email   Email Us: info@lupinepublishers.com phone   Call Us: +1 (914) 407-6109   57 West 57th Street, 3rd floor, New York - NY 10019, USA

Lupine Publishers Group

Lupine Publishers

lupinepublishers

ISSN: 2637-4706

Drug Designing & Intellectual Properties International Journal

Research Article(ISSN: 2637-4706)

An in vivo study for the effect of Citrus reticulata (Rutaceae) fruit peels extracts on the onset of toxicity of Cerastes cerastes venom in Albino mice Volume 3 - Issue 1

Mohamed A Gbaj1, Inass A Sadawe2, Nisreen H Meiqal2, Salah M Bensaber2, Massaud Salem Maamar3, Anton Hermann4 and Abdul M Gbaj2*

  • 1Department of Chemical Engineering, University of Tripoli, Libya
  • 2Department of Medicinal Chemistry, University of Tripoli, Libya
  • 3Zoology Department, Tripoli University, Libya
  • 4Department of Biosciences, University of Salzburg, Austria

Received: April 03, 2019;   Published: April 25, 2019

Corresponding author:Abdul M Gbaj, Department of Medicinal Chemistry, University of Tripoli, Libya

DOI: 10.32474/DDIPIJ.2019.03.000153

Abstract PDF

Abstract

Venom of Cerastes cerastes has been extracted and its toxicity was investigated in the presence of aqueous and methanolic extracts of Citrus reticulata (Rutaceae) fruit peels. The decline in the mean survival time of the male albino swiss mice were used to deduce the venom property in the presence and absence of aqueous and methanolic extracts of Citrus reticulata (Rutaceae) fruit peels. The aqueous and methanolic extracts of Citrus reticulata (Rutaceae) fruit Peels significantly decrease the mean survival time compared to the venom alone. From these results it was evident that the toxicity of Cerastes cerastes venomis increased significantly in the presence of Citrus reticulata in a dose dependent manner

Keywords: Citrus reticulata; Cerastes cerastes; Venom; Toxicity

Introduction

Snakebites are severe socio-medical difficulty that lead to morbid and fatal affect on victims in Libya and other North African countries [1,2]. Immediate antivenom treatment is crucial and vital to avoid morbidity and mortality [3]. The oxidative trauma condition, which result from snake bite envenomation is another measurement of kidney destruction and severe renal failure [4], connected with the antioxidant defense system, that might be subject for treatment by antioxidant therapy [5]. ROS (Reactive oxygen species) are engaged in many inflammatory reactions, thus influencing the physiology of the cells and participate a significant function in the pathological conditions [6]. As have been free radical, ROS are involved in harming cellular components, and they play an important function in venom induced toxicity, as reported among envenomed mice [7]. Ascorbic acid is an antioxidant that has been reported to have useful effects on a number of cancer types [8,9] and could be concerned in alleviation of Reactive oxygen species cellular damage, produced during exposure to toxins, metabolism and carcinogens [10]. In addition to augmentation of protease inhibitor effects concerned in preventing organ efficient injure [11,12]. Citrus reticulata (Rutaceae) is commonly known as narangi or santra (orange). It is a small spiny tree with thick top of slim branches, extensively grown in Egypt, Tunisia and Libya [13]. Mandarin is a collection name for this class of orange with thin, loose peel. The name ‘tangerine might be applied as an interchange name to the entire group, but in trade, it is usually limited to the types with red-orange skin. The fruit has aphrodisiac, laxative, tonic and astringent properties [14,15]. It is also used to alleviate vomiting [16,17]. The fruit peel controls the skin moisture, rough and softens hard skin and possess a cleaning effect on oily skin [18]. Chemical composition of the volatile oil of the fruit peels of this species has been reported [19-23]. The effects of the volatile oil of C. reticulata has been studied against Saccharomyces cerevisiae [24], pathogenic fungi, Paenibacillus larvae, Schistosoma mansoni, Aspergillus flavus , and other microorganisms [25-30]. Very recently, the volatile oil of C. reticulata also demonstrates anticancer activity [31-33]. The main aim of the current study is to investigate the effects of Citrus reticulata (Rutaceae) fruit peels extracts on the toxicity of Cerastes cerastes venom in albino mice.

