|Year : 2020 | Volume
| Issue : 69 | Page : 416-421
Antiprotease activity of indigenous medicinal plants against Pakistani Echis carinatus venom
Nazia Aslam1, Tariq Javed2, Sofia Khalid1, Nusrat Shaheen3, Syeda Fatima1, Muhammad Latif4, Khurram Afzal5, Samina Afzal6, Khizar Abbas6, Muhammad Arfat Yameen7, Yasser M. S A. Al-Kharaman7, Muhammad Imran Amirzada7, Ryan J R. McCleary8, Albert A Rizvanov9, Muhammad Hassham Hassan Bin Asad10
1 Department of Environmental Sciences, Fatima Jinnah Women University, Rawalpindi, Pakistan
2 Department of Pharmacy, LMDC, University of Health Sciences, Lahore, Pakistan
3 Department of Chemistry, Abbottabad University of Science and Technology, Abbottabad, Pakistan
4 Department of Zoology, Division of Science and Technology, University of Education, Multan Campus, Pakistan
5 Institute of Food Sciences, Bahauddin Zakariya University, Multan, Pakistan
6 Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan
7 Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Pakistan
8 Department of Biology, 421 N, Woodland Blvd, Stetson University, DeLand, Florida, United States
9 Department of Genetics, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia
10 Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Pakistan; Department of Genetics, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia
|Date of Submission||10-Aug-2019|
|Date of Decision||10-Oct-2019|
|Date of Acceptance||09-Jan-2020|
|Date of Web Publication||15-Jun-2020|
Muhammad Hassham Hassan Bin Asad
Department of Genetics, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan; Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Abbottabad
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Snakebite is a commonly neglected tropical disease globally. According to the World Health Organization, tens of thousands of deaths have been reported due to the venomous snakes previously. Among such snakes, vipers of the genus Echis are extremely important clinically. In folk medicine, plants are commonly used for the treatment of snakebites even though snake venom antiserum is the only efficacious treatment currently known. Objectives: This study was conducted to investigate the inhibitory potential of extracts from Pakistani medicinal plants against protease enzyme abundant in Echis carinatus venom. Materials and Methods: Organic extracts from different indigenous plant species and parts were used for in vitro determination of their inhibition against protease activity in snake venom. Active methanolic extracts were further fractioned using different solvents, and these fractions were also tested for antiprotease activity. Results: Results of this study show that Calotropis procera (Wild.) R. Br., Matthiola incana (L.) R. Br., and Terminalia arjuna Wight and Arn were able to neutralize the protease enzyme by 63%, 71%, and 66%, respectively. Trichodesma indicum (L.) R. Br. showed 51% inhibition of protease activity. Conclusion: The present study indicated about C. procera (Wild.) R. Br., M. incana (L.) R. Br., and T. arjuna Wight and Arn possessed inhibitor (s) against protease enzyme present in E. carinatus venom and would be worthwhile for development as treatment against envenomation in future.
Keywords: Calotropis procera , Echis carinatus , Matthiola incana , natural remedy, Pakistani, Terminalia arjuna
|How to cite this article:|
Aslam N, Javed T, Khalid S, Shaheen N, Fatima S, Latif M, Afzal K, Afzal S, Abbas K, Yameen MA, A. Al-Kharaman YM, Amirzada MI, R. McCleary RJ, Rizvanov AA, Bin Asad MH. Antiprotease activity of indigenous medicinal plants against Pakistani Echis carinatus venom. Phcog Mag 2020;16:416-21
|How to cite this URL:|
Aslam N, Javed T, Khalid S, Shaheen N, Fatima S, Latif M, Afzal K, Afzal S, Abbas K, Yameen MA, A. Al-Kharaman YM, Amirzada MI, R. McCleary RJ, Rizvanov AA, Bin Asad MH. Antiprotease activity of indigenous medicinal plants against Pakistani Echis carinatus venom. Phcog Mag [serial online] 2020 [cited 2020 Jul 7];16:416-21. Available from: http://www.phcog.com/text.asp?2020/16/69/416/286747
- Snake envenomation is a common yet neglected problem worldwide including Pakistan. Medicinal plants possess compounds that act differently (either individually or synergistically) to overcome snake venom potency by inhibiting its distribution and reducing physiological reactions, thus leading to complete neutralization of venom toxicity. The purpose of this study was to evaluate the inhibitory potential of selected medicinal plants against the protease enzyme of Echis carinatus venom. Results indicate that Calotropis procera (Wild.) R. Br., Matthiola incana (L.) R. Br., and Terminalia arjuna Wight and Arn have the ability to neutralize protease enzyme.
