Introduction: The application of herbal plants because of their lower side-effects has been

increasing in recent years.

Objectives: we aimed to study the effects of hydro-alcoholic extract of Momordica charantia

(bitter melon) on liver function and tissue structure in mice.

Materials and Methods: This study was conducted on 70 male mice which were randomly

designated into 7 groups of 10 and were injected with single doses of 0, 100, 500, 1000,

2000 and 4000 mg/kg and multiple daily dose of 500 mg/kg for 7 days, intraperitoneally.

Finally, liver tissues were taken out for histological examinations. Serum samples were assayed

for liver enzymes activities of alkaline phosphatase, aspartate aminotransferase and alanine

aminotransferase. Also the antioxidant activity of blood and bitter melon were measured.

Subsequently, data were analyzed by analysis of variance (ANOVA) and Bonferroni tests using

Stata software.

Results: The results of the present study showed that a single dose of Momordica charantia

fruit extract at doses up to 4000 mg/kg extract does not significant adverse effects on the liver

enzymes and tissue structure.

Conclusion: Using this drug for short time and low dose has not toxicity effects on liver enzymes

activity and tissue structure, hence, it is safe in this range of doses

Ahmadi, A.

Kazemi, E.

Khodadadi, S.

Mardani, S.

Rafieian-Kopaei, M.