Materials and Methods

Collection of plant material and preparation of aqueous extract

The oranges were bought from a shop in Tripoli (February 2019), and the Citrus reticulata was identified and authenticated by a botanist. Orange rinds were peeled off carefully with the help of a sharp razor blade, and each rind sample was cut into smaller pieces and 30g mass of the sample was taken. The sample was initially rinsed with distilled water, and the fresh peels (30g) were added to 30ml hot distilled water. In addition, another 30g of the fresh peels were macerated in cold 99% methanol for three hours at room temperature (28-30 °C), the mixture was then filtered under vacuum and the filtrate was stored at 4 °C and used when appropriate [34].

Experimental models

Albino mice (Swiss type) of either sex weighing approximately 18–28g (2 to 2.6 month old) were utilized for investigational purpose. They were kept in cages made from polypropylene in airconditioned room with the temperature retained at 25±2 °C, and twelve hours sporadicing dark and light cycles. The mice were supplied with drinking water ad libitum and an adequate diet during the study. The authorization for the experimental procedures was obtained from the Animal Ethics Committee.

Venoms

Cerastes cerastes venom was extracted by means of physical stimulation and was gained in liquid forms, from the Faculty of Science, Zoology Department, University of Tripoli (Libya) and kept at –20 °C until utilize. A 7.5μl aliquot from the venoms was added to eight hundreds microliter of normal saline. A dosage of hundred microliter (100 nanogram) was administered to the male Swiss Albino mice.

Acute toxicity study

Acute toxicity was commonly performed to determine the LD50 value in experimental animals. The intend of doing acute toxicity study is to establish the therapeutic index of a methanolic and aqueous extracts of Citrus reticulate and to guarantee the in-vivo safety. The acute toxicity experiment was done in mice, in which all animals were overnight fasted prior to treatment and given food one hour after aqueous and methanolic extracts administration, with the period observation of common behavior at 0.5, 1, 8, 12 and 24 hours. The number of animals that died after taken the extracts was monitored daily for 7 days [35,36].

Intoxication of venom by Citrus reticulata extracts The animals (albino mice) used in this study were divided to ten groups, each of them is of six mice (male or female). Five groups were used to investigate the aqueous extracts, while the other were used for methanolic extract. The first group received only hundred microliter (hundred microgram of total protein) of the Cerastes cerastes venom (LD99 5μg/kg). Groups 2 to 4 were used as treatment groups and given an equivalent amount of the Cerastes cerastes venom with 50μl, 100μl and 200μl of aqueous Citrus reticulate extracts intraperitoneally (30g/30ml), respectively. Group 5 was given 100μl of the Cerastes cerastes venom and polyvalent anti-snake venom (ASV) was bought from India from Haffkine Bio-Pharmaceuticals Company. The number of death was recorded within twenty-four hours. Similar experiments were repeated in the same manner with the methanolic extract using groups 6 to 10.

Statistical analysis

The difference among various control group and treated groups were analyzed using ANOVA method of one-way. The obtained results were dealt with using unpaired Student’s test. All results were articulates as the mean±SEM of the number of experiments performed, with P value less than 0.05 showing significant difference among groups.

Results and discussion

Acute toxicity study

With the growing amount of research about naringin as a component of the orange and its potential utilize within the pharmacological and food industries, illuminating its toxicological outline becomes increasingly significant. In the present study, the Citrus reticulata extracts were found to be safe up to 200mg/kg orally. This present study is compared with other previous studies in which an oral single dose of 16g/kg of naringin did not produce acute oral toxicity in rats [37].