Abbreviations used: E. carinatus : Echis carinatus ; EDTA: Ethylenediaminetetraacetic acid; SVMPs: Snake venom metalloproteases; NIH: National Institutes of Health.
| Introduction|| |
Snakebite is a common yet neglected medical problem throughout some parts of the world. It has a close association with rural communities and is often regarded as a “disease of poverty.” Epidemiological data concerning snakebites show that about 1.8 million venomous snakebites occur annually, which leads to nearly 125,000 deaths. Effects of snake venom range from minor consequences, including edema and pain, to more severe conditions such as permanent tissue damage, renal failure, hemorrhage, hematuria, hemoptysis, anemia, melena, hypotension, and hematemesis. It is a public health issue, particularly in Asian and African countries such as Pakistan, Indonesia, Mali, and Nigeria predominantly facing 20,000 deaths/year; Bangladesh about 6000 deaths/year; and Cameroon, India, and Ghana about 50,000 deaths/year.,
Among numerous snakes, vipers of the genus Echis are extremely important clinically and have been added to the WHO Category 1 list of neglected tropical diseases. In South Asia, bites caused by Echis carinatus is a severe threat as it leads to a significant number of deaths. In Pakistan, E. carinatus is generally found throughout the country. However, it has high population densities in Astola Island (Makran Coast, Balochistan) as well as in the deserts of Cholistan and Thar.
E. carinatus is a concoction of bioactive molecules attributed for degradation of tissue structure as well as hemorrhage. The majority of proteins that have been studied extensively in E. carinatus venom are metalloproteinase, disintegrins, and phospholipases A2. Metalloproteinases present in venom are involved in different hemostatic activities including inhibition of platelet aggregation through binding with glycoprotein IIb–IIIa receptors, enzymatic degradation of basement membrane (i.e., laminin, collagen IV, and fibronectin), hydrolysis of endothelial cell proteins (i.e., integrins and cadherins) as well as activation of prothrombin. Metalloproteases from E. carinatus venom have been defined as prothrombin activators. These activators inhibit clotting factors that induce coagulation, thus ultimately leading to microvascular thrombosis.
Indeed, antiserum is the only known efficacious therapy for the management of snakebites. However, venom antiserum has some limitations associated with it, including cost, significant preparation time, and special storage conditions. It has the potential to neutralize systematic but not local effects, and sometimes, it causes adverse reactions in victims after administration. Most snakebite cases happen in a rural area where snake venom antiserum is not available. Due to all these limitations, the management of snakebites through antiserum is quite a challenge for medical professionals in affected areas. Because of this, plant-based management of snakebite could be quite advantageous. Pakistan has an incredible diversity of plants. Plants are rich sources of active compounds that are currently used by people to treat numerous diseases, including snakebites. Numerous studies have reported plants neutralizing the effects of E. carinatus venom components.,,,,, The present research was conducted to evaluate the antiprotease activity of various medicinal plants that are traditionally used as an antivenom against snakebite ubiquitously present in Pakistan.
| Materials and Methods|| |
Snake venom and chemicals
Pakistani E. carinatus venom was gifted from the National Institutes of Health (NIH), Islamabad, Pakistan. Lyophilized venom was stored in light-resistant bottles at 2°C–8°C. The rest of all chemicals were purchased from Merck unless and otherwise described.