shahrekord university of medical scinces

Copyright © 2016 The Author(s); Published by Society of Diabetic Nephropathy Prevention. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The effects of Momordica charantia on liver function and histological structureAnn Res Antioxid. 2016;1(1):e12                                                                                                                          OriginalAnnals of Research in Antioxidants Saeed Mardani1, Samaneh Khodadadi2, Ali Ahmadi3, Elahe Kazemi4, Mahmoud Rafieian-Kopaei5* 1Department of Internal Medicine, Isfahan University of Medical Sciences, Isfahan, Iran2Nickan Research Institute, Isfahan, Iran3Department of Epidemiology and Biostatistics, Faculty of Health, Shahrekord University of Medical Sciences, Shahrekord, Iran4Department of Internal Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran5Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, IranCorrespondence to:Prof. Mahmoud Rafieian-Kopaei;  Email: rafieian@yahoo.com Received: 12 October 2015 Accepted: 29 November 2015ePublished: 3 December 2015Keywords: Momordica charantia, liver, Hypoglycemia, Medicinal plantsIntroduction: The application of herbal plants because of their lower side-effects has been increasing in recent years. Objectives: we aimed to study the effects of hydro-alcoholic extract of Momordica charantia (bitter melon) on liver function and tissue structure in mice.Materials and Methods: This study was conducted on 70 male mice which were randomly designated into 7 groups of 10 and were injected with single doses of 0, 100, 500, 1000, 2000 and 4000 mg/kg and multiple daily dose of 500 mg/kg for 7 days, intraperitoneally. Finally, liver tissues were taken out for histological examinations. Serum samples were assayed for liver enzymes activities of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase. Also the antioxidant activity of blood and bitter melon were measured. Subsequently, data were analyzed by analysis of variance (ANOVA) and Bonferroni tests using Stata software. Results: The results of the present study showed that a single dose of Momordica charantia fruit extract at doses up to 4000 mg/kg extract does not significant adverse effects on the liver enzymes and tissue structure.Conclusion: Using this drug for short time and low dose has not toxicity effects on liver enzymes activity and tissue structure, hence, it is safe in this range of doses.Abstract Citation: Mardani S, Khodadadi S, Ahmadi A, Kazemi E, Rafieian-kopaei M. The effects of Momordica charantia on liver function and histological structure. Ann Res Antioxid. 2016;1(1):e12. Introduction Since ancient times the use of medicinal plants has been common in the treatment of many diseases (1). But, some constituents of these herbs may lead to severe and on oc-casion fatal poisoning. Hence we are today faced with case reports of toxic herbs, con-stantly (2). A lot of herbal products are used to reduce blood sugar in different cultures, among them Momordica charantia is the most common medical plant (3).Bitter melon belongs to the Cucurbitace-ae family, Momordica genus and it is also known as Bitter ground in England, Karela in India, Paharkia in Tamil, Pinyin in China and in Malaysia (4).It has various effects such as anti-diabetic, anti-viral, anti-tumor, anti-ulcer, anti-in-flammatory, lowering cholesterol, lower-ing triglycerides, blood pressure, immune stimulant, insect repellent, prevention of pregnancy, antimalarial, anti-eczema, prop-erty menstrual stimulant, anti-pneumonia, Core tip Administration of Momordica charantia for short time and low dose has not toxicity effects on liver enzymes activity and tissue structure, hence, it is safe in this range of doses.anti-rheumatoid arthritis, anti-scabies, an-ti-psoriasis, and cytotoxic properties (5).Hypoglycemic activity is one of the most obvious medicinal properties of this herb so that all parts of the plant such as fruits, seeds, leaves and roots have this feature and because of the long-term history of use, this plant is known as herbal insulin. Over the years several mechanisms have been introduced to explain the hypoglycemic activity of this drug. For example, one of these proposed mechanisms is inhibition of glucose absorp-tion in intestinal tract by drugs (6). Other evidences suggest that Momordica charantia reduces gluconeogenesis, increases glycogen synthesis in the liver and also rises glucose http annresantioxidants.comMardani S et al Annals of Research in Antioxidants   2 Volume 1, Issue 1, January 2016oxidation in peripheral erythrocytes and fat cells (7).Some studies have reported that Momordica charantia in-creases the secretion of insulin from the pancreas. Hence, this hypothesis was created that Momordica charantia ex-tract increases the production of beta cells in the pancre-as, although this mechanism has not been confirmed by studies (8). ObjectivesThere are some unofficial reports denoting the possible liver toxicity of this plant. While, there are not sufficient published reports about side effects of bitter melon, we in-tended to investigate the effects of this herbal medicine on liver enzymes activity and histological changes. Materials and MethodsMomordica charantia fruits were obtained from India and approved by expert botanist in Medical Plants Research Center of Shahrekord University of Medical Science, Shahrekord, Iran.Extraction