This study obtained ethical approval from The Ethical Committee of Medical Research, Faculty of Medicine, The University of Lambung Mangkurat with No. 240/KEPK-FK UNLAM/EC/VII/2020

Forty-two female healthy Wistar rats (Rattus novergicus), weighing 120–170 grams and age of 45 days, were purchased from Animal Breeding House Unit, University of Gadjah Mada, Yogyakarta, Indonesia. The animals were acclimatized to the laboratory conditions for 7 days and maintained under 12 hours light and 12 hours dark conditions. The animals were kept in polypropylene cages in the Animal House of Center for Food and Nutrition Studies at room temperature 22 °C±3 °C with free access to standard rat pellets and water ad libitum.

Experimental animals were divided into seven groups with each group consisting of six animals: three control groups, namely G_normal (non-cancer control group/normal control), G_cancer (cancer without treatment group) and G_doxo (cancer with standard doxorubicin treatment group), and four treatment with C. odorata extract with a dose of 500, 1000, 2000 and 4000 mg/kg body weight (BW), assigned as G_Co500, G_Co1000 G_Co2000, and G_Co4000.

This range of doses was selected based on previous acute toxicity studies. Although other study reported acute toxicity on 2700 mg/kg BW (Ijioma et al. 2014), many other studies found that the ethanol extract of C. odorata leaves was well-tolerated by adult mice between 10–5000 mg/kg BW (Haji Jasnie 2009; Aba et al. 2015; Asomugha 2015). Another guideline provides the use of in vitro cytotoxicity IC50 (concentration at which cell viability is inhibited by 50%) values to estimate acute in vivo toxicity LD50 (the dose that produces lethality in 50% of the animals tested) values using Registry of Cytotoxicity (RC) prediction model (NTP 2001). However, the guideline was based on known chemicals and cannot be extrapolated to the crude extract as in our study.

After acclimatization, the body weight of all experimental animals was measured and followed by breast cancer induction by DMBA in all groups, except G_normal (assigned as time 1, T1). The induction was performed by feeding 20 mg/kg BW of DMBA suspended in CMC-Na 0.5% orally three times per week for 5 weeks. At the end of T5, breast palpation was performed to calculate the number, diameter, and volume of the nodules formed. The administration of doxorubicin and C. odorata extract was started on T6. Doxorubicin was given for 11 weeks with a dose of 15 mg/kg BW once a week intraparenterally for G_doxo group, while the treatment group received the ethanol extract of C. odorata leaves every day according to the dose, orally for 11 weeks. Data on the body weight, breast palpation, and nodule volume measurement were collected on T11 and T17 (completion of the experiment). On T17, after all data was collected, the animal was euthanized by injecting 2 mg/kg BW ketamine to the experimental animal, followed by nodule weight measurement and breast tissue collection.

C. odorata leaves was carefully identified at Biology Research Center of the Indonesian Institute of Sciences “Herbarium Bogoriense” or the Biology Laboratory of the Faculty of Mathematics and Natural Sciences, Syiah Kuala University, Banda Aceh. Fresh twenty-five kilograms of C. odorata leaves were collected, washed with running water three times, dried for two weeks, and then grounded using a grinder mill. Ten kilograms of C. odorata grounded leaves were then extracted using 80% ethanol with frequent stirring for 24 hours. The liquid extract was then filtered using Whatman filter paper and the residue was re-extracted three times with fresh solvent every 24 hours. The extract was filtered and concentrated by using a rotary evaporator at 40 °C. The final extract (220 gr) was then stored in a wide-mouthed and tightly closed bottle at 4 °C until used.

The phytochemical analysis of the ethanol extract of C. odorata used in this study had been previously conducted by using Gas chromatography–mass spectrometry (GC-MS) and published in different study (Yusuf et al. 2021). The result was presented in Suppl. material 1: Table S1.

The tumor nodule was calculated by breast palpation. The breast palpation was performed three times: after induction (at the end of T5), and at the end of T11 and T17 (at the end of the experiment).

The number of nodules was measure by breast palpation of the Wistar rats on T11 and T17. The diameter of the cancer nodule was measured by using callipers with an accuracy of 0.05 cm. This nodule diameter data is used to calculate the nodule volume using the following formula (Kubatka et al. 2014): V = π. (S₁)². S₂/12 (S₁, S₂ are tumor diameters; S₁ < S₂). The nodule weight was measured on T17 after euthanasia by weighing the nodule after surgery on each group.

Tumors were removed from euthanized rats, washed with 0.9% NaCl and fixed in 10% formalin fixative for 24 h. The tissues were then dehydrated in ascending series of alcohol (from 70% to absolute alcohol), cleared with xylol and embedded in paraffin wax with a melting point of 56–58 °C. The blocks were cut to obtain 4- to 5-μm-thick serial sections using a rotary microtome, stained with hematoxylin-eosin, and observed under a light microscope with 10–40 × magnification (BX51, Olympus company, Japan).

