J. Pharm. Pharmacogn. Res., vol. 11, no. 3, pp. 437-447, May-June 2023.

DOI: https://doi.org/10.56499/jppres23.1609_11.3.437

Original Article

Acute and repeated dose 28-day oral toxicity of Ruta angustifolia Pers. leaves ethanolic extract in Wistar rats

[Toxicidad oral aguda y por dosis repetidas durante 28 días del extracto etanólico de hojas de Ruta angustifolia Pers. en ratas Wistar]

Tutik Sri Wahyuni^1,2*, Hilkatul Ilmi², Ade Syamsi Kristiaji³, Martiana Candra Dewi³, Hanifah Khairun Nisa², Achmad Fuad Hafid^1,2, Aty Widyawaruyanti^1,2

¹Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Surabaya, 60115, East Java, Indonesia.

²Center for Natural Product Medicine Research and Development, Institute of Tropical Diseases, Universitas Airlangga, Surabaya, 60115, East Java, Indonesia.

³Undergraduate Program of Faculty of Pharmacy, Universitas Airlangga, Surabaya, 60115, East Java, Indonesia.

*E-mail: tutik-s-w@ff.unair.ac.id; wahyuni.tutiksri@yahoo.com

Abstract

Context: Ruta angustifolia Pers. has been used as a traditional herb among counties. The ethanol extract of R. angustifolia has proven active as an anti-hepatitis C virus in vitro. Due to the potency of this plant, a safety evaluation is necessary.

Aims: To examine the acute and sub-chronic toxicity of ethanol extract of R. angustifolia leaves in rats.

Methods: An acute toxicity study was done by treating 70% ethanol extract of R. angustifolia (doses of 5, 50, 300, and 2000 mg/kg of BW), while the control group received 1% Tween-80. Toxicity signs and mortality of rats for 14 days were observed. While sub-chronic (28-days repeated dose) toxicity was evaluated with 4 groups, both males and females. The three groups were treated with 70% ethanol extract of R. angustifolia at doses of 50, 500, and 1000 mg/kg BW daily for 28 days, and one group served as a control group. Biochemical, hematological, and histopathological parameters were measured after the period of treatment.

Results: The acute toxicity was no demonstrated toxicity signs or mortality effects up to 14 days of the experiment. Therefore, the LD₅₀ value of the extract was estimated to be higher than 2000 mg/kg BW. The sub-chronic assay revealed no mortality and toxicity effects, including behavior parameters, relative body weight, hematology, and biochemical at 50 and 500 mg/kg BW doses. However, a significant change was seen in the testis of all doses and the liver of the male rats treated at 1000 mg/kg BW. Histopathology assay observed the presence of activated Kupffer cells, sinusoidal dilatation, and infiltration of lymphocytes at the portal vein in the liver with 1000 mg/kg BW. Those may indicate possible toxicity in the liver at the dose of 1000 mg/kg BW. However, no toxic effect at the lower doses tested.

Conclusions: These results suggest that the use of Ruta angustifolia leaves ethanolic extract is relatively safe. The application of high doses needs to be evaluated.

Keywords: health; medicine; medicinal plants; Ruta angustifolia; toxicity assay.

Resumen

Contexto: Ruta angustifolia Pers. se ha utilizado como una hierba tradicional entre los condados. El extracto etanólico de R. angustifolia demostró ser activo como virus anti-hepatitis C por in vitro. Debido a la potencia de esta planta, es necesaria una evaluación de seguridad.

Objetivos: Examinar la toxicidad aguda y subcrónica del extracto etanólico de hojas de R. angustifolia en ratas.

Métodos: Se realizó un estudio de toxicidad aguda tratando extracto etanólico al 70% de R. angustifolia (dosis de 5, 50, 300 y 2000 mg/kg de PC), mientras que el grupo control recibió Tween-80 al 1%. Se observaron signos de toxicidad y mortalidad de ratas durante 14 días. Mientras que se realizó una evaluación de la toxicidad subcrónica (dosis repetida de 28 días) con 4 grupos, tanto de machos como de hembras. Los tres grupos fueron tratados con extracto etanólico al 70% de R. angustifolia en dosis de 50, 500 y 1000 mg/kg PC diariamente durante 28 días, y un grupo sirvió como grupo de control. Los parámetros bioquímicos, hematológicos e histopatológicos se midieron después del período de tratamiento.

Resultados: La toxicidad aguda no demostró signos de toxicidad ni efectos de mortalidad hasta los 14 días del experimento. Por lo tanto, el valor DL₅₀ del extracto se estimó superior a 2000 mg/kg de peso corporal. El ensayo subcrónico no reveló efectos de mortalidad ni toxicidad, incluidos los parámetros de comportamiento, el peso corporal relativo, la hematología y la bioquímica en dosis de 50 y 500 mg/kg de peso corporal. Sin embargo, se observó un cambio significativo en los testículos de todas las dosis y en el hígado de las ratas macho tratadas con 1000 mg/kg de peso corporal. El ensayo de histopatología observó la presencia de células de Kupffer activadas, dilatación sinusoidal e infiltración de linfocitos en la vena portal hepática con 1000 mg/kg BW. Estos pueden indicar una posible toxicidad en el hígado a la dosis de 1000 mg/kg de peso corporal. Sin embargo, no se observaron efectos tóxicos a las dosis más bajas ensayadas.Conclusiones: Estos resultados sugieren que el uso del extracto etanólico de hojas de Ruta angustifolia es relativamente seguro. La aplicación de dosis altas necesita ser evaluada.

