Research Article |
Corresponding author: Syafruddin Ilyas ( syafruddin6@usu.ac.id ) Academic editor: Rumiana Simeonova
© 2024 Cheryl Grace Pratiwi Rumahorbo, Syafruddin Ilyas, Salomo Hutahaean, Cut Fatimah Zuhra.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rumahorbo CGP, Ilyas S, Hutahaean S, Fatimah Zuhra C (2024) Bischofia javanica and Phaleria macrocarpa nano herbal combination on blood and liver-kidney biochemistry in Oral Squamous Cell Carcinoma-induced rats. Pharmacia 71: 1-8. https://doi.org/10.3897/pharmacia.71.e117398
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This research explores a contemporary approach to managing Oral Squamous Cell Carcinoma (OSCC) by integrating nano-technology into herbal production, combining Bischofia javanica leaves and Phaleria macrocarpa fruits. Benzo[a]pyrene (BaP)-induced OSCC rats were treated with nano herbals from Bischofia javanica leaves, Phaleria macrocarpa fruits, a combination of both, and Vitamin C as a control. Blood and organ analyses were compared with negative and positive controls. BaP induction in OSCC revealed cell transformation into carcinoma, confirmed by Papanicolaou staining. OSCC induction also caused significant changes in Complete Blood Count (CBC) and serum lipid profile. Nano herbals from both plants showed potential in reducing hematological and lipid damage in OSCC, especially in white blood cells, red blood cells, hemoglobin, and platelets. Statistical analysis also indicated that the combination of nano herbals was more effective in reducing (Low-Density Lipoprotein) LDL and total cholesterol levels than uncombined use while increasing (High-Density Lipoprotein) HDL levels comparable to Vitamin C. Liver and kidney functions were also significantly affected by OSCC, with nano herbals showing potential in normalizing albumin levels and positively impacting liver enzymes. In conclusion, both individually and in combination, nano herbals from Bischofia javanica leaves, and Phaleria macrocarpa fruits have the potential to be a promising treatment strategy for OSCC.
Oral Squamous Cell Carcinoma, nano herbal, Bischofia javanica, Phaleria macrocarpa
Oral Squamous Cell Carcinoma (OSCC) poses a significant global health challenge. According to recent data from the International Agency for Research on Cancer (IARC) recorded in the Global Cancer Observatory, OSCC accounted for approximately 377.713 new cases and 177.757 deaths in 2020 (
This study evaluates OSCC-induced rats using Benzo[a]pyrene (BaP) and treated with a combination of nano herbals. Vitamin C is used as an alternative comparator to assess the effectiveness of nano herbal testing against OSCC. Vitamin C possesses strong antioxidant properties, which can help protect body cells from damage caused by free radicals. Free radicals can contribute to cancer development by damaging DNA and triggering abnormal genetic changes (
Changes in enzyme activities or specific chemical levels can reflect the impact of OSCC on these organs or the side effects of treatment (
The nano herbals utilized consist of Bischofia javanica leaf (NDS), Phaleria macrocarpa fruit (NMD), and a combination of Bischofia javanica leaf with Phaleria macrocarpa fruit (KSMD). Both plants were harvested from the local plantations in the Simalungun Regency, North Sumatra Province, Indonesia. The High Energy Milling (HEM) equipment produces these three nano herbals. The specific type of HEM utilized is the Emax High Energy Ball Mill, manufactured by Retsch. Although the processing for NDS, NMD, and KSMD is conducted separately, the methodology remains consistent. This method is generally adapted from the procedure outlined by
This research involved 36 male rats (Rattus norvegicus) of the Wistar strain, weighing 180–250 g, and divided into six groups, each comprising six rats acclimatized for one week. Six rats without OSCC induction served as the negative control (K0). They were only given standard daily feed (Lab Diet - 5008 Formulab 23% Protein Rodent Diet). In comparison, the other 30 rats were initially induced with OSCC by injecting benzo[a]pyrene (Sigma-Aldrich Catalog number B1760) at 0.04 mg/0.04 ml corn oil (Brand Mazola) into the buccal mucosa of the proper oral cavity three times a week for four weeks. Each test subject underwent a swab test followed by a PAP smear (Papanicolaou Stain OG 6, Sigma-Aldrich Catalog Number HT40180) for Exfoliative cytopathology to ensure that the mucosal tissue had undergone differentiation towards carcinoma. Rats that tested positive for OSCC were grouped into five categories: K1 (Positive control), P1 (treated with 800 mg/kg/bw NDS), P2 (treated with 500 mg/kg/bw NMD), P3 (treated with 650 mg/kg/bw KSMD), and P4 (treated with 40 mg/kg/bw Vitamin C (Brand Allergy Research Group Pure Vitamin C 100 Vegetarian Capsules). All doses were determined based on previous research described in the “Preparation of the Nano Herbals” section. All treatment substances were administered orally by suspending them in a 0.3% Carboxymethyl Cellulose Sodium (Brand Merck - 419273-1KG - Sodium Carboxymethyl Cellulose) solution once daily for four weeks. Blood samples were collected periodically due to the extensive requirements. A one-week interval separated the first and second blood collection batches. The handling of animals followed the technical specifications outlined in NOM-062-ZOO-1999 for the production, care, and use of laboratory animals, as well as complying with ARRIVE guidelines and the UK Animals (Scientific Procedures) Act, 1986, and related guidelines, and the EU Directive 2010/63 on animal experiments.