Acute toxicity of Cerastes cerastes venom and its reaction with aqueous and methanolic Citrus reticulata extracts and antivenom The Cerastes cerastes venom at the dose five micrograms per kilogram (LD99) produces 100% mortality in mice. The aqueous and methanolic Citrus reticulata extracts significantly decrease the mean survival times by 3, 5 and 6 times for 50, 100 and 200µl (30g /30mL), respectively when compared with the venom alone which was 3.1±0.3 hours. ASV was established to be efficient and showing mean survival of 2-days for 5-mice and absolute survival of one mouse. The Cerastes cerastes toxins contain of cardiotoxin, neurotoxin, proteins and enzymes. The victim may die from respiratory troubles which is the main cause of death. Assisted ventilation and ASV can save life in a lot of cases [38-40].

It has been reported that the citrus species contain glycosides and flavonones in huge amounts, and they play a main function in treating a range of pathological conditions. Hesperidin and naringein, are the major components of the citrus fruits. Intestinal microorganism are able to convert naringin into naringenin (an aglycone part). They established to have metal chelating effect, antioxidant, antidiabetic, antiviral, antiallergic, antiestrogenic, antimicrobial, ischemic heart disease adipolytic activity, anti-inflammatory, antiobesity, hypoxia, anti-cancer and hepatoprotective activity. Because of all these pharmacological action, both naringenin and naringin are assumed to be useful as a food supplement [41-47]. The accelerated death could be related to the interactions of Citrus reticulata components (which were mainly polyphenolic components) with snake venom which is not consistent with the previous studies reporting that secondary metabolites polyphenol are competent to inhibit PLA2 [48]. In the literature, it has been reported that naringin which is a flavonoid that is contained in grapefruit and recognized for its various biochemical activities and pharmacological effects on a secretory phospholipase A (sPLA2 ) of Crotalus durissus cascavella, is concerned in the releasing of arachidonic acid in phospholipid membranes [48]. sPLA2 was incubated with naringin in a ratio of 1:1 mole at 37 °C and a distinct decrease in the ultraviolet absorption signal and a changes of the circular dichroism spectra suggesting a significant effect of PLA2 structure and function [48]. The obtained results are for the whole extract of Citrus reticulate and not for naringin or naringenin and this could be explained for the lack of association between pharmacological and enzymatic activities in which the chemical modification of some amino acids induced by naringin, in particular aromatic amino acids and histidines, affected the toxin’s ability to interact with the pharmacological receptor, but did not lead to eliminate of this function. Our results and those described by Cardoso et al. expressed that enzymatic activity of sPLA2 is not crucial for pharmacological activities of this sPLA2 which was isolated from C. d. cascavella venom [49].

Conclusion

The present study confirmed that the aqueous extract of peeled Citrus reticulate accelerate the onset of toxicity of Cerastes cerastes venomis in a dose-dependent effect.

Acknowledgement

The authors gratefully acknowledge the technical support and valuable suggestions obtained from Ms Amira Abdul Gbaj.

