWWDFADYGCYCGR 67172 37.76 50.42 13.4 CCQVHDNCYDEAEK
105 TYSYECSQGTLTCK GGSGTPVDDLDR NLYQFK vNGF EDHPVHNLGEHSVCDSVSAWVTK 128164 39.86 79.31 13 GIDSSHWNSYCTETDTFIK GNTVTVMENVNLDNK ALTMEGNQASWR CKNPNPEPSGCR IDTACVCVITK 3FTX LVPLFSKTCPAGK 117682 18.05 91.67 6.6 MFMVAAPHVPVK (CTX- 2) YVCCNTDKCN RGCIDVCPK SSLLVK NLCYK 2i Vespryns ADVTFDSNTAFESLVVSPDKK 32363235 31.74 69.44 12 TVENVGVSQVAPDNPER /Thaicobrin FDGSPCVLGSPGFR EWAVGLAGK HFFEVK IWQK GYLR 2j Helvepryn LGPPCGDCPSACDNGLCTNPCTIYNK 71041970 136.90 61.54 24.9 VLEGIQCGESIYMSSNAR WANTCSLNHSPDNLR TWTEIIHLWHDEYK MEWYPEAASNAER QKEIVDLHNSLR RVSPTASNMLK NVDFNSESTRR VSPTASNMLK EIVDLHNSLR SNCPASCFCR TATPYK 2k 5’-ND YDAMALGNHEFDNGLNGLLDPLLK Oh-7801 43.98 28.22 62.8 HPDDNEWNHVSMCIVNGGGIR FHECNLGNLICDAVVYNNLR VLLPSFLAAGGDGYYMLK HGQGTGELLQVSGIK QVPVVQAYAFGK VVSLNVLCTECR VPTYVPLEMEK FPILSANIRPK QAFEHSVHR ISGYILPYK 3a 3FTX TGVDIQCCSTDDCDPFPTR 128947 5.95 46.48 7.8
106 RVDLGCAATCPTVK (LNTX) 3b No significant ------hit 3c Kunitz RPGFCELPAAK 125050 6.88 26.32 6.4 type DSHR serine protease inhibitor 3FTX GCGCPKVKPGVNLNCCR 128973 25.13 90.16 6.9 LECHNQQSSQPPTTK (SNTX) VKPGVNLNCCR TCSGETNCYK KWWSDHR GTIIER 3d 3FTX CFITPDITSKDCPNGHVCYTK 128930 95.70 80.28 7.8 TGVDIQCCSTDNCNPFPTR (α-EPTX/ GKRVDLGCAATCPTVK LNTX) CFITPDITSKDCPNGHVCYTK 128932 87.07 91.55 7.8 TGVDIQCCSTDDCDPFPTR (LNTX) RVDLGCAATCPTVR TWCDGFCSIR GCGCPKVKPGVNLNCCR 128973 9.67 62.30 6.9 LECHNQQSSQPPTTK (SNTX) VKPGVNLNCCR GTIIER 3e 3FTX TGVDIQCCSTDDCDPFPTR 128932 65.36 92.96 7.8 RVDLGCAATCPTVR (LNTX) DCPNGHVCYTK IRCFITPDITSK TWCDGFCSIR TGVDIQCCSTDNCNPFPTRK 128930 60.01 80.28 7.8 RVDLGCAATCPTVK (α-EPTX/ DCPNGHVCYTK LNTX) IRCFITPDITSK 3f 3FTX GCAATCPIAENRDVIECCSTDK 12230755 49.82 92.31 7.3 SIFGVTTEDCPDGQNLCFK (MTLP-2) TRGCAATCPIAENR DVIECCSTDKCNL WHMIVPGR LTCVK 3g 3FTX RVDLGCAATCPTVKPGVNIKCCSTDNCNP- 128942 11.05 46.48 7.8 FP (LNTX)
107 3h PLA2 LAAICFAGAPYNDNDYNINLK 443187 7.46 37.29 13.1 SWWDFADYGCYCGR ISGCWPYFK
3i PLA2 TYSYECSQGTLTCKGDNNACAASVCDCDR 129514 78.67 96.64 13.3 GGSGTPVDDLDRCCQVHDNCYNEAEK LAAICFAGAPYNDNNYNIDLK SWWDFADYGCYCGR ISGCWPYFK NLYQFK CTVPSR NMIK NMIQCTVPNRSWWHFADYGCYCGR 804794 24.72 58.22 16.2 LAAICFAGAPYNDNNYNIDLK CCQIHDNCYNEAEK TYSYECSQGTLTCK GGSGTPVDDLDR 3j 3FTX TGVDIQCCSTDDCDPFPTR 128947 9.86 45.07 7.8 VDLGCAATCPTVK (LNTX) 3k No significant ------hit 3l Helvepryn VLEGIQCGESIYMSSNAR 71041970 44.21 42.99 24.9 WANTCSLNHSPDNLR MEWYPEAASNAER QKEIVDLHNSLR RVSPTASNMLK NVDFNSESTR SNCPASCFCR EIVDLHNSLR TATPYK 4a & 4b No significant ------hit 4c 3FTX GCGCPKVKPGVNLNCCR 128973 49.20 90.16 6.9 LECHNQQSSQPPTTK (SNTX) TCSGETNCYKK KWWSDHR GTIIER LECHDQQSSQTPTTTGCSGGETNCYKKR 786434 9.89 64.52 7.9 NGIEINCCTTDR (Post- synaptic- NTX) 4d 3FTX TGVDIQCCSTDDCDPFPTR 128932 76.50 94.37 7.8
108 RVDLGCAATCPTVR (LNTX) DCPNGHVCYTK IRCFITPDITSK TWCDGFCSIR TGVDIQCCSTDNCNPFPTRK 128930 74.24 80.28 7.8 RVDLGCAATCPTVK (α-EPTX/ VDLGCAATCPTVK LNTX) DCPNGHVCYTK IRCFITPDITSK CFITPDITSK TGVDIQCCSTDDCPFPTR 229595 50.14 92.86 7.7 RVDLGCAATCPTVR (LNTX) TWCDGFCSSR CFITPDITSK DCPNGHVCYTK IRCFITPDITSK GCAATCPEAKPREIVQCCSTDK 136565 34.39 75.38 7.6 RGCAATCPEAKPR (WNTX) LTCLNCPEVYCR 4e-g No significant ------hit 5a & 5b No significant ------hit 5c Helvepryn CQKFIYGGCGGNANRFR 125050 43.05 85.96 6.4 FIYGGCGGNANR RPGFCELPAAK AHKPAFYYNK TIDECNR 3FTX WWSDHRGTIIERGCGCPK 128973 14.48 90.16 6.9 GCGCPKVKPGVNLNCCR (SNTX) LECHNQQSSQPPTTK VKPGVNLNCCR TCSGETNCYKK 5d EIVECCSTDKCNH 136562 33.16 69.23 7.4 GCAATCPEAKPR (WNTX) RFYEGNLLGK FYEGNLLGKR LTCLICPEK EIVQCCSTDKCNH 136564 31.78 61.54 7.6 GCADTCPVR (WNTX) VHTCLNGEK LTCLICPEK RGCAATCPEAKPR 136565 27.41 81.54 7.6
109 FQICRDGEKICFK (WNTX) LTCLNCPEVYCR EIVQCCSTDK 5e-h No significant ------hit 6a-6c No significant ------hit 6d 3FTX NSLLVKYECCNTDRCN 298351639 39.99 83.33 6.8 LIPLAYKTCPAGK (CTX- 7) LKCNKLIPLAYK RGCIDVCPK YECCNTDR MYMVSNK MFMVAAPHVPVK 117682 28.31 81.67 6.6 YVCCNTDKCN (CTX-2) RGCIDVCPK LVPLFSK MFMVSTSTVPVKR 117664 19.16 60.00 6.7 YVCCSTDKCN (CTX- 1) RGCIDVCPK NLCYK YVCCNTDRCS 1345874 15.55 49.38 8.9 CNQLIPPFYK (CTX-8) RGCIDVCPK SSLLVK NLCYK 6e 3FTX TCPAGKNLCYKMFMVAAPHVPVKR 261716 78.54 80 6.7 YVCCNTDRCN (CTX) KLVPLFSK SSLLVK All spectra acquired were searched against non- redundant NCBI database while protein families were classified on the basis protein scores.
110
Fig 3.47 Sequence Alignment of Unique 3FTX (MTLP-3) Detected in Naja naja karachiensis Venom with Recently Discovered Novel Haditoxin (Ophiophagus hannah). Highlighted Green Color Indicates Homology in Amino Acids Sequence between Two Toxins. Red Letters Represent Actually Matched Amino Acids Sequence from Database While Black Letters is adopted to complete the Sequence of Toxins. Disulfide Linkages are shown by Black Solid Line between Conserved Cysteine (C) Residues Whilst Black Arrows Suggest Contributing Segments among Three Loops of Haditoxin (adopted from Roy et al., 2010).
111
Fig 3.48 The Summarized Proteome Picture of Naja naja karachiensis Venom. Overview of Different Components Found in Naja naja karachiensis Crude Venom (a). Relative Abundance of Protein Families Identified by LC-MS/MS Analysis from RP-HPLC Fractions (b). Various Three Finger Toxins Abundant in Cobra
Venom (c). 3FTX: Three Finger Toxin, PLA2: Phospholipase A2, SVMP: Snake Venom Metalloproteinase, LAAO: L-Amino Acid Oxidase, CVF: Cobra Venom Factor, 5’ ND: 5’-Nucleotidase, vNGF: Venom Nerve Growth Factor, CTX: Cytotoxin, LNTX: Long Neurotoxin, WNTX: Weak Neurotoxin, SNTX: Short Neurotoxin, MTLP: Muscarinic Like Toxin Protein, Post Synaptic NTX: Post Synaptic Neurotoxin.
112
Fig 3.49 Standard Curve for Bovine Serum Albumin (BSA) at its Various Concentrations for Determination of Protein Components in Naja naja karachiensis Venom.
113
3.2 Elemental Analysis of Naja naja karachiensis Venom
3.2.1 Quantitative Estimation of Inorganic Constituents
ICP-OES technique indicated about metallic and non-metallic constituents of Naja naja karachiensis venom. Cobra venom was found to possess 95% both monovalent and divalent cations while phosphorus (non-metal) constituted 5% of inorganic material. Inorganic material comprised of sodium (30%), potassium (13%), zinc (23%), magnesium (20%), calcium (9%), manganese (0.05%) and copper (0.003%) ions. Furthermore, attempts were made to detect cobalt and iron however remained unsuccessful uniformly. Complete detail about elements found in Naja naja karachiensis venom is discussed in table 3.18 & fig 3.50. Moreover lowest possible detection limits (LOD) for each element was also calculated as described in table 3.19. Efficiency of the adopted procedure was evaluated with certified reference material (NIST 1515, apple leaf) and an excellent agreement was attained with certified reference values for Na, K, Cu, Ca, Co, Fe, Mg, Mn, Zn and P (Table 3.20).
Table 3.18 Quantitative Estimation of Different Elements (Metal & Non metal) Found in Naja naja karachiensis Venom (100 mg) via ICP-OES Analysis.
Elements Monitored wavelength Quantity SEM %CV detected (nm) (µg/g) (n=3) Na 589.620 4519 2 0.06 K 766.528 2013 5.5 0.3 Cu 324.771 0.6 0.09 23 Ca 315.902 1442 19 1.8 Co 238.902 0 0 0 Fe 238.213 0 0 0 Mg 279.090 3047 31 1.5 Mn 257.621 6.5 0.65 14 Zn 213.867 3473 28 1 P 213.626 718 8.5 1.6
114
Table 3.19 LOD for Different Elements Detected in Naja naja karachiensis Venom. Elements Monitored LOD* (**analytical LOD (***method detected wavelength (nm) blank, µg/l) , n=23 blank, µg/l), n=9 Na 589.620 4 4 K 766.528 305 146 Cu 324.771 2 2 Ca 315.902 13 94 Co 238.902 0 0 Fe 238.213 0 1 Mg 279.090 3 2 Mn 257.621 0 0 Zn 213.867 0 3 P 213.626 19 12 *LOD (limit of detection) = 3× standard deviation of sample blank. ** Analytical blank = to evaluate cross contamination between samples. *** Method blank = to evaluate digestion and other analysis procedures.
115 Table 3.20 Accuracy and Other Parameters for the Concentration of Elements in Certified Reference Material (NIST 1515) Apple Leaf Measured After Closed Pressurized Digestion.
Element Reference (CRM, NIST 1515) Apple $Drift (CRM, NIST 1515) Apple leaves λ, nm leaves SD RSD CRM Accuracy SD RSD CRM Accuracy µ % µg/g %CRC % µg/g %CRC µg/g g/g n=9 n=12 Na 53 2 4 24 218 54 1 2 24 222 589.620 K 1664 248 1 16100 103 1679 197 1 16100 104 766.528 1 7
Cu 6 0 4 6 106 6 0 2 6 107 324.771 Ca 1640 417 3 15260 107 1652 227 1 15260 108 315.902 3 5 Co 2 0 5 0 1815 2 0 3 0 1887 238.902 Fe 76 4 5 83 91 81 4 5 83 98 238.213 Mg 2798 70 3 2710 103 2865 44 2 2710 106 279.090 Mn 54 1 3 54 100 56 2 3 54 105 257.621 Zn 12 1 6 13 13 12 0 3 13 93 213.867 P 1488 41 3 1590 94 1503 28 2 1590 94 213.626
116 : Average value; $Drift sample: to monitor any fluctuation in results after ten samples; SD: standard deviation; CRM: certified reference material; RSD: relative standard deviation; CRC: certified reference material concentration.
Fig 3.50 Relative Abundance of Elementals (Metal & Non metal) Found in Naja naja karachiensis Crude Venom.
117 3.3 Biodistribution and Kinetic Study of Naja naja karachiensis Venom via Radio Tracer (99mTc) Binding Technique
3.3.1 Optimization of Radio Labeling Procedure and its Percentage Yield
Naja naja karachiensis venom was labeled (97.7%) successfully with radioactive technetium-99m. Among four different concentrations of stannous chloride dihydrate percentage labeling was highest at 100 µg therefore served as optimum concentration for radio labeling (Table 3.21). Variation in pH influenced considerably on labeling efficiency however optimum percentage binding was achieved at pH 6 as discussed in table 3.22.
3.3.2 Stability Profile of Technetium Labeled Naja naja karachiensis Venom
99mTc labeled cobra venom complex was found stable via both In vitro and In vivo experiments separately. In vivo experiments revealed that radioactive technetium was 99m tagged (96%) with snake venom proteins even after 4h (˃ 1 t1/2 of Tc) of injection in rabbits. Similarly In vitro studies indicated 94% binding after 4h of incubation with human serum and saline solution individually. All the data was obtained after applying radioactive decay corrections via decay constant equation (1× e-λ t). Complete detail about stability profile of technetium labeled Naja naja karachiensis venom is mentioned in table 3.23.
3.3.3 Technetium Labeled Cobra Venom and its Toxic (Hemolytic) Potential
Unlabeled venom was found 50%, 57% and 71% hemolytic at the doses of 125µg/ml, 250µg/ml and 500µg/ml respectively. Consistent with unlabeled cobra venom results 99mTc labeled venom was observed 50%, 55% and 71% hemolytic at the same doses with p˃0.5, p˃0.1 and p˃0.1 respectively (Table 3.24).
3.3.4 Blood Kinetics Profile of Technetium Labeled Naja naja karachiensis Venom
Blood clearance was monitored post administration of radioactive bolus (technetium labeled venom) via periodic record of radioactivity from different serum samples in
118 rabbits. Rapid elimination (1.3%ID/gram) was observed after an initial hour of intravenous bolus. Subsequently rate of elimination was descended down (0.89%ID/g for 2h, 0.56%ID/g for 3h and 0.42%ID/g for 6h) and apparently completed within 24h however 0.01% activity was present at the end. Complete description about blood clearance of technetium labeled venom bolus is shown in fig 3.51.
3.3.5 Bio-distribution of Technetium Labeled Naja naja karachiensis Venom After 3h of intravenous injections in rabbits highest activity (77%ID/g) was found in Urinary system (kidneys: (53.7%ID/g); urinary bladder (23.7%ID/g) followed by lungs (14.2%ID/g) and liver (4.3%ID/g). Least activity was observed in brain (0.14%ID/g) and rests of all tissues were found intermediate as shown in fig 3.52. 3.3.6 Gamma Scintigraphy and SPECT Images Post intravenous administration (2 min), venom was detected in heart, lungs, kidneys and liver however it was noticed in urinary bladder in 5 min. After 1h of envenomation urinary bladder was found much saturated with radioactive venom. Moreover 99mTc labeled venom was not detected in brain via gamma scintigraphic approach. Complete description about venom injection blood flow study (2h with 15 min intervals) is shown in fig 3.53. Venom distribution in the middle compartment of the animal was accessed by R/L ratio which was approximately equal (1.08) (Fig 3.54). Furthermore at 360° transverse, sagittal and coronal single photon images were captured by single photon emission computed tomography approach as shown in fig 3.55.
119 Table 3.21 Effect of Various Concentrations of Stannous Chloride Dihydrate in Optimization of Radio Labeling Procedure (Binding of 99mTc with Naja naja karachiensis Venom) In Terms of Labeling Percentage at pH 7.
Sr. Optimization parameter Paper strip Radioactive % radio- No. Concentration of concern counts activity
SnCl2.2H2O (µg) (per 30 sec) 1. 50 1 3223 15.2 2 11343 53.7 3 1867 8.8 4 493 2.3 5 715 3.3 6 249 1.2 7 3106 14.7 8 045 0.2 9 041 0.19 10 034 0.161 Total 21116 68 2. 100 1 108649 40.56 2 128922 48.46 3 2628 0.98 4 1819 0.67 5 3659 1.36 6 2031 0.75 7 3528 1.31 8 16511 6.16 9 46 0.01 10 48 0.01 Total 267841 89 3. 150 1 18624 38.7 2 20302 42.28
120 3 415 0.86 4 552 1.14 5 844 1.75 6 636 1.32 7 1306 2.72 8 5226 10.88 9 41 0.08 10 66 0.13 Total 48012 81 4. 200 1 15630 59 2 1545 5.8 3 609 2.3 4 1580 6 5 2170 8.2 6 3327 12.5 7 1523 5.7 8 032 0.1 9 044 0.1 10 043 0.1 Total 26473 65
121 Table 3.22 Effect of Different pH for Optimization of Radio Labeling Procedure In
Terms of Labeling Percentage at 100 µg SnCl2.2H2O. Sr. Optimization Paper strip Radioactive % radio activity No. parameter concern counts (pH) (per 30 sec) 1. 5 1 11278 16 2 51413 73.1 3 550 0.78 4 464 0.65 5 654 0.93 6 593 0.84 7 1247 1.77 8 4019 5.71 9 46 0.06 10 58 0.08 Total 70323 89.0 2. 6 1 448 0.23 2 185290 97.7 3 501 0.26 4 481 0.25 5 472 0.24 6 510 0.26 7 1440 0.75 8 205 0.10 9 192 0.10 10 100 0.05 Total 189639 97.7 3. 7 1 10876 17.15 2 45649 72 3 517 0.81 4 441 0.69
122 5 628 0.99 6 519 0.81 7 1194 1.88 8 3525 5.55 9 50 0.07 10 38 0.05 Total 63437 89.15
123 Table 3.23 Stability Profile of Technetium Labeled Naja naja karachiensis Venom Complex via Both In vivo and In vitro Experimentation (Adopted from Asad et al., 2015).