The procedure involved gently rubbing the mucosal surface in the suspected area using a cytology brush moistened with sterile physiological saline solution. The brush was then gently scraped onto a clean glass slide. This procedure was repeated up to three times, and the glass slides were subsequently placed in a staining dish containing Sitofix solution. This process was carried out for all test animals undergoing a PAP smear. Next, the samples of mucosal epithelium were stained with Papanicolaou (PAP Smear) stain and immersed in the staining solution for 30–40 seconds. After rinsing in running water and differentiation in 70% ethanol, the tissues were rapidly dehydrated through increasing alcohol concentrations up to absolute alcohol. The tissue slides were cleared in xylene, air-dried, and mounted using a mounting medium and coverslips. The evaluation was performed at 400x magnification, with suspicious cells being evaluated at higher magnifications. The evaluation results were reported as a grading system, ranging from Grade 1 to Grade 5, indicating the severity of the cells. The grading of the observed preparations was based on the guidelines in the study by
Following the equipment’s guidelines, blood samples from ethylenediaminetetraacetic acid (EDTA) tubes were tested in a hematology analyzer (Mindray Hematology Analyzer BC-700 Series). Hematological parameters measured included white blood cell count (Leukocytes), red blood cell count (Erythrocytes), hematocrit (HCT), hemoglobin (Hb), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Blood Cell Distribution Width (RDW), platelets (Platelets), neutrophils, lymphocytes, monocytes, eosinophils, and basophils. For the biochemical profile analysis, blood without EDTA was used. Blood samples were inserted into the analysis equipment. Parameters tested in the blood biochemical profile included Low-Density Lipoprotein (LDL), High-Density Lipoprotein (HDL), total cholesterol, and levels of albumin, globulin, complete protein, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), total bilirubin, creatinine, Blood Urea Nitrogen (BUN), uric acid, and blood sugar levels.
The research data were analyzed using Sigmaplot and Prism software. If the data met the normal distribution and variance homogeneity criteria, an ANOVA test was conducted at a significance level of 5%. If the ANOVA test results indicated significant differences (p < 0.05), the Post Hoc-Duncan test was carried out. a, b, c, and d represent significant group differences. In cases where the data did not meet the criteria for normal distribution and/or variance homogeneity, the non-parametric Kruskal-Wallis test was employed. For comparisons between the two treatments, the Mann-Whitney test was used. The symbols (*) indicate that the p-value is < 0.05; (**) for p < 0.01; (***) for p < 0.001; and (****) for p < 0.0001.
The count of the number of stained cells and their classification into the appropriate grade is recorded in Table
Group | Grade I | Grade II | Grade III | Grade IV | Grade V |
---|---|---|---|---|---|
K0 | 64.6 ± 3.21e | 4.6 ± 3.51a | 2.2 ± 1.64a | 0 ± 0.00a | 0 ± 0.00a |
K1 | 2.2 ± 1.48a | 2.2 ± 2.59a | 5.2 ± 1.92b | 12.4 ± 3.05b | 62.2 ± 3.27c |
P1 | 37.8 ± 1.48b | 24.8 ± 2.64c | 15.4 ± 2.70bc | 8.8 ± 3.77b | 6.6 ± 4.51b |
P2 | 44.8 ± 4.38c | 21.8 ± 3.42c | 24.4 ± 3.65c | 3.5 ± 1.30a | 6.4 ± 3.78b |
P3 | 57.4 ± 3.97d | 24.4 ± 3.65c | 5.8 ± 3.77b | 2.8 ± 1.48a | 1.6 ± 0.89a |
P4 | 59.4 ± 2.07d | 15.4 ± 2.70b | 5.6 ± 3.91b | 4.2 ± 1.10a | 2.2 ± 0.84a |
Duncan’s post-hoc test showed that grade 1 in groups P3 and P4 showed no significant difference. (The same annotation, namely notation ‘d’) and only slightly between P3 and P4 with the control group (K0). This result indicates that the number of cells still classified as usual is significant in groups P3 and P4. The group with a cell grade distribution similar to group K1 was group P1. Compared to the two nano herbals in a single administration (not combined), the Phaleria macrocarpa nano herbal is better than the Bischofia javanica nano herbal in differentiating normal cells into carcinoma in oral mucosal tissue. Fig.
The mucosal tissue in K0 appears to have mild cell changes (regular and healthy epithelial cells). It has not led to cancer (Fig.