References

  1. Tianyi FL, Agbor VN, Tochie JN, Kadia BM, Nkwescheu AS (2018) Community-based audits of snake envenomations in a resourcechallenged setting of Cameroon: case series. BMC Res Notes 11(1): 317.
  2. Goncalves DV, Martinez-Freiria F, Crochet PA, Geniez P, Carranza S, et al. (2018) The role of climatic cycles and trans-Saharan migration corridors in species diversification: Biogeography of Psammophis schokari group in North Africa. Mol Phylogenet Evol 118: 64-74.
  3. Lavonas EJ, Tomaszewski CA, Ford MD, Rouse AM, Kerns WP (2002) Severe puff adder (Bitis arietans) envenomation with coagulopathy. J Toxicol Clin Toxicol 40(7): 911-918.
  4. Yamasaki SC, Villarroel JS, Barone JM, Zambotti-Villela L, Silveira PF (2008) Aminopeptidase activities, oxidative stress and renal function in Crotalus durissus terrificus envenomation in mice. Toxicon 52(3): 445- 454.
  5. Al Asmari AK, Khan HA, Manthiri RA, Al Yahya KM, Al Otaibi KE (2014) Effects of Echis pyramidum snake venom on hepatic and renal antioxidant enzymes and lipid peroxidation in rats. J Biochem Mol Toxicol 28(9): 407-412.
  6. Carroll IM, Andrus JM, Bruno-Barcena JM, Klaenhammer TR, Hassan HM, et al. (2007) Anti-inflammatory properties of Lactobacillus gasseri expressing manganese superoxide dismutase using the interleukin 10-deficient mouse model of colitis. Am J Physiol Gastrointest Liver Physiol 293(4): G729-G738.
  7. Dousset E, Carrega L, Steinberg JG, Clot-Faybesse O, Jouirou B, et al. (2005) Evidence that free radical generation occurs during scorpion envenomation. Comp Biochem Physiol C Toxicol Pharmacol 140(2): 221-226.
  8. Mirmohammadsadeghi M, Mirmohammadsadeghi A, Mahmoudian M (2018) Preventive Use of Ascorbic Acid For Atrial Fibrillation After Coronary Artery Bypass Graft Surgery. Heart Surg Forum 21(5): E415-E417.
  9. Kitahata K, Matsuo K, Hara Y, Naganuma T, Oiso N, et al. (2018) Ascorbic acid derivative DDH-1 ameliorates psoriasis-like skin lesions in mice by suppressing inflammatory cytokine expression. J Pharmacol Sci 138(4): 284-288.
  10. Banerjee P, Bhattacharyya SS, Bhattacharjee N, Pathak S, Boujedaini N, et al. (2009) Ascorbic acid combats arsenic-induced oxidative stress in mice liver. Ecotoxicol Environ Saf 72(2): 639-649.
  11. Choudhury M, Senthilvadivel V, Velmurugan D (2018) Inhibitory effects of ascorbic acid toward snake venom metalloproteinase (SVMP) from Indian Echis carinatus venom: Insights from molecular modeling and binding studies. J Biochem Mol Toxicol 32(12): e22224.
  12. Shamsi TN, Parveen R, Afreen S, Azam M, Sen P, et al. (2018) Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant, Anti-Inflammatory, and Antibacterial Activity. J Diet Suppl 15(6): 939-950.
  13. Sharif SI, Ali BH (1994) Effect of grapefruit juice on drug metabolism in rats. Food Chem Toxicol 32(12): 1169-1171.
  14. Tseng SH, Lee HH, Chen LG, Wu CH, Wang CC (2006) Effects of three purgative decoctions on inflammatory mediators. J Ethnopharmacol 105(1-2): 118-124.
  15. McGuire RG, Hagenmaier RD (2001) Shellac formulations to reduce epiphytic survival of coliform bacteria on citrus fruit postharvest. J Food Prot 64(11): 1756-1760.
  16. Yin OQ, Gallagher N, Li A, Zhou W, Harrell R, et al. (2010) Effect of grapefruit juice on the pharmacokinetics of nilotinib in healthy participants. J Clin Pharmacol 50(2): 188-194.
