Objective: The objective of the study was to evaluate the toxicological potential of the ethanolic extract of leaves of Mirabilis jalapa linn through acute and subacute toxicity studies in albino Wistar rats.
Methods: For acute toxicity studies, the ethanolic extract of M. jalapa was given up to 2000 mg/kg and then the animals were observed for 14 days to find out any adverse effect or death. For sub-acute toxicity studies, the exact was given for 28 days and the following parameters were observed such as changes in body weight, food intake, water intake, hematological parameters, biochemical parameters, lipid profile, urine analysis, and histopathological studies were undertaken.
Results: Single oral administration of 2000 mg/kg of the ethanolic extract of M. jalapa produced no mortality or signs of toxicity. During subacute toxicity there were no changes in body weight, food intake and water intake were observed. There were no changes in lipid profile, hematological parameters, and biochemical parameters. In histopathological changes, there were no structural changes in treated groups when compared to control.
Conclusion: The leaves of ethanolic extract of M. jalapa is safe when administered for 28 days.
There were no deaths or signs of toxicity in treated rats during acute toxicity studies and subacute toxicity studies

C, SENTHIL KUMARI

KESAVELU, DHANASEKHAR

Innovare Academic Sciences: E-Journals

Vol 14, Issue 6, 2021Online - 2455-3891 Print - 0974-2441ACUTE AND SUBACUTE TOXICITY STUDIES OF ETHANOLIC EXTRACT OF MIRABILIS JALAPA LINN IN WISTAR ALBINO RATSSENTHIL KUMARI C1*, DHANASEKHAR KESAVELU21Department of Pharmacology, K.K. College of Pharmacy, The Tamil Nadu Dr. M.G.R Medical University, Chennai, Tamil Nadu, India. 2Department of Paediatric Gastroenterology, Apollo Children’s Hospital, Tamil Nadu, India. Email: senmmc@yahoo.comReceived: 09 April 2021, Revised and Accepted: 15 May 2021ABSTRACTObjective: The objective of the study was to evaluate the toxicological potential of the ethanolic extract of leaves of Mirabilis jalapa linn through acute and subacute toxicity studies in albino Wistar rats.Methods: For acute toxicity studies, the ethanolic extract of M. jalapa was given up to 2000 mg/kg and then the animals were observed for 14 days to find out any adverse effect or death. For sub-acute toxicity studies, the exact was given for 28 days and the following parameters were observed such as changes in body weight, food intake, water intake, hematological parameters, biochemical parameters, lipid profile, urine analysis, and histopathological studies were undertaken.Results: Single oral administration of 2000 mg/kg of the ethanolic extract of M. jalapa produced no mortality or signs of toxicity. During subacute toxicity there were no changes in body weight, food intake and water intake were observed. There were no changes in lipid profile, hematological parameters, and biochemical parameters. In histopathological changes, there were no structural changes in treated groups when compared to control.Conclusion: The leaves of ethanolic extract of M. jalapa is safe when administered for 28 days.There were no deaths or signs of toxicity in treated rats during acute toxicity studies and subacute toxicity studies.Keywords: Mirabilis jalapa, Hematological parameters, Histological changes, Toxicity studies.INTRODUCTIONMirabilis jalapa linn is a herbal medicine commonly called Four O Clock or Marble of Peru [1].Synonyms are Mirabilis lindheimeri, Mirabilis dichotoma, Mirabilis odorata, and Common Names are Clavillia, four-o’clocks, jalap, maravilla, bonina. In Tamil Nadu, it is known as andhi mandhaarai and in Andhra Pradesh it is called as Chandrakantha indraganti, and in Karnataka it is called as sanje mallige, in Malayalam it is called as Naalumani poovu, in Maharashtra it is called as gulabakshi, in Odisha it is called as rangani, and in Punjab it is called as sham di sohnap, which means “evening beauty” [2]. Branching stems