Z. acanthopodium fruits (family Rutaceae) were collected from Kabanjahe Regency, Berastagi Indonesia (30°17'50"N to 3°18'39"N and 98°36'0"E to 98°36'36"E). The voucher was identified and authenticated by Dr N. Pasaribu (an Indonesian botanist), and deposited in the Herbarium Medanense (registration number 5940/MEDA/2022), at Universitas Sumatera Utara, Indonesia. MMP-9 (matrix metalloproteinase 9) ELISA Kit, catalogue number: E-EL-R3021 (Elabsciences, Houston, Texas, United State); rabbit polyclonal GLUT1 IHC antibody, catalogue number: IW-PA1120 (IHC WORLD, LLC Ellicott City, MD 21042, USA); rabbit polyclonal MMP9 antibody (ab237782), catalogue number: EPR22140-154 and BSA- and azide-free rabbit polyclonal antibody for cellular apoptosis susceptibility/CSE1L (ab96755) (Abcam, Cambridge Biomedical Campus Cambridge CB2 0AX, UK) were used in this study.

Zanthoxylum acanthopodium (andaliman) fruits were cleaned and then dried in the drying room and ground to a powder. The extract was prepared by macerating 10 kg of dried Zanthoxylum acanthopodium fruit in 10 litres of 96% methanol for 1 night. Then, it was filtered and evaporated to produce the dry extract. The phytochemical analysis results for Zanthoxylum acanthopodium were confirmed by subsequent studies (Wijaya et al. 2019; Situmorang et al. 2020).

The University of Sumatera Utara’s Animal House Laboratory provided 36 female rats (Rattus norvergicus) of the Wistar strain, weighing 180–200 g (8–12 weeks old) for this study. During the study, the rats were fed standardized food pellets and given sufficient water every day. They were acclimated to laboratory settings for 4 weeks. Cervical cancer was induced by injecting benzopyrene 50 mg/kg BW into their cervices and then the tumour was allowed to grow for 3 months (Simanullang et al. 2022).

The research was conducted at the University of Sumatera Utara’s Biology Laboratory, the Pathology and Anatomy Laboratory of the Faculty of Medicine, Universitas Methodist Indonesia and STIKes Murni Teguh. Doses of 100, 200 and 400 mg/kg were selected based on the acute toxicity test results of previous studies (Situmorang et al. 2020) and other researchers (Alam et al. 2020). There were five treatment groups consisting of 6 rats each: the control group (Group C-), the cancer model group (Group C+) without treatment, the ZAM100 group of cancer-bearing rats given a dose of 100 mg/kg BW of ZAM, the ZAM200 group of cancer-bearing rats given a dose of 200 mg/kg BW of ZAM and the ZAM400 group of cancer-bearing rats given a dose of 400 mg/kg BW of ZAM.

Each group was given its respective dose of extract orally for 30 days. On day 31, the rats were euthanised with chloroform and then dissected to collect blood and the cervix. Immunohistochemistry and a TUNEL assay were used to stain cervical tissue. The Ethics Committee for Handling Experimental Animals, Faculty of Mathematics and Natural Sciences USU approved this study (ethical clearance: No. 0262/KEPH-FMIPA/2022).

The analysis was conducted at Universitas Methodist Indonesia. First, 2–4 mg/ml of blood and SOD standard sample was added to the reaction mixture in the presence and absence of 1mM cyanide to measure SOD activity. Second, lipid oxidation was identified with the thiobarbituric acid reactive test (TBARS), which measures the products of the unsaturated fatty acid endoperoxides produced by lipid oxidation using an ELISA reader at 450 nm.

A microtome was used to cut 4-micrometre-thick slices of the paraffin-embedded cervical tissue (Qin et al. 2018). For pre-treatment, the tissue was heated in citrate buffer at a pH of 6.0. After a phosphate-buffered saline (PBS) wash, the tissue was incubated with antibodies at 37 °C according to the manufacturer’s instructions before being treated with avidin-biotin-peroxidase. 3,3-diaminobenzidine (DAB) hydrochloride was used for the chromogenic visualisation reaction. The slices were then stained with haematoxylin, after Mayer (Situmorang et al. 2021a). The stained cervical tissue score was calculated by multiplying the positive result by the staining intensity, where 0 indicated that less than 10% of the cells were stained, 1 indicated that 10–25% of the cells were stained (negative), 2 indicated that 25–50% of the cells were stained (weak), 3 indicated that 50–75% of the cells were stained (moderate) and 4 indicated that more than 75% of the cells were stained (strong).

