Research Article |
Corresponding author: Rony Abdi Syahputra ( rony@usu.ac.id ) Academic editor: Valentina Petkova
© 2023 Adi Muradi Muhar, Adrian Joshua Velaro, Arya Tjipta Prananda, Sony Eka Nugraha, Gamze Çamlik, Siddhanshu Wasnik, Satirah Zainal Abidin, Osfar Sjofjan, Muhammad Andika Yudha Harahap, Muhammad Faridz Syahrian, Nurpudji Astuti Taslim, Nelly Mayulu, Happy Kurnia Permatasari, Fahrul Nurkolis, Putri Cahaya Situmorang, Rony Abdi Syahputra.
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:
Muhar AM, Velaro AJ, Prananda AT, Nugraha SE, Çamlik G, Wasnik S, Abidin SZ, Sjofjan O, Harahap MAY, Syahrian MF, Taslim NA, Mayulu N, Permatasari HK, Nurkolis F, Situmorang PC, Syahputra RA (2023) Polyscias scutellaria: An emerging source of natural antioxidants and anti-inflammatory compounds for health. Pharmacia 70(4): 1463-1470. https://doi.org/10.3897/pharmacia.70.e112502
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Polyscias scutellaria (PS), an increasingly recognized botanical marvel, has emerged as a remarkable reservoir of natural antioxidants and anti-inflammatory compounds, holding immense potential for enhancing health and promoting overall well-being. In this comprehensive investigation, we meticulously examined the multifaceted properties of PS through various parameters, including DPPH scavenging activity, total phenol, and total flavonoid content in its ethanol extract (EEPS), ethyl acetate extract (EAPS), and n-hexane extract (nhPS). Additionally, we conducted in-depth assessments of cellular responses to EEPS and EAPS, encompassing cell viability, nitric oxide (NO) production, and the modulation of pivotal pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-12 (IL-12), utilizing the RAW 264.7 cell line as a model system.Our findings illuminate the exceptional antioxidant prowess of PS extracts, with EEPS, EAPS, and nhPS displaying noteworthy DPPH scavenging activities. These results underscore their potential in quenching harmful free radicals and mitigating oxidative stress. Furthermore, our investigation unveils the promising anti-inflammatory attributes of EEPS and EAPS, as evidenced by their capacity to preserve cellular viability, dampen NO production, and suppress the secretion of critical pro-inflammatory mediators (TNF-α, IL-6, IL-1β, and IL-12) in RAW 264.7 cells. These remarkable anti-inflammatory effects hint at the therapeutic potential of PS in ameliorating chronic inflammatory conditions and bolstering the body’s immune response.In conclusion, Polyscias scutellaria stands as an emerging botanical champion, offering a wealth of natural antioxidants and anti-inflammatory compounds that hold great promise for optimizing health and well-being. This study opens exciting avenues for future research to elucidate the precise bioactive constituents within PS and unravel their intricate mechanisms of action, paving the way for the development of innovative therapeutic interventions and wellness-enhancing products. The remarkable properties of PS underscore its potential as a cornerstone of holistic health and a valuable asset in the pursuit of a vibrant and balanced life.
Graphical abstract
Antioxidant, antiinflammation, Polyscias scutellaria
Generate oxidative stress in biological systems, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important in the pathophysiology of many chronic degenerative diseases. ROS and RNS levels rise above normal, causing oxidative stress. Thus, antioxidant systems cannot balance this discrepancy, resulting in unpaired free radicals (
Polyscias scutellaria were collected from the Faculty of Pharmacy, Universitas Sumatera Utara, Indonesia (coordinates 30°33'36.5"N, 98°039'12.5"E), ethanol 96% were purchased from bratachem Medan, n-hexane were purchased from bratachem Medan, etyhl acetate were purchased from bratachem Medan, 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from sigma aldrich, Tris-HCl buffer (pH 7.4) were purchased from sigma aldrich, Gallic acid were purchased from sigma aldrich, Gallic acid were purchased from sigma aldrich, phosphate-buffered saline (PBS) were purchased from sigma aldrich, lipopolysaccharide (LPS)were purchased from sigma aldrich, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from sigma aldrich, sodium nitrite (NaNO₂) were purchased from sigma aldrich, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and IL-12 were purchased from ABclonal Wuhan, China.
The total gram of dry of Polyscias scutellaria (PS) is 700 g in a powder that was macerated with 10 L n-hexane. Firstly the powder was dried and dissolved with Ethyl acetate for three days then stirred occasionally at a room-temperature. Lastly, the powder was dried and dissolved with Ethanol for three days stirred occasionally at a room temperature. Each filtrate was collected and evaporated under pressure.
The assessment of the antioxidant activity of the extracts encompassed the amalgamation of approximately 1.0 mL of a 0.1 M solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with 0.9 mL of a 50 mM Tris-HCl buffer (pH 7.4). Subsequently, a volume of 0.1 mL was added to the combination, which could either consist of the sample extract or deionized water serving as a reference. The resulting solution underwent complete homogenization and was thereafter incubated at room temperature for a duration of 30 minutes. Following the incubation period, the absorbance was quantified utilizing a UV-Vis spectrophotometer set at a specific wavelength of 517 nm.
