Effect of Cannabis Sativa Leaf on Oxidative Stress and the Recovery Period in Male Wistar Rats

Effect of Cannabis Sativa Leaf on Oxidative Stress and the Recovery Period in Male Wistar Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of Cannabis Sativa Leaf on Oxidative Stress and the Recovery Period in Male Wistar Rats Oluwasola Amuda, L.N. Usman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4686688/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigated the effect of CS on oxidative stress and the recovery period in male rats. Forty rats (170 g ± 1.24) were separately assigned into four groups of ten animals each, such that the rats in groups 1, 2, 3 and 4 received orally 1 ml of distilled water, 2mg, 4mg and 6mg of CS respectively for two weeks. Catalase, superoxide dismutase (SOD), Glutathione peroxidase (GPx), Glutathione reductase (GSH), malondialdehyde (MDA), total antioxidant capacity (TAC) and lactate dehydrogenase (LDH) were determined using standard methods. High dose (6 mg) and low doses (2mg and 4 mg) of CS significantly decrease catalase, SOD, GPx, GSH, TAC and significantly increase MDA and LDH levels when compared with the control. However, all the groups treated with low doses showed no significant difference in all the parameters when compared with the control after treatment. In conclusion, it could be deduced that these alterations in oxidative stress biomarkers were dependent on the doses of CS consumed. However, groups treated with low doses were able to recover from the damages caused by CS after treatment. This study recommends that people should abstain from the consumption of CS due to its detrimental effect in the body. Nutrition & Dietetics Cannabis sativa Oxidative stress Dose dependent Recovery period Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cannabis sativa (CS) has been utilized for medicinal purposes since ancient times due to its abundant phytochemical content. [ 1 ], hence the quest for harnessing its pharmacological potential by scientists. This substance is widely used as an illicit drug globally [ 2 ] Cells' normal redox state can be disrupted, leading to the production of peroxides and free radicals, damaging all cell components, including proteins, lipids, and DNA [ 3 ]. Oxidative stress (OS) is caused by a balance between pro-oxidants and antioxidants [ 4 ]. The ratio can be influenced by elevated levels of reactive oxygen species (ROS) or a decrease in antioxidant defense mechanisms [ 5 ]. OS can arise from an imbalance in the body's oxidizing system, primarily composed of free radicals, reactive oxygen species (ROS), and reactive nitrogen species (RNS) [ 6 ] and Antioxidant systems are essential in neutralizing free radicals that can have numerous harmful effects [ 7 , 8 ]. OS is involved in aging [ 9 ] and is present in certain chronic diseases like diabetes, cancers, hypertension, and coronary heart disease., etc. [ 10 ] and certain infections, particularly by the RNA viruses [ 11 ], a family to which belong corona viruses [ 12 ]. However few researches have been done on effect of different doses of CS in Wistar. In lieu of this, the research work examined the short term effect of different concentrations of CS in male wistar rats and the ability of the rats treated with CS to recover from the oxidative stress caused during administration of CS before abstinence. Materials and methods 2.1 Sample collection Cannabis sativa (CS) leaves were donated by National Drug Law Enforcement Agency (NDLEA), Nigeria, for research purpose only. 2.2 Extraction of Cannabis sativa leaves Extraction of Cannabis sativa (CS), was done with Soxhlet apparatus by soaking 800 grams of CS in 98% ethanol for 48 hours. It was filtered and the filtrate was poured into a round bottom conical flask which was fixed with a rotary evaporator. It was then evaporated and cooled. The dried yield of the extract was 55g. 2.3 Experimental animals Twenty male rats (160g ± 0.98) that were used for this research were obtained from Temilade Animal Venture, Ogbomoso, Oyo State, were housed at room temperature with unrestricted access to diet and water and maintained on a daily light/dark cycle. Principles of laboratory animal care (NIH publication No. 85 − 23, revised 1985) were followed. The experimental protocol was approved by Ethical Committee of Al-Hikmah University, Ilorin, Nigeria. 2.4 Experimental protocol After 2 weeks of acclimatization, the animals were separately assigned into four groups of ten animals each, such that the rats in groups 1, 2, 3 and 4 respectively received orally 1 ml of distilled water, 2 mg/kg body weight (bw) of CS, 4 mg/kg bw of CS and 6 mg/kg bw of CS respectively for two weeks. The animals were sacrificed on the 15th day with access to food and water. 