Subacute toxicological evaluation of Catharanthus pusillus Methanolic extract in wistar rats

Introduction

India is globally renowned for its rich heritage of traditional medicine and remarkable biodiversity of medicinal plants. Approximately 85% of traditional medicines worldwide are plant-derived, and nearly 7,500 species are used in India by various ethnic communities (Farnsworth et al., 1985) [9] . As the world's largest producer of medicinal herbs, India is often referred to as the "Botanical Garden of the World" (Ahmedulla & Nayar, 1999) [1] .

Medicinal plants contain diverse bioactive compounds-including alkaloids, glycosides, terpenes, phenolics, flavonoids, tannins, essential oils, and toxalbumins-that can exert both therapeutic and toxicological effects depending on dose, chemical composition, and mode of use (Satish, 2002; Groten, 2013) [19,11] . Plant poisoning remains a significant cause of economic loss in Indian livestock production due to mortality, reduced productivity, abortions, and associated veterinary costs (Khandare, 2015) [15] . Catharanthus pusillus (CP), a perennial herb of the family Apocynaceae, is notable for both its medicinal and toxicological significance (Navitha et al., 2012) [18] . The plant is characterized by white latex, a carrot-shaped taproot, quadrangular reddish stems, and oblong leaves (Figure 1). Traditionally, CP is used for its antimicrobial, antineoplastic (Subbaiyan et al., 2013) [20] , antioxidant (Thingujam et al., 2015) [22] , anthelmintic, and wound-healing properties (Gajalakshmi et al., 2013) [10] . Its roots, leaves, and latex are also employed in the treatment of skin and liver disorders, leprosy, ulcers, tumors, rheumatism, asthma, and cardiac ailments (El-Sayed & Cordell, 1981; Chandran & Saj, 2015) [6,7,5] . These effects are largely attributed to its alkaloid content, which also contributes to its potential toxicity. Field observations indicate that CP toxicity is common in livestock in parts of Gujarat, particularly the Virampur region of Banaskantha district, where it is locally known as "Marchadi." Outbreaks in cattle and buffaloes are reported annually, with high morbidity and production loss, and in severe cases, progression to coma and death. In the absence of a specific antidote, only symptomatic treatment is available. Despite frequent incidents, scientific data on the toxicodynamics, target organ effects, and LD₅₀ of CP in animals are scarce. Therefore, the present 28-day oral toxicity study in Wistar rats was undertaken to Evaluate clinical and behavioural alterations following CP extract administration, assess hematological and biochemical changes, and Investigate pathomorphological alterations in major organs.

Materials and Methods

The experimental protocol (Protocol No. VETCOLL/IAEC/2021/17/PROTOCOL-12) was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of the College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar, Gujarat, India. All experimental procedures were conducted in accordance with the guidelines of the Committee for the Control and Supervision of Experiments on Animals (CCSEA), New Delhi.

Experimental Animals and Management

A total of 40 healthy Wistar rats (20 males and 20 females) were procured from the Animal Facility of Cadila Pharmaceuticals Ltd., Dholka, Gujarat, India. Animals were acclimatized prior to experimentation and maintained under standard laboratory conditions (12 h light-dark cycle, temperature 22±2 °C, relative humidity 50±10%) with free access to pelleted feed and water ad libitum. All management and treatment procedures adhered to CCSEA guidelines.

Collection and Authentication of Plant Material

Catharanthus pusillus (Murray) G. Don (CP), locally known as "Marchadi" is commonly found around Virampur village,

Banaskantha

district, Gujarat-particularly during September to October, when incidences of bovine toxicity are highest. The plant was collected from a farm reporting toxicity in buffaloes and subsequently identified and authenticated by the Department of Botany, Hemchandracharya North Gujarat University, Palanpur.

Preparation of Alcoholic Plant Extract

Collected plants were washed, shade-dried, powdered, and sieved to obtain a fine powder. The powdered material was packed in a thimble and extracted using methanol in a Soxhlet apparatus, with three successive extractions over 8 hours at the boiling point of methanol (56 °C), following the method described by Harborne (1964) [12] . The extract was concentrated to dryness and stored in airtight sample bottles at 4 °C until use.

Experimental Design

The 40 rats were randomly assigned to four groups (I-IV), each consisting of five males and five females: Group I (Control): Received distilled water orally for 28 days. Group II: Received CP extract (CPE) at 750 mg/kg body weight orally for 28 days. Group III: Received CPE at 1500 mg/kg body weight orally for 28 days. Group IV: Received CPE at 3000 mg/kg body weight orally for 28 days. All animals were observed daily for clinical signs and behavioural alterations throughout the experimental period.

