AIP Conference Proceedings 2092, 030014 (2019); https://doi.org/10.1063/1.5096718 2092, 030014
© 2019 Author(s).
Reduction of blood IL-6 level on aged
Sprague-Dawley rats treated with Acalypha indica Linn ethanolic extract
Cite as: AIP Conference Proceedings 2092, 030014 (2019); https://doi.org/10.1063/1.5096718 Published Online: 09 April 2019
Matheus Nathanael, Rani Wardani Hakim, Siti Farida, Erni H. Purwaningsih, and Desak Gede Budi Krisnamurti
ARTICLES YOU MAY BE INTERESTED IN
The effect of Acalypha indica Linn. extract on insulin resistance in Sprague-Dawley rats after induction of high cholesterol-high fructose diet
AIP Conference Proceedings 2092, 030018 (2019); https://doi.org/10.1063/1.5096722
The reduction effect of blood interleukin-6 concentration on aged Sprague-Dawley rats after Centella asiatica ethanolic extract administration
AIP Conference Proceedings 2092, 030013 (2019); https://doi.org/10.1063/1.5096717 Protection effect of Acalypha indica L. extract versus combination of Acalypha indica L.- gemfibrosil against fatty renal induced high-fructose and fat diets in rats
AIP Conference Proceedings 2092, 030025 (2019); https://doi.org/10.1063/1.5096729
Reduction of Blood IL-6 Level on Aged Sprague-Dawley Rats Treated with Acalypha indica Linn Ethanolic Extract
Matheus Nathanael
3, Rani Wardani Hakim
1,2, Siti Farida
1,2, Erni H.
Purwaningsih
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: [email protected]
Abstract. Aging is a condition of impaired function due to the decrease of physiological integrity that can be a risk factor for another significant disease. The aging process is caused by free radicals that can damage the surrounding molecules.
This process can cause oxidative stress and increase the level of proinflammatory mediators, such as interleukin-6 (IL-6).
Acalypha indica Linn (AI), is widely used as an herbal medicine for various diseases. Several studies have shown that Acalypha indica Linn have antioxidant and anti-inflammatory effects. We aimed to study the impact of AI on aged Sprague- Dawley (SD) rats. The rats were divided into 4 groups consists of 7 rats on each group. The groups are treated with placebo, the ethanol extract of AI (250 mg/kg BW) and vitamin E (6 IU). The last group consists of young SD rats. After 28 days, rats were terminated, and laboratory tests were performed to see blood-IL-6 levels using enzyme-linked immunosorbent assay. The results showed AI significantly reduced blood IL-6 level compared to negative control group (P = 0,0048) while there is no significant difference of blood IL-6 level between treatment group (AI) compared to positive control group.
These results prove that AI ethanol extract has an antioxidant effects on aged SD rats and can be a potential antioxidant agent to inhibit aging process.
Keywords: Acalypha indica Linn, Aged Sprague-Dawley rats, aging, free radicals, interleukin-6, oxidative stress
INTRODUCTION
During the aging process, a person will experience a decrease in physical ability and increase chances of developing chronic diseases such as asthma, arthritis, cancer, dementia, depression, osteoporosis, and stroke [1].
Aging process can cause changes in skin layers and show signs of aging such as loss of volume and density of the skin layers and wrinkles. This process can be caused by oxidative stress, the release of free radicals in the body that accumulate over time. The cell structure of the skin including lipids and proteins can be damaged by this reactive nature of free radicals and can accelerate the aging process.
Antioxidant molecules play an important role in neutralizing free radicals that caused by exposure to sunlight, pollution, cigarette smoke, etc [2]. Free radicals can trigger inflammation in the body and at the molecular level mediated by mediators such as interleukin 1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) [3].
Antioxidants can directly neutralize free radicals, reduce peroxide concentrations and membrane repair, reduce ROS production, and neutralize ROS through lipid metabolism [2].
The body produces its antioxidants enzymes (endogenous antioxidants), such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) [4]. The first line of defense against superoxide (O2-) radical is Superoxide dismutase (SOD), it can also prevents cellular damage [5]. Glutathione peroxidase (GPx) eliminates hydrogen peroxide (H2O2)
and prevent the formation of hydroxyl radical (OH) [5]. When free radicals are accumulated, the body needs external (exogenous) supply of antioxidants.