Palabras Clave: ensayo de toxicidad; medicamento; plantas medicinales; Ruta angustifolia; salud.

Citation Format: Wahyuni TS, Ilmi H, Kristiaji AS, Dewi MC, Nisa HK, Hafid AF, Widyawaruyanti A (2023) Acute and repeated dose 28-day oral toxicity of Ruta angustifolia Pers. leaves ethanolic extract in Wistar rats. J Pharm Pharmacogn Res 11(3): 437–447. https://doi.org/10.56499/jppres23.1609_11.3.437

References

Bachar SC, Mazumder K, Bachar R, Aktar A, Al Mahtab M (2021) A review of medicinal plants with antiviral activity available in Bangladesh and mechanistic insight into their bioactive metabolites on SARS-CoV-2, HIV and HBV. Front Pharmacol 12: 732891. https://doi.org/10.3389/fphar.2021.732891

Del Castillo JB, Secundino M, Luis FR (1986) Four aromatic derivatives from Ruta angustifolia. Phytochemistry 25(9): 2209–2210. https://doi.org/10.1016/0031-9422(86)80093-0

Del Castillo JB, Luis FR, Secundino M (1984) Angustifolin, a coumarin from Ruta angustifolia. Phytochemistry 23(9): 2095–2096. https://doi.org/10.1016/S0031-9422(00)84991-2

Fan X, Song H, Xu X, Lux X, Wang Y, Duan X (2022) Subchronic oral toxicity of sodium p-hydroxybenzoate in Sprague-Dawley rats. Front Pharmacol 13: 843368. https://doi.org/10.3389/fphar.2022.843368

Haddouchi F, Chaouche TM, Zaouali Y, Ksouri R, Attou A, Benmansour A (2013) Chemical composition and antimicrobial activity of the essential oils from four Ruta species growing in Algeria. Food Chem 141(1): 253–258. https://doi.org/10.1016/j.foodchem.2013.03.007

Harris RBS, Zhou J, Youngblood BD, Rybkin II, Smagin GN, Ryan DH (1998) Effect of repeated stress on body weight and body composition of rats fed low- and high-fat diets. Am J Physiol 275(6): R1928–R1938. https://doi.org/10.1152/ajpregu.1998.275.6.R1928

Hidayat V, Elisma, Lestari I (2021) Toxicity test of inggu (Ruta angustifolia (L) ethanol leaves extract to male white mice (Mus musculus). BiGME 1(2): 21–28. https://doi.org/10.22437/bigme.v1i2.15412

Jothy SL, Zakaria Z, Chen Y, Lau YL, Latha LY, Sasidharan S (2011) Acute oral toxicity of methanolic seed extract of Cassia fistula in mice. Molecules 16(6): 5268–5282. https://doi.org/10.3390/molecules16065268

Kaur G (2019) Hepatic toxicity biomarkers. In: Biomarkers in toxicology. Elsevier, pp. 251–266. https://doi.org/10.1016/B978-0-12-814655-2.00014-1

Kifayatullah M, Mustafa MS, Sengupta P, Sarker MMR, Das A, Das SK (2015) Evaluation of the acute and sub-acute toxicity of the ethanolic extract of Pericampylus glaucus (Lam.) Merr. in BALB/c mice. J Acute Dis 4(4): 309–315. https://doi.org/10.1016/j.joad.2015.06.010

Nalawade SM, Tsay HS (2004) In vitro propagation of some important chinese medicinal plants and their sustainable usage. In Vitro Cell Dev Biol Plant 40(2): 143–154. https://doi.org/10.1079/IVP2003504

OECD (2008) Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents. OECD. https://doi.org/10.1787/9789264070684-en

OECD (2022) Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure. OECD. https://doi.org/10.1787/9789264071049-en

Pariyani R, Safinar II, Azam AA, Abas F, Shaari K, Sulaiman MR (2015) Phytochemical screening and acute oral toxicity study of Java tea leaf extracts. BioMed Res Int 2015: 742420. https://doi.org/10.1155/2015/742420

Parray SA, Bhat JU, Ahmad G, Jahan N, Sof G, Iqbal SMF (2012) Ruta graveolens: From traditional system of medicine to modern pharmacology: An overview. Am J PharmTech Res 2(2): 239-252.

Pollio A, De Natale A, Appetiti E, Aliotta G, Touwaide A (2008) Continuity and change in the Mediterranean medical tradition: Ruta spp. (Rutaceae) in Hippocratic medicine and present practices. J Ethnopharmacol 116(3): 469–482. https://doi.org/10.1016/j.jep.2007.12.013

Ramsay S (2002) WHO launches the first global strategy on traditional and alternative medicine. Cent Eur J Public Health 10(4): 145-156.

