AIP Conference Proceedings 2092, 030013 (2019); https://doi.org/10.1063/1.5096717 2092, 030013
© 2019 Author(s).
The reduction effect of blood interleukin-6 concentration on aged Sprague-Dawley
rats after Centella asiatica ethanolic extract administration
Cite as: AIP Conference Proceedings 2092, 030013 (2019); https://doi.org/10.1063/1.5096717 Published Online: 09 April 2019
Valdi Ven Japranata, Erni H. Purwaningsih, Rani Wardani Hakim, Adisti Dwijayanti, and Desak Gede Budi Krisnamurti
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The Reduction Effect of Blood Interleukin-6 Concentration on Aged Sprague-Dawley Rats after Centella asiatica
Ethanolic Extract Administration
Valdi Ven Japranata
3, Erni H. Purwaningsih
1,2, Rani Wardani Hakim
1,2, Adisti Dwijayanti
1,2, Desak Gede Budi Krisnamurti
1,2,a)1Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
2Drug Development Research Cluster, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
3Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
a)Corresponding author: desak.gede@ui.ac.id
Abstract. Aging is an inevitable time-related process leading to reduced cognition and physical strength also increased susceptibility of degenerative disorders. This study studies the aging process related to relatively high oxidative stress level and proinflammatory mediators such as interleukin-6 (IL-6). Centella Asiatica (CA), a traditional medicinal herb, has been widely reported to have an antioxidant and anti-inflammatory effect, both experimentally and clinically. The study investigates the impact of CA on aged Sprague-Dawley rats. Twenty-eight rats were divided into four groups: negative control, positive control (vitamin E 6 IU), treatment (CA ethanolic extract 300 mg/kg body weight), and regular group (young Sprague-Dawley rats). After 28 days of treatment, the rats were terminated, and their blood IL-6 concentrations were assessed with enzyme-linked immunosorbent assay. We found that CA significantly reduced blood IL-6 concentration in the treatment group compared to negative control (p = 0.001). There was also no significant difference of blood IL-6 concentration between treatment group and positive control, which indicated that CA and vitamin E may be equipotent as an antioxidant and anti-inflammatory agent. These results suggested that CA may be a potential candidate for an antiaging agent in the next future.
Keywords: aging, centella asiatica, interleukin-6, oxidative stress
INTRODUCTION
As time passes by, all individuals will undergo aging process, leading to cognition [1] and physical strength impairment[2], also increasing vulnerability of several degenerative disorders such as diabetes mellitus, cancer, neurodegenerative and cardiovascular disease[3]. Although it is an inevitable process, there is a trend of aging acceleration in human population. In 2013, a significant increase of prevalence of aging-related disorders such as diabetes mellitus and cancer was found in young Indonesian population [4].
Aging process is influenced by several factors, one of them is oxidative stress which is determined by the degree of free radicals and antioxidants in the body. Free radicals are reactive substances that may interact with cell components, causing inflammation mediated by interleukin (IL)-1, IL-6, and tumor necrosis factor-α (TNF-α). On the other hand, antioxidants are substances that offer protection from harmful effects of free radicals. Though human body produces endogenous antioxidants such as superoxide dismutase (SOD), catalase, glutathione (GSH), and glutathione peroxidase (GPX), it still needs exogenous antioxidants such as α-tocopherol (vitamin E), ascorbic acid (vitamin C), and carotenoid from various consumable sources [5-7].
Herb plants are known as one of the sources of anti-inflammatory and antioxidant agents. One of herb plants that is now utilized commercially is Centella asiatica (CA), a traditional medicinal herb which is largely found in Southeast Asia, including Indonesia [8]. The efficacy of CA has been widely studied and reported for its wound healing, vasculoprotective, neuroprotective, antioxidant and anti-inflammatory properties [9]. CA contains phenolic compounds that exhibit antioxidant effect by scavenging nitric oxide (NO), a free radical [10]. Triterpenoids, such as asiatic acid and asiaticoside, in CA are also known to improve cognitive function [11, 12] and induce antioxidants [9], respectively. Studies in rats with inflammation showed that CA provides anti-inflammatory effect by reducing blood IL-6 concentration [13, 14]. However, there is still no comprehensive study regarding CA in aged individuals to date.