Sr. Incubation In vivo percentage In vitro percentage labeling No. time labeling (mean ± Serum (mean ± Saline (mean ± (min) SEM), n=3 SEM), n=3 SEM), n=3 1. 0 97.6±0.643 97.2±0.635 97.7±0.649 2. 60 97.3±0.624 97.0±0.578 97.1±0.578 3. 120 97.0±0.867 96.1±0.851 96.5±0.777 4. 180 96.2±0.696 95.3±0.821 95.7±1.027 5. 240 96.0±0.882 94.1±0.317 94.3±1.011
Table 3.24 Comparison of Toxic (Hemolytic) Potential of 99mTc Labeled and Unlabeled Cobra Venom.
Sr. Observation Evaluated sample(s) No. Unlabeled crude venom 99mTc-labeled crude venom (µg/ml) (µg/ml) 125 250 500 125 250 500 1. Absorbance 0.075 ± 0.085 ± 0.107 ± 0.075 ± 0.082 ± 0.106 ± (mean ± 0.000 0.001 0.0005 0.0005 0.0008 0.0005 SEM) n=3 2. Hemolytic 50.3 57.0 71.8 50.3 55.0 71.1 percentage 3. Statistical select to select to select to p˃0.5 p˃0.1 p˃0.1 evidence compare compare compare
124
Fig 3.51 Blood Kinetics/Clearance Profile of 99mTc Labeled Naja naja karachiensis Venom Post Intravenous Injection in Rabbits (Adopted from Asad et al., 2015).
125
Fig 3.52 Biodistribution Profile of 99mTc Labeled Naja naja karachiensis Venom after Intravenous Injection in Healthy Male Rabbits (Adopted from Asad et al., 2015).
126
Fig 3.53 Whole Body Gamma Scintigraphic (2h Blood Flow Study) Scans after Intravenous Injection of 99mTc Labeled Cobra Venom in Rabbit (Adopted from Asad et al., 2015).
127
Fig 3.54 Distribution Profile of Technetium Labeled Naja naja karachiensis Venom in Middle Compartment of Rabbits In Terms of R/L Ratio (Adopted from Asad et al., 2015).
128
Fig 3.55 Acquired Single Photon Emission Computed tomography (SPECT) Images of Naja naja karachiensis Envenomed Rabbit at 360° (Adopted from Asad et al., 2015).
129 3.4 Toxic Biological/Biochemical Activities of Naja Naja Karachiensis Venom and their Neutralization with Medicinal Plants of Pakistan
3.4.1 Lethal Dose of Naja Naja Karachiensis Venom for 50% Population
(LD50)
Naja naja karachiensis venom was injected intraperitonially for determination of lethal dose required to kill half of a population in experimental mice. LD50 (least amount of venom that kills half of laboratory animals in unlimited time) was obtained after extrapolation of regression line where it intersected the ordinate scale. For this venom a straight line graph was obtained with positive correlation coefficient (r = 0.999) and y =
6.529x + 2.080. Therefore LD50 of Naja naja karachiensis venom was found to be 2.0 µg/g (2.0 mg/kg) intraperitonially (Fig 3.56).
Fig 3.56 LD50 Determination of Naja naja karachiensis Venom.
130 3.4.2 Hemolytic Potential of Naja Naja Karachiensis Venom and its Neutralization with Medicinal Plants
Naja naja karachiensis venom (100µg/ml) was found to destabilize human red blood corpuscles membranes that end up with hemolysis (Table 3.25). To exculpate this effect twenty six medicinal plants extract were evaluated and their efficiencies were compared with reference standard antidote (anti-sera). Sixty nine percent plants extract were not found beneficial against venom induced hemolysis. Among them Albizia lebbeck (L.) Benth (0.01˃P˃0.001), Allium sativum L (0.02˃P˃0.01), Bauhinia variegate L (p˂0.05), Brassica nigra (L Koch) (P˃0.001), Cuminum cyminum L (p˂0.05), Momordica charantia L (P˂0.001), Matthiloa incana (L) R.Br (P˂˂0.001), Nerium indicum Mill (P˃0.001), Ocimum sanctum (p˂0.05), Pinus roxburghii Sargent (p˂0.05), Pistacia integerrima (p˂0.05), Psoralea corylifolia L (P˃0.001), Rhazya stricta Dcne (0.02˃P˃0.01), Rubia cordifolia (P˂0.001), Sapindus mukorossi Gaertn (0.01˃P˃0.001), Terminalia arjuma Wight and Arn (p˂0.05), Trichodesma indicum (Linn) (0.02˃P˃0.01) and Zingiber officinale Rosc (p˂0.05) were included. They were failed to neutralize hemolysis at all concentrations (20-320 µg/ml) and assumed cytotoxic in nature as shown in table 3.26. Allium cepa L (P˂0.1), Althaea officinalis L (0.5˃P˃0.1), Citrus limon (L). Burm. F (0.5˃P˃0.1), Leucas capitata Desf (0.5˃P˃0.1) and Stenolobium stans (L) D. Don (0.5˃P˃0.1) together constituted 19% of plants and were found anti-hemolytic (partially) only at lower concentrations (˂160 µg/ml). Moreover 12% plants comprised of Cedrus deodara (Roxb. ex D. Don) G. Don (P˃0.5), Enicostemma hyssopifolium (Willd.) I. Verd (0.5˃P˃0.1) and Calotropis procera (Wild.) R.Br (0.5˃P˃0.1) was found valuable as anti-hemolytic. Anti-hemolytic potential was highest (72%) for Cedrus deodara (Roxb. ex D. Don) G. Don while reference standard antidote was 56% effective (Figure 3.57 & 3.58). Among all evaluated medicinal plants extract only Cedrus deodara (Roxb. ex D. Don) G. Don was proved to pose maximum protection at P˃0.5 therefore declared the best anti-hemolytic extract. Complete detail about medicinal plants extract is described in table 3.26.
131 Table 3.25 Aptitudes of Different Agents Towards Induction of Hemolysis In Terms of Hemoglobin Release Measured at 540 nm (Adopted from Asad et al.,
2104(e)).
Sr. Evaluated sample Quantity/strength Absorbance No. (Mean ± SD) 1. Saline (negative control) 0.89% 0.038 ± 0.002 2. Cobra venom (Naja naja 100 µg/ml 0.151 ± 0.002 karachiensis) 3. Hyposaline (positive control) 0.25% 4 ± 0.000
Table 3.26 Effect of Various Antidotes (Medicinal Plants Extract and Standard Antidote) Evaluated to Halt Venom (100 µg/ml) Induced Hemolysis (Adopted from
Asad et al., 2104(e)). Sr. Various evaluated antidote HRBCs membrane Conclusion(s) No. stability 1. Reference standard anti-sera Effective to stabilize Worked as anti- HRBCs at 20-320 µg / hemolytic ml (Fig 3.56)
132 2. Psoralea corylifolia L Zingiber officinale Rosc.
Matthiloa incana (L) R.Br. Albizia lebbeck (L.) Benth Pistacia integerrima.
Rhazya stricta Dcne. Trichodesma indicum (Linn). Failed to abolish venom Proved to Momordica charantia L. induce hemolysis (20- aggravate venom Sapindus mukorossi Gaertn 320 µg / ml) induced Allium sativum L. Hemolysis. Ocimum sanctum. Folklore claim as Brassica nigra (L Koch). anti-hemolytic Bauhinia variegate L. was not true. Cuminum cyminum L. Provoked Rubia cordifolia. cytotoxic like Nerium indicum Mill. effects on Terminalia arjuma Wight HRBCs. and Arn. Pinus roxburghii Sargent. 3. Althaea officinalis Linn. Leucas capitata Desf. Low doses (< 160 µg Higher concentrations Citrus limon (L). Burm. f. /ml) were found anti- (˃160 µg/ml) were Allium cepa L. hemolytic found to aggravate Stenolobium stans (L) D. venom response Don. (cytotoxicity) 4. Enicostemma hyssopifolium (Willd.) Verdoorn. Each dose was found Effective as anti- Cedrus deodara G. Don. anti-hemolytic hemolytic agents (Fig Calotropis procera (Wild.) (20-320 µg / ml) 3.57) R.Br.
133
Fig 3.57 Comparison of Short Listed Medicinal Plants (Enicostemma hyssopifolium (Willd.) I. Verd, Calotropis procera (Wild.) R.Br and Cedrus deodara (Roxb. ex D. Don) G. Don) Having Potentials to Neutralize Naja naja karachiensis Venom (100 µg/ml) Induced Hemolysis in Comparison with Reference Standard (Anti-Sera) at Various Concentrations: (a) 20 µg/ml; (b) 40 µg/ml; (c) 80 µg/ml; (d) 160 µg/ml; (e) 320 µg/ml.
134
Fig 3.58 Percentage Distribution of Plants for their Potential to Neutralize Hemolytic
Effect of Naja naja karachiensis Venom (Adopted from Asad et al., 2104(e)).
135 3.4.3 Anticoagulant Activity of Naja naja karachiensis Venom and its Neutralization with Medicinal Plants
Naja naja karchiensis venom (200µg/ml) was found to delay in general purpose coagulation tests. PT was delayed from 13±0.57 to 23±0.57 sec while aPTT was prolonged from 35±1.52 to 48±2.0 sec. TT was also delayed from 13±0.57 to 33±0.57 sec nevertheless all coagulation tests were found within 4.5% coefficient of variance (%CV) as mentioned in tables 3.27- 3.29. Various medicinal plants extract at minute concentration (5µg/ml) were found to minimize venom’s response of coagulopathy equally at high dose (640 µg / ml). Standard anti-serum was found to pose minimum protection (10%) against delay in PT however it was proved 92% and 70% effective to recover prolonged aPTT and TT tests respectively. Confidence limits (mean ± 2SD) of standard antidote for PT (20.59-23.41 sec), aPTT (33.5 – 38.5 sec) and TT (16.5 – 21.5 sec) were calculated and results were compared with it. Prolongation of PT was neutralized effectively (˃10%) by various evaluated medicinal plants extract. Among them Rubia cordifolia L, Bauhinia variegata L, Albizia lebbeck (L.) Benth, Citrullus colocynthis (L.) Schrad, Matthiloa incana (L.) W. T. Aiton, Citrus limon (L). Burm. f, Ocimum sanctum L, Terminalia arjuna (Roxb. ex DC.) Wight & Arn, Momordica charantia L, Sapindus mukorossi Gaertn, Calotropis procera (Aiton) W. T. Aiton (exudates), Stenolobium stans (L.) Seem and Trichodesma indicum (L.) Sm. was included. Rest of others was found with efficiency comparable to anti-sera (Table 3.27) while Stenolobium stans (L.) Seem was proved the most valuable (70%) to recover prolonged prothrombin time.
To compensate delay in aPTT many medicinal plants extract were found beneficial. Among them Enicostemma hyssopifolium (Willd.) I. Verd, Stenolobium stans (L.) Seem, Albizia lebbeck (L.) Benth, Cuminum cyminum L, Bauhinia variegata L, Calotropis procera (Aiton) W. T. Aiton (exudates & flowers), Cedrus deodara (Roxb. ex D. Don) G. Don, Allium cepa L, Fogonia cretica L, Leucas capitata Desf, Brassica nigra (L.) W. D. J. Koch, Trichodesma indicum (L.) Sm, Ocimum sanctum L, Pinus roxburghii Sarg, Matthiloa incana (L.) W. T. Aiton, Sapindus mukorossi Gaertn, Zingiber officinale Roscoe and Terminalia arjuna (Roxb. ex DC.) Wight & Arn were included. Stenolobium
136 stans (L) D. Don and Enicostemma hyssopifolium (Willd.) Verdoorn were declared the most useful while rest of others could not be short listed as standard anti-dote (Table 3.28). Two medicinal plants extract were found valuable to recover delay in TT as standard anti-dote. Among them Citrullus colocynthis (L.) Schrad and Enicostemma hyssopifolium (Willd.) I. Verd was included. Both of them were declared 70% effective as reference standard antidote however rest of all extracts were not proved equally effective. Complete detail about medicinal plants extract to combat delay in general purpose coagulation tests is mentioned in table 3.29.
Table 3.27 Anticoagulant Effect (Delay in PT) of Naja naja karachiensis Venom and its
Neutralization by Various Antidotes (Adopted from Asad et al., 2014(c)).
Evaluated samples: healthy plasma; different Prothrombin time (sec) antidotes (5 µg/ ml); snake venom (200 µg/ml) Mean ± SD % % (n =3) Efficiency CV Normal (healthy) plasma 13 ± 0.57$ 100 4.3 Standard antidote 22 ± 0.57* 10 2.6 Naja naja karachiensis 23 ± 0.57ᴪ 0 2.5 Althaea officinalis L 21 ± 0.57** 20 2.7 Calotropis procera (Aiton) W. T. Aiton (exudates) 18 ± 0.57*** 50 3.1 Calotropis procera (Aiton) W. T. Aiton (flowers) 22 ± 0.57** 10 2.6 Cuminum cyminum L 22 ± 1** 10 4.5 Allium cepa L 23 ± 0.57** 0 2.5 Trichodesma indicum (L.) Sm 18 ± 0.0*** 50 0.0 Cedrus deodara (Roxb. ex D. Don) G. Don 23 ± 0.57** 0 2.5 Psoralea corylifolia L 21 ± 0.0** 20 0.0 Pistacia integerrima J. L. Stewart 23 ± 0.57** 0 2.5 Stenolobium stans (L.) Seem 16 ± 0.57*** 70 3.5 Leucas capitata Desf 23 ± 0.57** 0 2.4 Rhazya stricta Dcne 22 ± 1.5** 10 6.7
137 Pinus roxburghii Sarg 23 ± 0.57** 0 2.4 Enicostemma hyssopifolium (Willd.) I. Verd. 22 ± 0.57** 10 2.5 Brassica nigra (L.) W. D. J. Koch 22 ± 0.00** 10 0.0 Allium sativum L 23 ± 1.00** 0 4.3 Bauhinia variegata L 18 ± 0.57*** 50 3.1 Matthiloa incana (L.) W. T. Aiton 20 ± 0.57*** 30 2.9 Citrus limon (L). Burm. f 19 ± 0.57*** 40 2.9 Nerium indicum Mill 21 ± 0.57** 20 2.7 Zingiber officinale Roscoe 21 ± 1.0** 20 4.7 Ocimum sanctum L 17 ± 0.57*** 60 3.5 Albizia lebbeck (L.) Benth 18 ± 1.00*** 50 5.5 Rubia cordifolia L 18 ± 1.0*** 50 5.5 Sapindus mukorossi Gaertn 20 ± 0.57*** 30 2.9 Fogonia cretica L 22 ± 0.57** 10 2.5 Terminalia arjuna (Roxb. ex DC.) Wight & Arn 18 ± 0.57*** 50 3.1 Citrullus colocynthis (L.) Schrad 19 ± 0.57*** 40 3.0 Momordica charantia L 20 ± 1*** 30 5.0 $ indicates respective coagulation test value of negative control; ᴪ indicates respective coagulation test value of positive control; * indicates respective coagulation test value of standard (set as reference); ** indicates that plant extract fall within 95% confidence limit of reference standard for PT (20.59-23.41 sec); *** indicates that plant extract don’t fall within 95% confidence limit of respective test for reference standard.
138 Table 3.28 Anticoagulant Effect (Delay in aPTT) of Naja naja karachiensis Venom and
its Neutralization by Various Antidotes (Adopted from Asad et al., 2014(c)).
Evaluated samples: healthy plasma; different Activated partial thromboplastin antidotes (5 µg/ ml); snake venom (200 µg/ml) time (sec) Mean ± SD % % (n =3) Efficiency CV Normal (healthy) plasma 35 ± 1.52$ 100 4.3 Standard antidote 36 ± 1.00* 92 2.7 Naja naja karachiensis 48 ± 2.00ᴪ 0 4.1 Althaea officinalis L 39 ± 0.57*** 69 1.4 Calotropis procera (Aiton) W. T. Aiton (exudates) 38± 0.00** 77 0 Calotropis procera (Aiton) W. T. Aiton (flowers) 37 ± 0.57** 84 1.5 Cuminum cyminum L 38 ± 1.00** 77 2.6 Allium cepa L 38 ± 2.00** 77 5.2 Trichodesma indicum (L.) Sm 37 ± 0.57** 84 1.5 Cedrus deodara (Roxb. ex D. Don) G. Don 38 ± 1.00** 77 2.6 Psoralea corylifolia L 45 ± 1.00*** 23 2.2 Pistacia integerrima J. L. Stewart 40 ± 0.57*** 61 1.4 Stenolobium stans (L.) Seem 36 ± 0.57** 92 1.5 Leucas capitata Desf 37 ± 0.57** 84 1.5 Rhazya stricta Dcne 47 ± 1.00*** 7 2.1 Pinus roxburghii Sarg 38 ± 1.00** 77 2.6 Enicostemma hyssopifolium (Willd.) I. Verd. 36 ± 1.00** 92 2.7 Brassica nigra (L.) W. D. J. Koch 38 ± 1.15** 77 3.0 Allium sativum L 39 ± 1.15*** 69 2.9 Bauhinia variegata L 37 ± 1.00** 84 2.7 Matthiloa incana (L.) W. T. Aiton 37 ± 0.57** 84 1.5 Citrus limon (L). Burm. f 42 ± 1.52*** 46 3.6 Nerium indicum Mill 40 ± 1.15*** 61 2.8 Zingiber officinale Roscoe 38 ± 0.57** 77 1.5
139 Ocimum sanctum L 37 ± 0.57** 84 1.5 Albizia lebbeck (L.) Benth 38 ± 0.57** 77 1.5 Rubia cordifolia L 46 ± 0.57*** 15 1.2 Sapindus mukorossi Gaertn 38 ± 1.00** 77 2.6 Fogonia cretica L 38 ± 1.73** 77 4.5 Terminalia arjuna (Roxb. ex DC.) Wight & Arn 37 ± 1.52** 84 4.1 Citrullus colocynthis (L.) Schrad 40 ± 1.00*** 61 2.5 Momordica charantia L 39 ± 0.57*** 69 1.4 $ indicates respective coagulation test value of negative control; ᴪ indicates respective coagulation test value of positive control; * indicates respective coagulation test value of standard (set as reference); ** indicates that plant extract fall within 95% confidence limit of reference standard for aPTT (33.5-38.5 sec); *** indicates that plant extract don’t fall within 95% confidence limit of respective test for reference standard.
Table 3.29 Anticoagulant Effect (Delay in TT) of Naja naja karachiensis Venom and its
Neutralization by Various Antidotes (Adopted from Asad et al., 2014(c)).
