AIP Conference Proceedings 2344, 040012 (2021); https://doi.org/10.1063/5.0048269 2344, 040012
© 2021 Author(s).
Administration of Centella asiatica ethanolic extract reduces tumor necrosis factor-alpha in hearts of aged sprague-dawley rats but not kidneys
Cite as: AIP Conference Proceedings 2344, 040012 (2021); https://doi.org/10.1063/5.0048269 Published Online: 23 March 2021
Jonathan Hartanto, Erni Hernawati Purwaningsih, and Desak Gede Budi Krisnamurti
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Administration of Centella asiatica Ethanolic Extract Reduces Tumor Necrosis Factor-Alpha in Hearts of Aged
Sprague-Dawley Rats but Not Kidneys
Jonathan Hartanto
1, Erni Hernawati Purwaningsih
2, 3, Desak Gede Budi Krisnamurti
2,3,a)1Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No 6, Jakarta 10430 Indonesia
2Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No 6, Jakarta 10430 Indonesia
3Drug Development Research Cluster, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No 6, Jakarta 10430 Indonesia
a)Corresponding author: [email protected]
Abstract. Aging is a time-related process leading to progressive deterioration of physiological bodily functions and increased vulnerability to degenerative disorders. The current trend of rapid growth in the global aging population poses a challenge for healthcare due to the increasing incidence of chronic diseases. In spite of this, preventive anti-aging agents such as vitamin supplements are not adequately available in many countries. Centella asiatica (CA), a traditional herb native to Southeast Asia, has been widely studied and demonstrated potent anti-inflammatory, antioxidant, neuroprotective, wound healing, and vasculoprotective effects in clinical studies. This study investigates the effect of CA treatment on aged Sprague-Dawley (SD) rats. Twenty-four rats were divided into four groups: positive control (vitamin E 6 IU), negative control (placebo), treatment group (CA 300 mg/kgBW), and comparison group (young SD rats with placebo). After 28 days of treatment, SD rats were terminated. TNF-α concentration in heart and kidney tissues were determined using enzyme-linked immunosorbent assay. We found that CA showed a significant decrease in heart TNF-alpha (p = 0.021) yet yields no statistically significant decrease in kidney TNF-alpha levels (p = 0.617). However, it was possible to identify a trend toward decreasing kidney TNF-α concentration in groups treated with CA as it was lower than the positive and negative control group. Our finding suggests different aging pathophysiology in different body organs and CA's potential as an anti-aging agent, corresponding to previous studies showing potent anti-inflammatory effects of CA in multiple organs. All in all, further research and exploration need to be made on aging pathophysiology and CA with variations of a more significant dose and longer time of administration.
Keywords: aging, Centella asiatica, tumor necrosis factor-alpha, oxidative stress
INTRODUCTION
Aging is a time-related process leading to progressive deterioration of physiological bodily functions and increased vulnerability to degenerative disorders, also known as 'diseases of aging' [1]. Today, there is a trend of rapid and continuous growth in the aging individual's global population. The same trend is also seen in Indonesia. In 2017, the elderly made up 9.03% of the Indonesian population, and this is projected to become 11.1% in 2025. More than a quarter of Indonesian elders (28.62%) are suffering from disease [2]. In spite of this, preventive anti-aging agents such as vitamin supplements are not adequately available in many countries, such as Indonesia.
There is not yet a consensus on what causes aging. The most widely accepted theory is the 'damage theory', where aging occurs from the accumulation of oxidative stress that increases with age [3]. Oxidative stress is determined by
the number of free radicals and antioxidants in someone's body. Free radicals are radical molecules generated during metabolism, leading to DNA, lipid, and protein damage. Meanwhile, antioxidants are substances that can neutralize free radicals. To combat oxidative stress, our bodies naturally produce endogenous antioxidants such as superoxide dismutase (SOD), glutathione, glutathione peroxidase (GPx), peroxiredoxin (POD), and catalase. However, there is still a need for exogenous antioxidants such as vitamin C (ascorbic acid), vitamin E (α-tocopherol), and carotenoids from nutritional sources [4].
Herbal plants are known as one source of antioxidants. Centella asiatica (CA) is a traditional herb native to Southeast Asia, widely studied and demonstrated potent anti-inflammatory, antioxidant, neuroprotective, wound healing, and vasculoprotective effects in clinical studies [5,6,7]. CA contains phenolic and flavonic constituents such as asiaticoside and caffeoylquinic acid. Asiaticoside will be transformed into asiatic acid, which can donate hydrogen, therefore being an antioxidant [5]. Caffeoylquinic acid, on the other hand, can activate endogenous antioxidant response pathway [6]. In rats with stressor-induced inflammations, CA treatment has shown decreased levels of TNF- α in major vital organs such as the kidney, heart, and brain [8,9,10]. However, to this date, there is no study discussing CA administration effects in the context of aging nor aging individuals. Thus, this study aims to observe CA extracts' effect on the kidney and heart of aged Sprague-Dawley rats.