processHydro-alcoholic extract of Momordica charantia was pre-pared by percolation method with 95% ethanol, followed by steam evaporation, after that, five different concentra-tions of extract were prepared. Phytochemical analysisTotal flavonoids content was estimated by aluminum chloride colorimetric method. The standard solutions of routine with concentrations of 25, 50, 100, 250 and 500 ppm from 60% methanol was prepared, then 1 mL of each solution was transferred to test tubes and 1 mL solution of aluminum chloride 2% and then 6 mL of potassium ace-tate 5% was added to the solution and after 40 minutes the absorbance was measured at 415 nm (9).Measurement of total phenolic contentTotal phenolic content was determined by folin-ciocalteu reagent method and the absorbance was measured at 760 nm. Total phenolic content in terms mg/g of extract was calculated (10).Antioxidant activity assaysΒ-carotene/linoleic acid-coupled oxidation reactionA solution of β-carotene was prepared by liquefying 2 mg in 10 mL of chloroform. Then 0.02 mL of linoleic acid and 0.2 mL of tween 40 was added, and the mixture was remained at 20°C for 15 minutes. After dehydration of the chloroform in a rotary evaporator at 40°C, 50 mL of oxygen-saturated distilled water at 25°C was added and the mixture was vortexed strongly (1 minute) to form an emulsion (β-carotene/linoleic acid emulsion). The re-quired wells were charged (of a 96-well microtiter plate) with each different amount of samples and 100 μL of emul-sion per well. The microplate was located on a horizontal shaker and shaken at 100 rpm (during 1 minute). A con-trol sample was also arranged similarly. Absorbance mea-surements (470 nm) at t = 0 minute were made after incu-bation at 50°C during 120 minutes. All experiments were done in triplicate. Antioxidant activity was mentioned as the percent of inhibition with regard to the control sample and calculated as follows:AA=100[1-A0-At1] /[A0-Atc].where SA0 and CA0 are the absorbance values of the sample and the control defined at 0 minute; the SAt and CAt were the absorbance values of test sample and control measured after 120 minutes. BHT was used as positive control (11).Animal and treatmentSeventy male db/db mice weighing 25-30 g and 3 weeks old were distributed into seven groups with 10 animals randomly in each group as follows: Group 1: control group (without drug)Group 2: obtained at 100 mg/kg extract as a single dose. Group 3: received at 500 mg/kg extract as a single dose. Group 4: gained 1000 mg/kg extract as a single dose. Group5: received 2000 mg/kg extract as a single dose. Group 6: obtained 4000 mg/kg extract as a single dose. Group 7: received 500 mg/kg/day extract for one week.All of the mice were maintained on a 12-hour light/dark cycle. All protocols for animal use were approved by the animal care committee of Shahrekord University of Med-ical Sciences animal care. All the experiments were done after one week in order for the mice to become accus-tomed to the new environment and they could freely ac-cess water and food (12). After one week on the eighth day various doses of the extract was injected intraperitoneally to mice. The mice were cared for 72 hours and then they were anesthetized and blood samples were stored in tubes for biochemical parameters estimation (13). Then blood samples were centrifuged at 3500 rpm for 15 minutes to evaluate serum liver enzymes. Liver enzymes were mea-sured with enzymatic method (Pars Azmoon kit) by Auto-matic Analyzer 902 (Hitachi, Germany).Ferric reducing ability Ferric reducing ability of plasma (FRAP) reagent was mixed with 90 μL of distilled water and 30 μL of test sam-ple solutions. The reaction mixture was then incubated at 37°C for 10 minutes and absorbance was recorded at 593 nm, using a spectrophotometer (uv3100 Shimadzu, Japan). The concentrations of FeSO4 were in turn plotted against concentrations of the standard antioxidants (14). Histological study After animals were euthanized by ether, liver tissues were collected and put into 10% buffered formalin for 48 hours. Subsequently, organs were embedded within paraffin. Solid sections of 5 μm thickness were made using a mi-crotome. The sections were stained with hematoxylin and eosin (H&E) and then observed by light microscopy for histopathological examination (15).Ethical issues The research followed the tenets of the Declaration of Hel-Momordica charantia and liver Annals of Research in Antioxidants   3 Volume 1, Issue 1, January 2016sinki. The research was approved by ethical committee of Shahrekord University of Medical Sciences. Prior to the experiment, the protocols were confirmed to be in accor-dance with the guidelines of Animal Ethics Committee of Shahrekord University of Medical Sciences.Statistical analysisData were analyzed by analysis of variance (ANOVA) and chi-square tests using SPSS software. P < 0.05 was consid-ered significant for all data.ResultsAntioxidant capacity of bitter melon (percent inhibition of peroxidation in linoleic acid production) was 68%. The amount of flavonoids was 54 mL/μg, flavonols 45 mL/μg and phenolic content 413 mL/μg in the extract. Antioxidant capacity of bloodAntioxidant capacity of blood was 568 µmol/L before the study. After the intervention the blood antioxidant capaci-ties in groups were as follows:Group 1: (control group) 564 µmol/L; group 2: 1108 µmol/L; group 3: 741 µmol/L; group 4: 553 µmol/L; group 5: 703 