Evaluated samples: healthy plasma; different Thrombin time (sec) antidotes (5 µg/ ml); snake venom (200 µg/ml) Mean ± SD % % (n =3) Efficiency CV Normal (healthy) plasma 13±0.57$ 100 4.5 Standard antidote 19 ±1.00* 70 5.2 Naja naja karachiensis 33 ±0.57ᴪ 0 1.7 Althaea officinalis L 32 ±0.57*** 5 1.7 Calotropis procera (Aiton) W. T. Aiton (exudates) 33 ±1.00*** 0 3.0 Calotropis procera (Aiton) W. T. Aiton (flowers) 26 ±2.00*** 35 7.6 Cuminum cyminum L 31 ±0.57*** 10 1.8 Allium cepa L 26 ±0.57*** 35 2.1 Trichodesma indicum (L.) Sm 25 ±1.00*** 40 4.0 Cedrus deodara (Roxb. ex D. Don) G. Don 22 ±0.57*** 55 2.5 Psoralea corylifolia L 27 ±1.52*** 30 5.5
140 Pistacia integerrima J. L. Stewart 23 ±0.57*** 50 2.4 Stenolobium stans (L.) Seem 29 ±0.57*** 20 2.0 Leucas capitata Desf 27 ±0.57*** 30 2.1 Rhazya stricta Dcne 27 ±0.57*** 30 2.4 Pinus roxburghii Sarg 26 ±1.15*** 35 4.3 Enicostemma hyssopifolium (Willd.) I. Verd. 19 ±0.57** 70 2.9 Brassica nigra (L.) W. D. J. Koch 33 ±0.57*** 0 1.7 Allium sativum L 27 ±2.08*** 30 7.6 Bauhinia variegata L 27 ±1.52*** 30 5.7 Matthiloa incana (L.) W. T. Aiton 26 ±1.00*** 35 3.8 Citrus limon (L). Burm. f 27 ±0.00*** 30 0.0 Nerium indicum Mill 28 ±0.57*** 25 2.0 Zingiber officinale Roscoe 24 ±1.52*** 45 6.2 Ocimum sanctum L 27 ±0.57*** 30 2.1 Albizia lebbeck (L.) Benth 24 ±1.00*** 45 4.1 Rubia cordifolia L 28 ±0.57*** 25 2.0 Sapindus mukorossi Gaertn 22 ±1.00*** 55 4.5 Fogonia cretica L 25 ±1.15*** 40 4.6 Terminalia arjuna (Roxb. ex DC.) Wight & Arn 28 ±0.57*** 25 2.0 Citrullus colocynthis (L.) Schrad 21 ±1.15** 60 5.5 Momordica charantia L 33 ±1.52*** 0 4.5 $ indicates respective coagulation test value of negative control; ᴪ indicates respective coagulation test value of positive control; * indicates respective coagulation test value of standard (set as reference); ** indicates that plant extract fall within 95% confidence limit of reference standard for TT (16.5-21.5 sec); *** indicates that plant extract don’t fall within 95% confidence limit of respective test for reference standard.
141
Fig 3.59 Schematic Overview of Different Stages of Blood Coagulation Pathway (I, II & III) and its Possible Hindrance by Three Ways (a, b & c) after Snake (Naja naja karachiensis) Bite Envenomation (Adopted from McCleary and Kini, 2013).
142 3.4.4 Biochemical Parameters for Heart, Liver and Kidney Damage and their Neutralization by Medicinal Plants
Naja naja karachiensis venom was found to provoke serum biochemical parameters attributed to the severe damage of heart, liver and kidneys. Snake venom at 400µg/kg was proved toxic with moderately increase in CK-MB (21 ± 1.5 U/L, 0.05˃p˃0.02), LDH (2064 ± 15.98 U/L, p˂0.001), AST (157 ± 24.24 U/L, 0.1˃p˃0.05), ALT (72 ± 4.70 U/L, 0.1˃p˃0.05), urea (42 ± 3.08 mg/dl, 0.05˃p˃0.02) and creatinine (1.74 ± 0.03 mg/dl, 0.01˃p˃0.001) levels. At high doses (800µg/kg) of venom severe toxicity to the heart, liver and kidneys were recorded in terms of significant release of CK-MB (77 ± 11.22 U/L, 0.05˃p˃0.02), LDH (2562 ± 25.14 U/L, p˂˂0.001), AST (251 ± 18.2 U/L, 0.01˃p˃0.001), ALT (86 ± 5.0 U/L, 0.05˃p˃0.02), urea (57.6 ± 3.84 mg/dl, 0.02˃p˃0.01) and creatinine (2.1 ± 0.10 mg/dl, 0.02˃p˃0.01) levels. Complete detail about venom toxicity and related (baseline measurement and control) information mentioned in table 3.30.
To combat cobra venom (800µg/kg) toxicity 28 medicinal plants extract were tried and their potentials compared with reference standard antidote. Taken as a whole only extract of Stenolobium stans (L.) Seem was proved the best anti-venom (p˃0.5) comparable to reference standard antidote. Among various plants extract Bauhinia variegata L, Althaea officinalis L, Cedrus deodara (Roxb. ex D. Don) G. Don, Allium cepa L, Fagonia cretica L, Momordica charantia L, Ocimum sanctum L and Leucas capitata Desf were observed equally effective (p˃0.5) as standard antidote (CK- MB=09±0.85U/L level) for neutralization of cardiotoxicity however rest of all was not observed as reference standard (0.5˃p˃0.1). Extracts of Althaea officinalis L, Leucas capitata Desf and Terminalia arjuna (Roxb. ex DC.) Wight & Arn were found somewhat valuable (0.5˃p˃0.1) to decline elevated levels of LDH as standard anti-sera (LDH=763±6.01U/L) however remaining extracts were not found beneficial (0.1˃p˃0.001) to combat cobra venom toxicity. Detailed description about biochemical parameters (CK-MB and LDH) for indication of cardiotoxicity and its neutralization is mentioned in table 3.31. Pinus roxburghii Sarg, Citrullus colocynthis (L.) Schrad, Psoralea corylifolia L, Allium sativum L, Rubia cordifolia L, Althaea officinalis L,
143 Leucas capitata Desf and Sapindus mukorossi Gaertn were short listed (p˃0.5) as hepatoprotective comparable to standard antidote (AST=69.5±18.55U/L & ALT=52.5±3.51U/L) however rest of all was not observed beneficial (0.5˃p˃0.05) to normalize AST and ALT levels (Table 3.32). Among twenty eight medicinal plants extract only Althaea officinalis L and Leucas capitata Desf was found valuable (p˃0.5) to decrease significantly high values of urea and creatinine as noticed for reference standard antidote (creatinine= 1.08±0.02mg/dl & urea=31.5±0.50mg/dl). Remaining plants extract could not be taken as guaranteed (0.5˃p˃0.01) to minimize kidney damage with cobra venom. A concise description about renal toxicity and its neutralization by medicinal plants is mentioned in table 3.33.
Table 3.30 Naja naja karachiensis Envenomation: Various Biochemical Parameters for Cardiac, Liver and Kidney Damage in Experimental Rabbits (Adopted from
Asad et al., 2014(d)). Biochemical markers for toxicity determination Groups Heart Liver Kidneys CK-MB LDH ALT AST Urea Creatinine (Mean± (Mean± (Mean± (Mean± (Mean± (Mean± SEM) SEM) SEM) SEM) SEM) SEM) (Group-I) 13.2 ± 2.0* 714±3.1* 52±3.46* 65±6.57* 28±1.73* 1.0±0.313* Base line U/L U/L U/L U/L mg/dl mg/dl (Group-II) 21±1.5 2064±15.9 72±4.70 157±24.24 42±3.08 1.74± 0.03 Envenomation U/L U/L U/L U/L mg/dl mg/dl at 0.05˃p˃0.0 p˂0.001 0.1˃p˃0.05 0.1˃p˃0.05 0.05˃p˃0 0.01˃p˃0.001 0.4 mg/kg 2 .02 (Group-III) 77±11.22 2562±25.1 86±5.0 251±18.2 57.6±3.8 2.1± 0.10 Envenomation U/L U/L U/L U/L mg/dl mg/dl at 0.05˃p˃0.0 p˂˂0.001 0.05˃p˃0.0 0.01˃p˃0.0 0.02˃p˃0 0.02˃p˃0.01 0.8 mg/kg 2 2 01 .01
144 (Group-IV) 13±0.56* 720±4.7* 52±3.48* 67±3.21* 28±0.33* 1±0.06* Control U/L U/L U/L U/L mg/dl mg/dl *Indicates that given value fall within normal reference range. Normal reference values for CK-MB, LDH, ALT, AST, urea and creatinine in growing healthy rabbits are (˂25) U/L, (559-2077) U/L, (48-80) U/L, (14-113) U/L, (10-28) mg/dl and (0.5-2.5) mg/dl respectively. Moreover CK-MB is ˂2% in rabbits nevertheless it is 10%-30% of complete CK activity.
Table 3.31 A List of Medicinal Plants Extract (100 mg/kg) used to Minimize Cardiotoxicity Posed by Naja naja karachiensis Venom (800 µg/kg) in
Rabbits (Adopted from Asad et al., 2014(d)). Group V Name of tested material CK-MB (U/L) LDH (U/L) (subgroup) (Mean ± (p-value) (Mean ± (p-value) SEM) SEM) V/2 Allium cepa L 14.8±1.65 p˃0.5 934±13.03 0.1˃p˃0.05 V/20 Pistacia integerrima J. L. 13.1±1.65 0.5˃p˃0.1 1135.5±0.5 0.02˃p˃0.01 Stewart V/11 Cuminum cyminum L 5.8±0.80 0.5˃p˃0.1 1589±22.56 0.02˃p˃0.01 V/17 Nerium indicum Mill 05±1.65 0.5˃p˃0.1 1268±12.03 0.02˃p˃0.01 V/22 Rhazya stricta Dcne 4.1±0.85 0.5˃p˃0.1 1538±20.05 0.02˃p˃0.01
V/7a Calotropis procera (Aiton) 6.6±3.30 0.5˃p˃0.1 1022±5.01 0.02˃p˃0.01 W. T. Aiton (exudates)
V/7b Calotropis procera (Aiton) 61.8 ±10.9 0.5˃p˃0.1 1114±1.00 0.02˃p˃0.01 W. T. Aiton (flowers) V/25 Stenolobium stans (L.) Seem 13±1.76 p˃0.5 787±28.08 p˃0.5 V/27 Trichodesma indicum (L.) 6.6±3.00 0.5˃p˃0.1 978.5±4.51 0.05˃p˃0.02 Sm V/5 Bauhinia variegate L 8.3±6.76 p˃0.5 1972±3.00 p˃0.001 V/6 Brassica nigra (L.) W. D. J. 9.0±0.85 0.5˃p˃0.1 855.5±0.50 0.05˃p˃0.02 Koch V/15 Matthiloa incana (L.) W. T. 08±1.66 0.5˃p˃0.1 1428±6.51 p˃0.001
145 Aiton V/16 Momordica charantia L 15.6± 2.45 p˃0.5 1475.5±3.51 p˃0.001 V/10 Citrullus colocynthis (L.) 05 ± 1.66 0.5˃p˃0.1 827 ± 6.51 0.1˃p˃0.05 Schrad V/26 Terminalia arjuna (Roxb. ex 6.6±0.00 0.5˃p˃0.1 798.5±14.54 0.5˃p˃0.1 DC.) Wight & Arn V/21 Psoralea corylifolia L 17.3± 2.50 0.5˃p˃0.1 1153.5 ± 0.5 p˃0.001 V/12 Enicostemma hyssopifolium 9.85±1.66 0.5˃p˃0.1 1615±1.51 p˃0.001 (Willd.) I. Verd V/14 Leucas capitata Desf 14±0.80 p˃0.5 783±10.02 0.5˃p˃0.1 V/18 Ocimum sanctum L 12.3± 2.45 p˃0.5 1335±12.03 0.02˃p˃0.01 V/3 Allium sativum L 6.6±3.30 0.5˃p˃0.1 1177±20.56 0.05˃p˃0.02 V/4 Althaea officinalis L 14.8± 3.30 p˃0.5 975.5±33.60 0.5˃p˃0.1 V/1 Albizia lebbeck (L.) Benth 4.1±0.85 0.5˃p˃0.1 1357±1.00 0.01˃p˃0.001 V/8 Cedrus deodara (Roxb. ex 41.2±31.6 p˃0.5 1230±23.57 0.05˃p˃0.02 D. Don) G. Don V/19 Pinus roxburghii Sarg 08±1.65 0.5˃p˃0.1 1050±1.00 0.02˃p˃0.01 V/23 Rubia cordifolia L 6.6±0.00 0.5˃p˃0.1 1078±16.04 0.05˃p˃0.02 V/9 Citrus limon (L). Burm. f 73±14.1 0.5˃p˃0.1 1831±65.69 0.05˃p˃0.02 V/24 Sapindus mukorossi Gaertn 4.1±0.80 0.5˃p˃0.1 1460.5±5.51 0.01˃p˃0.001 V/28 Zingiber officinale Roscoe 17.3± 0.80 0.5˃p˃0.1 888±2.00 0.05˃p˃0.02 V/13 Fagonia cretica L 11±2.52 p˃0.5 1418±13.03 0.02˃p˃0.01 V/29 Reference standard anti-sera 09±0.85 Standard 763±6.01 Standard drug drug
146 Table 3.32 A List of Medicinal Plants Extract (100 mg/kg) used to Minimize Hepatotoxicity Posed by Naja naja karachiensis Venom (800 µg/kg) in
Rabbits (Adopted from Asad et al., 2014(d)).
Group V Name of tested material AST (U/L) ALT (U/L) (subgroup) (Mean (p-value) (Mean (p-value) ± SEM) ±SEM) V/2 Allium cepa L 166.5±0.50 0.5˃p˃0.1 68±12.03 0.5˃p˃0.1 V/20 Pistacia integerrima J. L. 54.5±0.50 p˃0.5 41.5±1.5 0.5˃p˃0.1 Stewart V/11 Cuminum cyminum L 157±14.14 0.5˃p˃0.1 67±16.16 p˃0.5 V/17 Nerium indicum Mill 235±18.05 0.1˃p˃0.05 88±19.55 0.5˃p˃0.1 V/22 Rhazya stricta Dcne 132.5±12.62 0.5˃p˃0.1 66±24.24 p˃0.5
V/7a Calotropis procera (Aiton) 141±1.01 0.5˃p˃0.1 31.5±0.5 0.5˃p˃0.1 W. T. Aiton (exudates)
V/7b Calotropis procera (Aiton) 194±12.12 0.5˃p˃0.1 62.5±2.52 0.5˃p˃0.1 W. T. Aiton (flowers) V/25 Stenolobium stans (L.) 70.5±3.53 p˃0.5 46.5±6.56 p˃0.5 Seem V/27 Trichodesma indicum (L.) 125±4.01 0.5˃p˃0.1 52±0.00 p˃0.5 Sm V/5 Bauhinia variegate L 149.5±19.9 0.5˃p˃0.1 78±4.04 0.5˃p˃0.1 V/6 Brassica nigra (L.) W. D. J. 55±1.00 p˃0.5 45±0.00 0.5˃p˃0.1 Koch V/15 Matthiloa incana (L.) W. T. 121±5.05 0.5˃p˃0.1 62±8.08 0.5˃p˃0.1 Aiton V/16 Momordica charantia L 247±46.13 0.5˃p˃0.1 62.5±7.52 0.5˃p˃0.1 V/10 Citrullus colocynthis (L.) 74±9.09 p˃0.5 47.5±5.55 p˃0.5 Schrad V/26 Terminalia arjuna (Roxb. 78±6.06 p˃0.5 47.5±1.51 0.5˃p˃0.1 ex DC.) Wight & Arn
147 V/21 Psoralea corylifolia L 66.5±11.53 p˃0.5 54±1.00 p˃0.5 V/12 Enicostemma hyssopifolium 168.5±7.57 0.5˃p˃0.1 85.25±1.26 0.1˃p˃0.05 (Willd.) I. Verd V/14 Leucas capitata Desf 53.5±0.50 p˃0.5 45.5±7.52 p˃0.5 V/18 Ocimum sanctum L 158±10.02 0.5˃p˃0.1 61.5±7.52 0.5˃p˃0.1 V/3 Allium sativum L 82.5±18.55 p˃0.5 49±5.01 p˃0.5 V/4 Althaea officinalis L 74.5±14.54 p˃0.5 70±20.05 p˃0.5 V/1 Albizia lebbeck (L.) Benth 99±18.55 0.5˃p˃0.1 62±7.02 0.5˃p˃0.1 V/8 Cedrus deodara (Roxb. ex 72±2.02 p˃0.5 63.3±4.14 0.5˃p˃0.1 D. Don) G. Don V/19 Pinus roxburghii Sarg 57±13.03 p˃0.5 53±9.02 p˃0.5 V/23 Rubia cordifolia L 129±66.41 p˃0.5 65.5±17.67 p˃0.5 V/9 Citrus limon (L). Burm. f 49±24.24 p˃0.5 30.5±13.63 0.5˃p˃0.1 V/24 Sapindus mukorossi Gaertn 78.5±13.54 p˃0.5 56±6.01 p˃0.5 V/28 Zingiber officinale Roscoe 117.5±9.52 0.5˃p˃0.1 66.5±32.59 p˃0.5 V/13 Fagonia cretica L 170±13.13 0.5˃p˃0.1 80±7.82 0.5˃p˃0.1 V/29 Reference standard anti-sera 69.5±18.55 Standard 52.5±3.51 Standard drug drug
148 Table 3.33 A List of Medicinal Plants Extract (100 mg/kg) used to Minimize Nephrotoxicity Posed by Naja naja karachiensis Venom (800 µg/kg) in
Rabbits (Adopted from Asad et al., 2014(d)).
Group V Name of tested material Creatinine (mg/dl) Urea (mg/dl) (subgroup) (Mean± (p-value) (Mean± (p-value) SEM) SEM) V/2 Allium cepa L 1.50±0.17 0.5˃p˃0.1 51±11.0 0.5˃p˃0.1 V/20 Pistacia integerrima J. L. 1.68±0.10 0.5˃p˃0.1 66.5±0.5 0.02˃p˃0.01 Stewart V/11 Cuminum cyminum L 1.48±0.01 0.05˃p˃0.02 54±1.01 0.05˃p˃0.02 V/17 Nerium indicum Mill 1.24±0.10 0.5˃p˃0.1 47± 0.00 p˂0.05 V/22 Rhazya stricta Dcne 1.67±0.005 0.05˃p˃0.02 53.5±3.53 0.5˃p˃0.1
V/7a Calotropis procera (Aiton) 1.30±0.01 0.1˃p˃0.05 42.5±2.52 0.5˃p˃0.1 W. T. Aiton (exudate)
V/7b Calotropis procera (Aiton) 1.25±0.03 0.5˃p˃0.1 39±2.02 0.5˃p˃0.1 W. T. Aiton (flowers) V/25 Stenolobium stans (L.) 1.1±0.06 p˃0.5 25.5± 6.51 p˃0.5 Seem V/27 Trichodesma indicum (L.) 0.46±0.01 0.05˃p˃0.02 37±1.00 0.5˃p˃0.1 Sm V/5 Bauhinia variegate L 1.36 ± 0.24 0.5˃p˃0.1 50±12.6 0.5˃p˃0.1 V/6 Brassica nigra (L.) W. D. J. 1.07±0.11 p˃0.5 22.5±4.51 0.5˃p˃0.1 Koch V/15 Matthiloa incana (L.) W. T. 1.44±0.14 0.5˃p˃0.1 43.5± 3.53 0.5˃p˃0.1 Aiton V/16 Momordica charantia L 1.5±0.005 0.05˃p˃0.02 57±5.01 0.5˃p˃0.1 V/10 Citrullus colocynthis (L.) 1.52±0.15 0.5˃p˃0.1 49.5± 3.53 0.5˃p˃0.1 Schrad V/26 Terminalia arjuna (Roxb. 1.27±0.23 p˃0.5 38.5±1.51 0.5˃p˃0.1 ex DC.) Wight & Arn
149 V/21 Psoralea corylifolia L 1.27±0.15 0.5˃p˃0.1 42.5±2.50 0.5˃p˃0.1 V/12 Enicostemma hyssopifolium 1.35±0.05 0.5˃p˃0.1 39.5±4.54 0.5˃p˃0.1 (Willd.) I. Verd V/14 Leucas capitata Desf 1.07±0.05 p˃0.5 31.5±0.50 p˃0.5 V/18 Ocimum sanctum L 1.5±0.005 0.05˃p˃0.02 49.5±7.52 0.5˃p˃0.1 V/3 Allium sativum L 1.30±0.06 0.5˃p˃0.1 50±4.01 0.5˃p˃0.1 V/4 Althaea officinalis L 1.18±0.24 p˃0.5 26±5.05 p˃0.5 V/1 Albizia lebbeck (L.) Benth 1.6±0.20 0.5˃p˃0.1 57±0.0 0.02˃p˃0.01 V/8 Cedrus deodara (Roxb. ex 1.44±0.07 0.5˃p˃0.1 44±4.04 0.5˃p˃0.1 D. Don) G. Don V/19 Pinus roxburghii Sarg 1.4±0.05 0.5˃p˃0.1 45±1.00 p˃0.05 V/23 Rubia cordifolia L 0.93±0.48 p˃0.5 36±1.01 0.5˃p˃0.1 V/9 Citrus limon (L). Burm. f 1.40±0.03 0.1˃p˃0.05 39.5±2.52 0.5˃p˃0.1 V/24 Sapindus mukorossi Gaertn 1.43±0.12 0.5˃p˃0.1 44±1.00 0.1˃p˃0.05 V/28 Zingiber officinale Roscoe 1.30±0.01 0.5˃p˃0.1 47±1.00 0.05˃p˃0.02 V/13 Fagonia cretica L 1.23±0.13 p˂0.5 43±2.27 0.5˃p˃0.1 V/29 Reference standard anti-sera 1.08±0.02 Standard 31.5±0.50 Standard drug drug
150 3.5 Different Enzymatic Assays for Naja naja karachiensis Venom with their Neutralization by Medicinal Plants
3.5.1 Enzymatic Assays for Phospholipase Enzyme
This section comprised of findings regarding three different types of developed phospholipase assays.
