The Repeated Dose 28-Day Oral Toxicity Study of Combined Extract of <i>Cajanus cajan</i> Leaf and <i>Zingiber officinale</i> Rhizome in Male and Female Sprague-Dawley Rats


  • Tutik Wresdiyati School of Veterinary Medicine and Biomedical Science, IPB University, Bogor 16680, Indonesia
  • Siti Sa’diah School of Veterinary Medicine and Biomedical Science, IPB University, Bogor 16680, Indonesia
  • Made Astawan Department of Food Science and Technology, Faculty of Agricultural Engineering and Technology, IPB University, Bogor 16680, Indonesia.
  • Hamzah Alfarisi School of Veterinary Medicine and Biomedical Science, IPB University, Bogor 16680, Indonesia
  • Sandra A. Aziz Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Bogor 16680, Indonesia
  • Made Darawati Department of Nutrition, Health Polytechnic of Mataram, Mataram 83232, Indonesia
  • Mawar Subangkit 1School of Veterinary Medicine and Biomedical Science, IPB University, Bogor 16680, Indonesia


histopathology, biochemistry, repeated dose 28-day oral toxicity study, Zingiber officinale, Cajanus cajan


The combination of Cajanus cajan leaf and Zingiber oficinale rhizome extract resulted in a greater hypoglycemic effect than the single of both extracts. However, no research reported the toxic effect of both extracts. So, this study assessed the toxicity of a combination of C. cajan leaves and Z. officinale extract in male and female Sprague-Dawley rats. Thirty male and thirty female rats were used in this study. The rats were randomly assigned to six groups of ten rats per group (five males and five females): the control treatment group and three-dose extract treated groups for 28 days-treatment periods. The satellite control group and satellite group were extended for 14 daysrecovery periods. The combination doses of C. cajan leaves extracts, and Z. officinale extract were 200+100, 400+200 and 800+400 mg/kg bw. The number of dead rats, body weight, biochemistry, hematology, and histopathological observation of some organs were observed. The results demonstrated that orally administering the combination of C. cajan and Z. officinale extracts for
28 day did not show any toxic impacts on male and female rats. Moreover, no cumulative effects of the combined extracts were noted during the 14-day recovery period. These findings showed that there were insignificant alterations in body weight, hematology, biochemistry, and histopathological observations. The findings suggest the safety of the combined extract of C. cajan leaf and Z. officinale rhizome. 


Setyaningrum DR, Rumiyati, Purwestri YA, Saloko S, Nugroho AE, Pranoto Y, Widyastuti, S. α-Amilase Inhibitory Activity of Fraction of Ethanolic Extract of Lebui Seed (Cajanus cajan (L.) Millsp.) Grown in West Nusa Tenggara. J. Food Pharm Sci. 2020; 8(2):266–72. Doi:


Orni PR, Ahmed SZ, Monefa M, Khan T, Dash PR. Pharmacological and phytochemical properties of Cajanus cajan ( L .) Huth . (Fabaceae): A review. Int J. Pharm Sci Res. 2018; 3(2):27–37.

Salea R, Veriansyah B, Tjandrawinata RR. Optimization and scale-up process for supercritical fluids extraction of ginger oil from Zingiber officinale var. Amarum. J. Supercrit Fluids. 2017; 120(2):285–94. Doi: 10.1016/j.supflu.2016.05.035

Mahboubi M. Zingiber officinale Rosc. essential oil, a review on its composition and bioactivity. Clin Phytoscience. 2019; 5(1):1–12. Doi: 10.1186/s40816-018-0097-4

Wresdiyati T, Iskandar DC, Sa’diah S, Astawan M. In Vitro and In Vivo Hypoglycaemic Activity Test of Indonesian Cajanus cajan Leaves and Zingiber officinale Extracts. Malaysian J. Med Heal Sci. 2020; 16(Supp 13):13–14.

Wresdiyati T, Mayangfauni A, Sa’diah S, Astawan M. The Effect of Ethanolic Cajanus cajan Leaves and Zingiber officinale Extracts on Spermatogenic Cells, Interstitial Cells and Superoxide Dismutase in Testicular Tissues of Experimental Diabetic Rats. Malaysian J. Med Heal Sci.

; 16(Supp 13):11–12.

Wresdiyati T, Sa’diah S, Astawan M. Komposisi Herbal yang Mengandung Ekstrak Daun Undis (Cajanus cajan) dan Ekstrak Jahe (Zingiber officinal) untuk Antidiabetes dan Antioksidan. Indonesia; IDS000005645, 2023. 22–24 p.

Wresdiyati T, Sa’diah S, Astawan M. Metode Ekstraksi Daun Undis (Cajanus Cajan), Ekstraksi Jahe Merah (Zingiber Officinale Var Rubrum) dan Ekstraksi Jahe Emprit (Zingiber Officinale Var Amarum) sebagai Antihiperkolesterolemia. IDS000004902, 2022. 1–3 p.

Wresdiyati T, Stephany S, Handharyani E, Sa’diah S, Astawan M. Acute Toxicity Test of Pigeon Pea Leaves Extract (Cajanus cajan) in Rats. E3S Web Conf. 2020; 151:1–4. Doi: 10.1051/e3sconf/202015101043

Tang R, Tian RH, Cai JZ, Wu JH, Shen XL, Hu YJ. Acute and sub-chronic toxicity of Cajanus cajan leaf extracts. Pharm Biol. 2017; 55(1):1740–1746. Doi: 10.1080/13880209.2017.1309556

Kaban AN, Saleh DC. Uji Fitokimia, Toksisitas dan Aktivitas Antioksidan Fraksi N-Heksan dan Etil Asetat Terhadap Ekstrak Jahe Merah (Zingiber Officinale Var. Amarum.). J. Kim Mulawarman. 2016; 14(1):24–8.