Clinical Observations and Body Weight Monitoring

All animals were observed twice daily for clinical signs of morbidity and mortality throughout the study. Body weights were recorded on Day 1 and at weekly intervals thereafter. On Day 29, surviving rats were fasted overnight, euthanized, and terminal body weights were recorded prior to necropsy.

Organ Collection and Relative Organ Weights

Following euthanasia, a complete necropsy was performed. Organs including the liver, kidneys, lungs, heart, brain, spleen, adrenals, thymus, ovaries, epididymides, and testes were collected. All organs were fixed in 10% neutral buffered formalin, except testes and epididymides, which were initially preserved in modified Davidson's fluid for 48 h and then transferred to formalin. The liver, kidneys, adrenals, thymus, spleen, brain, heart, epididymis, and testes were trimmed and weighed immediately to determine absolute and relative organ weights.

Hematology and Serum Biochemistry

On Day 29, blood samples were collected from the retroorbital plexus of all surviving rats. Samples were placed in EDTA tubes for hematological analysis and clot activator tubes for biochemical analysis. Hematological parametersincluding TEC, Hb, HCT, MCV, MCH, MCHC, platelet count, WBC, TLC, and DLC-were analyzed using a fully automated hematology analyzer (Exigo™ EOS, Boule Diagnostics AB, Sweden). Giemsa-stained smears were prepared within 3 h of collection to assess platelet and erythrocyte morphology and basophil count. Biochemical parameters-including ALT, AST, ALP, total protein, albumin, urea, creatinine, triglycerides, phosphorus, calcium, magnesium, cholesterol, glucose, iron, uric acid, and GGT-were measured using an automated clinical chemistry analyzer (Randox Monaco, Randox Laboratories Ltd., UK).

Histopathology

Fixed tissues were trimmed, labeled, and washed for 2 h under running tap water. Dehydration was performed in graded isopropyl alcohol (30-100%), cleared in xylene, and paraffin-embedded using an automated tissue processor (Leica TP1020) and embedding station (Leica EG1160). Sections (4-5 µm) were cut with a rotary microtome (Leica RM2255) and mounted on poly-L-lysine-coated slides. Slides were deparaffinized, rehydrated, and stained with Harris' hematoxylin, differentiated in acid alcohol, blued in ammonia water, and counterstained with eosin. After dehydration and clearing in xylene, sections were mounted with DPX. Staining was performed using an automated stainer (Gemini AS, Thermo Scientific) following standard protocols (Luna, 1968; Suvarna et al., 2012) [16,21] .

Immunohistochemistry

Formalin-fixed, paraffin-embedded liver tissues from male rats were sectioned at 4 μm on poly-L-lysine-coated slides.

Sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in EDTA buffer (pH 8.5). Endogenous peroxidase activity was blocked with 3% H₂O₂, followed by incubation with primary antibodies against CYP3A1 (1:500) and CYP2E1 (1:200). After washing with Tris-buffered saline, slides were incubated with HRPconjugated secondary reagent (Dako REAL™ EnVision™) for 30 min at room temperature, and color was developed using DAB chromogen. Sections were counterstained with Gill's hematoxylin, dehydrated, cleared, and mounted for microscopic evaluation.

Statistical Analysis

Data on body weights, hematological and biochemical parameters, and organ weights were analyzed using twoway analysis of variance (ANOVA) followed by Dunnett's post hoc test. Statistical significance was set at p<0.05.

Results and Discussion Clinical Observations and Body Weight

No abnormal clinical signs were observed in Groups I and II throughout the 28-day study. One male in Group III and two males and one female in Group IV showed intermittent diarrhea from Day 9 onwards. One Group IV female exhibited temporary circling movements but recovered spontaneously. Similar clinical signs, including tremors and restlessness, have been reported in rats following administration of Catharanthus roseus extracts at high doses (Vutukuri et al., 2017; Kevin et al., 2012) [24,14] . No mortality was recorded in any group, aligning with previous findings in rats treated with Catharanthus extracts (Ukoha et al., 2017; Ajuru et al., 2019) [23,2] . Body weight remained unaffected at all dose levels (up to 3000 mg/kg), which agrees with earlier reports in rats exposed to Catharanthus extracts (Kevin et al., 2012) [14] .