3.5.1.1 Acidimetric Phospholipase (PLA2) Enzymatic Assay and its Neutralization
Phospholipase enzyme present in Naja naja karachiensis venom was found to hydrolyze egg yolk phospholipids in presence of sodium deoxycholate. It resulted in the liberation of free fatty acids dose dependently in terms of decline in pH of an egg yolk suspension. At 0.1 mg venom concentration 26.6 µmol of free fatty acids were released. On increasing venom concentration (0.3 mg, 0.5 mg, 1.0 mg, 2.0 mg and 4.0 mg) greater amount of fatty acids (53.2 µmol/min, 66.5 µmol/min, 79.8 µmol/min, 106.4 µmol/min and 119.7 µmol/min) were released respectively. At concentration of 8.0 mg of venom complete drop in 1.0 pH (from 8.0 to 7.0) was noticed tend to release in 133 µmol of fatty acids. Based on PLA2 hydrolytic action its activity was recorded as 133 units/mg, 266 units/mg, 332 units/mg, 399 units/mg, 532 units/mg, 598 units/mg and 665 units/mg at 0.1 mg, 0.3 mg, 0.5 mg, 1.0 mg, 2.0 mg, 4.0 mg and 8.0 mg of venom respectively as shown in detail in table 3.34.
In order to compensate hydrolytic action of PLA2 enzyme twenty eight medicinal plants extract were evaluated along with standard antidote in a concentration range of 0.1 to 0.6 mg/ml. Ocimum sanctum L, Bauhinia variegate L, Enicostemma hyssopifolium (Willd.) I. Verd, Citrus limon (L). Burm. f, Psoralea corylifolia L and Stenolobium stans (L.) Seem was found cent percent equally effective at low concentration (0.1mg/ml). However Sapindus mukorossi Gaertn, Althaea officinalis L, Terminalia arjuna (Roxb. ex DC.) Wight & Arn, Nerium indicum Mill, Citrullus colocynthis (L.) Schrad, Cuminum cyminum L, Pinus roxburghii Sarg and Momordica charantia L were found valuable (100%) at their high concentration (0.3 mg/ml or high dose). Extracts of Zingiber
151 officinale Roscoe, Pistacia integerrima J. L. Stewart and Matthiloa incana (L.) W. T. Aiton was found effective merely at low concentration (0.1 mg/ml) while at high doses
(˃0.1mg/ml) they were proved to mimic PLA2 action. Rest of other plants extract along with reference standard antidote was proved fifty percent effective regardless of the doses. Among them Fagonia cretica L, Allium sativum L, Cedrus deodara (Roxb. ex D. Don) G. Don, Allium cepa L, Rhazya stricta Dcne, Calotropis procera (Aiton) W. T. Aiton (exudates & flowers), Trichodesma indicum (L.) Sm, Brassica nigra (L.) W. D. J. Koch, Leucas capitata Desf, Rubia cordifolia L and Albizia lebbeck (L.) Benth were included. A concise description about various antidotes evaluated to neutralize Naja naja karachiensis venom PLA2 is mentioned in table 3.35.
Table 3.34 Dose Dependent Effect of Cobra Venom for the Release of Free Fatty Acids
In Terms of Variation in pH (Adopted from Asad et al., (2014) (b)).
Amount of venom Variation in pH Liberated fatty acids PLA2 activity (mg /0.1 ml) (Mean ± SEM) (µ mole) (Units/mg) 0.10 7.8 ± 0.02 26.6 133
0.30 7.6 ± 0.02 53.2 266
0.50 7.5 ± 0.02 66.5 332
1.00 7.4 ± 0.02 79.8 399
2.00 7.2 ± 0.00 106.4 532
4.00 7.1 ± 0.02 119.7 598
8.00 7.0 ± 0.02 133 665 Negative control 8.0 ± 0.00 0 0 (0.89%NaCl)
152 Table 3.35 List of Evaluated Medicinal Plants Extract Having Anti-Phospholipase A2
Property In Terms of Increase in pH (Adopted from Asad et al., (2014) (b)). Observed pH at Protection Name of evaluated antidote different offered concentration(mg/ml) (%) 0.1 0.3 0.6 Control (saline) 8.0 8.0 8.0 100 Allium cepa L 7.9 7.9 7.9 50 Pistacia integerrima J. L. Stewart 8.0 7.9 7.9 100 Cuminum cyminum L 7.9 7.9 8.0 100 Nerium indicum Mill 7.9 8.0 8.0 100 Rhazya stricta Dcne 7.9 7.9 7.9 50 Calotropis procera (Aiton) W. T. Aiton (exudates) 7.9 7.9 7.9 50 Stenolobium stans (L.) Seem 8.0 8.0 8.0 100 Calotropis procera (Aiton) W. T. Aiton (flowers) 7.9 7.9 7.9 50 Trichodesma indicum (L.) Sm 7.9 7.9 7.9 50 Bauhinia variegate L 8.0 8.0 8.0 100 Brassica nigra (L.) W. D. J. Koch 7.9 7.9 7.9 50 Matthiloa incana (L.) W. T. Aiton 8.0 7.9 7.9 100 Citrullus colocynthis (L.) Schrad 7.9 7.9 8.0 100 Momordica charantia L 7.8 7.8 8.0 100 Terminalia arjuna (Roxb. ex DC.) Wight & Arn 7.9 7.9 8.0 100 Psoralea corylifolia L 8.0 8.0 8.0 100 Enicostemma hyssopifolium (Willd.) I. Verd 8.0 8.0 8.0 100 Leucas capitata Desf 7.9 7.9 7.9 50 Ocimum sanctum L 8.0 8.0 8.0 100 Allium sativum L 7.9 7.9 7.9 50 Albizia lebbeck (L.) Benth 7.9 7.9 7.9 50 Althaea officinalis L 7.9 8.0 8.0 100 Cedrus deodara (Roxb. ex D. Don) G. Don 7.9 7.9 7.9 50 Pinus roxburghii Sarg 7.9 8.0 8.0 100
153 Rubia cordifolia L 7.9 7.9 7.9 50 Citrus limon (L). Burm. f 8.0 8.0 8.0 100 Sapindus mukorossi Gaertn 7.9 7.9 8.0 100 Zingiber officinale Roscoe 8.0 7.9 7.9 100 Fagonia cretica L 7.9 7.9 7.9 50 Reference standard anti-sera 7.9 7.9 7.9 50
154 3.5.1.2 Turbidimetric Phospholipase (PLA2) Enzymatic Assay and its Neutralization
Snake venom PLA2 has been found to decrease turbidity of the reaction mixture dose dependently at various incubation time (Table 3.36). A concentration of 20 mg/ml venom did reach to a steady state response after 20 min of incubation therefore reasoned to fix these values for further experimentation. Nevertheless rest of all venom concentrations (2.5 mg/ml, 5 mg/ml & 10 mg/ml) did reach maximum hydrolysis beyond 40 min of incubation (Fig 3.60).
Naja naja karachiensis venom (20 mg/ml) was proved to decrease turbidity of an egg yolk mixture and posed 100% PLA2 activity. When different samples were evaluated to mask PLA2 activity only metalloenzyme inhibitor (EDTA) was proved the best antidote (76%). Bauhinia variegate L was the only shortlisted (p˃0.01) extract having anti- phospholipase A2 potential (67%) comparable to the reference standard EDTA. However rest of other extracts (Allium cepa L, Pistacia integerrima J. L. Stewart, Cuminum cyminum L, Nerium indicum Mill, Rhazya stricta Dcne, Calotropis procera (Aiton) W. T. Aiton (exudates and flowers), Trichodesma indicum (L.) Sm, Brassica nigra (L.) W. D. J. Koch, Matthiloa incana (L.) W. T. Aiton, Citrullus colocynthis (L.) Schrad, Momordica charantia L, Terminalia arjuna (Roxb. ex DC.) Wight & Arn, Psoralea corylifolia L, Enicostemma hyssopifolium (Willd.) I. Verd, Leucas capitata Desf, Ocimum sanctum L, Allium sativum L, Albizia lebbeck (L.) Benth, Althaea officinalis L, Cedrus deodara (Roxb. ex D. Don) G. Don, Pinus roxburghii Sarg, Rubia cordifolia L, Citrus limon (L). Burm. f, Sapindus mukorossi Gaertn, Zingiber officinale Roscoe and Fagonia cretica L) were not proved beneficial (p˂˂0.001) compare to EDTA in neutralization of PLA2 activity of crude venom. Saline was served as control and unable to cause any effect on turbidity after 20 min of incubation. A complete discussion about antidotes and their effects as anti-PLA2 in comparison to EDTA is summarized in table 3.37.
155
Table 3.36 Data Points Acquired to Optimize Various Cobra Venom Concentrations at
Different Incubation Time for Validation of PLA2 (Turbidimetric) Assay.
Incubation time Absorbance (630nm) of mixture at various venom concentration (min) (Mean ± SD) where n=3 20 mg/ml 10 mg/ml 05 mg/ml 2.5 mg/ml 00 1.403±0.006 1.527±0.02 1.540±0.001 1.610±0.001
05 1.224±0.014 1.420±0.011 1.435±0.014 1.519±0.003
10 1.106±0.006 1.306±0.059 1.370±0.002 1.457±0.006
15 1.071±0.005 1.242±0.061 1.314±0.022 1.398±0.004
20 0.993±0.003 1.220±0.029 1.268±0.012 1.370±0.004 25 0.975±0.014 1.172±0.028 1.222±0.006 1.344±0.005
30 0.958±0.001 1.164±0.009 1.181±0.001 1.307±0.004
35 0.929±0.011 1.094±0.079 1.147±0.001 1.293±0.001
40 0.916±0.014 1.020±0.038 1.118±0.003 1.268±0.002 45 0.909±0.002 1.002±0.036 1.100±0.001 1.241±0.005
50 0.884±0.021 0.987±0.023 1.077±0.008 1.231±0.003
55 0.882±0.022 0.987±0.034 1.060±0.004 1.203±0.004
60 0.865±0.015 0.966±0.034 1.040±0.004 1.183±0.008
156
Fig 3.60 Represents Absorbance of the Turbidity at Various Venom Concentrations along with Incubation Time.
157 Table 3.37 Anti-Phospholipase A2 Potential of Various Antidotes in Neutralization of Decrease in Turbidity Posed by Naja naja karachiensis Venom.
Name of evaluated sample change in absorbance % PLA2 % PLA2 (0 to 20 min) activity activity mean ± SD retained inhibited
Venom (Naja naja karachisis) 0.352 ± 0.003 100 0** Allium cepa L 0.350 ± 0.002 99.4 0.6** Pistacia integerrima J. L. Stewart 0.351 ± 0.002 100 0** Cuminum cyminum L 0.352 ± 0.001 100 0** Nerium indicum Mill 0.351 ± 0.002 100 0** Rhazya stricta Dcne 0.350 ± 0.002 100 0** Calotropis procera (Aiton) W. T. 0.352 ± 0.002 100 0** Aiton (exudates) Stenolobium stans (L.) Seem 0.261 ± 0.002 74 26** Calotropis procera (Aiton) W. T. 0.350 ± 0.002 100 0** Aiton (flowers) Trichodesma indicum (L.) Sm 0.353 ± 0.002 100 0** Bauhinia variegate L 0.117 ± 0.002 33 67* Brassica nigra (L.) W. D. J. Koch 0.351 ± 0.002 100 0** Matthiloa incana (L.) W. T. Aiton 0.352 ± 0.002 100 0** Citrullus colocynthis (L.) Schrad 0.247 ± 0.002 70 30** Momordica charantia L 0.350 ± 0.003 100 0** Terminalia arjuna (Roxb. ex DC.) 0.349 ± 0.002 100 0** Wight & Arn Psoralea corylifolia L 0.302 ± 0.002 85 15** Enicostemma hyssopifolium (Willd.) 0.330 ± 0.002 92 08** I. Verd Leucas capitata Desf 0.351 ± 0.002 100 0** Ocimum sanctum L 0.280 ± 0.001 79 21** Allium sativum L 0.353 ± 0.002 100 0** Albizia lebbeck (L.) Benth 0.351 ± 0.002 100 0**
158 Althaea officinalis L 0.352 ± 0.002 100 0** Cedrus deodara (Roxb. ex D. Don) 0.204 ± 0.003 58 42** G. Don Pinus roxburghii Sarg 0.349 ± 0.002 100 0** Rubia cordifolia L 0.261 ± 0.002 74 26** Citrus limon (L). Burm. f 0.289 ± 0.001 82 18** Sapindus mukorossi Gaertn 0.350 ± 0.001 100 0** Zingiber officinale Roscoe 0.352 ± 0.000 100 0** Fagonia cretica L 0.353 ± 0.007 100 0** EDTA (metalloenzyme inhibitor) 0.084 ± 0.005 24 76***
Note: *indicates that value non-statistically different from reference (p˃0.01). ** indicates that value significantly different from reference (p˂˂0.001). *** indicates that value selected to compare.
159 3.5.1.3 Anticoagulant Phospholipase (PLA) Enzymatic Assay and its Neutralization
Naja naja karachiensis venom was found to cause anticoagulant effect on Hen’s egg yolk mixture owing to the prominent PLA2 enzymes. Snake venom (5 µg/200 µl) was attributed to delay in coagulation of an egg yolk mixture (125 ± 0.57 sec) when compared to venom free control (saline) sample (100 ± 1sec) in boiling water bath.
Different antidotes were tried to get rid anticoagulant effect of cobra venom. Among them reference standard antidote was proved cent percent effective (100 ± 0.57 sec) to reverse the effect of Naja naja karachiensis venom. However medicinal plants extract were proved to show wide range of antidotal effects. Rubia cordifolia L (p˃0.5) and Citrullus colocynthis (L.) Schrad (p˃0.5) was observed 100% efficient as reference standard while Stenolobium stans (L.) Seem was also found valuable (100%) with p˂0.5 (Table 3.38). Sapindus mukorossi Gaertn and Zingiber officinale Roscoe was found 80% and 92% effective respectively at 0.1˃p˃0.05. Similarly Cedrus deodara (Roxb. ex D. Don) G. Don and Psoralea corylifolia L were noticed 80% and 92% beneficial respectively at 0.05˃p˃0.02. Bauhinia variegate L, Calotropis procera (Aiton) W. T. Aiton (flowers) and Momordica charantia L were found 76% valuable at 0.02˃p˃0.01exactely similar to Ocimum sanctum L which was only 60% effective.
Rest of all extracts was included to show various anticoagulant effects (0%-80%) at lower probability thus couldn’t be taken as guaranteed. Among them Allium cepa L, Albizia lebbeck (L.) Benth, Allium sativum L, Brassica nigra (L.) W. D. J. Koch, Cuminum cyminum L, Citrus limon (L). Burm. f, Enicostemma hyssopifolium (Willd.) I. Verd, Fagonia cretica L, Leucas capitata Desf, Pinus roxburghii Sarg, Matthiloa incana (L.) W. T. Aiton, Pistacia integerrima J. L. Stewart, Terminalia arjuna (Roxb. ex DC.) Wight & Arn and Trichodesma indicum (L.) Sm were found at 0.01˃p˃0.001 while Calotropis procera (Aiton) W. T. Aiton (exudates), Althaea officinalis L, Nerium indicum Mill and Rhazya stricta Dcne were observed totally useless as p˂˂0.001. Complete detail about various tested material is discussed in table 3.38 while possible pharmacological illustration about snake venom and anti-venom effects is shown in fig 3.61.
160 Table 3.38 Reverse Anticoagulant Potential of Pakistani Medicinal Plants Extract Posed by Weak PLA Enzymes Abundant in Naja naja karachiensis Venom (Adopted from Asad et al., 2013).
Sample evaluated for Coagulation Percentage Percentage anticoagulant time (sec) PLA unmasked P-value activity (5µg/0.2ml) Mean ± SD inhibition activity Allium cepa L 106 ± 0.57 76 24 0.01˃p˃0.001 Pistacia integerrima J. L. Stewart 110 ± 1.00 60 40 0.01˃p˃0.001 Cuminum cyminum L 114 ± 2.00 44 56 0.01˃p˃0.001 Nerium indicum Mill 125 ± 0.57 0 100 p˂˂0.001 Rhazya stricta Dcne 125 ± 1.00 0 100 p˂˂0.001 Calotropis procera (Aiton) W. T. 114 ± 0.00 44 56 p˂˂0.001 Aiton (exudates) Stenolobium stans (L.) Seem 100 ± 0.57 100 0 0.5˃p˃0.1 Calotropis procera (Aiton) W. T. 106 ± 1.00 76 24 0.02˃p˃0.01 Aiton (flowers) Trichodesma indicum (L.) Sm 113 ± 0.57 48 52 0.01˃p˃0.001 Bauhinia variegate L 108 ± 1.52 76 24 0.02˃p˃0.01 Brassica nigra (L.) W. D. J. Koch 125 ± 1.52 0 100 0.01˃p˃0.001 Matthiloa incana (L.) W. T. Aiton 125 ± 1.52 0 100 0.01˃p˃0.001 Citrullus colocynthis (L.) Schrad 100 ± 0.57 100 0 p˃0.5 Momordica charantia L 106 ± 1.00 76 24 0.02˃p˃0.01 Terminalia arjuna (Roxb. ex DC.) 111 ± 1.15 56 44 0.01˃p˃0.001 Wight & Arn Psoralea corylifolia L 102 ± 0.57 92 8 0.05˃p˃0.02 Enicostemma hyssopifolium 110 ± 1.00 60 40 0.01˃p˃0.001 (Willd.) I. Verd Leucas capitata Desf 105 ± 0.00 80 20 0.01˃p˃0.001 Ocimum sanctum L 110 ± 2.00 60 40 0.02˃p˃0.01 Allium sativum L 115 ± 1.52 40 60 0.01˃p˃0.001 Albizia lebbeck (L.) Benth 125 ± 1.52 0 100 0.01˃p˃0.001
161 Althaea officinalis L 120 ± 0.57 20 80 p˂˂0.001 Cedrus deodara (Roxb. ex D. 105 ± 1.52 80 20 0.05˃p˃0.02 Don) G. Don Pinus roxburghii Sarg 114 ± 1.52 44 56 0.01˃p˃0.001 Rubia cordifolia L 100 ± 1.00 100 0 p˃0.5 Citrus limon (L). Burm. f 109 ± 1.00 64 36 0.01˃p˃0.001 Sapindus mukorossi Gaertn 106 ± 2.51 80 20 0.1˃p˃0.05 Zingiber officinale Roscoe 102 ± 1.00 92 8 0.1˃p˃0.05 Fagonia cretica L 113 ± 0.57 48 52 0.01˃p˃0.001 Reference anti-sera 100 ±0.57 100 0 Select to compare (standard drug)
162
Fig 3.61 Mechanism of Action of Secondary Metabolites (Plants Extract) to Neutralize Anticoagulant Effect of Phospholipase Enzyme Present in Naja naja karachiensis Venom (Adopted from Asad et al., 2013).