MATERIALS AND METHODS Study Design
An experimental study was conducted in laboratories of the Department of Medical Pharmacy and Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia (FMUI), Jakarta, Indonesia, in a period of one year (2019 – 2020).
Centella asiatica Ethanol Extract Preparation
Dried Centella asiatica (CA) leaves were obtained from The Indonesian Institute of Sciences (LIPI), Bogor, Indonesia. The leaves were formerly dried under direct sunlight to evaporate the water content. We ground the dried leaves to a fine powder using a grinding machine and macerated the powder repeatedly in 70% ethanol solution for 24 hours to extract the active substances. The powder was re-macerated three times with the same solvent. The collected extract was then evaporated using a rotary vacuum evaporator and dissolved in distilled water to reach 300 mg/kg body weight.
Experimental Animals Preparation
Sprague-Dawley rats were chosen as animal models due to their calmness, ease of handling, and overall availability. The minimum sample was calculated using the Federer formula. In this experiment, we used a total of 24 Sprague-Dawley (SD) rats, consisting of 18 aged (20-24 months old) and six young (8-12 weeks old) rats with relatively similar weights, in good health, free from any disease or handicap. The SD rats were bred in the National Agency of Health Research and Development laboratory breeding house, Indonesian Ministry of Health, Jakarta, Indonesia. Before treatment, all rats were acclimatized for a period of 7 days under controlled room temperature (25°C) and 12:12 hours light and dark cycle in the Department of Nutrition, Faculty of Medicine, Universitas Indonesia. The food pellet was given with a 10g/rat/day dose, while water was given ad libitum.
Experimental Animals Treatment
The experiment consisted of four groups of rats, with six in each group. The experimental groups were positive control, negative control, and treatment group. Rats in the positive control group were treated with vitamin E 6 IU twice daily, while rats in the negative control were given a placebo (water). Rats in the treatment group were given CA ethanolic extract with a concentration of 300 mg/kg body weight, twice daily. There is also an additional group that consists of six young SD rats treated with a placebo. After 28 days of the administration, animals were given sedative anesthesia (75-100 mg/kg ketamine and 5-10 mg/kg xylazine) and terminated. Animal hearts and kidneys were harvested in 20/50 cc pots with 10% formalin buffer and stored in a freezer (temp -20 °C).
Tumor Necrosis Factor-Alpha Analysis
The biochemical evaluation was done in the Laboratory of Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia. An enzyme-linked immunosorbent assay (ELISA) kit was used to evaluate levels of proinflammatory cytokine TNF-α in each kidney and heart specimen.
Statistical Analysis
Experiment data were analyzed using Statistical Package for the Social Sciences (SPSS) software version 24.0.
Values were reported as mean ± SEM (standard error of the mean). Data normality was determined using the Shapiro- Wilk test. The data were found to be parametric, and the significance of difference concerning controls was evaluated using the One-Way ANOVA test. A probability (P-value) level of less than 0.05 was considered as significant.
Ethical Statement
Before conducting the experiment, all necessary ethical clearance was obtained from Health Research Ethical Committee, Faculty of Medicine Universitas Indonesia – Cipto Mangunkusumo National Central General Hospital in accordance with Indonesian Health Act on Health Research and Development with registration number 307/UN2.F1/ETIK/PPM.00.02/2020.
RESULTS AND DISCUSSION
FIGURE 1. Effect of Centella asiatica (CA) on Heart and Kidney TNF-alpha concentration of Sprague-Dawley rats. * and **
indicates significant difference (p < 0.05 and p < 0.005, respectively). Bars without * or ** indicate insignificant difference.
The data of tissue TNF-α concentration between several experimental groups can be seen in Fig. 1. We found a higher concentration of tissue TNF-α in aged SD rats than young SD rats, both in hearts (16.50 ± 1.33 pg/mg in negative control vs 7.23 ± 0.90 pg/mg in young control) and kidneys (1.37 ± 0.41 pg/mg in negative control vs 1.09
± 0.17 pg/mg in young control), despite the insignificant difference in kidneys. Moreover, heart tissue TNF-α concentration was found higher than young control even in the positive control group.
It is also observed that the group treated with CA has lower TNF-α concentration than the positive and negative control, both in hearts (10.00 ± 1.10 pg/mg in the treatment group vs 16.50 ± 1.33 pg/mg in negative control and 11.72
± 1.52 pg/mg in positive control) and kidneys (0.88 ± 0.16 pg/mg in the treatment group vs 1.37 ± 0.41 pg/mg in negative control and 0.90 ± 0.28 pg/mg in positive control), albeit not significant in kidneys. No significant difference
was found in kidney TNF-α concentration among the groups. However, we found that TNF-α concentration in kidneys of the group treated with CA is still the lowest between all groups.