µmol/L; group 6: 624 µmol/L; group 7: 436 µmol/L.Effect of different doses of Momordica charantia on liver enzymes activityThere were no significant difference of alkaline phos-phatase (ALP), aspartate aminotransferase (SGOT), al-anine aminotransferase (SGPT) activity between groups (P > 0.05; Table 1).Effects of different doses of Momordica charantia on liver histologyHistological examination of the liver showed no patholog-ic findings and there was not significant different between liver tissue in control group and other treatment groups (P > 0.05).DiscussionIn this study, 70 mice were divided into 7 groups and were treated with different doses of Momordica charantia ex-tract. The smallest dose was 100 mg/kg, and the greatest was 4000 mg/kg. Also, a group treated for one week 500 mg/kg extract. In this study, liver enzymes activity includ-ing ALP, SGPT and SGOT was measured. During the in-tervention, liver enzymes were not significantly different between the control group and groups treated with extract.As well, there were not changes in histology of liver tissue in our study and there was not difference between treated groups and control group. The results in our study were in agreement with the results of another study which was carried out on the effects of Momordica charantia on key hepatic enzymes and indicat-ed that this valuable plant contain hepatotoxin capable of causing cellular damage but at molecular level without any significant histopatological change. While in many other investigations effects of Momordica charantia on kidney were confirmed because not only kidney is one of the most affected organs by diabetes but also in many researches the anti-diabetic properties of this worthy herbal drug has been proved currently like lots of other medicinal plant.A report about the effect of Momordica charantia on di-abetic mice showed that due to diabetes extra space be-tween hepatic sinusoids of liver disappeared after treat-ing with extract. The extract of bitter melon can protect damage tissue liver (16). Also in the study conducted by Nazrul-Hakim  et al liver pathology was examined and they observed a small amount of congestion and also found that there were no significant differences between control and other groups (17). Subsequently, the results of the present study remarkably showed that a single dose of Momordica charantia fruit extract at doses up to 4000 mg/kg extract does have not significant adverse effects on the liver contrary to effect on kidney structure and tissue. Be-cause of antioxidant activity of this medicinal plant and also its anti-diabetic characteristics we strongly recom-mend more research about its impact on all organs and tissues involved in diabetes.ConclusionMomordica charantia extract and low dose had not toxicity effects on liver enzymes activity and tissue for short time so long term treatment for patients with liver abnormal-ities should be checked regularly and if it has disrupting drug should be discontinued.Authors’ contribution SM, EK and MRK conducted the research. AA conducted the statistical analysis. SK Prepared the primary draft. MRK edited the final manuscript. All authors read and sign the final paper.Conflicts of interestThe authors declared no competing interests.Table 1. Comparison mean of liver enzyme activity in the study groupsGroups SGOT (mg/dL) SGPT (mg/dL) ALP (mg/dL)(Control) 165.5 ± 51 90 ± 70 200 ± 95(100 mg/kg) 196 ± 91 97.8 ± 68 206 ± 71(500 mg/kg) 143 ± 57 67.7 ± 45 243 ± 108(1000 mg/kg) 199 ± 92 77 ± 40 190 ± 110(2000 mg/kg) 173 ± 130 49.5 ± 28 159 ± 62(4000 mg/kg) 206 ± 38 88 ± 67 192 ± 99(500 mg/kg) 180 ± 63 85.7 ± 40 223 ± 49Total mean 180.8 ± 76 80.6 ± 54 204.7 ± 88Data are shown as the Mean ± SEM, (n = 7, P < 0.05).Mardani S et al Annals of Research in Antioxidants   4 Volume 1, Issue 1, January 2016Ethical considerations Ethical issues (including plagiarism, data fabrication, double publication) have been completely observed by the authors.Funding/Support This paper was financially supported by Research Deputy of Shahrekord University of Medical Sciences, Shahrekord, Iran (Grant No. 1182).AcknowledgmentsThis study was extracted from MD thesis of Elahe Kazemi.References1. Basch E, Gabardi S, Ulbricht C. Bitter melon (Momordica charantia): a review of efficacy and safety. Am J Health Syst Pharm. 2003;65:356-9.2. Grover JK, Yadav SP. Pharmacological actions and potential uses of Momordica Charantia: a review. J Ethnopharmacol. 2004;93:123-4.3. Jayasooriya AP, Sakono M, Yukizaki C, Kawano M, Yamamoto K, Fukuda N. Effects of Momordica charantia powder on serum glucose levels and various lipid parameters in rats fed with cholesterol free and cholesterol-enriched diets. J Ethnopharmacol. 2000;72:331-6.4. Bno F, Akhtar N. Effect of the aqueous extract of momordica charantia on body weight of rats. Journal of Basic and Applied Sciences. 2011;7:1-5.5. Shibib BA, Khan LA, Rahman R. Hypoglycaemic activity of Coccinia indica and momordica charantia in diabetic rats depression of the hepatic gluconeogenetic enzymes G6P and F1,6B and elevation of both liver and red cell shunt enzyme G6PD. Biochem J. 1993;292:267-70.6. Tennekoon KH, Jeevathayaparan S, Angunawala P, Karunanayake EH, Jayasinghe KS. 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The effects of Momordica charantia on liver function and histological structure

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