grows to an extent of 36” with tall and erect in size and it is a perennial herb which is bushy and tender. Generally grows in various colors such as pink, rose, red, magenta, yellow, and white and different color appears in same plant. The flower blossom around 4 O’ Clock and continue to blossom till morning. Size of the leaves is in Ovate and Dark Green color and generally appears as heart shape at the base [3]. It is used traditionally for purgative, emetic, genitourinary system disorders, muscle pain, abdominal colic, inflammation, in skin disease, as anesthetic, as anthelmintic, in bronchitis, in kidney problems, and in blood and vaginal disease [4].METHODSPlant collection and authentication of plant materialThe leaves of the plant of M. jalapa were collected from in and around Kanyakumari district. The plant was identified and authenticated by Dr. Jeyaraman, Assistant Professor of Botany, Chennai.Extraction processThe leaves were shade dried at room temperature. The dried leaves were subjected to size reduction to a coarse powder using dry grinder and pass through sieve. The powder was packed in Soxhlet apparatus and extracted successively with ethanol (60–80°C) [5]. The extract was dried at 45°C in hot air oven till solid to semi-solid mass is obtained and is stored in air tight container in a refrigerator below 10°C [6].Experimental animalsAlbino rats (Wistar rats) weighing (150–250 g) were placed in a polypropylene cages in a controlled room temperature 24±1°C and relative humidity of 60–70% in animal house. The animals were maintained in standard pellet diet and water ad libitum. They were acclimatized to laboratory condition for 7 days before commencement of the experiment [7].Animal experimentation protocols were approved by Institutional Animal Ethical Committee in K.K. College of Pharmacy, Chennai with the reference no. (Ref: KKCP/2015/038).Drug and preparation of stock solutionThe ethanolic extract of M. jalapa was prepared in 1% carboxymethylcellulose solution in distilled water before oral administration to animals. It was used within 7 days and stored at 8°C while for further use, freshly prepared solution was used. The vehicle alone served as control.Toxicity studiesAcute oral toxicity (OECD -423 Guidelines)Three animals are used for each step. The dose level used as the starting dose was selected from one of four fixed levels, 5, 50, 300, and 2000 mg/kg body weight [8]. Animals were observed individually for the signs of toxicity all observations are systematically recorded with individual records being maintained for each animal. Attention was directed to observations of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma.© 2021 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ajpcr.2021v14i6.40979. Journal homepage: https://innovareacademics.in/journals/index.php/ajpcrResearch Article81Asian J Pharm Clin Res, Vol 14, Issue 6, 2021, 80-85 Kumari and KesaveluSub-acute oral toxicity study (OECD – 407 guidelines)From the results of acute toxicity, studies in Wistar rats indicated that M. jalapa was nontoxic and no behavioral changes were observed up to the dose level of 2000 mg/kg body weight. The test doses were selected as 200 and 400 mg/kg [9]. Ten rats (Five Male and Five Female) were in each group randomly divided into four groups for dosing up to 28 days. The test substance was freshly Table 7: Lipid profileParameters Control 200 mg/kg 400 mg/kgBlood glucose (mg/dl) 126.20±0.02 126.42±1.24 126.12±1.24Triglycerides(mg/kg) 80.12±1.22 79.12±1.01 78.11±0.14High-density lipoprotein (mg/dl)12.08±1.02 12.44±1.20 12.88±1.28Total cholesterol (mg/kg)40.20±1.01 38.24±1.24 38.16±1.28Low-density lipoprotein (mg/dl)38.10±1.04 40.12±1.42 40.00±1.04Very-low-density lipoproteins (mg/dl)15.62±1.02 15.22±1.08 15.01±1.24Total cholesterol/High-density lipoprotein ratio (g/dl)2.02±0.22 2.44±1.20 3.12±0.04Values are mean of a six animals±S.E.M (Dunnett’s test) *p<0.05;**p<0.01.n=6Table 6: Renal function testDose (mg/kg) Control 