163 3.5.2 Enzymatic Assay for Alkaline Phosphatase (ALPase) and its Neutralization
Naja naja karachiensis venom was found abundant in alkaline phosphatase (ALPase) which hydrolyzed p-nitrophenyl phosphate dose dependently and ultimately liberated p- nitrophenol (p-NP). Standard curve for p-NP was constructed successfully with positive correlation coefficient equal to one (r = 0.999 & y = 0.009x +0.001) (Fig 3.62). High value of ALPae activity was noticed with high doses of cobra venom such that 2.87 U/mg, 4.75 U/mg, 6.70 U/mg and 8.6 U/mg were calculated in 0.2 mg, 0.4 mg, 0.6 mg and 0.8 mg of venom respectively. In addition ALPase activity was completely lost after heating however results were not changed even after cooling of it. A complete description about ALPase activity present in Naja naja karachiensis venom is shown in table 3.39.
In this study twenty eight medicinal plants extract were endeavored to neutralize fixed amount (0.4 mg) of cobra venom. Plants were found with varying effectiveness (80%- 93%) and potencies (0.625µg-2.5µg) to cope with deleterious effect of ALPase. Reference standard anti-dote was declared the best antidote (93% at 2.5µg) to neutralize ALPase activity while specific metalloenzyme inhibitor (EDTA) was found 91% effective. On comparison with reference standard only extract of Sapindus mukorossi Gaertn was found (93% at 2.5µg) equally effective (p˃0.5) as anti-sera. Among others Rhazya stricta Dcne (p˃0.5), Stenolobium stans (L.) Seem (p˃0.5), Brassica nigra (L.) W. D. J. Koch (p˃0.5), Enicostemma hyssopifolium (Willd.) I. Verd (p˃0.5) and Pinus roxburghii Sarg (p˃0.5) were recorded 91%, 86%, 87%, 91% and 90% effective respectively. Ocimum sanctum L, Psoralea corylifolia L, Allium cepa L, Rubia cordifolia L, Bauhinia variegata L, Zingiber officinale Roscoe, Citrus limon (L.) Burm. f, Matthiola incana (L.) W. T. Aiton, Albizia lebbeck (L.) Benth, Cuminum cyminum L, Pistacia integerrima J. L. Stewart, Allium sativum L and Citrullus colocynthis (L.) Schrad were found ≤91% beneficial at 0.5 ˃ P ˃0.1. Rest of all extracts (Calotropis procera (Aiton) W. T. Aiton (both exudates and flowers), Terminalia arjuna (Roxb. ex DC.) Wight & Arn, Cedrus deodara (Roxb. ex D. Don) G. Don, Trichodesma indicum (L.) Sm, Momordica charantia L, Nerium indicum Mill, Leucas capitata Desf, Althaea officinalis L, and Fagonia cretica L were recorded ≤86% valuable at 0.1 ˃P
164 ˃0.05. Complete description about anti-ALPase activity of different medicinal plants extract is mentioned in table 3.40.
Fig 3.62 Standard Curve for p-Nitrophenol In Terms of Absorbance at Different Concentrations.
165 Table 3.39 Effect of Various Concentration of Naja naja karchiensis Venom Abundant in Alkaline Phosphatase (ALPase) Enzyme in the Release of p-Nitrophenol
(Adopted from Asad et al., 2014 (a)). Sr. No. Venom concentration Absorbance (400 nm) ALPase activity (mg/0.1 ml) (Mean ± SEM) (Unit/mg) 1 0.2 0.312 ± 0.011 2.87 2 0.4 0.514 ± 0.013 4.75 3 0.6 0.721 ± 0.018 6.7 4 0.8 0.929 ± 0.021 8.6
Table 3.40 Medicinal Plants of Pakistan having Anti-Alkaline Phosphatase (Anti- ALPase) Activity Posed by Naja naja karachiensis (0.4 mg/0.1ml) Venom
(Adopted from Asad et al., 2014 (a)).
Evaluated sample Effective ALPase activity Protection amount of (U/mg) offered P-value antidote Masked unmasked (%) (µg/ml) Allium cepa L 2.5 4.12 0.63 87** 0.5 ˃ P ˃0.1 Pistacia integerrima J. L. 2.5 4.30 0.45 90* 0.5 ˃ P ˃0.1 Stewart Cuminum cyminum L 2.5 4.11 0.64 86** 0.5 ˃ P ˃0.1 Nerium indicum Mill 0.625 3.89 0.86 82** 0.1 ˃ P ˃0.05 Rhazya stricta Dcne 2.5 4.33 0.42 91* P ˃ 0.5 Calotropis procera
(Aiton) W. T. Aiton 0.625 4.08 0.67 86** 0.1 ˃ P ˃0.05 (exudates) Stenolobium stans (L.) 0.625 4.11 0.64 86** P ˃ 0.5 Seem Calotropis procera 0.625 3.86 0.89 81** 0.1 ˃ P ˃0.05 (Aiton) W. T. Aiton (flowers) Trichodesma indicum (L.) 0.625 4.01 0.74 84** 0.1 ˃ P ˃0.05 Sm Bauhinia variegate L 1.25 4.03 0.72 85** 0.5 ˃ P ˃0.1
166 Brassica nigra (L.) W. D. 1.25 4.15 0.60 87** P ˃0.5 J. Koch
Matthiloa incana (L.) W. 2.5 4.12 0.63 87** 0.5 ˃ P ˃0.1 T. Aiton Citrullus colocynthis (L.) 0.625 4.10 0.65 86** 0.5 ˃ P ˃0.1 Schrad Momordica charantia L 0.625 3.79 0.96 80** 0.1 ˃ P ˃0.05 Terminalia arjuna (Roxb. 2.5 3.79 0.96 80** P < 0.05 ex DC.) Wight & Arn Psoralea corylifolia L 0.625 4.25 0.50 89** 0.5 ˃ P ˃0.1 Enicostemma 0.625 4.35 0.40 91* P ˃ 0.5 hyssopifolium (Willd.) I.
Verd Leucas capitata Desf 1.25 3.93 0.82 83** 0.1 ˃ P ˃0.05 Ocimum sanctum L 1.25 4.10 0.65 86** 0.5 ˃ P ˃0.1 Allium sativum L 2.5 4.13 0.62 87** 0.5 ˃ P ˃0.1 Albizia lebbeck (L.) Benth 1.25 4.06 0.69 85** 0.5 ˃ P ˃0.1 Althaea officinalis L 0.625 4.03 0.72 85** 0.1 ˃ P ˃0.05 Cedrus deodara (Roxb. ex 1.25 3.89 0.86 82** 0.1 ˃ P ˃0.05 D. Don) G. Don Pinus roxburghii Sarg 1.25 4.27 0.48 90* P ˃ 0.5 Rubia cordifolia L 0.625 4.31 0.44 91* 0.5 ˃ P ˃0.1 Citrus limon (L). Burm. f 1.25 4.33 0.42 91* 0.5 ˃ P ˃0.1 Sapindus mukorossi 2.5 4.42 0.33 93* P ˃ 0.5 Gaertn Zingiber officinale Roscoe 1.25 3.98 0.77 84** 0.5 ˃ P ˃0.1 Fagonia cretica L 2.5 3.90 0.85 82** 0.1 ˃ P ˃0.05 Reference anti-sera Select to 2.5 4.4 0.36 93*** compare (standard drug) Note: * represents values non-significantly different from reference. ** Represents values significantly different from reference. *** represents value selected to compare.
167 3.5.3 Enzymatic Assay for 5ʹ-Nucleotidase and its Neutralization
Venom from Naja naja karachiensis was evaluated for 5ʹ-nucleotidase (5ʹ-ND) activity on its substrate adenosine monophosphate (AMP) dose dependently which resulted in the release of inorganic phosphate. Moreover standard curve for inorganic phosphate was obtained with positive value of correlation coefficient equal to one (r = 0.999 & y = 0.013x + 0.012) (Fig 3.63). Increase in 5’-nucleotidase activity was noticed with high doses of cobra venom such that 48 U/ml, 119 U/ml, 183 U/ml, 262 U/ml and 335 U/ml were found in 0.005 mg, 0.01 mg, 0.02 mg and 0.04 mg of venom respectively. Complete description about 5’-nucleotidase activity present in Naja naja karachiensis venom is summarized in table 3.41.
Present study described twenty eight medicinal plants extract were evaluated to neutralize fixed low absorbance dose (0.01 mg) of cobra venom. Reference standard anti-dote was declared the best antidote (94% at 0.08 mg) to neutralize 5ʹ-nucleotidase activity. However two medicinal plants extract (Citrus limon (L.) Burm. f and Bauhinia variegata L) were found extremely useful (94% at 0.16 mg) and effective (p˃0.5) comparable to the reference anti-sera (Fig 3.64). Pistacia integerrima J. L. Stewart, Rhazya stricta Dcne, Stenolobium stans (L.) Seem, Citrullus colocynthis (L.) Schrad, Terminalia arjuna (Roxb. ex DC.) Wight & Arn, Enicostemma hyssopifolium (Willd.) I. Verd, Cedrus deodara (Roxb. ex D. Don) G. Don, Zingiber officinale Roscoe and Fagonia cretica L were proved somewhat beneficial (p˂0.5) to halt 5ʹ-nucleotidase activity nevertheless could not be taken as guaranteed. Rest of all extracts (Allium cepa L, Cuminum cyminum L, Nerium indicum Mill, Calotropis procera (Aiton) W. T. Aiton (exudates and flowers), Trichodesma indicum (L.) Sm, Brassica nigra (L.) W. D. J. Koch, Matthiloa incana (L.) W. T. Aiton, Momordica charantia L, Psoralea corylifolia L, Leucas capitata Desf, Ocimum sanctum L, Allium sativum L, Albizia lebbeck (L.) Benth, Althaea officinalis L, Pinus roxburghii Sarg, Rubia cordifolia L and Sapindus mukorossi Gaertn) were failed (p˂0.001) to combat deleterious effect of 5’-nucleotidase as summarized in detail in table 3.42.
168
Fig 3.63 Standard Curve for Inorganic Phosphate In Terms of Absorbance at Various Concentrations (Adopted from Asad et al., 2016).
169 Table 3.41 Effect of Various Concentration of Naja naja karachiensis Venom Abundant in 5ʹ-Nucleotidase Enzyme in the Release of Inorganic Phosphate (Adopted from Asad et al., 2016).
Venom concentration Absorbance at 820nm Enzyme activity (U/ml) (mg/0.1ml) (Mean ± SEM), n=4 0.005 0.355± 0.101 48 0.010 0.850 ± 0.184 119 0.020 1.303 ± 0.269 183 0.030 1.856 ± 0.274 262 0.040 2.369 ± 0.261 335
Table 3.42 List of Medicinal Plants of Pakistan having Anti-5ʹ-Nucleotidase Activity Posed by Naja naja karachiensis (0.01mg/0.1 ml) Venom (Adopted from Asad et al., 2016).
Evaluated sample 5ʹ-nucleotidase 5ʹ-ND activity (effective concentration) inhibition P-value Masked activity Unmasked activity (%) in U/ml in U/ml Allium cepa L 0 All present 0 P˂ 0.001 Pistacia integerrima J. L. 104 (0.64 mg) 15 87.3 p˂ 0.01 Stewart Cuminum cyminum L 0 All present 0 P˂ 0.001 Nerium indicum Mill 0 All present 0 P˂ 0.001 Rhazya stricta Dcne 97.8 (0.64 mg) 21.2 82 P˂0.05 Calotropis procera
(Aiton) W. T. Aiton 0 All present 0 P˂ 0.001 (exudates) Stenolobium stans (L.) 96.7 (0.32 mg) 22.3 81 P˂0.05 Seem Calotropis procera 0 All present 0 P˂ 0.001 (Aiton) W. T. Aiton
170 (flowers) Trichodesma indicum (L.) 0 All present 0 P˂ 0.001 Sm Bauhinia variegate L 112 (0.16 mg) 7 94 P˃0.5 Brassica nigra (L.) W. D. 0 All present 0 P˂ 0.001 J. Koch Matthiloa incana (L.) W. 0 All present 0 P˂ 0.001 T. Aiton Citrullus colocynthis (L.) 114 (0.32 mg) 5 95.7 P˂0.05 Schrad Momordica charantia L 0 All present 0 P˂ 0.001 Terminalia arjuna (Roxb. 113.2 (0.16 mg) 5.8 95 P˂0.5 ex DC.) Wight & Arn Psoralea corylifolia L 0 All present 0 P˂ 0.001 Enicostemma hyssopifolium (Willd.) I. 109.5 (0.32 mg) 9.5 92 P˂0.5 Verd Leucas capitata Desf 0 All present 0 P˂ 0.001 Ocimum sanctum L 0 All present 0 P˂ 0.001 Allium sativum L 0 All present 0 P˂ 0.001 Albizia lebbeck (L.) Benth 0 All present 0 P˂ 0.001 Althaea officinalis L 0 All present 0 P˂ 0.001 Cedrus deodara (Roxb. ex 106.8 (0.16 mg) 12.2 90 P˂0.5 D. Don) G. Don Pinus roxburghii Sarg 0 All present 0 P˂ 0.001 Rubia cordifolia L 0 All present 0 P˂ 0.001 Citrus limon (L). Burm. f 112 (0.16 mg) 7 94 P˃0.5 Sapindus mukorossi 0 All present 0 P˂ 0.001 Gaertn Zingiber officinale Roscoe 112.4 (0.32 mg) 6.6 94.4 P˂0.5 Fagonia cretica L 102.5 (0.32 mg) 16.5 86 P˂0.05
171 Reference anti-sera 111 (0.08 mg) 8 94 Select to (standard drug) compare
Fig 3.64 Comparison of Two Valuable Medicinal Plants Extract with Reference Standard Antidote in Neutralization of 5ʹ-Nucleotidase Activity Posed by Naja naja karchiensis (0.01mg) Venom (Adopted from Asad et al., 2016).
172 3.5.4 Enzymatic Assay for Protease (Exclusion of the Protease Bioassay) Presence of protease (caseinolytic) activity was determined via digestion of casein into tyrosine that ultimately reduced Folin-Ciocalteu reagent and detected photometrically at 630 nm.
Even high (20 mg/ml) doses of Naja naja karachiensis venom induced minor (0.083) response in turbidity within fifty min of incubation as shown in Fig 3.65. Highest standard deviation was at 20 mg/ml which would be concentration of choice in order to conduct highest response of turbidity change, was ±0.026, three times standard deviation left an operational window of 0.006. This window was too narrow for validation of protease (caseinolytic) enzymatic assay therefore rejected.
Interestingly result of this study was in consistent with other cobras’ venom (e.g., Naja nigricollis) research led to deduce that proteases don’t digest casein effectively therefore present assay was not possible to conduct for Naja naja karachiensis venom (Nielsen, 2013). Additionally, low protease activity has already been noticed by Yap et al. (2011) for other Asiatic cobra’s venom such as Naja sumatrana, Naja sputatrix, Naja siamensis and Naja kaouthia (Yap et al., 2011).
Fig 3.65 Screening for Protease Enzymatic Activity in Naja naja karachiensis Venom.
173 3.5.5 Enzymatic Assay for Hyaluronidase and its Neutralization
Snake venom was found to decrease turbidity of insoluble complex dose dependently with prolonged incubation time (Fig 3.66). Cobra venom at the concentration of 10 mg/ml was proved to attain a steady state response after 10 min of incubation. Moreover,
Michaelis-Menten kinetics data was acquired (Km=19.0 & Vmax =0.050) for proper optimization of hyaluronidase assay (Fig 3.67).
Cobra venom at the dose of 10 mg/ml was proved to diminish turbidity of the reaction mixture and declared to show 100% hyaluroniadse activity. Rutin trihydrate (reference hyaluronidase inhibitor) was proved 99% effective to neutralize hyaluronidase activity. Among twenty eight medicinal plants extract Trichodesma indicum (L.) Sm was declared the best anti-venom (97%, p˃0.5) in comparison with reference standard antidote. However, sixteen medicinal plnats extract (Allium cepa L, Pistacia integerrima J. L. Stewart, Nerium indicum Mill, Calotropis procera (Aiton) W. T. Aiton (exudates), Brassica nigra (L.) W. D. J. Koch, Matthiloa incana (L.) W. T. Aiton, Citrullus colocynthis (L.) Schrad, Psoralea corylifolia L, Allium sativum L, Albizia lebbeck (L.) Benth, Althaea officinalis L, Cedrus deodara (Roxb. ex D. Don) G. Don, Pinus roxburghii Sarg, Citrus limon (L). Burm. f, Rubia cordifolia L and Bauhinia variegate L) were found completely useless (p˂0.01) to halt hyaluronidase activity (Table 3.43). Calotropis procera (Aiton) W. T. Aiton (flowers), Momordica charantia L, Sapindus mukorossi Gaertn, Cuminum cyminum L, Terminalia arjuna (Roxb. ex DC.) Wight & Arn, Zingiber officinale Roscoe and Ocimum sanctum L were also found ineffective (0.01˃P˃0.001) to abort toxicity posed by hyaluronidase enzyme. Antidotal property of Fagonia cretica L, Enicostemma hyssopifolium (Willd.) I. Verd and Stenolobium stans (L.) Seem was found little effective (p˂0.02). Rhazya stricta Dcne and Leucas capitata Desf were found to show limited beneficial (p˂0.5) effects however could not be shortlisted as valuable antidote. A concise overview about various tested antidotes to neutralize hyaluronidase activity is summarized in table 3.43.
174
Fig 3.66 Optimization of Hyaluroniadse Assay: Effect of Incubation Time at 37 °C on the Reaction Mixture Containing Hyaluroniadase Enzyme at Different Concentrations of Naja naja karachiensis Venom.
175
Enzyme Kinetics Data
0.04
60
40 Rate 0.02
20 1 /1 Rate
0 0 0.02 0.040.06 0.08 0.1 1 / [Substrate] 0 0 20 40 60 80 [Substrate]
Parameter Value Std. Error
Vmax 0.0503 0.0066 Km 19.0265 7.5304
Fig 3.67 Michaleous Menten Kinetics Data Obtained in Optimization of Hyaluroniadse Assay for Naja naja karachiensis venom.