Hematology and Biochemistry

No significant changes in hematological parameters were observed in males. In females, a significant increase in platelet count was noted in Groups II and IV, while other parameters were comparable to controls. Terpenoid indole alkaloids in Catharanthus species (e.g., vindoline, catharanthine, yohimbine) may have vincristine-like activity, contributing to thrombocytosis (Mackin et [17,3,26] . Biochemical parameters remained within normal limits in all groups, indicating no functional impairment of major organs, similar to findings in Catharanthus toxicity in sheep (Aydogan et al., 2015) [4] .

Organ Weights

No significant changes in absolute or relative organ weights were observed in females. In males, an increase in spleen (Groups II-IV) and relative liver weight (Group IV) was noted. These changes may reflect lymphoid hyperplasia and mild hepatic alterations. Sex-related physiological differences may explain the male-specific effects, as reported previously (Jothy et al., 2011; Yuet et al., 2013) [13,25] .

Gross and Histopathology

No gross lesions were observed in any group. Microscopically, male rats in Group IV showed minimal hepatocellular necrosis (Figure 2), vacuolation, and neutrophilic inflammation localized to periportal and midzonal regions (Figure 3). Lower dose groups showed no hepatic lesions. Splenic lymphoid hyperplasia (Figure 4) was observed in all Group IV males and in some Group II and III males, characterized by expansion of the periarteriolar lymphoid sheath (Figure 5) and prominent germinal centers. Other organs, including brain, kidneys, lungs, heart, adrenals, thymus, reproductive organs, GI tract, salivary glands, and lymph nodes, were unremarkable. Similar hepatic changes have been reported with Catharanthus roseus extracts in rats (Ajuru et al., 2019; Elshama et al., 2014) [2,8] . The observed lymphoid hyperplasia may reflect immunostimulation due to bisindole alkaloids in Catharanthus pusillus (El-Sayed & Cordell, 1981) [6,7] .

Immunohistochemistry

Because liver lesions occurred only in males, CYP expression was evaluated in this group. CYP2E1 showed strong pericentral cytoplasmic immunoreactivity in controls and increased midzonal and periportal staining in Groups II-IV, suggesting enzyme induction (Figure 6). CYP3A1 expression remained comparable among all groups. These findings suggest that CPE may induce or activate CYP2E1, contributing to its metabolism. Similar CYP modulation has been reported with vindoline in rats (Zhang et al., 2022) [27] .

Conclusion

This study demonstrated that 28-day oral administration of Catharanthus pusillus methanolic extract (CPE) up to 3000 mg/kg body weight did not produce significant changes in body weight, hematological, or biochemical parameters. However, dose-related increases in spleen and liver weights were observed, accompanied by minimal hepatocellular necrosis and splenic lymphoid hyperplasia. Immunohistochemical analysis revealed induction of CYP2E1 expression, indicating metabolic activation, while CYP3A1 remained unaffected. These findings suggest that CPE induces subacute toxic effects at higher doses. Therefore, a No Observed Adverse Effect Level (NOAEL) could not be established under the conditions of this study.

Acknowledgement

AcknowledgementAuthors are thankful to the Principal, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar, for providing necessary facilities and sanctions for conduct of research work.