For this study, plants were selected based on previous ethnobotanical evidence of anti-snake venom activity. Medicinal plants were collected from various regions of Pakistan; however, few were procured from the local market named Naswari Bazar in Rawalpindi. All plants were identified by expert botanist Dr. Zafar Ullah Zafar, and voucher specimens were submitted to the herbarium of the Department of Botany, B. Z. University, Multan, Pakistan. Complete detail about medicinal plants is summarized in [Table 1].
|Table 1: List of Pakistani medicinal plants to neutralize snakebite problem|
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Shade-dried parts of the plants were crushed using an electric grinder. Dried powder was soaked in methanol at ambient temperature for a period of 4 weeks. Filtration was done initially with ordinary filter paper and subsequently with Whatman filter paper 41. After filtration, methanol was allowed to evaporate at room temperature and prepared plant extracts were stored for further use.
Enzymatic assay for proteases
Antiprotease activity of medicinal plants was analyzed through casein as a substrate. Briefly, reaction mixture containing 0.5% casein (in Tris-HCl, pH 7.5) and venom (0.1–1.6 mg; phosphate buffer, pH 8) was incubated at 37°C for 10 min. After incubation, the reaction was stopped by the addition of trichloroacetic acid and mixture was filtered. Filtrate was used to estimate enzyme activity using L-tyrosine as standard. In the above analysis, one unit of enzyme activity was expressed as 0.02 μmole of tyrosine released per hour. For inhibition studies, venom was preincubated with plant extracts at 37°C for 15 min.,
Fractionation of active plant extracts
Active methanolic plant extracts were further fractioned using different solvents including n-hexane, chloroform, dichloromethane, and ethyl acetate. After drying, all fractions were again tested for antiprotease activity using the previously described assay.
Phytochemical screening was performed for both active methanolic plant extracts and their active fractions using standard analytical procedures. A complete picture of phytochemical screening is given in [Table 2].
| Results and Discussion|| |
The protease enzyme works by causing the breakdown of proteins into their structural amino acid units. Standard curves (absorbance vs. concentration) were constructed using different concentrations of L-tyrosine [Figure 1]. Various concentrations of E. carinatus venom were tested using casein as a standard substrate to check protease activity. Activities at venom concentration of 0.1, 0.2, 0.4, 0.8, and 1.6
|Figure 1:Standard curve for proteolytic activity. L-tyrosine was used as an indicator of protein degradation|
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mg were found to be 68, 75, 120, 178, and 259 units/ml, respectively [Table 3]. A standard concentration (0.8 mg) was then used to evaluate the antiprotease activity of Pakistani medicinal plants. In the current study, snake venom antiserum and ethylenediaminetetraacetic acid (EDTA) were used as reference standards. The results of antiprotease activity from medicinal plant extracts, EDTA, and snake venom are shown in [Table 4]. Different fractions of active plants' extract were further tested for their antiprotease activity against snake venom. The antiprotease activity of different fractions of C. procera (Wild.) R. Br., Matthiola incana (L.) R. Br., and T. arjuna Wight and Arn is presented in [Table 5], [Table 6], [Table 7] respectively. In case of M. incana (L.) R. Br., maximum inhibition was achieved for n-hexane and ethyl acetate fractions. However, maximum inhibition was achieved with C. procera (Wild.) R. Br. and T. arjuna Wight and Arn with n-hexane and ethyl acetate fractions, respectively. The phytochemical analysis of C. procera (Wild.) R. Br., M. incana (L.) R. Br., and T. arjuna Wight and Arn is shown in [Table 8]. Active fractions of plant extracts were also analyzed for their phytochemical content, and the results of this analysis are summarized in [Table 9].