Therefore, this present study is aimed to investigate the effect of CA on blood IL-6 concentration in aged Sprague- Dawley rats.
MATERIALS AND METHODS
Centella asiatica Ethanolic Extract Preparation
Dried Centella asiatica (CA) leaves was obtained from Indonesian Institute of Sciences, Bogor. We macerated the dried leaves with ethanol 70% for 24 hours. Subsequently, the residue was remacerated three times by the same solvent.
We then performed evaporation of the collected extract with rotary vacuum evaporator. The dosage of CA used in this study was 300 mg/kg body weight. The extract was dissolved in distilled water to get the appropriate dilution to be administered orally before the treatment exposure on subjects of this study.
Experimental Animals Preparation
A total of 28 Sprague-Dawley (SD) rats, consisting of 21 aged (20-24 months old) and 7 young (8-12 weeks old) rats with relatively similar weight were used in this experiment. The rats were obtained from National Institute of Health Research and Development, Ministry of Health Republic of Indonesia. Before treatment, all SD rats underwent one-week acclimation in same room under 24°C temperature and 12 hours light-dark cycles at Department of Nutrition, Faculty of Medicine, Universitas Indonesia. The rats were fed with standard chow and had free access to water ad libitum.
Animals Treatment
Aged SD rats were randomly distributed into three experimental groups with seven rats for each group. The experimental groups were negative control, positive control, and treatment group. Rats in negative control were given placebo (water), while rats in positive control group were treated with vitamin E 6 IU, twice daily. Rats in treatment group were given CA ethanolic extract with concentration of 300 mg/kg body weight, twice daily. There was an additional regular group which consisted of seven young SD rats treated with placebo.
The treatment was conducted for 28 days in the same room where the rats were acclimated. At the last day of treatment, the rats were all terminated under ketamine and xylazine anesthesia. The blood of SD rats was collected into different K3-EDTA tubes and then centrifuged at 3000 rpm for 10 minutes at 4°C.
Blood Interleukin (IL)-6 Measurement
Serum collected from centrifuged SD rats blood were assessed for IL-6 with enzyme-linked immunosorbent assay (ELISA) method. The measurement was taken place in Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia and was conducted according to ELISA manufacturer’s protocol. The results of SD rats blood IL-6 concentration were reported in mean ± standard error of mean (SEM) pg/mL serum.
Statistical Analysis
The results obtained were then analyzed for statistical significance of mean difference among experimental groups using analysis of variance (ANOVA) and Tukey multiple comparisons test. The graph visualization and statistical analysis for the results was performed with GraphPad Prism 7 for Microsoft Windows.
Ethical Consent
Ethical consent regarding the utilization of rats as experimental subjects in this study was approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Indonesia-Cipto Mangunkusumo Hospital in 2016.
RESULTS AND DISCUSSIONS
Effect of Centella asiatica on Blood IL-6 Concentration
FIGURE 1. Effect of Centella asiatica (CA) on blood IL-6 concentration of Sprague-Dawley rats. *** indicates significant difference (p = 0.001) compared to negative control. ns indicates insignificant difference compared to negative control.
The results of blood IL-6 concentration measurement among experimental groups are shown in Fig. 1. Blood IL- 6 concentration was found to be slightly higher in aged SD rats (negative control, 2197 ± 214.5 pg/mL) than young SD rats (regular group, 1836 ± 82.78 pg/mL) despite insignificant difference. In contrast, treatment with CA ethanolic extract in aged SD rats significantly reduced blood IL-6 concentration (1221 ± 116.1 pg/mL) compared to negative control. It is also observed that aged SD rats treated with CA ethanolic extract has similar concentration of blood IL- 6 with aged SD rats treated with vitamin E in positive control group (1164 ± 110.3 pg/mL, p = 0.9905).