Academies TN. Guide for the Care and Use of Laboratory Animals. 8th ed. Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Washington.: The National Academies Press; 2011.

BPOM. Peraturan Badan Pengawas Obat Dan Makanan Nomor 10 Tahun 2022 Tentang Pedoman Uji Toksisitas Praklinik Secara In Vivo. Badan Pengawas Obat dan Makanan Republik Indonesia Indonesia; 2022. 1–220 p.

OECD. OECD Guidelines for the testing of chemicals: Repeated Dose 28-day Oral Toxicity Study in Rodents. Paris: OECD Publishing; 2008. 1–13 p.

Alfarisi H, Subangkit M, Sa’diah S, Wresdiyati T. Acute Toxicity of Ethanolic Extract of Acalypha hispida Leaves in Female Rats: A Physiological and Histological Study. J. Kedokt Hewan - Indones J. Vet Sci. 2020; 14(3):48–53. Doi: 10.21157/j.ked.hewan.v14i3.16176

Abebe MS, Asres K, Bekuretsion Y, Abebe A, Bikila D, Seyoum G. Sub-chronic toxicity of ethanol leaf extract of Syzygium guineense on the biochemical parameters and histopathology of liver and kidney in the rats. Toxicol Reports. 2021; 8:822–888. Doi: 10.1016/j.toxrep.2021.03.032

Loha M, Mulu A, Abay SM, Ergete W, Geleta B. Acute and Subacute Toxicity of Methanol Extract of Syzygium guineense Leaves on the Histology of the Liver and Kidney and Biochemical Compositions of Blood in Rats. Evidencebased Complement Altern Med. 2019; 2019. Doi:


Wolfensohn S, Lloyd M. Handbook of Laboratory Animal Management and Welfare. Oxford: Blackwell Publishing; 2003. 1–319 p.

Zheng Y, Castro D, Gay D, Cai D. Mean Corpuscular Hemoglobin Concentration in Hemoglobin CC, SC, and AC. North Am J. Med Sci. 2015; 8(1):1–4. Doi: 10.7156/najms.2015.0801001

Delwatta SL, Gunatilake M, Baumans V, Seneviratne MD, Dissanayaka MLB, Batagoda SS, Udagedara AH, Walpola PB. Reference values for selected hematological, biochemical and physiological parameters of SpragueDawley rats at the Animal House, Faculty of Medicine,

University of Colombo, Sri Lanka. Anim Model Exp Med. 2018; 1(4):250–254.

Bhide RM, Bethapudi B, Chalichem NSS, Nithyanantham M, Murugan SK, Mundkinajeddu D. Acute and Subchronic Toxicity Study of Flavonoid Rich Extract of Glycyrrhiza glabra (GutGard®) in Sprague Dawley Rats. J. Toxicol. 2022; 2022. Doi: 10.1155/2022/8517603

Tran PNT, Tran TTN. Evaluation of Acute and Subchronic Toxicity Induced by the Crude Ethanol Extract of Plukenetia volubilis Linneo Leaves in Swiss Albino Mice. Biomed Res Int. 2021; 2021.

Abdurrahman GF, Ambi A, Bisallah M, Abubakar A, Yusuf A, Jajere UM, Yabagi IZ. Acute and sub-chronic toxicity studies on methanol stem bark extract of Cussonia barteri seeman (Araliaceae) in Wistar rats. Trop J. Nat Prod Res. 2020; 4(8):392–6. Doi: 10.26538/tjnpr/v4i8.12

Pham Q, Vuong T, Doan M, Nguyen H. View of Assessment of Acute and Sub-Chronic Toxicity of HYK Aqueous Extracts in Experimental Animals.pdf. Trop J. Nat Prod Res. 2021; 5(3):519–27. Doi: 10.26538/tjnpr/v5i10.25

Féres CAO, Madalosso RC, Rocha OA, Leite JPV, Guimarães TMDP, Toledo VPP, Tagliati CA. Acute and chronic toxicological studies of Dimorphandra mollis in experimental animals. J. Ethnopharmacol. 2006; 108(3):450–6. Doi: 10.1016/j.jep.2006.06.002

Andersson ER. In the zone for liver proliferation. Science. 2021; 371(6532):887–888. Doi: 10.1126/science.abg4864

Zhang Q, Zhou PH, Zhou XL, Zhang DL, Gu Q, Zhang SJ, et al. Effect of Magnesium gluconate administration on lipid metabolism, antioxidative status, and related gene expression in rats fed a high-fat diet. Magnes Res. 2018; 31(4):117–130. DoiL 10.1684/mrh.2019.0445



How to Cite

Wresdiyati, T., Sa’diah, S., Astawan, M., Alfarisi, H., Aziz, S. A., Darawati, M., & Subangkit, M. (2023). The Repeated Dose 28-Day Oral Toxicity Study of Combined Extract of <i>Cajanus cajan</i> Leaf and <i>Zingiber officinale</i> Rhizome in Male and Female Sprague-Dawley Rats: Tropical Journal of Natural Product Research (TJNPR), 7(8), 3706–3716. Retrieved from