2.5 Preparation of serum The male and female rats were sacrificed under ketamine anesthesia and blood was collected by cardiac puncture into sample bottles. The blood was left for 30 min to clot and thereafter centrifuged at 625× g for 10 min using a Uniscope Laboratory Centrifuge (Model SM800B, Surgifield Medicals, Essex, England). The serum was collected into plain bottles with the aid of a Pasteur pipette. Sera were stored in a freezer maintained at -5 ℃ and used within 12 hours of preparation. 2.6 Drug and assay kits Lactate dehydrogenase (LDH) activity was assayed spectrophotometrically (Spectramax Plus; Molecular Devices, Sunnyvale, CA, USA) following the kit manufacturer’s procedures (product code BXC0243; Fortress Diagnostics, UK). The determination of serum superoxide dismutase (SOD) concentration was done with SOD colorimetric assay kit (Fortress Diagnostics Ltd., Antrim, UK; Product code: BXC0531), following the manufacturer’s protocols. The determination of serum glutathione peroxidase (GPx) activity was done with GPx colorimetric assay kit (BioVision Inc., Milpitas, CA, USA), following the manufacturers protocols. Based on the manufacturer’s protocol, total anti-oxidant capacity (TAC) measurement in the serum was done with a spectrophotometric microplate reader (Spectramax Plus, Molecular Devices, Sunnyvale, CA, USA) using OxiSelect TAC assay kit that uses the single electron transfer mechanism (Cell Biolabs, Inc. San Diego, CA. cat no: STA-360). The continuous catalase activity was determined through spectrophotometric reading [ 13 ]. Reduced glutathione (GSH) was measured according to the method of [ 14 ]. The assay method of [ 15 ], modified by [ 16 ] was adopted for Malondiadehyde (MDA). 2.7 Statistical analysis Results were expressed as the mean ± standard error of mean. Data were analyzed using a two-way Analysis Of Variance, followed by the LSD post-hoc test to determine significant differences in all the parameters graph pad, version 9.0. Differences with values of P < 0.05 were considered statistically significant. Results Administration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p < 0.05) increase LDH level when compared with the control (Fig. 1). However, all the groups treated with low doses showed no significant difference in LDH but the group treated with high dose showed significant (p < 0.05) increase in LDH when compared with the control after treatment (Fig. 1). Administration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p < 0.05) decrease catalase level when compared with the control (Fig. 2 ). However, all the groups treated with low doses showed no significant difference in catalase but the group treated with high dose showed significant (p < 0.05) decrease in catalase when compared with the control after treatment (Fig. 2 ). Administration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p < 0.05) decrease SOD level when compared with the control (Fig. 4). However, all the groups treated with low doses showed no significant difference in SOD but the group treated with high dose showed significant (p < 0.05) decrease in SOD when compared with the control after treatment (Fig. 4). Administration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p < 0.05) decrease GPx level when compared with the control (Fig. 5). However, all the groups treated with low doses showed no significant difference in GPx but the group treated with high dose showed significant (p < 0.05) decrease in GSH when compared with the control after treatment (Fig. 5). Administration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p < 0.05) increase MDA level when compared with the control (Fig. 6). However, all the groups treated with low doses showed no significant difference in MDA but the group treated with high dose showed significant (p < 0.05) increase in MDA when compared with the control after treatment (Fig. 6). Administration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p < 0.05) decrease TAC level when compared with the control (Fig. 7). However, all the groups treated with low doses showed no significant difference in TAC but the group treated with high dose showed significant (p < 0.05) decrease in TAC when compared with the control after treatment (Fig. 7). Discussion When the quantity of reactive oxygen species (ROS) exceeds the amount of antioxidants that can scavenge them, oxidative stress takes place and might have harmful consequences. Reactive oxygen species' systemic manifestation and a biological system's capacity to quickly detoxify the reactive intermediates or repair the harm they cause are out of balance, which is what is known as oxidative stress [ 17 ]. When a cell's normal redox state is disturbed, peroxides and free radicals are produced, which harm various parts of the cell, including DNA, lipids, and proteins, and can have toxic effects [ 18 ]. In addition to base damage, oxidative stress resulting from oxidative metabolism also damages DNA strands [ 19 ]. The majority of base damage is