In previous studies, blood IL-6 concentrations in different age groups and the effect of CA to blood IL-6 concentration have been widely investigated, but reports regarding blood IL-6 concentration upon CA treatment in aged individuals remain limited. Therefore, in this study, we investigated blood IL-6 concentration in aged SD rats treated with CA. It has been long known that IL-6 in blood circulation was positively correlated with age [15]. Our present result also showed that aged SD rats (negative control) had higher IL-6 in their blood compared to young SD rats (regular group), although the difference was not significant. The probable explanation to this phenomenon is chronic low-grade inflammation related to aging process. This might be due to events observed in aged individuals, such as accumulation of damaged cells and macromolecules, mitochondrial dysfunction, leakage of microbial components of the human body into tissues and circulation, cellular and immune system senescence, ultimately resulting in more proinflammatory substances (such as IL-6) being produced [16].
This present study showed decrease in blood interleukin (IL)-6 concentration in aged SD rats receiving CA ethanolic extract compared to negative control. It indicates that CA may offer anti-inflammatory effect on aged Sprague-Dawley (SD) rats. In previous study, CA is known to have anti-inflammatory effect by reducing blood proinflammatory mediators’ concentration on young SD rats with liver injury (TNF-α, interferon-γ (IFN-γ), IL-1β, IL-2, IL-6, IL-10, and IL-12)[13],and mice with allergic dermatitis (TNF-α, IL-1β, and IL-6) [14]. This present result suggested that CA may act as anti-inflammatory agent in aged SD rats as well. One possible mechanism to explain this result is that CA contains asiaticoside and phenolic compound as some of its major constituents. Asiaticoside is known to induce enzymatic and non-enzymatic antioxidants, such as catalase, glutathione peroxidase, superoxide dismutase, vitamin C (ascorbic acid), and vitamin E, in rats at initial stage of excision-type cutaneous wounds [17].
Phenolic compound acts as antioxidant which provides protective effect against deleterious effects of oxidative stress and thus reducing inflammation [10]. In addition, the concentration of polyphenol of CA extracted with ethanol is
relatively high compared to other solvents such as hexane. Therefore, CA ethanolic extract may exert higher anti- inflammatory effect [18].
On the other hand, our study also revealed that blood IL-6 concentration in aged SD rats treated with CA ethanolic extract was similar with aged SD rats treated with vitamin E (positive control). Vitamin E is an exogenous substance that is widely known to have antioxidant and anti-inflammatory effect. Vitamin E exerts these effects by neutralizing free radicals before they could trigger NF-κB activation, a protein complex that regulates cytokine synthesis, and thus decreasing synthesis of proinflammatory mediators, such as IL-6 and C-reactive protein (CRP) [19-21]. Therefore, the result in present study suggested that CA and vitamin E may have the same efficacies as an antioxidant and anti- inflammatory agents.
CONCLUSIONS
To conclude, the present study showed that CA ethanolic extract treatment significantly reduced blood IL-6 concentration in aged SD rats (p = 0.001). The blood IL-6 concentration was similar with aged SD rats receiving vitamin E (p = 0.9905), which indicated that both CA and vitamin E may be equipotent in reducing inflammation.
Therefore, CA may be a potential antiaging candidate and we suggest further investigations regarding possible CA effects as anti-inflammatory and antiaging agent on human subject to be conducted in the future.
ACKNOWLEDGEMENTS
This study would not be possible without supports from National Institute of Health Research and Development, Ministry of Health Republic of Indonesia and Faculty of Medicine, Universitas Indonesia (Department of Medical Pharmacy and Department of Biochemistry and Molecular Biology). Our gratitude also goes for the financial aid from PITTA Grant, provided by the Directorate of Research and Community Engagement, Universitas Indonesia with contract number 2063/UN2.R3.1/HKP.05.00/2018.
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