To study apoptotic cells, the cervical tissue was stained with the TUNEL assay technique (Kyrylkova et al. 2012). Slides with mounted cervical slices were immersed in xylene for 5 minutes, then rehydrated with graded ethanol (70–100%) and rinsed with 0.85% PBS. Following the manufacturer’s instructions for antigen retrieval, the slides were rinsed in TBS-Tween (TBST) for 1 minute. A working solution of Proteinase K was applied to the slides and they were incubated for 10 minutes before being rinsed in 1X TBST for 1 minute. The diluted rTdT reaction mixture before endogenous peroxidase synthesis, the slide was submerged in citrate buffer at a pH of 6.0 for 15 minutes at room temperature and then rinsed with PBS. The tissue was then incubated at room temperature for 30 minutes. Then, 3,3-diaminobenzidine hydrochloride (DAB) was utilized for the chromogenic imaging reaction. The slides were immersed in ethanol and xylene before being covered with glass. The researcher used a light microscope to observe five fields of view.

A one-way ANOVA test was used for data analysis. If the p-value was less than 0.05, there was a significant difference between groups and if the p-value was higher than 0.05, there was no difference between groups. A Kruskal-Wallis test (for non-parametric data) was used to analyse the data with SPSS V.22.

Data on body and cervical weight from each experimental group are shown in Table 1. There was an insignificant difference (p > 0.05) between groups before the injection of 50 mg/kg BW benzopyrene in the cervix. After benzopyrene injection, there was a significant difference between group C- and C+ (p = 0.04). Table 1 also shows that cervical weight was significantly different in C- vs. C+ (p = 0.004) or in C+ vs. ZAM100 (p = 0.03), ZAM200 (p = 0.03) or ZAM400 (p = 0.004).The injection of benzopyrene and ZAM treatment affected body weight and cervical weight significantly.

No significant difference in the body weight of cervical-cancer-bearing rats was found between groups after the administration of ZAM at doses of 100 to 400 mg/kg BW. Tumour cells can proliferate indefinitely and exhibit excessive angiogenesis. However, in the cervical-cancer-bearing rats, the impact was not significant (Clarke et al. 2018). This could be due to the small size of cervical cancer, rats’ fat or excessive activity (Clarke et al. 2018). Cervical tumours could influence cervical weight in both the control and ZAM groups.

There was a significant difference in SOD and MDA levels in C+ rats compared to the C- group (p < 0.05), with cancer-bearing rats having lower SOD levels. SOD levels increased in cancer-bearing rats given a ZAM dose of 100 mg/kg BW, but decreased under a dose of 400 mg/kg BW (Fig. 1a). This contrasts with the increased MDA levels in cancer-bearing rats, for which there was a significant difference between C- and C+ (p > 0.01) (Fig. 1b), and ZAM administration at doses ranging from 100 to 400 reduced MDA levels in cancer-bearing rats. ZAM more effectively altered SOD levels in rats with cervical cancer at doses of 100 and 200 mg/kg BW and MDA levels at 200 and 400 mg/kg BW. Increased lipid peroxidation due to antioxidant deficiency is linked to increased circulating levels of MDA and decreased SOD activity in tissues in general, including in cervical cancer tissue (Thakur et al. 2015; Sherif et al. 2018). Furthermore, elevated MDA levels in tumour tissue may be linked to SOD deficiency. Long-term, superoxide anions, which are highly radical and can penetrate membranes, accumulate and cause negative effects away from the tumour (Sherif et al. 2018). The decreased MDA levels and increased SOD activity are thought to be because the andaliman fruit has certain substances that can affect their expression. Cells can be protected from oxidant interference and oxidative stress via ZAM administration, increasing SOD activity and reducing the effects of diseases, including cancer.

Figure 1. 

Levels of Superoxide Dismutase (SOD) and Malondialdehyde (MDA) in cancer model rats. a. SOD; b. MDA. C-: Control, C+: cancer-bearing rats ZAM100: cancer-bearing rats with a dose of 100mg/BW of ZAM, ZAM200: cancer-bearing rats with a dose of 200 mg/BW of ZAM, ZAM400: cancer-bearing rats with a dose of 400 mg/BW of ZAM (#p < 0.05 vs. C-, ##p < 0.01 vs. C-, *p < 0.05 vs. C+, **p < 0.01 Vs. C+, ^nsp > 0.05).