indirect and results from the generation of reactive oxygen species, such as superoxide radical (O2 −), hydroxyl radical (OH), and hydrogen peroxide (H2O2) [ 20 ]. According to study [ 21 ], oxidative stress is thought to play a role in the development of attention deficit hyperactivity disorder, cancer, Parkinson's disease, Lafora disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, sickle-cell disease, lichen planus, vitiligo, autism, infection, chronic fatigue syndrome, and depression. This oxidative stress can be prevented by the body system through the production of antioxidant enzymes [ 22 ]. According to our findings, CS significantly increased LDH dose-dependently when compared with the control. This is in agreement with the studies of [ 8 , 23 ] who observed increase in LDH following the administration of CS. The work also confirmed a previous study [ 24 ] which reported that LDH plays a significant role in the generation of NADPH, which could be due to nicotinamide adenine dinucleotide phosphate (NADPH), a fuel for ROS generation. The reduction in TAC observed in cannabis-treated rats when juxtaposed with the control indicated a role for oxidative stress in their gonadotoxic consequences. This is also in line with the study of [ 8 ] who showed significant decrease in TAC following administration of different doses of CS in rats. This could have contributed to the build-up of ROS because it is consistent with the increase in lactate dehydrogenase activity that was previously seen in the cannabis-treated rats. Additionally, it was shown that CS increased MDA level which was dose dependent. This finding corresponds to that of [ 25 ] who observed increase in MDA level following administration of CS in rats. This finding suggests that CS may cause lipid peroxidation of polyunsaturated fatty acids, which ROS break down [ 26 ]. More so, the results indicated a decrease in glutathione reductase (GSH), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). These were consistent with the increase in lactate dehydrogenase activity in these animals, which led to accumulation of ROS. These are in line with the findings of [ 8 , 27 ] who observed decrease in antioxidant enzymes following administration of CS in rats. However, when compared to the control group following treatment after two weeks, the groups treated with high dose exhibited significant differences, while the other groups that were treated with low doses did not demonstrate any significant difference in any of the parameters. This could mean that it might take the body longer period of time to recover from oxidative damage brought on by the high dose of CS before withdrawal. Conclusion This study revealed that CS could stimulate oxidative stress which was dose dependent. However, withdrawal from its consumption could ameliorate the damage caused. It may be recommended that people should stay away from the consumption of CS because of its detrimental effect most especially on the oxidative stress in the body. Declarations Consent It is not applicable. Competing interests Authors have declared that no competing interests exist. Acknowledgement We acknowledged Mr. Emeka for the laboratory analysis of this research work. This research work did not receive any financial support but solely financed by the authors. Authors’ contributions AO designed the experimental work and prepare the manuscript. LNU carried out the research work. All authors proof read the manuscript. References Arif M et al (2023) Cannabis sativa L.-An Important Medicinal Plant: A Review of its Phytochemistry, Pharmacological Activities and Applications in Sustainable Economy. Int J Pharma Professional’s Res (IJPPR) 14(3):43–59 Chouvy P-A (2019) Cannabis cultivation in the world: heritages, trends and challenges. EchoGéo, (48) Sadiq IZ (2023) Free radicals and oxidative stress: Signaling mechanisms, redox basis for human diseases, and cell cycle regulation. Curr Mol Med 23(1):13–35 Demirci-Cekic S et al (2022) Biomarkers of oxidative stress and antioxidant defense. J Pharm Biomed Anal 209:114477 Sachdev S et al (2021) Abiotic stress and reactive oxygen species: Generation, signaling, and defense mechanisms. Antioxidants 10(2):277 Aranda-Rivera AK et al (2022) RONS and oxidative stress: An overview of basic concepts. Oxygen 2(4):437–478 Ifeanyi OE (2018) A review on free radicals and antioxidants. Int J Curr Res Med Sci 4(2):123–133 Oluwasola A et al Effect of Acute Administration of Ethanol Extract of Cannabis sativa Leaf on Oxidative Stress Biomarkers in Male and Female Wistar Rats. Martemucci G et al (2022) Oxidative stress, aging, antioxidant supplementation and their impact on human health: An overview. Mech Ageing Dev 206:111707 Yaribeygi H et al (2020) Molecular mechanisms linking oxidative stress and diabetes mellitus. Oxidative Med Cell Longev 2020(1):8609213 Zhang F et al (2020) Global discovery