|Table 3: Protease activity posed by Pakistani snake Echis carinatus venom|
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|Table 4: Antiprotease activity of selected medicinal plants (0.8 mg/0.8 ml) from Pakistan to neutralize Echis carinatus (0.8 mg/0.8 ml) venom|
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|Table 5: Antiprotease activity of different fractions of Calotropis procera (Wild.) R. Br. extract|
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|Table 6: Antiprotease activity of different fractions of Matthiola incana (L.) R. Br.|
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|Table 7: Antiprotease activity of different fractions of Terminalia arjuna Wight and Arn|
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|Table 8: Phytochemicals analysis of Calotropis procera (Wild.) R. Br, Matthiola incana1 (L.) R. Br., and Terminalia arjuna Wight and Arn crude extracts|
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|Table 9: Phytochemicals analysis of active fractions of Calotropis procera (Wild) R. Br., Matthiola incana (L.) R. Br., and Terminalia arjuna Wight and Arn crude extracts|
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Bites caused by snakes of family Viperidae are attributed to the high morbidity and mortality rates globally. In Pakistan, an increase in snake bites is believed to be caused by deforestation and migration of poisonous snakes toward human settlements. Among numerous viper snakes, E. carinatus is the most dangerous one. E. carinatus venom is a rich source of serine and metalloproteases. Majority of the E. carinatus activities are due to the presence of metalloproteases (90%). Snake venom metalloproteases have a significant relation with extracellular membrane and coagulation cascades which results in a variety of hemostatic dysfunctions and tissue damage. Snake venom interaction with coagulation cascades leads to intravascular coagulation. Procoagulant effect of E. carinatus venom is due to the presence of ecarin and carinactivase, both of which are metalloproteases, which activates prothrombin activator. Mortality after snake venom envenomation is typically caused by systemic hemostatic disruption, which usually results in systemic hemorrhage. Hemorrhage is the most significant effect caused by snake bites of family Viperidae. The main event behind this lethal effect is microvasculature damage caused by snake venom.,,
Medicinal plants are the most accessible resource for the treatment of numerous diseases including snake bites. People of Pakistan usually rely on herbal medicines for the treatment of snake bites. These medicines are easily available by traditional practitioners in those areas. This extensive use of plant-based medicine for snakebite management is ascribed to a range of bioactive compounds in plants that have the ability to neutralize snake venom effects. Phytochemicals neutralize the venom effect either by reducing its diffusion or causing disintegration of venom components. In this regard, the present research was designed to evaluate the antiprotease activity of 18 medicinal plant extracts against E. carinatus venom.
The results of this study show that percentage inhibition for snake venom antiserum and EDTA was found to be 78.5% and 64%, respectively [Table 4]. Among 18 selected medicinal plants, three plants, i.e., C. procera (Wild.) R. Br., M. incana (L.) W. T. Aiton, and T. arjuna Wight and Arn, were able to significantly neutralize the protease activity of E. carinatus venom, which was comparable to standard inhibitors. C. procera (Wild.) R. Br. inhibited the activity by 63%, M. incana (L.) W. T. Aiton 71%, and T. arjuna Wight and Arn by 66%. Parentage inhibition for Trichodesma indicum (L.) R. Br. was found to be 51%. All other plants show inhibition activity < 50% [Table 4]. C. procera (Wild.) R. Br. n-hexane fraction was able to neutralize the protease enzyme close to the crude extract [Table 5]. Percentage inhibition for n-hexane fraction was 64.6%. For M. incana (L.) W. T. Aiton, two fractions, i.e., n-hexane and ethyl acetate, were effective. N-hexane fraction inhibits protease activity by 71.7% and ethyl acetate fraction by 74.8% [Table 6], whereas for T. arjuna Wight and Arn, ethyl acetate fraction exhibited the inhibitory effect (65.8%) close to crude extract [Table 7]. Various studies have reported such neutralizing potential of plants against protease activity of E. carinatus venom. Albizia lebbeck L. seeds' extract was able to inhibit hyaluronidase (IC50 =91.95 μg; P < 0.0001) and protease (IC50 =36.32 μg; P < 0.0001) activities throughin vitro evaluation.Cassia auriculata L. leave extracts neutralized the protease enzyme activity of E. carinatus venom by 96%. This study showed that extract of Cassia auriculata L. leaves also inhibited hemorrhagic, edematogenic, myotoxic, and lethal effects of E. carinatus venom. Another study revealed that Tabernaemontana alternifolia (Roxb) roots' extract showed the neutralization ability against the protease enzyme of E. carinatus venom. Girish et al . reported the root extract of Mimosa pudica L. against protease enzyme of E. carinatus venom. This study revealed that aqueous root extract was able to neutralize protease activity in a dose-dependent manner.