200 (mg/kg) 400 (mg/kg)Urea (mg/dl) 56.21±1.23 55.80±1.02 53.09±1.21Creatinine (mg/dl) 0.74±0.01 0.74±0.02 0.76±0.04Uric acid (mg/dl) 1.6±0.02 1.6±0.04 1.6±0.06Na m.mol 136.4±1.02 138.2±1.02 140.01±1.20K m.mol 18.01±1.14 19.02±1.08 19.42±1.22Cl m.mol 96.04±1.02 99.20±0.12 100.04±0.22Values are mean of a six animals±S.E.M (Dunnett’s test) *p<0.05;**p<0.01.n=6Table 5: Effect of treatment with ethanolic extract of Mirabilis jalapa Linn on biochemical parametersDose (mg/kg) Control 200 mg/kg 400 mg/kgTotal bilirubin  (mg/dl)0.210±0.11 0.214±0.10 0.216±0.16Bilirubin direct  (mg/dl)0.1±0.02 0.1±0.04 0.1±0.04Bilirubin indirect (mg/dl)0.1±00 0.1±00 0.1±00ALP (U/L) 382.02±10.22 360.46±10.12 340.12±12.04Serum glutamate oxaloacetate transaminase (U/L)160.02±1.06 160.11±1.04 159.26±1.12Serum glutamic-pyruvic transaminase (U/L)46.14±0.20 46.20±0.24 46.46±0.02Total protein (g/dl) 10.01±0.08 9.02±0.11 9.01±1.48Albumin (g/dl) 3.10±0.02 3.10±0.04 3.12±0.04Globulin (g/dl) 6.08±0.12 5.84±0.24 5.42±0.24LFT: Liver function test. Values are mean of a six animals±S.E.M (Dunnett’s test)* p<0.05;**p<0.01.n=6Table 4: Hematological parameters after 28 days treatment with ethanolic extract of Mirabilis jalapa Linn in ratsParameters Control 200 mg/kg 400 mg/kgRed blood cell (mm3)7.18±0.24 8.02±1.44 9.46±1.28HB (%) 14.22±0.10 15.42±0.28 16.48±1.0212Leukocyte (×106/ml)10144±200.12 10228±241.27 10302±252.24Platelets/ul 1432±21.22 1411±30.20 1462±32.01Mean corpuscular volume (gl)55.02±1.02 56.02±2.22 57.12±2.08Values are mean of a six animals ± S.E.M (Dunnett’s test)* p<0.05;**p<0.01.n=6Table 3: Food (g/day) intake of albino rats exposed to ethanolic extract of Mirabilis jalapa Linn for 28 daysDose (mg/kg/day) Days (g/rats)1 7 14 21 28Control 40.10±1.12 42.46±1.21 42.24±0.24 44.20±1.20 46.12±1.04200 mg/kg M.E 42.20±0.44 45.28±0.18 47.22±1.02 50.16±1.02 52.11±0.22400 mg/kg M.E 45.21±0.12 46.22±1.606 48.12±0.13 50.24±1.08 54.12±0.22Values are mean of a six animals±S.E.M (Dunnett’s test)*p<0.05;**p<0.01.n=6Table 2: Water (ml/day) intake of albino rats exposed to for 28 daysDose (mg/kg/day) Days (ml/rat)1 7 14 21 28Control 44.01±0.11 46.22±0.04 45.21±0.32 46.13±0.97 46.64±0.11200 mg/kg M.E 48.42±0.63 52.08±0.14 55.67±0.12 58.20±1.24 60.12±1.02400 mg/kg M.E 50.28±0.61 54.22±0.44 58.12±1.08 62.01±1.44 64.02±1.21Values are mean of a six animals±S.E.M (Dunnett’s test) *p<0.05;**p<0.01.n=6Table 1: Body weight (g) of albino rats exposed to ethanolic extract of Mirabilis jalapa Linn for 28 daysDose (mg/kg/day) Days1 7 14 21 28Control 1 40.24±1.02 142.22±1.06 148.04±1.46 150.11±0.44 152.01±0.21200 mg/kg M.E 144.11±0.01 142.24±1.65 140.22±1.21 138.20±0.11 136.12±0.01400 mg/kg M.E 142.01±0.02 140.20±1.02 139.01±1.12 138.01±1.04 136.22±0.48Values are mean of a six animals ± S.E.M (Dunnett’s test) *p<0.05;**p<0.01.n=682Asian J Pharm Clin Res, Vol 14, Issue 6, 2021, 80-85 Kumari and Kesaveluprepared every day for 28 days [10]. The control animals were administered vehicle only.Body weightWeight of each rat was recorded on day 0, at weekly intervals throughout the course of study and at termination to calculate relative organ weights. From the data, group mean body weights and percent body weight gain were calculated [11].Food and water consumptionThe quantity of food consumed by groups consisting of six animals for different doses was recorded at weekly interval. Food consumed per animal was calculated for control and the treated dose groups.Clinical signsAll animals were observed daily for clinical signs. The time of onset, intensity, and duration of these symptoms, if any, were recorded.MortalityAll animals were observed twice daily for mortality during entire course of study.Terminal studiesLaboratory investigationsFollowing laboratory investigations were carried out on day 29 in fasted animal’s over-night. On 29th day, the animals were anesthetized with ether and blood samples were collected from the retro-orbital plexus into two tubes: One with EDTA for immediate analysis of