As shown in Table 2, a statistically significant difference (p < 0.00) appeared among the treatment groups. According to the average, there was a significant difference in MMP-9 expression (p < 0.01) compared to C-. The lowest dose of ZAM (100 mg/kg BW) had no significant effect (p > 0.05), but doses of 200 and 400 mg/kg BW were significant (p < 0.05). Cervical cells in group C- had normal epithelial and nuclear layers (Fig. 2a). In contrast, as Fig. 2b (C+ group) shows, undifferentiated cells were confined to the lower layers of the epithelium and developed mitotic features. Lower epithelial cell changes were characterised by epithelial thickening and increased MMP-9 expression. Additionally, MMP-9 expression in cancer tissue decreased as the ZAM dose increased. ZAM (Fig. 2c, e) administration at different doses reduced the number of nuclei that were stained brown by immunohistochemistry, indicating a positive index of MMP-9 expression in cancer tissue. Carcinomas that were uncontrollably spreading in the C+ group slowed and no longer developed into the epithelium. Fig. 3 shows that serum MMP-9 levels in cancer-bearing rats differed significantly (p < 0.05) from C- rats, but doses of 100 and 200 mg/kg BW did not result in significant differences (p > 0.05) from C+ rats. The highest dose (400 mg/kg BW) produced significant results. As Table 2 and Fig. 2 show, administering ZAM, particularly at 400 mg/kg BW, can suppress the expression of MMP-9 cervical cancer histological changes.

Figure 2. 

MMP-9 Expression and Histology changes of cervical cancer model in rats. a. Control (C-); b. cancer-bearing rats (C+); c. cancer-bearing rats with a dose of 100mg/BW of ZAM (ZAM100); d. cancer-bearing rats with a dose of 200 mg/BW of ZAM (ZAM200); e. cancer-bearing rats with a dose of 400 mg/BW of ZAM (ZAM400). MMP-9 expression is indicated by a brown-black color in histology (400x).

Figure 3. 

MMP-9 Expression in serum of cervical cancer model rats. C-: Control, C+: cancer-bearing rats ZAM100: cancer-bearing rats with a dose of 100mg/BW of ZAM, ZAM200: cancer-bearing rats with a dose of 200 mg/BW of ZAM, ZAM400: cancer-bearing rats with a dose of 400 mg/BW of ZAM (#p < 0.05 vs. C-, *p < 0.05 vs. C+, ^nsp > 0.05).

ZAM, particularly at a dose of 400mg/kg BW, can suppress the expression of MMP-9 in cervical cancer. The greater the immunohistochemical positivity of MMP-9, measured as intensity and quantity, the more severe the cervical neoplastic lesions (Mondal et al. 2020). MMP-9 immunohistochemical expression is elevated in cervical tumours and contributes to carcinogenesis. Cervical carcinomas can be polypoid or infiltrative on the macroscopic level. Unlike polypoid tumours, infiltrative tumours will invade and damage the surrounding tissue. Microscopically, their enlargement, rough chromatin and prominent nucleoli can be seen throughout the thickness of the squamous epithelial layer (Tanaka et al. 2019). The body’s ability to degrade MMP depends strongly on the balance of active enzymes and natural inhibitors. MMP damages the basement membranes of blood vessel walls, allowing tumour cells to enter and exit the bloodstream (intravasation and extravasation) (Rajesh and Mandal 2017; Tanaka et al. 2019). MMPs also affect the modification of new microenvironments at metastasis sites, aiding metastatic tumour cell growth (Mondal et al. 2020). Zanthoxylum acanthopodium contains alkaloids, glycosides, tannins, phenols and flavonoids that act as antioxidant, anti-inflammatory and antibacterial substances (Wijaya et al. 2019: Li et al. 2020). These substances are suspected to drive ZAM’s MMP-9 expression control in the tissues and serum of cancer-bearing mice. Effective cancer treatment strategies may include applying appropriate antioxidant-containing compounds as inhibitors of free-radical-generating compounds.

Table 3 shows that both the Kruskal-Wallis test and the Mann-Whitney follow-up test revealed significant differences among groups. According to the mean values, there was a significant difference (p < 0.001) in GLUT-1 expression between the C+ and C- groups. The difference was not significant at the lowest ZAM dose (100 mg/kg BW) but was significant at 200 and 400 mg/kg BW (p < 0.01). The C+ group had the highest level of GLUT-1 expression, while the C- group had the lowest level, occasionally showing no expression at all. Fig. 4a shows that C+ rats exhibited normal histological changes, but the carcinoma had spread to the pelvic wall, there was no clear space between the tumour and the pelvic wall and the core was irregular (Fig. 4b). Cancer cell metabolism is reprogrammed to promote cancer cell proliferation (Sharen et al. 2017). Cancer cells frequently have high glucose metabolism values compared to normal cells to support their proliferative ability (Pragallapati and Manyam 2019). This contrasts starkly with the C- group’s histology, which showed that the cervical tissue still contained normal cells (Fig. 4a). The lesions in the group at the lowest ZAM dose (Fig. 4c) were larger than in the control group, but GLUT-1 expression began to decrease. The reduction in GLUT-1 expression at doses of 200 and 400 mg/kg BW (Fig. 4d, e) shows that andaliman could significantly reduce GLUT-1 expression. The space between tumours was reduced, the carcinoma stopped developing and the nuclei began to form normally. Fig. 5 shows that serum GLUT-1 levels in cancer-bearing rats were significantly different (p < 0.01) from C- rats, but not significantly different (p > 0.05) from C+ at 100 mg/kg BW.