Phytochemical analysis of active fractions of plants' extract enunciated about various phytochemicals such as alkaloids, flavonoids, phenols, saponins, and steroids. A complete summary of phytochemical screening is encompassed in [Table 8] and [Table 9]. Manyin vitro andin vivo studies were reported to confer potential of alkaloids, terpenoids, polyphenols, flavonoids, saponins, and glycosides as an antidote. The presence of these phytochemicals in active plant extracts could be the reason behind the neutralization of protease enzymes abundant in E. carinatus venom. Future investigation about isolation and characterization of active metabolites from potential plant extracts could play a tremendous role in the development of effective antidote against E. carinatus snake bite.
| Conclusion|| |
The present study indicated about C. procera (Wild.) R. Br., M. incana (L.) R. Br., and T. arjuna Wight and Arn possessed inhibitor(s) against protease enzyme present in E. carinatus venom and would be worthwhile for development as treatment against envenomation in the future.
The authors highly acknowledge Dr. Muhammad Hassham Hassan Bin Asad (KFU, Russia; CUI, Pakistan) and Dr. Ryan J. R. McCleary (Stetson University, USA) for their valuable support complete this work. Moreover, the authors are thankful to the NIH, Islamabad, Pakistan, for provision of E. carinatus venom.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Harrison RA, Hargreaves A, Wagstaff SC, Faragher B, Lalloo DG. Snake envenoming: A disease of poverty. PLoS Negl Trop Dis 2009;3:e569.
Slagboom J, Kool J, Harrison RA, Casewell NR. Haemotoxic snake venoms: Their functional activity, impact on snakebite victims and pharmaceutical promise. Br J Haematol 2017;177:947-59.
Maheshwari R, Kumar V, Verma HK. Neural network-based species identification in venom-interacted cases in India. J Venom Anim Toxins Incl Trop Dis 2007;13:766-81.
Ali G, Kak M, Kumar M, Bali SK, Tak SI, Hassan G, et al
. Acute renal failure following Echis carinatus
(saw-scaled viper) envenomation. Indian J Nephrol 2004;14:177-81.
Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM, et al
. Snakebite mortality in India: A nationally representative mortality survey. PLoS Negl Trop Dis 2011;5:e1018.
Whitaker R, Whitaker S. Venom antivenom production and the medically important snakes of India. Curr Sci 2012;103:635-43.
Casewell NR, Cook DA, Wagstaff SC, Nasidi A, Durfa N, Wüster W, et al
. Pre-clinical assays predict pan-African Echis
viper efficacy for a species-specific antivenom. PLoS Negl Trop Dis 2010;4:e851.
Khan MS. A Guide to the Snake of Pakistan. Frankfurt, Germany: Edition Chimaira; 2002.
Sunitha K, Hemshekhar M, Gaonkar SL, Sebastin Santhosh M, Suresh Kumar M, Basappa, et al
. Neutralization of haemorrhagic activity of viper venoms by 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-3-oxo-1,3-dihydroisobenzofuran-5-carbonitrile. Basic Clin Pharmacol Toxicol 2011;109:292-9.
Yamada D, Morita T. CA-1 method, a novel assay for quantification of normal prothrombin using a Ca2+
-dependent prothrombin activator, carinactivase-1. Thromb Res 1999;94:221-6.
Terra RM, Pinto AF, Guimarães JA, Fox JW. Proteomic profiling of snake venom metalloproteinases (SVMPs): Insights into venom induced pathology. Toxicon 2009;54:836-44.