Herbs have been known to have some medical effects because of their antioxidant property such as polyphenols, phenolic acids, and flavonoids [6].This phenolic compounds can interact with ROS and thus inhibit the inflammatory process which plays a role in chronic diseases [7,8]. Polyphenols are also known to have antioxidant effects on endothelial cells by reducing the expression and activity of NADPH oxidase [7]. Acalypha indica Linn, a traditional herb, has been used as a phytomedicine in tropical and subtropical countries [6,9]. Research has shown that Acalypha indica Linn can relieve oxidative stress by decreasing lipid peroxidation and increase cell proliferation in rats given Acalypha indica Linn extract [9]. Based on a study, Acalypha indica Linn, possess high levels of polyphenols and flavonoids [10]. Polyphenols contained in AI have been shown to decrease the secretion of proinflammatory cytokines (IL-6 and TNFα) by downregulating the NF-κB pathway [7]. However, the research on the effects of AI plants on individuals undergoing aging is still limited. This study aims to identify the effect of Acalypha indica Linn in the aging process, mainly related to the parameters of blood IL-6 level in aged Sprague-Dawley rats.
MATERIALS AND METHODS Acalypha indica Linn Extraction
The dried roots of Acalypha indica Linn (AI) were collected from Depok, West Java, Indonesia and had been identified at LIPI (Indonesian Institute of Sciences) Bogor, West Java, Indonesia. The dried roots of AI were macerated using ethanol 70% overnight and this process repeated 3 times with the same solvent. The solvent and the active ingredient are separated by evaporation using a rotary vacuum evaporator. The dose of AI in this study was 250 mg/kg body weight. The extract was dissolved in aquadest to get the dilution before being administered orally to the SD rats every day.
Animals Preparation for Experiment
A total of 28 Sprague-Dawley rats, consists of both aged (21 rats, 20-24 months old) and young (7 rats, 8-12 weeks old) with similar weight were used and obtained from National Institute of Health Research and Development, Ministry of Health Republic of Indonesia. Before treatment starts, both aged and young Sprague-Dawley rats were given a one-week acclimation, placed in a 24°C room, 12 hours of light-dark cycles. The rats fed with standard rat food and had access to water ad libitum.
Animals Treatment
Sprague-Dawley rats were randomly distributed into four treatment groups. Each group consists of aged (20-24 weeks old) Sprague-Dawley rats are given specific following treatment; (1) placebo/water (negative control), (2) vitamin E (6 IU) (positive control), and (3) ethanol extract of AI (250 mg/kg BW). Group consists of young (8-12 weeks old) Sprague-Dawley rats are given placebo treatment. Treatment was given for 28 days.
On the 29th day, all Sprague-Dawley rats were terminated under ketamine and xylazine anesthesia. Each of the blood samples was collected and placed into K3-EDTA tubes and centrifuged at 3000 rpm, 4°C temperature for 10 minutes.
Measurement of Blood Interleukin (IL)-6
Blood serum obtained from centrifugation were assessed for interleukin-6 (IL-6) blood level parameters using the enzyme-linked immunosorbent assay (ELISA) method. Laboratory measurements were taken place in Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia. The results of SD rats blood IL-6 concentration were reported in mean ± standard error of mean pg/mL serum.
Statistical Analysis
Laboratory results from blood samples of Sprague-Dawley rats will be analyzed for its statistical significance of mean difference among treatment groups using analysis of variance (ANOVA) and Tukey’s multiple comparison test.
Statistical analysis performed with GraphPad Prism 7 for Microsoft Windows and visualized into graphs.
Ethical Consent
Ethical consent regarding the usage 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 No.
1016/UN2.F1/ETIK/2016.
RESULTS AND DISCUSSION
Effect of Acalypha indica Linn Ethanolic Extracts on Blood IL-6 Level
The following are the results of statistical analysis based on laboratory examinations among all experimental groups;
Ne ga tive Control
Pos itiv e Control
AI (2 50 mg/kgBW) Norm
a l Group 0
1 0 0 0 2 0 0 0 3 0 0 0
T r e a t m e n t G r o u p s
Blood IL-6 Level (pg/mL)
n s
* *
* *
FIGURE 1. The effects of Acalypha indica Linn on blood IL-6 level of aged Sprague-Dawley rats. ** indicates a significant difference (p = 0.0048) compared to negative control. ns indicates insignificant difference compared to negative control.
Blood interleukin-6 (IL-6) level on aged SD rats group given Acalypha indica Linn ethanolic extract (1158±221.3) were significantly decreased (p = 0.0048) compared with the negative control group (2197±214.5). In contrast, there is no significant difference (p > 0.9999) between aged SD rats group given Acalypha indica Linn ethanolic extract compared with aged SD rats group given vitamin E (1164±110.3).
In this present study, we investigated and compared blood IL-6 level in aged Sprague-Dawley rats treated with ethanolic extract of Acalypha indica Linn with other treatment groups. Based on laboratory results, we found that blood level of IL-6 in aged Sprague-Dawley rats (negative control) were higher than blood IL-6 level in young Sprague-Dawley rats (standard group), although the difference was not significant (P = 0.2976). This phenomenon is caused by increasing of inflammatory mediators (IL-6, IL-1β, and TNF-α) across multiple age-related diseases [11].