hematological parameters, and the other without any anticoagulant and was centrifuged at 4000 rpm at 4°C for 10 min to obtain the serum. Serum was stored at 20°C until analyzed for biochemical parameters.Hematological investigationsBlood samples of control and experimental rats were analyzed for hemoglobin content, total red blood corpuscles, white blood corpuscles count, mean corpuscular volume, and platelets. Then, the mean values are calculated [12].Biochemical investigationsSerum and urine was used for the estimation of biochemical parameters. Samples of control and experimental rats were analyzed for protein, albumin, globulin, bilirubin, urea, uric acid, creatinine, potassium, chlorine, and sodium levels were carried using standard methods. Activities of glutamate oxaloacetate transaminase/aspartate aminotransferase, glutamate pyruvate transaminase/alanine amino transferase, and alkaline phosphatase were estimated as per the colorimetric procedure [13].Urine analysisUrine samples were collected in week 4 and in week 6 and for the estimation of normal parameters.NecropsyAll the animals were sacrificed on day 29. Necropsy of all animals was carried out and the weights of the organs including liver, kidneys, adrenals, spleen, brain, heart, uterus, and testes/ovaries were recorded.HistopathologyHistopathological investigation of the vital organs was done. The organs include stomach, heart, kidneys, liver, and spleen of the animals were preserved they were subjected to histopathological examination [14].Statistical analysisFindings such as clinical signs of intoxication, body weight changes, food consumption, hematology and biochemical parameters were subjected to One-way ANOVA followed by Dunnett’s test.RESULTSAcute oral toxicity studiesThere were no signs of toxicity up to 2000 mg/kg of the extract; it shows the absence of pilo erection, righting reflux, seizures, any stains of urine, loss of righting reflux, alopecia, edema, lacrimation, etc.Subacute oral toxicity studiesAdministration of ethanolic extract of mirabilis jalapa 200mg/kg and 400mg/kg during 28 days had no significant changes on body weight (Table 1), water intake (Table 2), food intake (Table 3) when compared ControlMirabilis jalapa Linn (200 mg/kg)Mirabilis jalapa Linn (400 mg/kg)83Asian J Pharm Clin Res, Vol 14, Issue 6, 2021, 80-85 Kumari and Kesaveluto control group. The effect of the extract on hematological parameters are shown in (Table 4) result in no significant changes when compared to the control group. The results of biochemical parameters (Table 5), showed no significant difference in all parameters when compared to control. Administration of the extract for 28 days had no significant changes in renal function test (Table 6), lipid profile (Table 7) urine analysis (Table 8) when compared to control group. The effect of different doses of administration of ethanolic extract of mirabilis jalapa on organ weight (Table 9) showed no significant changes when compared to control.  Histopathological StudiesKidneyHeartMirabilis jalapa Linn (200 mg/kg)ControlControlMirabilis jalapa Linn (200 mg/kg)Mirabilis jalapa Linn (400 mg/kg) Mirabilis jalapa Linn (400 mg/kg)84Asian J Pharm Clin Res, Vol 14, Issue 6, 2021, 80-85 Kumari and KesaveluLiverSpleenStomachDISCUSSIONIn acute toxicity studies, it showed no toxic signs up to 2000 mg/kg body weight and does not produce any mortality or toxic effect. In subacute toxicity studies, the extract 200 mg/kg and 400 mg/kg have been given for a period of 28 days. During this period, there was a gradual normal increase in the mean body weight of the extract and control group. There were no changes in food intake and water intake of treated groups. In assessment of hematological parameters, the readings are all within the reference range for rats. In biochemical parameter, the extract 200 mg/kg and 400 mg/kg of did not show any significant changes in extract Mirabilis jalapa Linn (200 mg/kg)Mirabilis jalapa Linn (400 