of human-infective RNA viruses: A modelling analysis. PLoS Pathog 16(11):e1009079 Eskandar K (2023) Treating COVID-19 using Micro RNA. J de la Faculté de Médecine 7(1):881–884 Claiborne A et al (1985) Antenatal diagnosis of cystic adenomatoid malformation: effect on patient management. Pediatr Radiol 15:337–339 Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70–77 Hunter C, Mestel L (1963) The structure and stability of self-gravitating disks. Mon Not R Astron Soc 126(4):299–315 Gutteridge JM, Wilkins S (1982) Copper-dependent hydroxyl radical damage to ascorbic acid: formation of a thiobarbituric acid-reactive product. FEBS Lett 137(2):327–330 Afzal S et al (2023) From imbalance to impairment: the central role of reactive oxygen species in oxidative stress-induced disorders and therapeutic exploration. Front Pharmacol 14:1269581 Valko M et al (2016) Redox-and non-redox-metal-induced formation of free radicals and their role in human disease. Arch Toxicol 90:1–37 Gonzalez-Hunt CP, Wadhwa M, Sanders LH (2018) DNA damage by oxidative stress: Measurement strategies for two genomes. Curr Opin Toxicol 7:87–94 Madkour LH (2019) Function of reactive oxygen species (ROS) inside the living organisms and sources of oxidants. Pharm Sci Anal Res J 2:180023 Verlaet AA et al (2019) Oxidative stress and immune aberrancies in attention-deficit/hyperactivity disorder (ADHD): A case–control comparison. Eur Child Adolesc Psychiatry 28:719–729 Bratovcic A (2020) Antioxidant enzymes and their role in preventing cell damage. Acta Sci Nutr Health 4:01–07 Mustafa T (2018) Enzymatic Oxidative Stress Biomarkers Alterations in Treated Addicted Female Rats. Polytechnic J 8(2):487–495 Ying M et al (2021) Lactate and glutamine support NADPH generation in cancer cells under glucose deprived conditions. Redox Biol 46:102065 Kubiliene A et al (2021) The effects of cannabis sativa L. Extract on oxidative stress markers in vivo. Life 11(7):647 Collodel G et al (2015) Semen characteristics and malondialdehyde levels in men with different reproductive problems. Andrology 3(2):280–286 Umoren E et al (2020) The effect of ethanolic extract of Cannabis sativa leaves from Nigeria on the antioxidants markers in albino wistar rats. Int J Biochem Res Rev 29(9):58–65 Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4686688","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322726616,"identity":"7e3e1dba-4dfb-4d62-87a3-78650b8cc6d2","order_by":0,"name":"Oluwasola Amuda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABGklEQVRIie3QMUsDMRTA8RcCd0tq1xw5/QwB4RwK3lfJITTLDRVBDhQ8EK6L4CaI+B106ZwS0CWIo1KHm27qoEtxkGKuXUPLbSL5Ty/Dj8cLgM/3F1OA1GoIAdUwUoQCBPaJNxFYEwyYA+9IAmoJbCU7M610rzpM+xg3xTf/iKMb3dRQDLKSjV9dJHoZCkuOstvLIHm/4g1hbHjAwcisjM3IRbghXPcmWHANyZudyR4TCUWVzkqaiw3kIuU6XBz/rIhcULTcSjR60CTB7RbGcrulbIlUzltMIKb3y2d7CzlhsSXRXX5KxZPcr2ju/jGDp/XcnKX9cPz4NS90SmdyQj/PB7vXVNZO4649IgDCO5B1YZctPp/P93/7BQhZYkr8XS/JAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0002-3288-7585","institution":"Al-Hikmah University","correspondingAuthor":true,"prefix":"","firstName":"Oluwasola","middleName":"","lastName":"Amuda","suffix":""},{"id":322727664,"identity":"9f192d46-1620-4c0f-9940-cb632903dc54","order_by":1,"name":"L.N. 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[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], hence the quest for harnessing its pharmacological potential by scientists. This substance is widely used as an illicit drug globally [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Cells' normal redox state can be disrupted, leading to the production of peroxides and free radicals, damaging all cell components, including proteins, lipids, and DNA [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Oxidative stress (OS) is caused by a balance between pro-oxidants and antioxidants [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The ratio can be influenced by elevated levels of reactive oxygen species (ROS) or a decrease in antioxidant defense mechanisms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. OS can arise from an imbalance in the body's oxidizing system, primarily composed of free radicals, reactive oxygen species (ROS), and reactive nitrogen species (RNS) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and Antioxidant systems are essential in neutralizing free radicals that can have numerous harmful effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. OS is involved in aging [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and is present in certain chronic diseases like diabetes, cancers, hypertension, and coronary heart disease., etc. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and certain infections, particularly by the RNA viruses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], a family to which belong corona viruses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However few researches have been done on effect of different doses of CS in Wistar. In lieu of this, the research work examined the short term effect of different concentrations of CS in male wistar rats and the ability of the rats treated with CS to recover from the oxidative stress caused during administration of CS before abstinence.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection\u003c/h2\u003e \u003cp\u003eCannabis sativa (CS) leaves were donated by National Drug Law Enforcement Agency (NDLEA), Nigeria, for research purpose only.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Extraction of \u003cem\u003eCannabis sativa\u003c/em\u003e leaves\u003c/h2\u003e \u003cp\u003eExtraction of \u003cem\u003eCannabis sativa\u003c/em\u003e (CS), was done with Soxhlet apparatus by soaking 800 grams of CS in 98% ethanol for 48 hours. It was filtered and the filtrate was poured into a round bottom conical flask which was fixed with a rotary evaporator. It was then evaporated and cooled. The dried yield of the extract was 55g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental animals\u003c/h2\u003e \u003cp\u003eTwenty male rats (160g\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98) that were used for this research were obtained from Temilade Animal Venture, Ogbomoso, Oyo State, were housed at room temperature with unrestricted access to diet and water and maintained on a daily light/dark cycle. Principles of laboratory animal care (NIH publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1985) were followed. The experimental protocol was approved by Ethical Committee of Al-Hikmah University, Ilorin, Nigeria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental protocol\u003c/h2\u003e \u003cp\u003eAfter 2 weeks of acclimatization, the animals were separately assigned into four groups of ten animals each, such that the rats in groups 1, 2, 3 and 4 respectively received orally 1 ml of distilled water, 2 mg/kg body weight (bw) of CS, 4 mg/kg bw of CS and 6 mg/kg bw of CS respectively for two weeks. The animals were sacrificed on the 15th day with access to food and water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Preparation of serum\u003c/h2\u003e \u003cp\u003eThe male and female rats were sacrificed under ketamine anesthesia and blood was collected by cardiac puncture into sample bottles. The blood was left for 30 min to clot and thereafter centrifuged at 625\u0026times;\u003cem\u003eg\u003c/em\u003e for 10 min using a Uniscope Laboratory Centrifuge (Model SM800B, Surgifield Medicals, Essex, England). The serum was collected into plain bottles with the aid of a Pasteur pipette. Sera were stored in a freezer maintained at -5 ℃ and used within 12 hours of preparation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Drug and assay kits\u003c/h2\u003e \u003cp\u003eLactate dehydrogenase (LDH) activity was assayed spectrophotometrically (Spectramax Plus; Molecular Devices, Sunnyvale, CA, USA) following the kit manufacturer\u0026rsquo;s procedures (product code BXC0243; Fortress Diagnostics, UK). The determination of serum superoxide dismutase (SOD) concentration was done with SOD colorimetric assay kit (Fortress Diagnostics Ltd., Antrim, UK; Product code: BXC0531), following the manufacturer\u0026rsquo;s protocols. The determination of serum glutathione peroxidase (GPx) activity was done with GPx colorimetric assay kit (BioVision Inc., Milpitas, CA, USA), following the manufacturers protocols. Based on the manufacturer\u0026rsquo;s protocol, total anti-oxidant capacity (TAC) measurement in the serum was done with a spectrophotometric microplate reader (Spectramax Plus, Molecular Devices, Sunnyvale, CA, USA) using OxiSelect TAC assay kit that uses the single electron transfer mechanism (Cell Biolabs, Inc. San Diego, CA. cat no: STA-360). The continuous catalase activity was determined through spectrophotometric reading [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Reduced glutathione (GSH) was measured according to the method of [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The assay method of [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], modified by [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] was adopted for Malondiadehyde (MDA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eResults were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean. Data were analyzed using a two-way Analysis Of Variance, followed by the LSD post-hoc test to determine significant differences in all the parameters graph pad, version 9.0. Differences with values of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003cp\u003eAdministration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase LDH level when compared with the control (Fig.