Pap Smear results of the oral mucosa of OSCC-Induced Rats; A. Grade I from K0; B. Grade II from P1; C. Grade III from P2; D. Grade IV from P3, E. Grade V from K1, and F. Grade I from P4. K0: Normal, K1: OSCC, P1: OSCC treated with nano herbal Bischofia javanica, P2: OSCC treated with nano herbal Phaleria macrocarpa, P3: OSCC treated with a combination of nano herbal Bischofia javanica and Phaleria macrocarpa, P4: OSCC treated with Vitamin C.
Fig.
Hematological parameter values. a, b, c, d represent significant differences between groups. ns = p > 0.05/Not substantial. K0: Normal, K1: OSCC, P1: OSCC treated with nano herbal Bischofia javanica, P2: OSCC treated with nano herbal Phaleria macrocarpa, P3: OSCC treated with a combination of nano herbal Bischofia javanica and Phaleria macrocarpa, P4: OSCC treated with Vitamin C.
Based on the findings of this study on hematological profiles, it is concluded that OSCC induction using BaP can lead to impairment in the physiological functions expressed through the complete blood count profile. An exciting aspect of this research is the condition of leukocytes. Certain types of cancers, such as leukaemia, lymphoma, and myeloma, can cause an increase in leukocyte count because these cancers develop in the blood system or bone marrow, where white blood cells are produced. However, in many non-blood-related cancers, there may not always be an increase in the white blood cell count. Conversely, some cancers can cause a decrease in the white blood cell count by inhibiting the production of normal white blood cells or causing damage to the organs that produce white blood cells (
Although the body’s response to cancer generally increases the leukocyte count, in the case of OSCC, specific factors can lead to a decrease in leukocyte count. OSCC can produce certain growth factors or cytokines that inhibit the production and function of leukocytes (
Fig.
The serum lipid profile. Panels a, b, c, and d represent statistically significant group differences. K0: Normal, K1: OSCC, P1: OSCC treated with nano herbal Bischofia javanica, P2: OSCC treated with nano herbal Phaleria macrocarpa, P3: OSCC treated with a combination of nano herbal Bischofia javanica and Phaleria macrocarpa, P4: OSCC treated with Vitamin C.
Exposure to polycyclic aromatic hydrocarbons (PAH), such as BaP, disrupts various biochemical pathways, including lipid metabolism and cholesterol regulation, triggering oxidative stress and chronic inflammation (
Fig.
Blood Biochemical Values for Assessing Liver, Kidney, and Pancreas Functions. Panels a, b, c, and d denote statistically significant group differences. K0: Normal, K1: OSCC, P1: OSCC treated with nano herbal Bischofia javanica, P2: OSCC treated with nano herbal Phaleria macrocarpa, P3: OSCC treated with a combination of nano herbal Bischofia javanica and Phaleria macrocarpa, P4: OSCC treated with Vitamin C.
Meanwhile, liver enzymes AST, ALT, and ALP exhibited differences compared to the positive control group, with ALP showing similarity. On the other hand, BaP exposure significantly increased blood sugar levels. However, administering nano herbals from both plants, individually or in combination, and Vitamin C did not affect the elevated blood sugar levels induced by BaP. It’s worth noting that the increase in blood sugar levels remained within normal limits based on Rattus norvegicus standards. This study contradicts previous research indicating that ethanol extract from Bischofia javanica leaves affects blood sugar levels (
OSCC, as oral cancer, may indirectly influence liver function. Liver dysfunction in OSCC may be related to the systemic effects of the disease, such as inflammation and metastasis, rather than the direct involvement of cancer in liver function. The metastasis of oral cancer to the liver can directly impact liver function, but usually, oral cancer spreads to lymph nodes before affecting other organs. Therefore, the study results suggest that BaP-induced metastasis of OSCC may have reached the liver, affecting the biochemical profile of the liver measured from the blood and urine of OSCC-induced rats in this study.
Active cancer can induce metabolic stress, including in the liver, reflected in changes in the blood’s albumin, globulin, total protein, and other parameters. Low albumin and total protein levels may indicate a decrease in liver protein synthesis. Although the primary reasons for low albumin levels in cancer patients are not fully understood, some mechanisms have been investigated. For instance, cancer cells can produce cytokines like interleukin-6 (IL-6), influencing albumin production. Cancer and inflammation-related processes can also affect liver enzymes such as ALT and AST, which increase in response to inflammation (
The research findings indicate that nano herbals effectively mitigate hematological and lipid damage induced by BaP in OSCC rats. Nano herbals also exhibit the potential to alleviate liver damage, particularly in reducing LDL and total cholesterol levels. This study provides valuable insights into the potential use of a nano herbal combination of Bischofia javanica leaves and Phaleria macrocarpa fruits as an innovative approach to cancer treatment. The detailed approach to nano herbal preparation and toxicity testing contributes to understanding the safety and efficacy of nano-based herbal drug applications.