mg/kg)ControlMirabilis jalapa Linn (400 mg/kg)Mirabilis jalapa Linn (200 mg/kg)Control85Asian J Pharm Clin Res, Vol 14, Issue 6, 2021, 80-85 Kumari and Kesavelutreated groups. In histopathogical studies, liver was excised from all treated groups to analyze the injuries in bile ducts, hepatic vein, and portal area. It showed that there was no modification detected when compared to control group. In spleen, heart, intestine, there were no structural changes when compared to control. Kidney tissues also examined for any morphological changes. There were no morphological changes when compared to control group.CONCULSIONIn administration of the ethanolic extract of M. jalapa 200 mg/kg and 400 mg/kg for a period of 28 days, there was no changes in food intake, water intake, biochemical parameters, lipid profile, renal function, and urine analysis when compared to control. There were no signs of toxicity observed in heart, kidney, stomach, spleen, and liver. The ethanolic extract of M. jalapa is a safer and nontoxic and further studies have to be carried out for its medicinal and therapeutic efficacy.ACKNOWLEDGEMENTThe author would like to acknowledge the Department of Pharmacology, K.K. college of Pharmacy, Chennai.AUTHOR’S CONTRIBUTIONSAll authors have contributed equally.CONFLICTS OF INTERESTThe authors declare no conflict of interest.FUNDINGNil.REFERENCES1. Liya FI, Yasmin MF, Chowdhury NS. Mirabilis jalapa: A review of ethno and pharmacological activities. Adv Med Plant Res 2021;9:1-10.2. Nidavani RB, Mahalakshmi AM. An ethanopharmacological review of four o’clock flower plant (Mirabilis jalapa Linn). J Biol Sci Opin 2014;6:344-8.3. Saha S, Deb J, Deb NK. Review on Mirabilis jalapa L., (nyctaginaceae): A medicinal plant. Int J Herbal Med 2020;8:14-8.4. Singh M, Garg A, Mittal SK, Kalia AN. Mirabilis jalapa-a review. Int J Pharm Chem Appl Sci 2012;1:1-22.5. Satheeskumar KG. Significant role of Soxhlet extraction process in phytochemical. Mintage J Pharm Med Sci 2018;7:43-7.6. Azwanida NN. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med Aromatic Plants 2015;4:2-4.7. Ekanayake CP, Thammitiyagodage MG, Padumadasa S, Seneviratne B, Padumadasa C. Abeysekera AM. Acute and subacute toxicity studies of the ethyl acetate soluble proanthocyanidins of the immature inflorescence of Cocos nucifera L. in female Wistar rats. Biomed Res Int 2019;2019:8428304.8. OECD Ilibrary, OECG Guidelines for the Testing of Chemicals Section 4; 2008.9. 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Patel SS, Verma S, Nayak G, Singhai AK, Ganesh N. Acute and sub-acute toxicity studies of Passiflora nepalensis in rats. Rev Bras Farmacogn 2011;21:730-6.Table 9: Effect of oral administration of ethanolic extract of Mirabilis jalapa Linn on organ weightDose (mg/kg) Control 200mg/kg 400mg/kgLiver (g) 4.22±0.20 4.84±0.12 5.10±0.89Heart (g) 0.60±0.01 0.60±0.02 0.62±0.02Lung (g) 1.44±0.02 1.42±0.04 1.42±0.06Spleen (g) 0.54±0.02 0.54±0.04 0.56±0.08Ovary (g) 1.54±0.08 1.56±0.02 1.58±0.02Testes (g) 1.26±0.06 1.28±0.26 1.32±0.18Brain (g) 1.42±0.12 1.44±0.01 1.52±0.14Kidney (g) 0.64±0.01 0.66±0.02 0.68±0.02Stomach (g) 1.34±0.1 1.38±0.10 1.38±0.11Values are mean of a six animals±S.E.M (Dunnett’s test) *p<0.05;**p<0.01. n=6Table 8: Urine analysisParameters Control 200 mg/kg 400 mg/kgTransparency Clear Slightly turbid Slightly turbidSpecific gravity 1.010 1.010 1.010PH >7.2 >7.4 >7.4Protein Nil 2+ 2+Glucose Nil Nil NilBilirubin −ve −ve −veKetones −ve −ve −veBlood Absent Absent AbsentUrobilinogen Normal Abnormal AbnormalPus cells 0-cells/HPF 0-cells/HPF 1-cells/HPFRed blood corpuscles Nil Nil 1-cells/HPFEpithelial cells Nil 1-cells/HPF NilCrystals Nil Nil NilCasts Nil Nil NilOthers Bacteria seen Bacteria seen Bacteria seenColor Yellow Yellow YellowValues are mean of a six animals±S.E.M (Dunnett’s test)* p<0.05;**p<0.01.n=6

ACUTE AND SUBACUTE TOXICITY STUDIES OF ETHANOLIC EXTRACT OF MIRABILIS JALAPA LINN IN WISTAR ALBINO RATS

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