Escalante T, Rucavado A, Fox JW, Gutiérrez JM. Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases. J Proteomics 2011;74:1781-94.
Gupta YK, Peshin SS. Do herbal medicines have potential for managing snake bite envenomation? Toxicol Int 2012;19:89-99.
] [Full text]
Amog PU, Manjuprasanna VN, Yariswamy M, Nanjaraj Urs AN, Joshi V, Suvilesh KN, et al. Albizia lebbeck
seed methanolic extract as a complementary therapy to manage local toxicity of Echis carinatus
venom in a murine model. Pharm Biol 2016;54:2568-74.
Guerranti R, Aguiyi JC, Errico E, Pagani R, Marinello E. Effects of Mucuna pruriens
extract on activation of prothrombin by Echis carinatus
venom. J Ethnopharmacol 2001;75:175-80.
Hasson SS, Al-Balushi MS, Said EA, Habbal O, Idris MA, Mothana RA, et al
. Neutralisation of local haemorrhage induced by the saw-scaled viper Echis carinatus sochureki
Venom using ethanolic extract OF Hibiscus aethiopicus
L. Evid Based Complement Alternat Med 2012;2012:540671.
Mahadeswaraswamy YH, Nagaraju S, Girish KS, Kemparaju K. Local tissue destruction and procoagulation properties of Echis carinatus
venom: Inhibition by Vitis vinifera
seed methanol extract. Phytother Res 2008;22:963-9.
Nanjaraj Urs AN, Yariswamy M, Joshi V, Suvilesh KN, Sumanth MS, Das D, et al
. Local and systemic toxicity of Echis carinatus
venom: Neutralization by Cassia auriculata
L. leaf methanol extract. J Nat Med 2015;69:111-22.
Vineetha MS, Bhavya J, Mirjakar KM, More SS.In vitro evaluation of active phytochemicals from Tabernaemontana alternifolia
(Roxb) root against the Naja naja
and Echis carinatus
Indian snake venom. JBAPN 2014;4:286-94.
Butt MA, Ahmad M, Fatima A, Sultana S, Zafar M, Yaseen G, et al
. Ethnomedicinal uses of plants for the treatment of snake and scorpion bite in Northern Pakistan. J Ethnopharmacol 2015;168:164-81.
Husain SZ, Malik RN, Javaid M, Bibi S. Ethonobotanical properties and uses of medicinal plants of Morgah biodiversity park Rawalpindi. Pak J Bot 2008;40:1897-911.
Samy RP, Thwin MM, Gopalakrishnakone P, Ignacimuthu S. Ethnobotanical survey of folk plants for the treatment of snakebites in Southern part of Tamilnadu, India. J Ethnopharmacol 2008;115:302-12.
Razi MT, Asad MH, Khan T, Chaudhary MZ, Ansari MT, Arshad MA, et al
. Antihaemorrhagic potentials of Fagonia cretica
against Naja naja
Karachiensis (black Pakistan cobra) venom. Nat Prod Res 2011;25:1902-7.
Jabeen A, Rani S, Ibrahim M, Mohammad AS. A review on Lepidium sativum
. Indo Am J Pharm Sci 2017;4:2223-7.
Bin Asd MH, Iqbal M, Akram MR, Khawaja NR, Muneer S, Shabbir MZ, et al
. 5'-nucleotidases of Naja naja
Karachiensis snake venom: Their determination, toxicities and remedial approach by natural inhibitors (medicinal plants). Acta Pol Pharm 2016;73:667-73.
Dey A, De JN. Traditional use of plants against snakebite in Indian subcontinent: A review of the recent literature. Afr J Tradit Complement Altern Med 2012;9:153-74.
Kumar V, Van Staden J. A review of Swertia chirayita
(Gentianaceae) as a traditional medicinal Plant. Front Pharmacol 2015;6:308.
Janardhan B, Shrikanth VM, Mirajkar KK, More SS.In vitro screening and evaluation of antivenom phytochemicals from Azima tetracantha
Lam. leaves against Bungarus caeruleus
and Vipera russelli
. J Venom Anim Toxins Incl Trop Dis 2014;20:12.