  17. Glasscock SG, Friman PC, O’Brien S, Christophersen ER (1986) Varied citrus treatment of ruminant gagging in a teenager with Batten’s disease. J Behav Ther Exp Psychiatry 17(2): 129-133.
  18. Khan MA, Ali M, Alam P (2010) Phytochemical investigation of the fruit peels of Citrus reticulata Blanco. Nat Prod Res 24(7): 610-620.
  19. Fayek NM, Farag MA, Abdel Monem AR, Moussa MY, Abd-Elwahab SM, et al. (2019) Comparative Metabolite Profiling of Four Citrus Peel Cultivars via Ultra-Performance Liquid Chromatography Coupled with Quadrupole-Time-of-Flight-Mass Spectrometry and Multivariate Data Analyses. J Chromatogr Sci.
  20. Guo Q, Liu K, Deng W, Zhong B, Yang W, et al. (2018) Chemical composition and antimicrobial activity of Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) peel essential oils. Food Sci Nutr 6(6): 1431-1437.
  21. Xu C, Zhang S, Zhang Y, Pu Y, Yin L, et al. (2018) [Determination of spirotetramat and its four metabolites in citrus by ultra-high performance liquid chromatography-triple quadrupole-ion trap mass spectrometry]. Se Pu 36(4): 339-344.
  22. Kealey KS, Kinsella JE (1978) Orange juice quality with an emphasis on flavor components. CRC Crit Rev Food Sci Nutr 11(1): 1-40.
  23. Dunlap WJ, Wender SH (1960) Purification and identification of flavanone glycosides in the peel of the sweet orange. Arch Biochem Biophys 87: 228-231.
  24. Singh P, Shukla R, Kumar A, Prakash B, Singh S, et al. (2010) Effect of Citrus reticulata and Cymbopogon citratus essential oils on Aspergillus flavus growth and aflatoxin production on Asparagus racemosus. Mycopathologia 170(3): 195-202.
  25. Lemes RS, Alves CCF, Estevam EBB, Santiago MB, Martins CHG, et al. (2018) Chemical composition and antibacterial activity of essential oils from Citrus aurantifolia leaves and fruit peel against oral pathogenic bacteria. An Acad Bras Cienc 90(2): 1285-1292.
  26. Uckoo RM, Jayaprakasha GK, Vikram A, Patil BS (2015) Polymethoxyflavones Isolated from the Peel of Miaray Mandarin (Citrus miaray) Have Biofilm Inhibitory Activity in Vibrio harveyi. J Agric Food Chem 63(32): 7180-7189.
  27. Mehmood B, Dar KK, Ali S, Awan UA, Nayyer AQ, et al. (2015) Short communication: in vitro assessment of antioxidant, antibacterial and phytochemical analysis of peel of Citrus sinensis. Pak J Pharm Sci 28(1): 231-239.
  28. Mahadwar G, Chauhan KR, Bhagavathy GV, Murphy C, Smith AD, et al. (2015) Swarm motility of Salmonella enterica serovar Typhimurium is inhibited by compounds from fruit peel extracts. Lett Appl Microbiol 60(4): 334-340.
  29. Rakholiya K, Kaneria M, Chanda S (2014) Inhibition of microbial pathogens using fruit and vegetable peel extracts. Int J Food Sci Nutr 65(6): 733-739.
  30. Min KY, Kim HJ, Lee KA, Kim KT, Paik HD (2014) Antimicrobial activity of acid-hydrolyzed Citrus unshiu peel extract in milk. J Dairy Sci 97(4): 1955-1960.
  31. Nair SA, Sr RK, Nair AS, Baby S (2018) Citrus peels prevent cancer. Phytomedicine 50: 231-237.
  32. Arora S, Mohanpuria P, Sidhu GS, Yadav IS, Kumari V (2018) Cloning and Characterization of Limonoid Glucosyltransferase from Kinnow Mandarin (Citrus reticulata Blanco). Food Technol Biotechnol 56(2): 228-237.
  33. Tahsin T, Wansi JD, Al Groshi A, Evans A, Nahar L, et al. (2017) Cytotoxic Properties of the Stem Bark of Citrus reticulata Blanco (Rutaceae). Phytother Res 31(8): 1215-1219.
  34. Gray AM, Flatt PR (1999) Insulin-releasing and insulin-like activity of the traditional anti-diabetic plant Coriandrum sativum (coriander). Br J Nutr 81(3): 203-209.