References

1. Ahmedulla M, Nayar MP. Red data book of Indian plants. Vol. 4. Calcutta: Botanical Survey of India; 1999.
2. Ajuru MG, Ajuru G, Nmom FW, Worlu CW, Igoma PG. Toxicological studies on ethanolic extract of Catharanthus roseus leaves in Wistar albino rats. Journal of Applied Sciences. 2019;19(3):217-222. doi:10.3923/jas.2019.217.222
3. Allen DG, Houston RH, Mackin AJ. Vincristineinduced thrombocytosis in dogs: Case series and literature review. Veterinary Clinical Pathology. 2021;50(4):567-574. doi:10.1111/vcp.13047
4. Aydogan A, Sezer K, Ozmen O. Clinical and pathological investigations of accidental Catharanthus roseus toxicity in sheep. Israel Journal of Veterinary Medicine. 2015;70(4). Available from: https://www.ivis.org/library/israel-journal-ofveterinary-medicine
5. Chandran R, Saj A. Ethnopharmacological relevance of Catharanthus pusillus. Journal of Ethnopharmacology. 2015;172:408-415. doi:10.1016/j.jep.2015.06.045
6. El-Sayed M, Cordell GA. Catharanthus alkaloids. Journal of Natural Products. 1981;44(3):289-299. doi:10.1021/np50015a001
7. El-Sayed M, Cordell GA. Catharanthus alkaloids. XXXIII. Alkaloids of Catharanthus pusillus. Journal of Natural Products. 1981;44(2):179-183. doi:10.1021/np50014a003
8. Elshama SS, Moustafa RH, Abdel-Aziem SH. Toxic effects of aqueous extract of Catharanthus roseus on liver and kidney functions in rats. Journal of Applied Pharmaceutical Science. 2014;4(12):57-62. doi:10.7324/JAPS.2014.41210
9. Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo Z. Medicinal plants in therapy. Bulletin of the World Health Organization. 1985;63(6):965-981.
10. Gajalakshmi S, Vijayalakshmi S, Devi RV. Phytochemical and pharmacological properties of Catharanthus species: A review. International Journal of Pharmaceutical Sciences Review and Research. 2013;23(1):333-340.
11. Groten C. Toxic plants and livestock poisoning. In: Gupta RC, editor. Veterinary toxicology: Basic and clinical principles. 2nd ed. Elsevier; 2013. p. 12051223.
12. Harborne JB. Phytochemical methods. London: Chapman & Hall; 1964.
13. Jothy SL, Zakaria Z, Chen Y, Lau YL, Latha LY, Sasidharan S. Acute oral toxicity of methanolic seed extract of Cassia fistula in mice. Molecules. 2011;16(6):5268-5282. doi:10.3390/molecules16065268
14. Kevin LY, Hussin AH, Zhari I, Chin JH. Sub-acute oral toxicity study of methanol leaves extract of Catharanthus roseus in rats. Journal of Acute Disease. 2012;1(2):130-134. doi:10.1016/S2221-6189(13)600305
15. Khandare AL. Economic impact of livestock poisoning by toxic plants. Journal of Veterinary Science and Animal Husbandry. 2015;2(3):34-40.
16. Luna LG. Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. 1968.
17. Mackin AJ, Callan MB, Henry CJ, Couto CG. Effects of vincristine and prednisone on platelet numbers and function in clinically normal dogs. American Journal of Veterinary Research. 1995;56(2):261-267. PMID:7745595
18. Navitha A, Sridevi V, Subramanian R. Catharanthus pusillus: A review on phytochemistry and pharmacology. International Journal of Pharmacy and Pharmaceutical Sciences. 2012;4(2):45-48.
19. Satish S. Phytochemicals as bioactive compounds in medicinal plants. Indian Journal of Experimental Biology. 2002;40(6):708-713.
20. Subbaiyan R, Arumugam T, Gopalakrishnan VK. Antineoplastic activity of Catharanthus pusillus. Asian Journal of Pharmaceutical and Clinical Research. 2013;6(4):186-189.
21. Suvarna M, Anuradha C, Kumar KK, Sekhar PC, Chandra KLP, Reddy B R. Cytomorphometric analysis of exfoliative buccal cells in type II diabetic patients. Journal of Dr. NTR University of Health Sciences. 2012;1(1):33.
22. Thingujam I, Singh MK, Sharma A. Antioxidant potential of Catharanthus pusillus. Journal of Applied Pharmaceutical Science. 2015;5(5):92-96. doi:10.7324/JAPS.2015.50516
23. Ukoha AI, Okereke SC, Arunsi UO, Ngwogu AC, Jack AB, Nwachukwu DC, Uwaezuoke JC. Sub-lethal assessment of aqueous dried leaf extract of Catharanthus roseus (Linn.) G. Don in male albino rats. MOJ Toxicology. 2017;3(5):00068. doi:10.15406/mojt.2017.03.00068
24. Vutukuri VR, Das MC, Reddy M, Prabodh S, Sunethri P. Evaluation of acute oral toxicity of ethanol leaves extract of Catharanthus roseus in Wistar albino rats. Journal of Clinical and Diagnostic Research. 2017;11(3):FF01-FF04. doi:10.7860/JCDR/2017/24937.9325
25. Yuet PK, Chen Y, Sasidharan S, et al. Toxicological evaluation of Cassia spectabilis leaf extract in rats. Journal of Ethnopharmacology. 2013;150(1):453-458. doi:10.1016/j.jep.2013.08.037
26. Zárate LG, Filippini R, De Luca V. Terpenoid indole alkaloid profile changes in Catharanthus pusillus during development. Phytochemistry. 2001;58(5):717722. doi:10.1016/S0031-9422(01)00340-5
27. Zhang X, Li J, Wang Y, Zhou Y, Chen X. Vindoline inhibits rat CYP2D1 activity in vitro: Implications for herb-drug interactions of Catharanthus alkaloids. Xenobiotica. 2022;52(10):1119-1128. doi:10.1080/00498254.2022.2031845