176
Table 3.43 Medicinal Plants of Pakistan (0.1mg, 10 mg/ml) having Anti-hyaluronidase Enzymatic Activity Posed by Naja naja karachiensis (0.1mg, 10 mg/ml) Venom.
Name of evaluated material Absorbance Hyaluronidase P-Value (Mean ± SD) Inhibition (%) Naja naja karachiensis venom 0.170±0.002 0** P˂0.001 Saline (negative control) 0.370±0.003 100* P˃0.5 Calotropis procera (Aiton) W. T. Aiton 0.271±0.014 50** 0.01˃P˃0.001 (flowers) Cedrus deodara (Roxb. ex D. Don) G. Don 0.151±0.002 -9** P˂˂0.001 Momordica charantia L 0.308±0.004 69** 0.01˃P˃0.001 Psoralea corylifolia L 0.194±0.001 12** P˂0.001 Sapindus mukorossi Gaertn 0.216±0.008 23** 0.01˃P˃0.001 Albizia lebbeck (L.) Benth 0.096±0.006 -37** P˂˂0.001 Pistacia integerrima J. L. Stewart 0.029±0.001 -70** P˂˂0.001 Pinus roxburghii Sarg 0.224±0.001 27** P˂0.001 Rubia cordifolia L 0.167±0.001 -1** P˂˂0.001 Stenolobium stans (L.) Seem 0.236±0.036 33** 0.02˃P˃0.01 Enicostemma hyssopifolium (Willd.) I. Verd 0.285±0.019 58** 0.02˃P˃0.01 Bauhinia variegate L 0.007±0.001 -81** P˂˂0.001 Brassica nigra (L.) W. D. J. Koch 0.171±0.010 0.5** P˂0.001 Cuminum cyminum L 0.157±0.016 -6** 0.01˃P˃0.001 Matthiloa incana (L.) W. T. Aiton 0.095±0.024 -37** P˂0.001 Terminalia arjuna (Roxb. ex DC.) Wight & 0.324±0.002 77** 0.01˃P˃0.001 Arn Citrullus colocynthis (L.) Schrad 0.069±0.014 -50** P˂0.001 Allium cepa L 0.025±0.004 -72** P˂˂0.001 Trichodesma indicum (L.) Sm 0.364±0.044 97* P˃0.5 Zingiber officinale Roscoe 0.163±0.030 -3** 0.01˃P˃0.001
177 Fogonia cretica L 0.209±0.030 20** 0.02˃P˃0.01 Rhazya stricta Dcne 0.327±0.041 79** 0.5˃P˃0.1 Althaea officinalis L 0.218±0.002 24** P˂0.001 Leucas capitata Desf 0.295±0.058 63** 0.5˃P˃0.1 Nerium indicum Mill 0.064±0.052 -53** P˂0.01 Ocimum sanctum L 0.073±0.032 -48** 0.01˃P˃0.001 Allium sativum L 0.032±0.002 -69** P˂˂0.001 Calotropis procera (Aiton) W. T. Aiton 0.138±0.001 -16** P˂˂0.001 (exudates) Citrus limon (L). Burm. f 0.083±0.002 -43** P˂˂0.001 Rutin trihydrate (2.5 mM) 0.368±0.001 99*** Select to (Reference hyaluronidase inhibitor) compare Note: * represents values non-significantly different from reference. ** Represents values significantly different from reference. *** represents value selected to compare.
178 3.6 Identification of Specific Enzyme(s) Inhibitor(s)
3.6.1 Anti-PLA2 Activity of the Most Efficient Plant Extract Among all evaluated plants material Bauhinia variegata L extract was found maximally (100%, 76% and 67%) protective in neutralization of acidimetric, anti-coagulant and turbidimetric PLA2 enzymatic assays respectively (section: 3.5.1. for detail).
3.6.2 RP-HPLC Profile of Bauhinia variegata L Extract
Chromatographic separation of various components of Bauhinia variegata L extract was performed via newly developed method for RP-HPLC. Certainly methanolic extract of
Bauhinia variegata L was found to show anti-PLA2 activity attributed to the tannins (polyphenols) therefore excluding them from structure determination for this study as shown in fig 3.68 & 3.69.
Presence of tannins was confirmed visually via RP-HPLC chromatogram where they appeared as broad lump of unresolved peaks (broad spectrum of closely related molecule) between 25%- 65% gradient of mobile phase B at 8 min to 22 min retention time (Fig 3.68) . They were seemed to elute with more apolar mobile phase however they still be easily extracted with water.
3.6.3 Screening of Fractionated Extract of Bauhinia variegata L
Turbidimetric PLA2 was the only developed assay in microplate therefore subsequently performed in each well to localize the effect. The inhibitory effect on PLA2 enzyme was plotted against chromatographic retention time to acquire high-resolution biochromatogram as shown in fig 3.70.
Crude Bauhinia variegata L extract was proved 67% effective to abort PLA2 enzymes (Table 3.37) however fractionated samples in microplate wells were not found effective however 15% inhibition was recorded maximally. It was observed that inhibitory effect of the extract was lost during fractionation via RP-HPLC. It was therefore deduced that isolation and structure elucidation of the inhibitor(s) was not possible due to the loss of anti-PLA2 activity after passing through RP-HPLC system.
179 3.6.4 Dose Response Relationship of Bauhinia variegata L Dose response relationship was evaluated which confirmed anti-phospholipase activity of the extract. IC50 value of the methanolic Bauhinia variegata L extract was found to be
0.652 mg/ml (mean of three replicates) as shown in fig 3.71. Moreover IC50 value of the specific metalloenzyme inhibitor (EDTA) was also determined (0.1513 mg/ml) as shown in fig 3.72 (as a mean of three replicates).
Fig 3.68 RP-HPLC Chromatogram of Ethanolic Bauhinia variegata L Extract (280 nm) with Detail Change in Percentage Gradient of Buffer B.
180
Fig 3.69 General representation of RP-HPLC Chromatogram of Bauhinia variegata L Extract.
181
Fig 3.70 High Resolution Biochromatogram of Bauhinia variegata L Extract.
182
150
100
50 Inhibition(%) -2.0 -1.5 -1.0 -0.5 0.5
-50
Log concentration (mg/ml)
Fig 3.71 Dose Response Curve for Bauhinia variegata L in the Turbidimetric PLA2 Assay.
183
150
100
50 Inhibition(%)
-3 -2 -1 1
-50 Log concentration (mg/ml)
Fig 3.72 Dose Response Curve for EDTA in the Turbidimetric PLA2 Assay.
184
Chapter 4
Discussion
185 In this chapter results that were obtained from laboratory work will be discussed.
4.1 Proteomic Characterization of Naja naja karachiensis Venom
Simple proteomic approach has clearly elucidated proteome picture of snakes’ venom which is crucial for depth insights about envenomation. Protein content for Naja naja karachiensis venom was 94% as high enough as documented previously by for colubrid venoms (Hill and Mackessy, 1997). Generally 70% to 90% protein contents were reported for snake’s venom however significant amount in Naja naja karachiensis venom represented a goldmine of proteins. Snake venom was investigated primarily via universally recognized, rapid and cost effective SDS-PAGE technique (Vejayan et al., 2010). Low molecular weight proteinous bands (3FTX) were clearly visible with a polyacrylamide gel (stained with CBB) while bands of high molecular weight (˃95 KDa) were not obvious. However problem was alleviated with silver stain detected proteins down to 1 ng (Wilson and Walker, 2005). Proteinous bands were lower at reduced state argued about complexes of protein(s) present in this venom. Aberrant profiles of venom (at reduced and non-reduced states) strongly recommended disulfide linkages (hetero or homo dimeric/trimeric complexes or absence of single polypeptide) possibly due to
PLA2, 3FTXs or SVMPs (P-IId, P-IIe and P-IIId) interactions. Appearance of bands at reduced condition (9 KDa to 12 KDa) further suggested about proteins in oligomeric form as reported previously for other venoms. (Zelanis et al., 2010; Rusmili et al., 2014). Different isomeric forms of a protein and tendency to form multimeric complexes with other toxins might be one of the reasons for different profiles of venom at different states (Malih et al., 2014; Zelanis et al., 2010).
Crude venom and its SEC separated RP-HPLC fractions were fragmented by shotgun tryptic digestion followed by LC-MS/MS analysis. Detailed study revealed that majority of the RP-HPLC fractions were heterogeneous due to structural isomers and closely related molecular sizes and charge of proteins (Li et al., 2004; Malih et al., 2014). Consistent with earlier reports from genus Naja, venom from Naja naja karachiensis contained 3FTX, PLA2, SVMP, CRISP, Kunitz, LAAO , CVF and vNGF (Ali et al., 2013). Nevertheless it additionally possessed 5ʹ-ND, vespryn, cobra serum albumin and phosphodiesterase. Literature review revealed that few Asiatic cobras have been reported
186 previously for complete identification of their toxic proteins such as 124 for Naja naja atra, 61 for Naja kaouthia and 28 for Pakistani Naja naja (Li et al., 2004; Malih et al., 2014; Ali et al., 2013; Yap et al., 2014). In this study for the first time venom from Naja naja karahiensis was evaluated and 43 toxic proteins were identified from its various trypsin digested (SEC separated RP-HPLC) fractions.
Among various toxic proteins 3FTXs were abundant (58%) as reported earlier for Pakistani Naja naja and Naja haje venom (Malih et al., 2014; Ali et al., 2013). 3FTXs are known to induce cardiotoxicity, neurotoxicity and cytotoxicity therefore cardiac damage attributed to Naja naja karachiensis bite is due to the rich pool of 3FTXs. CTXs (32%) were numerous among 3FTXs as reported previously for different cobra venoms like Naja katiensis, Naja mossambica, Naja nigricollis, Naja nubiae and Naja pallid (Ali et al., 2013). CTXs are main contributing factors toward necrosis however their toxic potentials vary from species to species. MTLPs (8%) were found as a unique component among 3FTXs. To the best of our knowledge it is the first report for the presence of MTLPs (MTLP-2 and MTLP-3) in Naja naja karachiensis venom. MTLPs are protein ligands interacted with muscarinic acetylcholine receptors (mAChRs) and mediated their intracellular response(s) via G-protein coupled receptors (GPCRs). mAChRs have been recognized earlier as potential drug targets for depression, drug addiction, Alzheimer’s disease, schizophrenia and Parkinsonism. Previously reported MTLPs were non toxic and showed less affinity towards acetylcholine receptors (AChE) however varieties of conventional ligands available now a days are non-specific in nature (Roy et al., 2010; Malih et al., 2014; Roy et al., 2011; Ali et al., 2013). In this study sequence alignment of novel haditoxin (dimeric SNTX for neuronal and homodimeric 3FTX for muscular nAChRs) with MTLP-3 further revealved the significance of later as 78% amino acids were in homology (Fig 3.47). Nevertheless remaining 3FTXs (LNTX, WNTX, SNTX and post synaptic NTX) were usual as mentioned in table 3.18.
Phospholipase A2 was found the second largest (19%) component of Naja naja karahiensis venom. Taken together both of the families (3FTX & PLA2) account for ~80% proteins for this venom. Multimeric complex formation with other toxins is an intrinsic aptitude of this enzyme and further contributes in the pathogenesis of cobra
187 ++ venom (Asad et al., 2014(d); Malih et al., 2014). Zn depended metalloproteinases (ZMP) and hemorrhagic metalloproteinase (HMP) together constituted another 5% of total venom proteins (SVMPs). Pathogenic role of SVMPs is quite convincing in viperidae or colibridae envenomation while it is debatable for elapids (cobras) envenomation. According to literature survey Naja naja karchiensis venom is little hemorrhagic in nature therefore HMP is attributed for its hemorrhagic lesion (Razi et al., 2011; Malih et al., 2014). LAAO was another enzyme identified in Naja naja karchiensis venom. It showed 5% of total venom proteins in several isoformic forms (Table 16 & 17). It is involved in prey immobilization and to produce hypotension (Malih et al., 2014).
5'-ND was another group of proteins detected in this venom and was found to constitute 2% of total proteins. This enzyme has been reported previously from different cobra species such as Indian Naja naja, Naja kauthia, Naja melanoleuca and Naja naja sputatrix. They are known to cause anticoagulant effect through the liberation of multitoxin adenosine (Dhananjaya et al., 2010). Cysteine rich secretory proteins (CRISPs) / helvepryns were found to constitute second largest (3%) non enzymatic protein family. LC-MS/MS detected several peptides with homology with CRISPs found in elapid (Naja atra) species. Toxicological role of CRISPs is not clearly known however natrin an important member of CRISP family is known to block BKCa channels (Malih et al., 2014; Yap et al., 2014). Vespryn (2%) constituted another non enzymatic family for this venom. LC-MS/MS detected peptides with homology with thaicobrin from Naja kaouthia venom. Vespryn found in two isomeric forms: ohanin like proteins (Lachesis muta); thaicobrin (Naja kaouthia). Ohanin is a vespryn isolated from Ophiophagus hannah venom and known to produce hyperalgesia and hypolocomotion in mice (Yap et al., 2014; Malih et al., 2014). CVF (2%) was another non enzymatic group of proteins found in Naja naja karachiensis venom. It is a mutated form of C3 and plays an important role as an activator of complement proteins. It promotes local inflammation and tissue damages after cobra venom envenomation (Ali et al., 2013; Malih et al., 2014; Yap et al., 2014). vNGF was found to constitute 2% of total proteins of this venom. It is ubiquitously present in all snake venoms. Snake venom NGFs contribute sequence homology with
188 mammalian’s nerve growth factors and are related to the normal functioning of neurons (Yap et al., 2014; Malih et al., 2014). Kunitz type serine protease inhibitors constituted 2% of total proteins found in Naja naja karachiensis venom. LC-MS/MS detected its peptides with homology with kunitz type inhibitors of elapids venoms. It is predominantly reported from Asiatic cobras and from African Naja nivea venom. Its role in snake bite envenomation is still unclear (Malih et al., 2014). Cobra serum albumin (monocled cobra matched peptides) and phosphodiesterase (Ophiophagus hannah transcriptomic database matched peptides) were detected in crude venom while remained unidentified by chromatographic approach (Table 3.16). Cobra serum albumin is known to show anti-toxin property while phosphodiesterase plays a pivotal role in signal transduction and to degrade polynucleotide chains (Yap et al., 2014; Mamillapalli et al., 1998). A general summary of proteomes of Naja naja karachiensis venom is discussed in table 3.16. On a more practical level, proteomics study about Naja naja karachiensis venom will be helpful to the clinician in manifestation of an intriguing mechanism of envenomation. Currently there is no single anti-venom available in Pakistan created against its own species. In future it will have an impact on effective production of anti-venom against Pakistani snakes species as dire need of it has been felt since long period of time.
4.2 Elemental Analysis of Naja naja karachiensis Venom
ICP-OES is one of the mostly recognized techniques to evaluate quantitatively various elements present in different biological samples particularly snake venoms. Certified reference material (apple leaf) was found suitable in agreement with certified reference values due to lower lipid, starch and protein contents and smaller particle size (~75 µm). Collectively these features led to attain good efficiency of the whole developed digestion procedure. Moreover it was also due to appropriate final dilution volume which enabled the sample(s) to aspirate directly and fitted well into standard auto samplers (Behera et al., 2010; Hansen et al., 2009).
To neutralize charged protein molecules (electrostatically), Naja naja karachiensis venom was found a mixture of inorganic components as reported earlier for Viperdae,
189 Elapidae and Crotalidae venoms (Friederich and Tu, 1971). Sodium ion (4519±2 µg/g) was detected in largest amount while potassium (2013±5.5 µg/g) was recorded the second most abundant monovalent cation (Table 18). According to literature survey the highest quantity of sodium ions were detected from earlier reported venoms such as, V. russelli siamensis (34100 µg/g), C. horridus atricaudatus (49900 µg/g), N. naja atra (43600 µg/g), C. horridus horridus (53000 µg/g), N. naja (60200 µg/g), N. naja (60200 µg/g), C. atrox (57300 µg/g) and B. arietans (41500 µg/g) (Anthony, 1983; Friederich and Tu, 1971). Monovalent potassium was observed in high amount particularly when compared with previously reported B. fasciatus (391 µg/g), C. adamenteus (750 µg/g), C. horridus atricaudatus (350 µg/g), C. atrox (410 µg/g), A. acutus (1070 µg/g), B. gabonica (220 µg/g), V. russelli siamensis (760 µg/g), C. basciliscus (670 µg/g), ), N. naja (150 µg/g), C. horridus horridus (420 µg/g), B. arietans (500 µg/g), C. viridis viridis (710 µg/g), N. naja atra (300 µg/g) and C. durissus totonacus (590 µg/g) snake venoms. Toxic role of divalent cations is prominent as a cofactor (activation or deactivation of enzymes) while monovalent cations didn’t play any poisonous role in snake venom (Friederich and Tu, 1971; Anthony, 1983). Among divalent cations copper, manganese, calcium, magnesium and zinc were quantified (in ascending order) from Naja naja karachiensis venom.
Zinc was found the most abundant divalent cation (3473±28 µg/g) as documented earlier from lot of venoms. Among those S. milarius barbouri (2010 µg/g), C. horridus horridus (980 µg/g), A. acutus (1200 µg/g), C. viridis viridis (1847 µg/g), N. naja atra (380 µg/g), C. durissus (1203 µg/g), B. gabonica (690 µg/g), B. arietans (1000 µg/g), C. durissus terrificus (1856 µg/g), V. russelli siamensis (1800 µg/g), C. atrox (1394 µg/g), C. horridus atricaudatus (680 µg/g), C. adamenteus (773 µg/g), B. fasciatus (196 µg/g), C. durissus totonacus (840 µg/g), N. naja (1600 µg/g) and C. basciliscus (1400 µg/g) were included (Friederich and Tu, 1971; Anthony, 1983). Zinc has been known to activate multicatalytic NADase (AA-NADase from Agkistrodon acutus), protease (acutolysin D from Agkistrodon acutus) and PDE however it deactivated vNGF, PLA2, clavata (spider insecticidal compound) and 5’-ND enzyme (Xu et al., 2006; Xu et al., 2010; Yoshioka et al., 1994; Anthony, 1983). All of these reports clearly indicated significant role of zinc after cobra bite envenomation.
190 The second largest divalent cation in Naja naja karachiensis venom was found to be magnesium ion (3047±31 µg/g). It is present in large amount when compared with the previously documented B. fasciatus (810 µg/g), A. acutus (450 µg/g), C. durissus totonacus (117 µg/g), N. naja atra (650 µg/g), C. viridis viridis (240 µg/g), N. naja (840 µg/g), C. basciliscus (376 µg/g), C. durissus (1470 µg/g), C. atrox (701 µg/g), V. russelli siamensis (976 µg/g), C. durissus terrificus (342 µg/g), B. arietans (700 µg/g), C. adamenteus (107 µg/g), S. milarius barbouri (446 µg/g), C. horridus atricaudatus (129 µg/g), B. gabonica (636 µg/g) and C. horridus horridus (973 µg/g) venoms (Anthony, 1983; Friederich and Tu, 1971). Magnesium ion has been proved to deactivate insecticidal clavata compound however it helps in substantial binding (factor Xa and PDE) and activation of snake venom enzymes (5’-ND, proteolytic acutolysin D, LAAO and PLA2) (Yoshioka et al., 1994; Shen et al., 2011; Anthony, 1983; Xu et al., 2006).