Figure 4. 

GLUT-1 Expression in Histology changes of cervical cancer model rats. a. Control (C-); b. cancer-bearing rats (C+); c. cancer-bearing rats with a dose of 100mg/BW of ZAM (ZAM100); d. cancer-bearing rats with a dose of 200 mg/BW of ZAM (ZAM200); e. cancer-bearing rats with a dose of 400 mg/BW of ZAM (ZAM400). GLUT-1 expression is indicated by a brown-black color in histology (400x).

Figure 5. 

GLUT-1 Expression in serum of cervical cancer model rats. C-: Control, C+: cancer-bearing rats ZAM100: cancer-bearing rats with a dose of 100mg/BW of ZAM, ZAM200: cancer-bearing rats with a dose of 200 mg/BW of ZAM, ZAM400: cancer-bearing rats with a dose of 400 mg/BW of ZAM (##p < 0.01 vs. C-, *p < 0.05 vs. C+, ^nsp > 0.05).

ZAM administration can reduce GLUT-1 expression and improve histology in cervical cancer. The expression of GLUT-1 in cancer can indicate a tumour’s metabolic and vascular requirements, which have clinical implications for survival and treatment plans. Given the importance of GLUT-1 in oncogenesis, several studies have been conducted to investigate its prognostic value in tumours (Pragallapati and Manyam 2019). GLUT-1 overexpression may be linked to increased glucose metabolism in cancer cells (Zambrano et al. 2019). Andaliman doses from 200 to 400 mg/kg BW produced significant results, and serum GLUT-1 analysis affirms that ZAM can reduce GLUT-1 expression in cervical cancer. Thus, ZAM doses of 200 and 400 mg/kg BW can reduce GLUT-1 expression in the serum and improve histology in cervical-cancer-bearing rats, as the data in Table 3, Figs 4, 5 show.

The data on apoptotic cells from each experimental group is presented in Table 4. They show that both the Kruskal-Wallis test and the Mann-Whitney follow-up test revealed significant differences. There was a significant difference (p < 0.05) in the mean values of apoptotic cells between the C+ and C- groups. The difference was not significant at 100 mg/kg BW ZAM but was significant at 200 mg/kg BW (p < 0.01) and 400 mg/kg BW (p < 0.001). The C+ group had the highest level of apoptotic cell expression, while the C- group had the lowest level.

Fig. 6 depicts the apoptotic histology of the rat cervix after benzopyrene injection and ZAM administration at various doses. The histology of cervical tissue in group C- (Fig. 6a) was healthy and normal, but it changed dramatically after benzopyrene injection in group C+. Fig. 6b depicts the histology of C+ cervices with irregular cell nuclei forming bubbles known as apoptotic bodies. Apoptosis has been linked to increased ROS production and oxidative stress, thus contributing to cancer pathogenesis and aetiology (Situmorang and Ilyas 2018). The environment within the cell nucleus appears disjointed and karyorrhexis occurs. Because the protein structures that comprise the cytoskeleton are digested by a specific peptidase enzyme (caspase) that is activated in the cell, the cell becomes circular (red arrow). The absence of apoptotic regulation lengthens cancer cells’ lifespans and allows more time for mutations to accumulate, which can increase invasiveness during tumour progression, induce angiogenesis, deregulate cell proliferation and interfere with differentiation (Chen et al. 2004). Apoptosis can be triggered with herbal medicine to repair tissue (Situmorang and Ilyas 2018). Histological features differed significantly between treatments (p < 0.05). According to the statistical data (Table 4), ZAM100 and ZAM200 rats showed higher apoptosis than ZAM400 rats (Fig. 6c, e). This is because the 400 mg/kg BW dose was the highest, so there was little apoptosis in the tissue.

Figure 6. 

Apoptotic cells in Histology changes of cervical cancer model rats. a. Control (C-); b. cancer-bearing rats (C+); c. cancer-bearing rats with a dose of 100mg/BW of ZAM (ZAM100); d. cancer-bearing rats with a dose of 200 mg/BW of ZAM (ZAM200); e.cancer-bearing rats with a dose of 400 mg/BW of ZAM (ZAM400). Yellow arrows indicate low expression of apoptosis in tissues, while red arrows indicate high expression (400x).