  35. Ni H, Peng L, Gao X, Ji H, Ma J, (2019) Effects of maduramicin on adult zebrafish (Danio rerio): Acute toxicity, tissue damage and oxidative stress. Ecotoxicol Environ Saf 168: 249-259.
  36. Wheeler MW (2018) Bayesian additive adaptive basis tensor product models for modeling high dimensional surfaces: an application to highthroughput toxicity testing. Biometrics.
  37. Li P, Wang S, Guan X, Cen X, Hu C (2014) Six months chronic toxicological evaluation of naringin in Sprague-Dawley rats. Food Chem Toxicol 66: 65-75.
  38. Abdel-Aty AM, Salama WH, Ali AA, Mohamed SA (2019) A hemorrhagic metalloprotease of Egyptian Cerastes vipera venom: Biochemical and immunological properties. Int J Biol Macromol.
  39. Ozverel CS, Damm M, Hempel BF, Gocmen B, Sroka R, et al. (2019) Investigating the cytotoxic effects of the venom proteome of two species of the Viperidae family (Cerastes cerastes and Cryptelytrops purpureomaculatus) from various habitats. Comp Biochem Physiol C Toxicol Pharmacol.
  40. Lin CC, Wang PJ, Liu CC (2019) Venom concentrations in blisters and hemorrhagic bullae in a patient bitten by a Taiwan habu (Protobothrops mucrosquamatus). Rev Soc Bras Med Trop 52: e20180160.
  41. Abdel-Magied N, Shedid SM (2019) The effect of naringenin on the role of nuclear factor (erythroid-derived 2)-like2 (Nrf2) and haem oxygenase 1 (HO-1) in reducing the risk of oxidative stress-related radiotoxicity in the spleen of rats. Environ Toxicol.
  42. Xu C, Chen J, Zhang J, Hu X, Zhou X, et al. (2013) Naringenin inhibits angiotensin II-induced vascular smooth muscle cells proliferation and migration and decreases neointimal hyperplasia in balloon injured rat carotid arteries through suppressing oxidative stress. Biol Pharm Bull 36(10): 1549-1555.
  43. Hermenean A, Ardelean A, Stan M, Herman H, Mihali CV, et al. (2013) Protective effects of naringenin on carbon tetrachloride-induced acute nephrotoxicity in mouse kidney. Chem Biol Interact 205(2): 138-147.
  44. Annadurai T, Thomas PA, Geraldine P (2013) Ameliorative effect of naringenin on hyperglycemia-mediated inflammation in hepatic and pancreatic tissues of Wistar rats with streptozotocin- nicotinamideinduced experimental diabetes mellitus. Free Radic Res 47(10): 793- 803.
  45. Tarun EI, Kurchenko VP, Metelitsa DI (2006) Flavonoids as effective protectors of urease from ultrasonic inactivation in solutions. Bioorg Khim 32(4): 391-398.
  46. Kanno S, Tomizawa A, Ohtake T, Koiwai K, Ujibe M, et al. (2006) Naringenin-induced apoptosis via activation of NF-kappaB and necrosis involving the loss of ATP in human promyeloleukemia HL-60 cells. Toxicol Lett 166(2): 131-139.
  47. Misty R, Martinez R, Ali H, Steimle PA (2006) Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration. Biochem Biophys Res Commun 345(1): 516-522.
  48. Santos ML, Toyama DO, Oliveira SC, Cotrim CA, Diz-Filho EB, et al. (2011) Modulation of the pharmacological activities of secretory phospholipase A2 from Crotalus durissus cascavella induced by naringin. Molecules 16(1): 738-761.
  49. Cardoso DF, Lopes-Ferreira M, Faquim-Mauro EL, Macedo MS, Farsky SH (2001) Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions. Mediators Inflamm 10(3): 125-133.
Close

Online Submission System

Drag and drop files here

or

Browse Files
( For multiple files submission, zip them in a single file to submit. For file zipping software Download )