The third largest divalent cation in Naja naja karachiensis venom was found to be calcium ion (1442±19 µg/g). It is present in greater amount when compared with the previously reported C. horridus atricaudatus (150 µg/g), N. naja atra (1000 µg/g) and N. naja (1000 µg/g) venoms. On the other hand it was found in small amount when compared with venoms obtained from C. horridus horridus (4930 µg/g), S. milarius barbouri (4000 µg/g), C. atrox (4196 µg/g), C. adamenteus (1610 µg/g), V. russelli siamensis (1987 µg/g), B. gabonica (2900 µg/g), C. basciliscus (1989 µg/g), B. arietans (2306 µg/g), B. fasciatus (1620 µg/g), A. acutus (3000 µg/g), C. durissus (3003 µg/g), C. durissus totonacus (1633 µg/g), C. viridis viridis (4560 µg/g) and C. durissus terrificus (2390 µg/g) (Friederich and Tu, 1971; Anthony, 1983). Calcium was found essential for proteolytic acutolysin D, PLA2, PDE and insecticidal (clavata) activity however for structural stability and binding of factor Xa with ACF II it is still considered valuable. At the dose of 10 mM it was recorded to worsen (99.5%) hemolytic activity posed by Acanthaster planci spines. However, it was found to deactivate LAAO activity in literature. (Peng et al., 2013; Shen et al., 2011; Anthony, 1983; Xu et al., 2006; Yoshioka et al., 1994; Lee et al., 2013).
The fourth most abundant divalent cation was found to be manganese (6.5±0.65 µg/g) ion. It was present in much smaller amount when compared with the previously reported
191 cobra venoms such as N. naja atra (13µg/g) and N. naja (200 µg/g) (Friederich and Tu, 1971; Anthony, 1983). According to the literature survey, it was proved to deactivate proteolytic activity of acutolysin D, however, it was found to aggravate enzymatic actions of NADase, PDE and AT(D)Pase (AA-NADase from Agkistrodon acutus) (Peng et al., 2013; Xu et al., 2010; Xu et al., 2006). Copper ion was found the least in concentration (0.6±0.09 µg/g) from Naja naja karachiensis venom. With few exceptions of Crotalidae such as, S. milarius barbouri (200 µg/g) and A. acutus (175 µg/g), it has not been previously reported from other species of snake venom (Anthony, 1983; Friederich and Tu, 1971). It was found to inhibit acutolysin D (caseinolytic), hemolytic and insecticidal (clavata) activity; however, it is reported in activation of AA-NADase and PDE enzymatic activities in literature (Yoshioka et al., 1994; Lee et al., 2013; Peng et al., 2013; Xu et al., 2010; Xu et al., 2006). The only non-metallic constituent in Naja naja karachiensis venom was found to be phosphorous (718±8.5 µg/g). It was first reported in 1968 by Devi in different species of snake venom. Due to the lack of physiological/pathophysiological functions it was not studied in detail previously. It is reported in snake venom due to the normal tissue degradation in snake venom glands (Bieber, 1979). Complete description about inorganic elements detected is summarized in table 3.18.
All these findings collectively deduced significant role of inorganic elements particularly zinc, magnesium, calcium, manganese and copper for activation or deactivation of different enzymes present in Naja naja karachiensis venom. Other elements such as molybdenum, bismuth, selenium, platinum, palladium, silver, gold and cobalt were not detected and reported up till now from any snake venom.
192 4.3 Biodistribution and Kinetic Study of Naja naja karachiensis Venom via Radio Tracer (99mTc) Binding Technique
In clinical assessment of snake bite cases pharmacokinetics (biodistribution and kinetics) of toxins have prime importance to discover the mysterious of this neglected disease. Radioisotope based compounds are very effective to perform pharmacokinetics experiments for clinical trials. Previously lots of techniques have been reported for labeling of different compounds particularly proteins such as indirect and direct labeling and chelate approach (Shirmardi et al., 2010). However direct labeling is the most feasible way due to ease in application and without need of modifications (blocking or deblocking) of functional groups. In direct labeling technique reducing agent is used to convert disulfide linkages into sulfahydril groups to tag with technetium-99m.
In this study for the first time Naja naja karachiensis venom was labeled with 99mTc (97.7%) as reported earlier for others such as Scorpaena plumier, Mesobuthus eupeus and Crotalus venom (Asad et al., 2015; Shirmardi et al., 2010; Pujatti et al., 2005). Attempts were made to attain optimum concentration of stannous chloride dihydrate (100 µg at pH 6) for utmost sustainable tagging of 99mTc to the snake venom proteins. Stability experiments (In vitro and In vivo) revealed that Naja naja karachiensis venom was stable with at least more than 94% binding led to extend our views to evaluate its pharmacokinetics parameters in laboratory rabbits (Murugesan, 1999). Additionally technetium labeled venom was found to possess hemolytic behavior as similar as unlabeled venom strongly deduced about radio labeling procedure didn’t mask (significantly) any toxicity of Naja naja karachiensis venom.
After intravenous dose injection it was revealed that venom was highest in urinary system (77% ID/g), strongly suggested its excretion through kidneys however sufficient amount in lungs (14.2%ID/g) and liver (4.3%ID/g) represented these places as metabolic sites (Asad et al., 2015). Heart and brain was the least saturated areas of the scarified rabbits. Heart is a pumping organ and transferred venom to the other tissues via blood might be one of the reasons for minute amount. In contrast negligible concentration in brain was fascinating. It might be due to some hydrophobic component(s) (as an impurity) in
193 venom or lesion of blood brain barrier (BBB) attributed to some component(s) present in this venom (Asad et al., 2015). Technetium labeled Naja naja karachiensis venom was monitored for its localization at various tissues via gamma scintigraphic approach (2 hours study with 15 minutes intervals).
At the beginning clearance of venom from blood was rapid and detected in urinary bladder after 5min however complete excretion was observed in 24h of envenomation. 99mTc labeled venom was also evaluated for its evenly distribution with right over left ratio (R/L). It was 1.08 (~1) clearly indicated equal distribution in the middle compartment of rabbits (heart, liver and lungs). Moreover single photon emission computed tomographic (SPECT) images at sagittal, transverse and coronal sections further clarified complete discrimination of venom distribution at various tissues from one another (Asad et al., 2015). For effective treatment of Naja naja karchiensis bite standard protocols (for serum) still not available therefore resulted in lack of pharmacokinetic data for this venom. In future this study can help to establish standard serum therapy protocols and to develop effect antidote against Pakistani cobra venoms.
4.4 Toxic Biological/Biochemical Activities of Naja Naja Karachiensis Venom and their Neutralization with Medicinal Plants of Pakistan
4.4.1 LD50 of Naja naja karachiensis Venom
Health hazards due to the toxic chemical substances have become a great concern to differnt communities in the word. It was therefore reasoned to introduce LD50 test (first time in 1927) to acquire biological profile of venemous chemical agents (Zbinden and Flury-Roversi, 1981). Since long period of time poisonous snakes venom have been recognized for high rate of morbidity and mortality. These reports led to investigate variety of venoms to access and compare toxic venom potentials via LD50 values. LD50 is widely recognized not only to compare different venoms but also to access potency of anti-sera for an effective anti-venom production (Oukkache et al., 2014; Asad et al.,
2014(e)). Literature review revealed that LD50 value is greatly influenced with: (1) method of extraction of venom; (2) method of collection of venom; (3) number of animal used for
194 LD50 determination; (4) route of administration of venom. Among various routes of injection intravenous (i.v), intramuscular (i.m), subcutaneous (sc), intraperitoneal (i.p) and intracerebroventricular (i.c.v) are widely acceptable however highest value is obtained with subcutaneous while intracerebroventricular route gives the lowest value (Oukkache et al., 2014).
In this study attention was focused for the first time to determine lethal toxic dose (LD50) of Pakistani cobra venom. LD50 of Naja naja karachiensis venom was found to be 2.0µg/g (2.0mg/kg) intraperitoneally. It was found much potent than Indian cobra i.e.,
Naja naja naja (LD50 = 2.8 mg/kg, i.p) while king cobra (Ophiophagus hannah) was revealed more potent (LD50 =1.644 mg/kg, i.p) when compared to Naja naja karachiensis venom (Bhat and Gowda, 1989; Spawls and Branch, 1995; www.avur.org). Pakistani cobra venom was seemed less potent when compared with other Naja species such as
Naja naja atra (LD50 = 0.62 mg/kg, i.p), Naja nigricollis (LD50 = 0.44 mg/kg, i.p), Naja haje (LD50 = 0.185 mg/kg, i.p), Naja kaouthia (LD50 = 0.225 mg/kg, i.p), Naja melanoleuca (LD50 = 0.324 mg/kg, i.p) and Naja nivea (LD50 = 0.4 mg/kg, i.p) (Liu et al., 2010; Abdou et al., 2015; Spawls and Branch, 1995; www.avur.org). However literature review revealed that potency of Naja naja karachiensis venom is comparable to the red spitting African cobra i.e., Naja pallida (LD50 = 2.0 mg/kg, i.p) in the world (Spawls and
Branch, 1995; www.avur.org). It is therefore the need of the time to estimate LD50 with remaining routes of administration to encompass complete glimpse of toxicity posed by Naja naja karachiensis venom.
4.4.2 Hemolytic Potential of Naja Naja Karachiensis Venom and its Neutralization with Medicinal Plants
One of the most devastating occupational perils is snake bite envenomation since it has been unnoticed by various medical and health organizations. Snake bite cases have been awfully noticed from different regions of the world in particular southern Asian countries like Pakistan. Among various complications (produced by cobra venom) hemolysis is the most common but still considered very far short to produce a severe lethality. According to literature survey cobra bites induced moderate hemolysis with high levels of serum bilirubin, urine urobilinogen and RBCs counts with decline in hemoglobin level (Asad et
195 al., 2014(e); Condrea, 1979). Certainly destruction of RBCs membrane resulted in hemolysis attributed to the presence of various toxic protein(s). Among them PLA2 has been reasoned predominantly for hemolysis as reported by Asad et al. (2014) e for Naja naja karachiensis venom (subspecies of Pakistani Naja naja). More precisely phospholipases A2 attached with HRBCs membrane by the formation of disulfide, covalent or non-covalent bonds. Subsequently intact phospholipids hydrolyzed and broken down into lysophospholipids and free fatty acids. All these changes posed a variant environment for target protein(s) and led to provoke an agonistic or antagonistic effect by interrupting the function(s) of physiologic ligands. Collectively these effect(s) paved the way for venom to induce hemolysis via destruction of HRBCs membranes. On the other hand hyposaline (positive control) induced stress to form transient resealing fissures in HRBCs which ultimately resulted in hemolysis (Asad et al., 2014(e)). Various protein binding compounds have been recognized previously to assuage lethal potentials of different toxins present in snake’s venom. Among these natural inhibitors particularly medicinal plants extract have been claimed since long period of time to neutralize such toxins (proteins/peptides). On these grounds when Pakistani medicinal plants extract were evaluated some of them (Cedrus deodara (Roxb. ex D. Don) G. Don, Enicostemma hyssopifolium (Willd.) I. Verd and Calotropis procera (Wild.) R.Br) proved useful to minimize and abolish toxin(s) induced hemolysis. However it was assumed that secondary metabolites (phenols, quinonoids, terpenoids, xanthenes and flavonoids) are mainly responsible for such results. Oxidative hemolysis posed by production of free radicals (superoxide or peroxide) after bimolecular degradation (HRBCs lyses) was additionally circumvented due to antioxidant potential of medicinal plants extract (Asad et al., 2011; Asad et al., 2014(e)). Therefore it is the need of time to isolate and characterize active constituent(s) from effective plants extract for complete treatment of snake bite in future.
196 4.4.3 Anticoagulant Activity of Naja naja karachiensis Venom and its Neutralization with Medicinal Plants Snake bite envenomation is one of the most alarming tropical and subtropical illness resulted in several awful complications. Among them coagulopathy is the most familiar serious issue reported in the victims of Naja naja karchiensis bite. Unfortunately complete cause of it is still unknown however often marked with prolonged coagulation time in clinics (Asad et al., 2012). According to literature survey prolonged coagulation tests reflected strong or weak anticoagulant aptitude of venom. Generally snake venoms obstruct blood coagulation pathway at three different stages: (I) extrinsic tenase complex;
(II) prothrombinase complex; (III) fibrin thread formation (Asad et al., 2014(c)). In order to diagnose the interruption posed by snake venom envenomation different coagulation tests (PT, aPTT or TT) have been introduced previously. PT is used to ascertain interference of venom at extrinsic tenase or prothrombinase complex while aPTT is used to determine disorder of factors VIII, IX, XI and XII (intrinsic prothrombin activators). Moreover TT is widely employed to pinpoint deficiency of fibrinogen level or its conversion to fibrin thread formation as shown in detail in fig 3.59 (Asad et al., 2012). Various toxins from snake’s venom were assumed to delay PT, aPTT or TT due to interference at blood coagulation cascade (Fig 3.59) by different ways (factor IX/X inhibition, protein C activation, direct thrombin inhibition and degradation of membrane phospholipids). According to literature review similar reports have been observed for other venoms such as Agkistrodon contortrix contortrix, Deinagkistrodon acutus, Trimeresurus flavoviridis and Echis carinatus leucogaster. American Agkistrodon species (Agkistrodon contortrix contortrix) was reported to produce anticoagulant effect by protein C activation while Trimeresurus flavoviridis, Deinagkistrodon acutus and Echis carinatus leucogaster were found to halt blood coagulation via factor IX/X binding inhibition (Asad et al., 2014(c)). Thrombin inhibitors directly attacked to the thrombin or its (α) subunit and posed a hindrance in binding with fibrinogen consequently provoked anticoagulant effect. Phospholipases enzymes found in Naja nigricollis, Naja. Naja, Naja melanoleuca, N. m. mossambica and Trimeresurus mucrosquamatus venoms degraded membrane phospholipids and block pothrombinase complex resulted in reverse coagulation. Similar
197 effects (prolonged PT, aPTT and TT) have been reported by Asad et al., (2012) about Naja naja karachiensis venom. For this venom In vitro prolongation of PT and TT is quite aberrant (usually not possible and only reported for Naja haje) attributed to the actions of Naja haje like phospholipases A2, fibrinogenolytic or plasminogen activating enzymes (Osipov et al., 2010; Asad et al., 2014(c)). Recent trends in natural products about toxin(s) neutralizing compounds have introduced a new arena for the effective, alternate and cheap treatment of snake bite. Medicinal plants have long history for their therapeutic potentials particularly to neutralize snakes venom owing to the presence of secondary metabolites such as flavonoids, polyphenols, quinonoids, terpenoids and xanthenes (Asad et al., 2011). On these grounds when medicinal plants of Pakistan were evaluated some of them (Stenolobium stans (L) D. Don and Enicostemma hyssopifolium (Willd.) Verdoorn) proved valuable as standard antidote. They produced maximum useful effect (at equimolar concentration) by posing a hindrance in binding of toxin(s) with their potential target(s) (Edwards, 1985). It is therefore the need of the time to further characterize these valuable plants extract for effective and complete eradication of coagulopathies in future.
4.4.4 Biochemical Parameters for Heart, Liver and Kidneys Damage and their Neutralization by Medicinal Plants
Inhabitants of tropical and subtropical regions are more susceptible to snake bite envenomation often end up in serious complications and amputation. Like other areas of the world snake (Naja naja karachiensis) bite cases have been abundantly reported from Pakistan and resulted in approximately 20, 000 deaths annually (Razi et al., 2011). Indeed snake venom is a complex mixture of various toxic proteins along with inorganic constituents. Among different toxic proteins phospholipase A2 is the most abundant and noxious component of Naja naja karachiensis venom predominately reported for cardiac, liver and kidney damage in the victims. Among all cardiac toxicities posed by Naja naja subspecies, systolic heart arrest is the most frequent complication. Consistent with earlier reports Naja naja karachiensis venom further provoked myocyte injury In vivo with the release of cystolic enzymes (CK-MB & LDH). Cobra venom 3FTXs (CTX) and
198 phospholipases enzymes are actually reasoned for induction of cytotoxicity (cardiac damage) and necrosis. Furthermore 3FTXs being the most abundant component of this venom reached to the microvasculature and produce thrombus led to halt the way of anti- sera towards site of action (Ali et al., 2013; Asad et al., 2014(d)).
Dose dependent elevation of ALT and AST further described hepatotoxic effect of cobra venom attributed to the immunological or its direct action. As a matter of fact immunological reactions are not dose dependent therefore indicated hepatic
(mitochondrial and cytoplasmic membrane) damage after snake bite envenomation. PLA2 enzymes have been claimed previously for hepatic cells death and inflammation by disorganization of membranous structure with the influx of water and Na+ ions (decrease + + in Na /K ATPase). Hydrolysis of membranous phospholipids (via PLA2) further fortified the concept of hepatic cells injury as noticed in snake bite cases. Furthermore anticoagulant aptitude of cobra venom (due to PLA2 and others) often resulted in liver damage idiosyncratically as reported in literature for other anticoagulants (Asad et al.,
2014(d)). Snake bite envenomation has been known to cause kidney damage as reported for Hemiscorpius lepturus and Naja naja subspecies. In the present study Naja naja karachiensis venom was found to increase in urea and creatinine level dose dependently.
Among various toxic proteins PLA2 enzyme has been proved for glomerular and tubular damage with significant increase in vascular permeability therefore resulted in lesions and hemorrhage. Furthermore odema and lymphocytes accumulation at cortical and modularly regions further supported the concept of renal damage in snake bite cases. Beside these reports Asad et al. (2015) evaluated that technetium-99m labeled Naja naja karachiensis venom was enormous in kidneys and urinary bladder after intravenous injection in rabbits (Asad et al., 2015; Asad et al., 2014(d)). All these findings strongly suggested detrimental effects of Pakistani cobra venom towards heart, liver and kidneys in the victims of snake bite.
Medicinal plants (natural inhibitors) have been used since long period of time to treat different poisons. On these bases twenty eight medicinal plants of Pakistan (having ethnobotanical claims as anti-venom) were evaluated for their potential to combat snake venom toxicity. Extract of Stenolobium stans (L.) Seem was found the best as reference
199 standard as reported beneficial against phospholipases induced anticoagulation (Asad et al., 2013; Asad et al., 2014(c)). Literature review revealed that anti-venom potentials of Stenolobium stans (L.) Seem owing to the presence of secondary metabolites (terpenoids, phenols, xanthenes, flavonoids and tannins). Anti-venom constituent(s) offers a hindrance in binding of toxic protein(s) to its potential target therefore produced anti-venom effect. Extract of Stenolobium stans (L.) Seem may be used as an alternate first aid treatment to minimize the diffusion of toxins before proper medical treatment (hospitalization). However it is the need of the time to further characterize this valuable plant extract for effective treatment of snake bite in future (Asad et al., 2011; Asad et al., 2014(d)) .