\u0026nbsp;1). However, all the groups treated with low doses showed no significant difference in LDH but the group treated with high dose showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in LDH when compared with the control after treatment (Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eAdministration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease catalase level when compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, all the groups treated with low doses showed no significant difference in catalase but the group treated with high dose showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in catalase when compared with the control after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAdministration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease SOD level when compared with the control (Fig.\u0026nbsp;4). However, all the groups treated with low doses showed no significant difference in SOD but the group treated with high dose showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in SOD when compared with the control after treatment (Fig.\u0026nbsp;4).\u003c/p\u003e\u003cp\u003eAdministration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease GPx level when compared with the control (Fig.\u0026nbsp;5). However, all the groups treated with low doses showed no significant difference in GPx but the group treated with high dose showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in GSH when compared with the control after treatment (Fig.\u0026nbsp;5).\u003c/p\u003e\u003cp\u003eAdministration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase MDA level when compared with the control (Fig.\u0026nbsp;6). However, all the groups treated with low doses showed no significant difference in MDA but the group treated with high dose showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in MDA when compared with the control after treatment (Fig.\u0026nbsp;6).\u003c/p\u003e\u003cp\u003eAdministration of high dose (6 mg) and low doses (2mg and 4 mg) of CS significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease TAC level when compared with the control (Fig.\u0026nbsp;7). However, all the groups treated with low doses showed no significant difference in TAC but the group treated with high dose showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in TAC when compared with the control after treatment (Fig.\u0026nbsp;7).\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003cp\u003eWhen the quantity of reactive oxygen species (ROS) exceeds the amount of antioxidants that can scavenge them, oxidative stress takes place and might have harmful consequences. Reactive oxygen species' systemic manifestation and a biological system's capacity to quickly detoxify the reactive intermediates or repair the harm they cause are out of balance, which is what is known as oxidative stress [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When a cell's normal redox state is disturbed, peroxides and free radicals are produced, which harm various parts of the cell, including DNA, lipids, and proteins, and can have toxic effects [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition to base damage, oxidative stress resulting from oxidative metabolism also damages DNA strands [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The majority of base damage is indirect and results from the generation of reactive oxygen species, such as superoxide radical (O2 \u0026minus;), hydroxyl radical (OH), and hydrogen peroxide (H2O2) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. According to study [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], oxidative stress is thought to play a role in the development of attention deficit hyperactivity disorder, cancer, Parkinson's disease, Lafora disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, sickle-cell disease, lichen planus, vitiligo, autism, infection, chronic fatigue syndrome, and depression. This oxidative stress can be prevented by the body system through the production of antioxidant enzymes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to our findings, CS significantly increased LDH dose-dependently when compared with the control. This is in agreement with the studies of [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] who observed increase in LDH following the administration of CS. The work also confirmed a previous study [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] which reported that LDH plays a significant role in the generation of NADPH, which could be due to nicotinamide adenine dinucleotide phosphate (NADPH), a fuel for ROS generation. The reduction in TAC observed in cannabis-treated rats when juxtaposed with the control indicated a role for oxidative stress in their gonadotoxic consequences. This is also in line with the study of [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] who showed significant decrease in TAC following administration of different doses of CS in rats. This could have contributed to the build-up of ROS because it is consistent with the increase in lactate dehydrogenase activity that was previously seen in the cannabis-treated rats.