Rhiouani H, El-Hilaly J, Israili ZH, Lyoussi B (2008) Acute and sub-chronic toxicity of an aqueous extract of the leaves of Herniaria glabra in rodents. J Ethnopharmacol 118(3): 378–386. https://doi.org/10.1016/j.jep.2008.05.009

Richardson JSM, Sethi G, Lee GS, Malek SNA (2016) Chalepin: Isolated from Ruta angustifolia L. Pers induces mitochondrial mediated apoptosis in lung carcinoma cells. BMC Complement Alternat Med 16(1): 389. https://doi.org/10.1186/s12906-016-1368-6

Salawu O, Chindo B, Tijani AY, Ezenyi IO (2009) Acute and sub-acute toxicological evaluation of the methanolic stem bark extract of Crossopteryx febrifuga in rats. Afr J Pharm Pharmacol 3(12): 621–626.

Schlatt S, Ehmcke J (2014) Regulation of spermatogenesis: An evolutionary biologist’s perspective. Semin Cell Dev Biol 29: 2–16. https://doi.org/10.1016/j.semcdb.2014.03.007

Sellers RS, Mortan D, Michael B, Roome N, Johnson JK, Yano BL, Perry R, Schafer K (2007) Society of toxicologic pathology position paper: Organ weight recommendations for toxicology studies. Toxicol Pathol 35(5): 751–755. https://doi.org/10.1080/01926230701595300

Shuib NA, Iqbal A, Sulaiman FA, Razak I, Susanti D (2015) Antioxidant and antibacterial activities of Ruta angustifolia extract. J Teknol 77(25): 101-105. https://doi.org/10.11113/jt.v77.6747

Singh T, Sinha N, Singh A (2013) Biochemical and histopathological effects on liver due to acute oral toxicity of aqueous leaf extract of Eclipta alba on female Swiss albino mice. Indian J Pharmacol 45(1): 61–65. https://doi.org/10.4103/0253-7613.106437

Vaghasiya YK, Shukla VJ, Chanda SV (2011) Acute oral toxicity study of Pluchea arguta Boiss extract in mice. J Pharmacol Toxicol 6(2): 113–123. https://doi.org/10.3923/jpt.2011.113.123

Vysakh A, Jayesh K, Helen LR, Jyothis M, Latha MS (2020) Acute oral toxicity and anti-inflammatory evaluation of methanolic extract of Rotula aquatica roots in Wistar rats. J Ayurveda Integr Med 11(1): 45–52. https://doi.org/10.1016/j.jaim.2017.09.007

Wahyuni TS, Khoiriyah N, Tumewu L, Ekasari W, Hafid AF, Widyawaruyanti A (2023) Microscopic and physicochemical evaluation of Ruta angustifolia leaves. J Public Health Afr 14(SI): 2520. https://doi.org/10.4081/jphia.2023.2520

Wahyuni TS, Mahfud H, Permatasari AA, Widyawaruyanti A, Hafid AF (2019) Synergistic anti-hepatitis C virus activity of Ruta angustifolia extract with NS3 protein inhibitor. J Basic Clin Physiol Pharmacol 30(6): 20190348. https://doi.org/10.1515/jbcpp-2019-0348

Wahyuni TS, Permanasari AA, Tumewu L, Widyawaruyanti A, Hafid AF (2021) Qualitative and quantitative analysis of 70% ethanol extract from Ruta angustifolia for developing anti-hepatitis C agents. Pharmacogn J 13(3): 682–687. https://doi.org/10.5530/pj.2021.13.87

Wahyuni TS, Widyawaruyanti A, Lusida MI, Hafid AF, Soetjipto, Fuchino H, Kawahara N, Hayashi Y, Aoki C, Hotta H (2014) Inhibition of hepatitis C virus replication by chalepin and pseudane IX isolated from Ruta angustifolia leaves. Fitoterapia 99: 276–283. https://doi.org/10.1016/j.fitote.2014.10.011

Yam MF, Lim, CP, Fung AL, Por LY, Wong ST, Asmawi MZ, Basir R, Ahmad M (2013) Antioxidant and toxicity studies of 50% methanolic extract of Orthosiphon stamineus Benth. Biomed Res Int 2013: 351602. https://doi.org/10.1155/2013/351602

Yang M, Wang Y, Fan Z, Xue Q, Njateng GSS, Liu Y, Cao J, Zhao T, Cheng G (2020) Acute and sub-acute toxicological evaluations of bioactive alkaloidal extract from Melodinus henryi and their main chemical constituents. Nat Prod Bioprospect 10(4): 227–241. https://doi.org/10.1007/s13659-020-00252-2

Yi-Chen, Wu MX, Jie-Liu, Ma XJ, Shi JL, Wang SN, Zheng ZQ, Guo JY (2018) Acute and sub-acute oral toxicity studies of the aqueous extract from radix, radix with cortex and cortex of Psammosilene tunicoides in mice and rats. J Ethnopharmacol 213: 199–209. https://doi.org/10.1016/j.jep.2017.11.011

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