Different Compositions of Heart and Kidney TNF-α Concentration
Our present study demonstrated the usage of TNF-α as a biological measurement for aging. Our results showed that aged SD rats have higher tissue TNF-α levels in multiple vital organs than young SD rats, although the kidney's difference was not significant. This correlates to previous studies reporting the role of TNF-α in the pathophysiology of aging. Innumerable studies have shown that TNF-α as an inflammatory marker is close-linked to inflammaging, chronic, and sterile inflammation due to prolonged oxidative stress processes [11,12]. Inflammaging is believed to accelerate the process of natural aging and contributes to the pathogenesis of age-related diseases, with studies showing a correlation of TNF-α levels along with the severity of degenerative diseases such as heart failure and chronic kidney disease [11-14]. Therefore, TNF-α and other inflammatory markers might be upregulated in an aging individual.
The current study was the first to explore aging biomarker composition in the tissue of multiple organs. Our results displayed that TNF-α composition is found higher in cardiac tissue in contrast to kidney tissue (16.50 ± 1.33 pg/mg in heart negative control vs 1.37 ± 0.41 pg/mg in kidney negative control). This is a nod to previous studies having more widely reported usage of TNF-α as a biomarker of aging-related cardiac disease such as heart failure [14]. One possible explanation for this result is that there is a different pathophysiologic mechanism and/or different timeframe of aging in each bodily organ, which will be discussed in the following discussions.
Effects of Centella asiatica on Heart and Kidney TNF-α Concentration
This present study demonstrated lower TNF-α concentration in the heart tissue of aged SD rats treated with CA ethanolic extract compared to the negative control group. Previous studies have demonstrated CA’s anti-inflammatory effect in reducing blood pro-inflammatory mediators’ concentration on SD rats induced with various stressors, such as diabetes and internal organ injury [8,9,10]. Our results suggested that CA’s anti-inflammatory effect might also be applicable in the context of inflammaging. A plausible explanation for this result is due to the active substances contained in CA, such as asiaticoside, a phenolic compound that has shown the capacity to act as a free radical scavenger [5]. Another active substance in CA, caffeoylquinic acid, has also shown the capacity to activate endogenous antioxidant response pathway through increased mRNA expression of antioxidant response genes [6].
Previous studies showed significantly lower TNF-α concentration in kidney tissue of rats treated with CA ethanolic extract. However, our study showed an insignificant result for CA on kidney TNF-α concentration (p = 0.584). Several explanations could be the answer for this discrepancy. First of all, cardiovascular aging might happen in an earlier timeframe compared to nephrotic aging. Atherosclerotic lesions, which are the most notable age-related cardiovascular system changes, are thought to already occur in utero and increase steadily with age [15,16]. Due to this reason, the duration of our present study might be too short for kidney aging to be represented in our experiment.
Another explanation is that the anti-inflammatory response of kidney tissue in our present study is inadequate.
Preceding animal studies evaluating potentials of CA has used longer administration time. Zhao et al. and Grey et al.
demonstrated significant results upon administration duration of 45 days [17,18]. A study by Hussin et al. even implemented a longer duration of 25 weeks [19] as opposed to our administration time of 28 days. Moreover, a lack of CA dosage concentration might be another possible explanation. For example, Rochmah et al. showed a significant decrease of hippocampal TNF-α at 600 mg/kgBW dosage, and Masola et al. showed a decrease of renal TNF-α at 500 mg/kgBW dosage [20,21] compared to our dosage of 300 mg/kgBW.
Lastly, TNF-α might not be the correct aging biomarker for a kidney. Some studies done in human subjects showed a correlation of TNF-α with age and its reliability as an aging biomarker, especially in cardiac diseases. However, previous studies evaluating CA treatment potentials in the kidney tend to use other biomarkers, such as IL-6 and high- sensitivity C-reactive protein (hsCRP), to evaluate inflammation. Other studies demonstrated the usage of non- inflammatory biomarkers (such as hematological, lipid, or endocrinal biomarkers of aging) and non-serological biomarkers (such as tissue volume and histopathological changes) [22].
CONCLUSIONS
Based on the present study, we conclude that CA ethanolic extract treatment significantly reduced heart TNF-α concentration in aged SD rats (p = 0.021). In contrast, CA does not demonstrate a significant decrease in kidney TNF- α concentration despite a slight decrease in the treatment group than the negative control group. This might be caused by several factors, from different aging pathophysiology and timeframe in different body organs to lack of administration time duration and dosage concentration resulting in inadequate anti-inflammatory effects. All in all, our results suggest the potential of CA as an herbal antiaging agent. As the prevention of aging is still a mostly growing field, we urge additional investigations on CA and aging pathophysiology to develop an affordable and effective antiaging agent in the future.
ACKNOWLEDGMENTS
Our study would not be possible without support from National Institute of Health Research and Development, Ministry of Health Republic of Indonesia and Faculty of Medicine, Universitas Indonesia, especially Department of Medical Pharmacy and Department of Biochemistry and Molecular Biology.
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