Hussain J, Ali L, Khan AL, Rehman NU, Jabeen F, Kim JS, et al
. Isolation and bioactivities of the flavonoids morin and morin-3-O-ß-d-glucopyranoside from Acridocarpus orientalis
– A wild Arabian medicinal plant. Molecules 2014;19:17763-72.
María R, Shirley M, Xavier C, Jaime S, David V, Rosa S, et al
. Preliminary phytochemical screening total phenolic content and antibacterial activity of thirteen native species from Guayas province Ecuador. J King Saud Univ Sci 2018;30:500-5.
Godghate A, Sawant R, Sutar A. Phytochemical analysis of ethanolic extract of roots of Carrisa carandus
Linn. Rasayan J Chem 2012;5:456-59.
Bansode TS, Salalkar D. Phytochemical analysis of some selected Indian medicinal plants. Int J Pharma Bio Sci 2015;6:550-6.
Alabri TH, Al Musalami AH, Hossain MA, Weli AM, Al-Riyami Q. Comparative study of phytochemical screening antioxidant and antimicrobial capacities of fresh and dry leaves crude plant extracts of Datura metel
L. J King Saud Univ Sc 2014;26:237-43.
Ayoola GA, Coker HA, Adesegun SA, Adepoju-Bello AA, Obaweya K, Ezennia EC, et al
. Phytochemical screening and antioxidant activities of some selected medicinal plants used for malaria therapy in Southwestern Nigeria. Trop J Pharm Res 2008;7:1019-24.
Jaradat N, Hussen F, Ali A. Preliminary phytochemical screening quantitative estimation of total flavonoids total phenols and antioxidant activity of Ephedra alata
Decne. J Mater Environ Sci. 2015;6:1771-8.
Ismail AM, Mohamed EA, Marghany MR, Abdel-Motaal FF, Abdel-Farid IB, El-Sayed MA. Preliminary phytochemical screening, plant growth inhibition and antimicrobial activity studies of Faidherbia albida
legume extracts. J Saudi Soc Agric Sci 2016;15:112-17.
Nasim MJ, Asad MH, Sajjad A, Khan SA, Mumtaz A, Farzana K, et al
. Combating of scorpion bite with Pakistani medicinal plants having ethno-botanical evidences as antidote. Acta Pol Pharm 2013;70:387-94.
Yamada D, Sekiya F, Morita T. Isolation and characterization of carinactivase, a novel prothrombin activator in Echis carinatus
venom with a unique catalytic mechanism. J Biol Chem 1996;271:5200-7.
Warrell DA, Davidson NMcD, Greenwood BM, Ormerod LD, Pope HM, Watkins BJ, et al
. Poisoning by bites of the saw-scaled or carpet viper (Echis carinatus
) in Nigeria. Q J Med 1977;46:33-62.
Boyer L, Alagón A, Fry BG, Jackson TN, Sunagar K, Chippaux JP. Signs, symptoms and treatment of envenomation. In: Fry BG, editor. Venomous Reptiles and Their Toxins: Evolutionary, Pathophysiological and Biodiscovery Implications. New York: Oxford University Press; 2015. p. 32-60.
Gomes A, Das R, Sarkhel S, Mishra R, Mukherjee S, Bhattacharya S, et al
. Herbs and herbal constituents active against snake bite. Indian J Exp Biol 2010;48:865-78.
Girish KS, Mohanakumari HP, Nagaraju S, Vishwanath BS, Kemparaju K. Hyaluronidase and protease activities from Indian snake venoms: Neutralization by Mimosa pudica
root extract. Fitoterapia 2004;75:378-80.
Nanjaraj AN, Yariswamy M, Joshi V, Nataraju A, Gowda TV, Vishwanath BS. Implications of phytochemicals in snakebite management: Present status and future prospective. Toxin Rev 2014;33:60-83.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]