4.5 Various Enzymatic Assays for Naja naja karachiensis Venom and their Neutralization by Medicinal Plants of Pakistan
4.5.1 Enzymatic assays for phospholipase enzyme and its Neutralization
Snake bite envenomation is an awful occupational hazard often accounts for tens of thousands of deaths annually. Like other regions of the world South Asian countries particularly Pakistan bear much burden of it (20,000 deaths annually) often end up with severe complications and sequelae. Certainly deadly venomous proteins abundant in snake (Naja naja karachiensis) venom play a tremendous role in emergence of toxicities post envenomation. Among them PLA2 is the most copious enzyme and play a pivotal role in hemolysis, necrosis, coagulopathies along with severe damage to the liver, heart and kidneys (Razi et al., 2011; Asad et al., 2014(d)). Toxicities due to PLA2 enzyme produced by enzymatic (substrate binding mechanism) and non enzymatic actions. PLA2 is rich in both pharmacologic and catalytic sites (specific PLA2) or catalytic (non-specific
PLA2) site to recognize their target cells (phospholipids/glycoproteins). Specific regions of PLA2 (e.g., 54-77 anticoagulant residues/Asp49 variants) hydrolyzed membrane phospholipids and liberated free fatty acids and lysophospholipids led to produce toxic pharmacological effects. Moreover PLA2 has intrinsic ability to form complex with target protein(s) via disulfide, covalent or non covalent linkages. It caused delay in binding of physiological ligands with the target protein(s) therefore provoked agonistic or antagonistic effects. All these effects were the contributing factors in acidimetric,
200 turbidimetric and anticoagulant assays developed for PLA2 present in Naja naja karachiensis venom (Asad et al., 2014(b); Asad et al., 2013). According to literature survey different protein binding compounds have been recognized previously to minimize toxicities posed by cobra venom envenomation. Among them natural inhibitors (medicinal plants) have been widely used to treat snake bite cases. Pakistan is a hub of medicinal plants where most of the rural populations relay on the folklore remedies to cure snake bite due to the presence of secondary metabolites such as flavonoids, tannins, terpenoids, quinonoids and xanthenes (Asad et al., 2011). Present study revealed that pre- incubation of venom with different medicinal plants extract resulted in coating of secondary metabolites on PLA2. It caused obstacle in binding with target protein(s) therefore neutralized phospholipase actions (Asad et al., 2013). In this study for the first time folklore claims about medicinal plants of Pakistan as anti-venom (anti-PLA2) were evaluated via three different PLA2 assays. Nevertheless it is the need of the time to further characterize potential medicinal plant(s) extrat for effective and complete treatment of snake bite in future.
4.5.2 Enzymatic Assay for Alkaline Phosphatase (ALPase) and its Neutralization
Diverse species of snakes and spiders have been recognized for variety of enzymes particularly alkaline phosphatases (ALPases). They were reported in significant amount especially in the venom of elapids, viperids and crotalids. On this ground Naja naja karachiensis (being elapid) was studied for the first time to assure the presence of ALPase and to map out its toxicity post envenomation. Initially ALPases were engrossed less attention by toxinologists owing to their digestive and non toxic role. However recent studies revealed that ALPases hydrolyze phosphate esters (ribo and deoxy ribonucleotides) non-specifically therefore provoke deleterious effects (Dhananjaya and
D’Souza, 2010; Asad et al., 2014(a)).
Naja naja karcahiensis venom was found abundant in ALPase enzyme consequently produced dose dependent effect. Like other toxic protein ALPase was denatured permanently with heat (100 °C) attributed to the destruction of its locus. Among different evaluated plants sample Sapindus mukorossi Gaertn was declared the best antidote
201 (equally potent) comparable to the reference standard antidote (anti-sera). It was due to the snake venom which reacted as an antigen while anti-sera/plant sample behaved like an antibody. Most favorable results were obtained at equivalence point (when neither antibody/extract nor antigen in excess) and rendered maximum protection (93%) at equimolar concentration to the ALPase (Edwards, 1985; Asad et al., 2014(a)).
Each medicinal plant extract offered various protection (80%-93%) at most effective concentration (0.625µg-2.5µg) therefore indicated about their respective potencies. At high dose of an antidote (10mg/ml) maximum protection was not observed owing to the unachievable equivalence point. Furthermore high dose of a plant extract have intrinsic color which interrupted absorbance pattern consequently end up with erroneous results. All these factors might be possible explanation for effectiveness of different antidotes at low concentration. However on the other hand specific metalloenzyme inhibitor (EDTA) was found effective (anti-ALPase) due to the sequestration of various metal ion(s) indispensable for optimum enzymatic activity. Therapeutic relevance of secondary metabolites (quinonoids, flavonoids, xanthenes, phenols and terpenoids) has been recognized since long period of time to abolish toxic effects of snake’s venom (Asad et al., 2014(a); Edwards, 1985; Chakrabarty et al., 2000). Therefore anti-ALPase potential of Sapindus mukorossi Gaertn was owing to the secondary metabolites as reported earlier for various valuable anti-venom plants extract (Asad et al., 2011). It is therefore the need of the time to isolate and identify active component(s) of Sapindus mukorossi Gaertn extract for complete eradication of snake bite in future.
4.5.3 Enzymatic Assay for 5ʹ-Nucleotidase and its Neutralization
Since long time 5ʹ-nucleotidase (enzyme commission number 3.1.3.5) has been considered as a key component in different venoms (Vipera russellii, Agkistrodon piscivorus, Bungarus fasciatus, Vipera aspis, Trimeresurus graminus, Crotalus adamanteus and Naja naja) although little information is available about its toxic mechanism (Dhananjaya et al., 2011). In this study 5ʹ-nucleotidase was found to dephosphorylate adenosine-5-phosphate while it remained inert for other substrates (α- or β-glycerophosphate, adenosine-3-phosphate and monophenylphosphate) as reported previously by other researchers.
202 Naja naja karachiensis envenomation provoked hydrolytic enzymes to generate AMP/adenosine which subsequently resulted in cardiac arrest, inflammation, redness, cognitive impairment, renal damage, neurotransmitter disturbances and anti-platelet aggregation via different adenosine (A1 and A2) receptors. Indeed 5ʹ-nucleotidase acts synergistically with other enzymes particularly phospholipase, disintegrin and ADPase to produce marked anti-coagulant effect (coagulopathies) via A2A and A2B receptors stimulation (Dhananjaya et al., 2011; Asad et al., 2014(c)). All these aspects could be possible explanation about the toxicities posed by 5ʹ- nucleotidase enzyme present in Naja naja karachiensis venom (Asad et al., 2016; Dhananjaya et al., 2011).
Protein binding compounds particularly natural inhibitors (medicinal plants) have been accepted earlier to minimize toxicities posed by different cobra venom enzymes. Consistent with previous reports present study confirmed neutralization of 5ʹ- nucleotidase (pre-incubation of venom with different plants material) by the virtue of secondary metabolites (quinonoids, flavonoids, xanthenes, phenols and terpenoids). Secondary metabolites posed a hindrance in binding of target protein(s) with receptor(s) therefore masked enzymatic activity. Anti-5ʹ-nucleotidase activities of Citrus limon (L.) Burm. f and Bauhinia variegata L was due to the secondary metabolites as reported earlier for different plants extract (Asad et al., 2011; Asad et al., 2013). It was therefore concluded that extract of Citrus limon (L.) Burm. f and Bauhinia variegata L have intrinsic ability to nullify 5ʹ-ND activity might be effective as an alternate first aid therapy for snake bite patients. However it is inevitable to further characterize these valuable plants extract for complete and effective treatment of snake bite cases in future.
4.5.4 Enzymatic Assay for Hyaluronidase and its Neutralization
Tropical and subtropical areas of the world are more prone to snake bite envenomation. It has been estimated that more than 20,000 snake bite cases endup with painful deaths annually (Yap et al., 2011). Venomous enzymatic proteins particularly PLA2, LAAO, PDE, 5ʹ-ND, ALPase and hyaluronidase are mainly responsible for such fierceful deaths. Venom hyaluronidase commonly known as ‘spreading factor’ is one of the key enzymes attributed for degradation of extracellular matrix (ECM) in the victims. It increases the potency of different toxins and furthermore causes local tissue damage (necrosis) and
203 inflammation via cleavage of internal glycosidic bonds of hyaluronic acid, which acts as a substrate (Kreil, 1995).
Literature review depicted that less attension was given previously to the hyaluronidase enzyme (due to its digestive role) however, recent studies revealved its important role in the diffusion of various toxin(s) via degradation of hyaluronic acid in the ECM. It is therefore essential to inhibit systemic distribution of venom and to curtail local tissue damage post envenomation (Kemparaju and Girish, 2006). Hyaluronidase enzymatic activity has been reported earlier from various Southeast Asian cobras such as Naja naja, Naja sumatrana, Naja sputatrix, Naja siamensis and Naja kaouthia (Gopi et al., 2014; Yap et al., 2011). On these bases, Naja naja karachiensis venom for the first time was subjected to evaluate its hyaluronidase activity. A simple turbidity method was adopted as turbidity is directly proportional to the amount of hyaluronic acid in the reaction mixture (Song et al., 2009).
Conventional ways of treatment are very popular among the people to mitigate health related issues particularly snake bite in Pakistan. It is therefore essential to rationalize medicinal plants extract for their anti-venom effect towards toxicities posed by different snake venom enzymes (Asad et al., 2011). In this study for the first time medicinal plants extract were evaluated to combat hyaluroniadse activity measured in Naja naja karachiensis venom. Among twenty eight evaluated extracts only Trichodesma indicum (L.) Sm was found equally potent as Rutin trihydrate (reference standard) attributed to the presence of active metabolite(s). Medicinal plants are the rich source of various secondary metabolites, such as tannins, quinonoids, flavonoids, xanthenes, phenols and terpenoids that have been documented previously to diminish snake bite effects (Asad et al., 2014(d)). Anti-hyluronidase activity of Trichodesma indicum (L.) Sm might be due to the presence of these secondary metabolites. However, it is crucial to characterize further potential medicinal plant extract for complete and effective treatment of snake bite in future.
204 4.6 Identification of Specific Enzyme(s) Inhibitor(s) from Bauhinia variegata L Extract against Turbidimetrimic Phospholipase Activity
HPLC coupled bioassay technique is a time efficient method which segregates and collects all components of an extract to perform bioactivity in vitro. It helps in identification of bioactive chromatographic peak(s) and facilitates throughput. Present assay was performed with strict time slicing and fraction collection for reproducible separation and retention of components without splitting into multiple wells. Less than 300 µl volume was collected in 20 sec in each well for efficient mobile phase volume and peak splitting. Efficient speedvac was used to force down the components to the bottom of the well (during evaporation) however reconstitution volume was crucial for proper response of the bioassay. Plate vortexing method (1 h period) was adopted for improved inhibition and to acquire stable baseline response (Nielsen, 2013).
HPLC coupled bioassay technique revealed that selected crude Bauhinia variegata L extract was effective (anti-PLA2) due to the presence of secondary metabolites particularly tannins. Tannins are usually poly-phenolic chromophores of different size and ultimately form insoluble compounds with snake venom protein(s) non-specifically (Toft, 2012). Since tannins chromophores of different polarity are present in Bauhinia variegata L extract (visually assessed) therefore reasoned for broad merge of overlapping peaks as reported previously for other plants extract (Lannea acida A. Rich, Parkia biglobosa (Jacq.) G. Don, Ximenia americana L, Cyperus textilis Thunb, Aristolochia trilobata L, Cyperus ligularis L and Cyperus ustulatus A. Rich) in literature (Nielsen, 2013; Toft, 2012). Tannins have been reported previously in literature abundant in Bauhinia variegata L methonolic extract ranges from 15-102 µg/mg of tannic acid equivalents (Negi et al., 2012). Due to these findings Bauhinia variegata L methanolic extract was not further investigated because our primary aim was to highlight specific
PLA2 inhibitor(s). Furthermore there was always a risk of possibility of specific PLA2 inhibitor(s) besides the suspected tannins which would certainly require further investigations to validate this claim.
205 RP-HPLC fractionation was resulted in loss of PLA2 activity in separated wells might be due to the active constituent(s) degradation (by the developed HPLC procedure) or their retention on RP- HPLC column. It was also a possibility that active compound is highly polar and washed out with solvent front therefore was not collected in any well. Anti-
PLA2 activity of Bauhinia variegata L extract might be represented by the last signal of HPLC chromatogram which was not included due to the limitations in microplate capacity (presumed highly non-polar compound(s) in the evaluated extract). The most promising illustration about significant loss in anti-PLA2 activity is synergy among different compounds. Crude extract was active due to synergistic effect of various compounds abundant in it however fractionation resulted in loss of anti-PLA2 activity (Toft, 2012).
In spite of loss of maximum response with fractionation Bauhinia variegata L extract showed an expected dose response relationship. The term IC50 (half maximal inhibitory concentration) is used to estimate antagonist (plant extract) potency by examining its effect (enzyme inhibition) with various concentration in neutralization of agonist activity
(enzyme activity). IC50 value of the crude methanolic Bauhinia variegata L extract was found to be 0.652 mg/ml which indicates whether the response is partial or full, competitive, non- competitive or un-competitive. IC50 value depends on several factors (amount of substrate & inhibitor, time and temperature) in acquisition of an optimum response certainly if specific inhibitor is isolated from an extract (Toft, 2012).
On the whole present study enunciated that isolation and structure elucidation of an inhibitor was not possible due to the loss of effect of Bauhinia variegata L extract after passing through RP- HPLC system. It would be more realistic to re-evaluate the system to highlight the cause for loss in anti-PLA2 activity.
206 Conclusion
The primary aim of this study was to identify various toxic (protenous & non-protenous) components of Naja naja karachiensis venom and to determine its LD50. To map out complete picture of envenomation, biodistribution (maximum profusion in various envenomed tissues), kinetic, toxic biological (hemolytic & anticoagulant) and biochemical (AST, ALT, CK-MB, LDH, urea & creatinine) parameters were evaluated.
Pakistani cobra venom was also assessd for its PLA2, ALP, 5ʹ-ND, hyaluronidase and protease enzymatic activities in vitro. However, their deleterious effects were endeavored to neutralize with twenty eight folklorie claimed methanolic plants extract. Finally efforts were made to characterize specific PLA2 enzyme inhibitor(s) from the most effective anti-PLA2 (Bauhinia variegate L) extract.
Proteomic characterization of Naja naja karchiensis venom was carried out by adopting two strategies: (1) SDS-PAGE; (2) SEC, RP-HPLC and LC-MS/MS. Among different toxic proteins 3FTXs (CTX, WNTX, LNTX, SNTX, MTLP and post synaptic-NTX),
PLA2, SVMP, HMP, LAAO, CVF, 5ʹ-ND, vNGF, helvepryn, vespryn, and kunitz type serine protease inhibitor were identified and sequenced. PLA2 is the most abundant proteinous enzyme while MTLP is a unique novel 3FTX present in this venom (MTLP-3 amino acids sequence is 78% analogus to novel Haditoxin). However cobra serum albumin, PDE and hyaluronidase were not identified from any RP-HPLC fractions. Total protein cconcentration was found to be 188 ± 0.011 µg per 200 µg of dry venom that constituted 94% of total venomous protein. Among non-proteinous (inorganic) components both metals and non-metals are present. ICP-OES technique was used to quantify these elements. Among them sodium, potassium, zinc, magnesium, calcium, manganese, copper were quantified. Phosphorous was the only observed non-metallic component while selenium, platinum, bismuth, gold, palladium, silver, molybdenum, iron and cobalt were not identified in this venom.
To observe biodistribution and kinetic, venom was labled (97.7%) for the first time with radio active isotope 99mTc. In vivo and in vitro experiments revealed that radio labeled venom were 96% and 94% stable respectively after 4h of injection/incubation.
207 Furthermore, it was proved that direct labeling technique didn’t alter any toxic biological property of this venom. Rapid elimination was observed after an initial hour of intravenous dose however it slows down subsequently and apparently completed within 24h of envenomation. After 3h of i.v dose highest activity was reported in urinay system followed by lungs and liver while the least fraction was observed in brain. Venom distribution in the middle compartment of the rabbit was almost equal however, SPECT images were additionally acquired to encompass comple picture of biodistribution.
LD50 for Naja naja karachiensis venom was found to be 2.0 µg/g or 2.0 mg/kg intraperitonially in mice. In vitro experiments revealved that Pakistani cobra venom is hemolytic and anti-coagulant in nature. Cedrus deodara (Roxb. ex D. Don) G. Don was proved to pose maximum protection against venom induced hemolysis while Enicostemma hyssopifolium (Willd.) I. Verd and Stenolobium stans (L.) Seem were declared the best antidote to normalize prolonged PT, aPTT & TT tests. Cobra venom was found extremely venomous in terms of significant release of surrogate markers for liver (AST, ALT), heart (CK-MB, LDH) and kidneys (urea, creatinine) damage. The only medicinal plant extract proved to reverse these toxicities was Stenolobium stans (L.)
Seem. Among different enzymatic assays PLA2 activity was accessed via acidimetric, turbidimetric and anticoagulant ways. Against acidimetric PLA2 activity Ocimum sanctum L, Bauhinia variegate L, Enicostemma hyssopifolium (Willd.) I. Verd, Citrus limon (L). Burm. f, Psoralea corylifolia L and Stenolobium stans (L.) Seem were seemed beneficial. However, to neutralize turbidimetric PLA2 activity only Bauhinia variegate L was found the best. To minimize PLA2 anticoagulant affects Rubia cordifolia L and Citrullus colocynthis (L.) Schrad was declared valuable however, Bauhinia variegate L was found 76% effective. Extract of Bauhinia variegate L was also found excellent against deleterious activity posed by 5ʹ-ND enzyme. Furthermore, Sapindus mukorossi Gaertn and Trichodesma indicum (L.) Sm extracts were noticed to abort toxic ALPase and hyaluronidase activity respectively. Regarding proteolytic activity, high dose (20 mg/ml) of venom was found to induce a minor response in turbidity change; three times SD left an operational window of 0.006. This window was too narrow for validation of this assay therefore excluded.
208 Against each PLA2 biochemical assay, Bauhinia variegate L extract (IC50 = 0.652 mg/ml) was found excellent and subjected for detailed characterization of bioactive constituent(s). HPLC coupled bioassay fractionation of Bauhinia variegata L extract was resulted in loss of PLA2 activity and showed anti-PLA2 effect mainly due to the tannins (secondary metabolite). Tannins chromophores of different polarity are present in Bauhinia variegata L extract therefore reasoned for broad merge of overlapping peaks (as a large hump). Tannins are poly-phenolic chromophores of different sizes and form insoluble compounds with snake venom protein(s) thus impart anti-PLA2 activity.
.
209 Future Perspective
Further studies are relevant to expand upon the finding of this thesis as a consequence of the limited conclusiveness of the results presented. These include:
Further study to isolate and map out complete sequence of newly identified novel toxin (MTLP-3) via Edman degradation (N-terminal sequencing) and related techniques.
Complete characterization of novel PLA2 responsible for concurrent prolongation of PT and TT tests in vitro. Futher studies to detect and characterize hyaluronidase enzyme since it was not being detected in this venom (insufficient database depository). Further studies using for example MS and NMR systems to confirm the presence
of tannins in the extract of Bauhinia variegata L and to investigate specific PLA2 inhibitor(s) in this extract. Validation of the HPLC system is necessary to explain the loss of effect of the methanolic extract of Bauhinia variegata L and to find out better applicable
system. This might lead to the identification and characterization of specific PLA2 inhibitor(s). Future studies to characterize active constituent(s) from the extracts displaying significant activity in this study against venom induced hemolysis, anticoagulation, alkaline phosphatase, hyaluronidase, 5ʹ-nucleotidase and elevated biochemical parameters for cardiac, renal and hepatic damage. If in vitro results are promising, in vivo tests, toxicology and safty tests could be considered, in order to develop a standardized extract(s) that could be used by those coexisting with Naja naja karachiensis.
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