Previous study state that Interleukin-6 now is used commonly as a marker of inflammatory status [11]. Dysregulation of this proinflammatory mediator considered as the first step in the development of frailty in the aging process and seems to be strongly linked in the pathophysiology of chronic disease, and decreased physical function in the elderly age group [12]. This may be the result of the accumulation of damaged and dysfunctional cells, and decreased immunity in aged individuals, resulting in increased production of proinflammatory mediators such as IL-6 [11].
In the previous study, Acalypha indica Linn is known to have antioxidative potential by improving cellular proliferation, increasing rate of wound contraction, epithelialization, and enhancing tensile strength in the healing process of rats cutaneous wound [9]. AI also shows hepatoprotective activity in streptozotocin-induced diabetic rats [6].In the present study, it is showed that Acalypha indica Linn had a potential to be an antioxidant agent with an anti- inflammatory effect. It is indicated by blood IL-6 level which is significantly decreased in aged Sprague-Dawley rats receiving Acalypha indica Linn ethanolic extract compared to the negative control group.
A possible mechanism that can explain this result is because AI, as one of the traditional herbs, posses high levels of polyphenols and flavonoids [10]. These compounds show the contribution to the defense of endothelial cell antioxidants by reducing the expression of NADPH oxidase and its activity directly. Polyphenol can reduce the secretion of pro-inflammatory cytokines (IL-6 and TNFα) through downregulation of the NF-κB pathway [7]. AI can increase antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) activities and improving antioxidant status in diabetic rats liver [6]. Therefore, AI ethanolic extract may be considered as a potent antioxidant agent.
It is also shown in present study that blood IL-6 level in aged SD rats group given AI ethanolic extract have no significant difference with aged SD rats treated with vitamin E (positive control group). The tocopherol content of vitamin E has a unique antioxidant and anti-inflammatory activity by inhibiting cyclooxygenase and suppress proinflammatory signaling such as NF-kB [13]. It is also shown that vitamin E also can reduce proinflammatory mediators such as IL-6 and TNFα [14]. Therefore, the results of this study suggest that AI and vitamin E may have same roles and functions as antioxidants and anti-inflammatory agents.
CONCLUSION
In conclusion, this present study showed that Acalypha indica Linn (AI) ethanolic extract can significantly reduce blood IL-6 concentration in aged Sprague-Dawley (SD) rats (p = 0.0048). At the end of the treatment, there is no significant difference of blood IL-6 concentration between SD treated with AI and vitamin E (p > 0.9999). This result indicates Acalypha indica Linn may be equipotent with vitamin E for reducing inflammation and can be considered as a potential anti-aging and as a source of anti-aging drug discovery. We suggest further investigations regarding the possibility of Acalypha indica Linn as an anti-inflammatory and antiaging to be conducted on human subject in the future .
ACKNOWLEDGMENTS
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). We also would like to express our gratitude 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.
REFERENCES
1. C. Lopez, M. Blasco, L. Partridge, M. Serrano, G. Kroemer, Cell 153 (6), 1194-1217 (2013) 2. D. Fusco, G. Colloca, M.R. Monaco and M. Cesari, Clin Interv Aging 2 (3), 377-387 (2007).
3. R. A. Harvey and D. R. Ferrier, Biochemistry (Wolters Kluwer-Lippincott William & Wilkins, Baltimore, 2013), pp. 203-215
4. A. Rahal, A. Kumar, V. Singh, B. Yadav, R. Tiwari, S. Chakraborty and K. Dhama, Biomed Res Int 2014, 761264 (2014).
5. S. Ekarattanawong, C. Tanprasertkul, C. Somprasit, P. Chamod, R. Tiengtip, K. Bhamarapravatana and K.
Suwannarurk, Int J Womens Health 9, 711-716 (2017).
6. C. L. Priya and K. V. B. Rao, Pharmacogn mag 12 (Suppl 4), S475-S481 (2016).
7. C. Tangney and H. E. Rasmussen, Curr Atheroscler Rep 15 (5), 324 (2013).
8. B. L. Queen and T. O. Tollefsbol, Curr Aging Sci 3(1), 34-42 (2010).
9. M. Ganeshkumar, T. Ponrasu, R. Krithika, K. Iyappan, V. S. Gayathri and L. Suguna, J Ethnopharmacol 142 (1), 14-22 (2012).
10. C.L. Priya and K. V. B. Rao, Res J Biotechnol 9 (4), 60-68 (2014).
11. C. Franceschi and J. Campisi, J Gerontol A Biol Sci Med Sci 69 (Suppl 1), S4-9 (2014).
12. M. Maggio, J. M. Guralnik, D. L. Longo and L. Feruci, J Gerontol A Biol Sci Med Sci 61(6), 575-584 (2006).
13. Q. Jiang, Free Radic Biol Med 72, 76-90 (2014)
14. H. Sarir, G. Emdadifard, H. Farhangfar, H. TaheriChadorneshin, J Res Med Sci 20(12), 1177-1181 (2015).