\u003c/p\u003e \u003cp\u003eAdditionally, it was shown that CS increased MDA level which was dose dependent. This finding corresponds to that of [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] who observed increase in MDA level following administration of CS in rats. This finding suggests that CS may cause lipid peroxidation of polyunsaturated fatty acids, which ROS break down [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. More so, the results indicated a decrease in glutathione reductase (GSH), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). These were consistent with the increase in lactate dehydrogenase activity in these animals, which led to accumulation of ROS. These are in line with the findings of [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] who observed decrease in antioxidant enzymes following administration of CS in rats.\u003c/p\u003e \u003cp\u003eHowever, when compared to the control group following treatment after two weeks, the groups treated with high dose exhibited significant differences, while the other groups that were treated with low doses did not demonstrate any significant difference in any of the parameters. This could mean that it might take the body longer period of time to recover from oxidative damage brought on by the high dose of CS before withdrawal.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed that CS could stimulate oxidative stress which was dose dependent. However, withdrawal from its consumption could ameliorate the damage caused. It may be recommended that people should stay away from the consumption of CS because of its detrimental effect most especially on the oxidative stress in the body.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledged Mr. Emeka for the laboratory analysis of this research work. This research work did not receive any financial support but solely financed by the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAO designed the experimental work and prepare the manuscript. LNU carried out the research work. All authors proof read the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArif M et al (2023) Cannabis sativa L.-An Important Medicinal Plant: A Review of its Phytochemistry, Pharmacological Activities and Applications in Sustainable Economy. Int J Pharma Professional\u0026rsquo;s Res (IJPPR) 14(3):43\u0026ndash;59\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChouvy P-A (2019) \u003cem\u003eCannabis cultivation in the world: heritages, trends and challenges.\u003c/em\u003e EchoG\u0026eacute;o, (48)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadiq IZ (2023) Free radicals and oxidative stress: Signaling mechanisms, redox basis for human diseases, and cell cycle regulation. Curr Mol Med 23(1):13\u0026ndash;35\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemirci-Cekic S et al (2022) Biomarkers of oxidative stress and antioxidant defense. J Pharm Biomed Anal 209:114477\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSachdev S et al (2021) Abiotic stress and reactive oxygen species: Generation, signaling, and defense mechanisms. Antioxidants 10(2):277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAranda-Rivera AK et al (2022) RONS and oxidative stress: An overview of basic concepts. Oxygen 2(4):437\u0026ndash;478\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIfeanyi OE (2018) A review on free radicals and antioxidants. Int J Curr Res Med Sci 4(2):123\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOluwasola A et al \u003cem\u003eEffect of Acute Administration of Ethanol Extract of Cannabis sativa Leaf on Oxidative Stress Biomarkers in Male and Female Wistar Rats.\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartemucci G et al (2022) Oxidative stress, aging, antioxidant supplementation and their impact on human health: An overview. Mech Ageing Dev 206:111707\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYaribeygi H et al (2020) Molecular mechanisms linking oxidative stress and diabetes mellitus. Oxidative Med Cell Longev 2020(1):8609213\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang F et al (2020) Global discovery of human-infective RNA viruses: A modelling analysis. PLoS Pathog 16(11):e1009079\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEskandar K (2023) Treating COVID-19 using Micro RNA. J de la Facult\u0026eacute; de M\u0026eacute;decine 7(1):881\u0026ndash;884\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClaiborne A et al (1985) Antenatal diagnosis of cystic adenomatoid malformation: effect on patient management. Pediatr Radiol 15:337\u0026ndash;339\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70\u0026ndash;77\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunter C, Mestel L (1963) The structure and stability