AgNOR staining was performed according to the guidelines (Aubele et al. 1994; Ofner et al. 1994). Each section was soaked in sodium citrate buffer (at pH 6.0) and incubated in an autoclave at 120 °C for 20 minutes. The slide was allowed to cool down to 37 °C followed by soaking the slide in a freshly prepared silver staining solution containing one part by volume of 0.5% gelatine in 1% formic acid and two parts of 50% aqueous silver nitrate solution, incubated at 37 °C for 11 minutes. The reaction was stopped by washing the slides with double‐distilled deionized water. All stained sections were dehydrated in increasing grades of concentration of ethanol and then clarified in xylene.

For each AgNOR stained slide, at least 100 nuclei per microscopic field was calculated for three microscopic fields. The observation was performed under a light microscope with 1000 × magnification (BX51, Olympus company, Japanese). The AgNOR counting was performed by dividing the total number of silver-stained dots per cell by the total of cells observed. We also calculated the average silver-stained dot in the treatment group with G_cancer and G_doxo.

Results are presented as means ± standard deviation (SD). Data was analyzed using the one-way analysis of variance (ANOVA) with 95% confidence interval (05% CI). Multiple comparisons were carried out with the Least Significant Difference (LSD) test. Statistical significance of differences was considered at a p-value < 0.05.

The success of breast cancer induction was indicated by palpation of the mass during breast examination of the rats. The number of breast nodules was decreased in all groups, except animals in Gcancer, which continued to increase each week (Fig. 1). After week 6^th, the number of nodules in G_Co2000 and G_Co4000 decreased significantly when compared to G_cancer (p < 0.05). A decrease of tumor nodules was also observed at week 11^th\in all treatment groups (except G_Co500) when compared with G_normal and G_cancer (p < 0.05 in all comparisons). At the end of treatment (week 17^th), the number of nodules in the treatment group (G_Co500, G_Co1000 G_Co2000, and G_Co4000.) also significantly decreased when compared to G_cancer (p < 0.001). However, there was no statistically significant decrease in the number of nodules between the G_doxo and the treatment group. There was also no statistically significant reduction in the number of nodules with different doses among all treatment groups.

Figure 1. 

The means of total nodules observed on T5, T6, T11, and T17.

The volume of cancer nodules at week 6^th in all treatment groups decreased significantly when compared with G_cancer (p < 0.0001) except G_Co2000 (Fig. 2). Meanwhile, when compared with G_doxo, a significant reduction in the nodule volume was observed only at G_Co1000 and G_Co4000 (p < 0.05). At week 11^th, the nodule volume decreased significantly only in G_Co2000 when compared to G_doxo (p < 0.0001).

Figure 2. 

The means of nodules volume observed on T1, T6, and T1.

The weight of the cancer nodule at week 16^th was also significantly reduced in G_Co2000 when compared to G_doxo (P < 0.0001). The weight of cancer nodules also differed significantly when comparing G_Co500 with G_Co1000, G_Co2000 and G_Co4000 (p < 0.001). However, there was no statistically significant decline of nodule weight among all treatment groups with different extract doses (Fig. 3). The most significant decrease in the mean weight of cancer nodule was observed in G_Co2000.

Figure 3. 

The means of nodules weight observed on week 16^th.

On weight observation at week 6^th, G_Co2000 and G_Co4000 showed significant weight gain compared to G_doxo (p < 0.001 in both comparisons). Whereas at week 11^th, body weight of all treatment groups increased significantly compared to G_doxo (p < 0.001), except G_Co500. At the end of the treatment, the body weight of the treatment group increased significantly compared to G_doxo, where p < 0.05 for G_Co500 & G_Co1000 and p < 0.001 for G_Co2000 and G_Co4000 (Fig. 4). A significant increase in body weight was also observed among the treatment groups with the increasing dose of C. odorata extract, the body weight of the experimental animals increased significantly, especially at week 11^th (p < 0.05 in all comparisons) and week 16^th (p < 0.001 in all comparisons).

Figure 4. 

The means of body weight of experimental animals observed during the experiment.

There was a significant reduction in the AgNOR point in breast cancer cells in all treatment groups when compared with G_cancer(p < 0.05 in all comparisons). Among the treatment groups, there was a significant difference of the AgNOR points at G_Co1000, G_Co2000, and G_Co4000 when compared to G_Co500 (p < 0.001). AgNOR points in cancer cells of experimental animals in G_Co4000 (group with maximum C. odorata extract dose) decreased although not statistically significant when compared with G_Co1000 & G_Co2000. The highest decrease in AgNOR point was observed in G_Co2000, although not statistically significant when compared to G_doxo (Fig. 5).

Figure 5. 

The means of AgNOR point on breast cancer tissue of experimental animals.