of self-gravitating disks. Mon Not R Astron Soc 126(4):299\u0026ndash;315\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGutteridge JM, Wilkins S (1982) Copper-dependent hydroxyl radical damage to ascorbic acid: formation of a thiobarbituric acid-reactive product. FEBS Lett 137(2):327\u0026ndash;330\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfzal S et al (2023) From imbalance to impairment: the central role of reactive oxygen species in oxidative stress-induced disorders and therapeutic exploration. Front Pharmacol 14:1269581\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValko M et al (2016) Redox-and non-redox-metal-induced formation of free radicals and their role in human disease. Arch Toxicol 90:1\u0026ndash;37\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez-Hunt CP, Wadhwa M, Sanders LH (2018) DNA damage by oxidative stress: Measurement strategies for two genomes. Curr Opin Toxicol 7:87\u0026ndash;94\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadkour LH (2019) Function of reactive oxygen species (ROS) inside the living organisms and sources of oxidants. Pharm Sci Anal Res J 2:180023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerlaet AA et al (2019) Oxidative stress and immune aberrancies in attention-deficit/hyperactivity disorder (ADHD): A case\u0026ndash;control comparison. Eur Child Adolesc Psychiatry 28:719\u0026ndash;729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBratovcic A (2020) Antioxidant enzymes and their role in preventing cell damage. Acta Sci Nutr Health 4:01\u0026ndash;07\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustafa T (2018) Enzymatic Oxidative Stress Biomarkers Alterations in Treated Addicted Female Rats. Polytechnic J 8(2):487\u0026ndash;495\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYing M et al (2021) Lactate and glutamine support NADPH generation in cancer cells under glucose deprived conditions. Redox Biol 46:102065\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKubiliene A et al (2021) The effects of cannabis sativa L. Extract on oxidative stress markers in vivo. Life 11(7):647\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollodel G et al (2015) Semen characteristics and malondialdehyde levels in men with different reproductive problems. Andrology 3(2):280\u0026ndash;286\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmoren E et al (2020) The effect of ethanolic extract of Cannabis sativa leaves from Nigeria on the antioxidants markers in albino wistar rats. Int J Biochem Res Rev 29(9):58\u0026ndash;65\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cannabis sativa, Oxidative stress, Dose dependent, Recovery period","lastPublishedDoi":"10.21203/rs.3.rs-4686688/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4686688/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the effect of CS on oxidative stress and the recovery period in male rats. Forty rats (170 g\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24) were separately assigned into four groups of ten animals each, such that the rats in groups 1, 2, 3 and 4 received orally 1 ml of distilled water, 2mg, 4mg and 6mg of CS respectively for two weeks. Catalase, superoxide dismutase (SOD), Glutathione peroxidase (GPx), Glutathione reductase (GSH), malondialdehyde (MDA), total antioxidant capacity (TAC) and lactate dehydrogenase (LDH) were determined using standard methods.\u003c/p\u003e \u003cp\u003eHigh dose (6 mg) and low doses (2mg and 4 mg) of CS significantly decrease catalase, SOD, GPx, GSH, TAC and significantly increase MDA and LDH levels when compared with the control. However, all the groups treated with low doses showed no significant difference in all the parameters when compared with the control after treatment.\u003c/p\u003e \u003cp\u003eIn conclusion, it could be deduced that these alterations in oxidative stress biomarkers were dependent on the doses of CS consumed. However, groups treated with low doses were able to recover from the damages caused by CS after treatment. This study recommends that people should abstain from the consumption of CS due to its detrimental effect in the body.\u003c/p\u003e","manuscriptTitle":"Effect of Cannabis Sativa Leaf on Oxidative Stress and the Recovery Period in Male Wistar Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-08 03:52:27","doi":"10.21203/rs.3.rs-4686688/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91d95c57-8bde-4908-aee2-f230646a91ed","owner":[],"postedDate":"July 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34249041,"name":"Nutrition \u0026 Dietetics"}],"tags":[],"updatedAt":"2024-07-08T03:52:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-08 03:52:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4686688","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4686688","identity":"rs-4686688","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}