The calculation of the DPPH scavenging activity was performed using the following formula:
The determination of total phenolic content (TPC) was performed using the Folin–Ciocalteu technique. In this experiment, a 20 µL aliquot of the desiccated sample-extract solution, which had been produced in a methanol solution at a weight-to-volume ratio of 1:10, was combined with 1.58 mL of distilled water and 100 µL of the Folin-Ciocalteu reagent. Subsequently, a 5% sodium carbonate solution with a volume of 300 µL was introduced into the mixture. Following this, the resulting solution was exposed to a controlled environment with limited light, while being kept at a constant temperature of 25 °C, for a duration of 2 hours. The measurement of absorbance was conducted at a specific wavelength of 765 nm. Additionally, a control sample was created by employing distilled water in accordance with the identical approach. The determination of the overall phenolic content (TPC) was performed by employing gallic acid equivalents (GAE) and quantified in milligrams per gram of dry extract. The calibration curve was generated by employing solutions of Gallic acid with varying concentrations (5, 10, 20, 40, and 80 mg/L). The calculated coefficient of determination (R2) was found to be 0.9871.
The determination of total flavonoid content (TFC) was conducted using a simplified methodology. The experimental procedure involved combining 1 mL of the extract with 300 μL of a 5% NaNO2 solution and 300 μL of a 10% aluminum chloride solution. The resulting combination was then subjected to incubation at a temperature of 25 °C for a duration of 5 minutes. Subsequently, a 2 mL aliquot of a 1 N sodium hydroxide solution was introduced into the amalgamation. The solution was diluted with water in order to achieve a final volume of 10 mL. Subsequently, it was exposed to agitation using a vortex mixer to ensure thorough homogenization. The measurement of absorbance was conducted at a specific wavelength of 510 nm. A calibration curve was established in order to ascertain the content of catechin, yielding a coefficient of determination (R2) value of 0.974. The determination of the total flavonoid content (TFC) in the sample was conducted by quantifying it in milligrams of equivalents (CE) per gram of sample, utilizing the dry weight.
The RAW 264.7 cell line, which originates from murine macrophages, was cultivated in Dulbecco’s Modified Eagle’s Medium (Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea) supplemented with 10% fetal bovine serum (Welgene), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Grand Island, NY, USA). The cells underwent incubation at a temperature of 37 °C within a regulated humidity environment and a carbon dioxide concentration of 5%.
The RAW 264.7 cell line was cultured by inoculating them overnight in a 96-well plate at a density of 5 × 104 cells per well. A volume of 100 µL of cell culture medium was added to each well. The cells were exposed to various doses of PE, spanning from 0 to 250 μg/mL, during a duration of 4 hours. Following this, the cells were washed with cold phosphate-buffered saline (PBS) and subsequently exposed to lipopolysaccharide (LPS) obtained from Sigma-Aldrich, a business headquartered in St. Louis, MO, USA, at a concentration of 1 μg/mL. The duration of the session of stimulation lasted for a total of 20 hours. A 10 microliter amount of a solution containing 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) was added to each well. Following a time period of 5 hours, a quantity of 100 μL of a hydrochloric acid (HCl) solution with a concentration of 0.04 N, dissolved in isopropanol, was added. The quantification of live cells was performed by measuring the optical density at a wavelength of 540 nm using an EMax microplate reader produced by Molecular Devices, situated in Sunnyvale, CA, USA.
The RAW 264 cell line is frequently employed as a murine macrophage cell line in the field of biomedical research.A total of seven cells were exposed to ALE treatment for a period of four hours. After the completion of the aforementioned treatment, the cells were subjected to a cold phosphate-buffered saline (PBS) rinse. Subsequently, the cells were stimulated with lipopolysaccharide (LPS) for a duration of 20 hours. Subsequently, a quantity of 50 microliters of culture supernatant was acquired and mixed with an equivalent volume of Griess reagent, sourced from Sigma-Aldrich. The optical density was measured at a wavelength of 540 nm. The quantification of nitric oxide (NO) production was conducted using sodium nitrite (NaNO₂) sourced from Junsei Chemical CO., a company situated in Chuo-ko, Tokyo, Japan. This was done to establish a standard reference curve for nitrite.
RAW 264.7 cells were treated with OS and incubated for 48 h. The supernatant was collected by centrifugation (15,000 rpm, 10 min, 4 °C). The immunomodulatory effects of OS in RAW 264.7 cells were evaluated by ELISA. The concentrations of cytokines (IL-1β, IFN-γ, and TNF-α) in the cell supernatant were measured by IL-1β (ab197742), IFN-γ (ab282874), and TNF-α (ab285327) according to each ELISA kit manual, respectively. An amount of 50 μL of supernatant and 50 μL of cytokine antibody cocktail were added into a 96 well plate coated with antibody and were incubated at RT for 1 h. The plate was washed three times and 100 μL of TMB solution was added to each well. It reacted for 10 min and the reaction was stopped with 100 μL of a stop solution. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices). Each cytokine concentration was calculated by a standard curve.
Data were presented as the mean ±Standard error of means (SEM). GraphPad Prism (version 10) was used for statistical analysis of all data. T-test was used to compare the in vitro antioxidant activities of extract and fractions. For all other data, the one-way analysis of variance (ANOVA) followed by Dunnet’s post hoc test was used to compare the means of one group with every other group. The correlation coefficient was calculated using Microsoft office Excel 2010. Statistical significance was set at 5% thus p value ≤0.05 was considered significant.
The DPPH method is widely recognized as a prominent technique for evaluating antioxidant activity in vitro. The scavenging activities of AS on the DPPH radical were compared with those of VC, as depicted in Fig.
Table
Cytotoxicity of PE on RAW 264.7 macrophage cells in the presence of LPS. Cells were treated with various concentrations of EEPS and EAPS (10, 20, 30, 40, and 50 μg/mL) for 24 h followed by LPS (1 µg/mL). While the effect of PE in NO production RAW 264.7 macrophage cells pre-treated with 10, 20, 30, 40, and 50 μg/mL PE for 1 hours and subsequently simulated with LPS) at a concentration of 1 μg/mL for a period of 24 hours. (ns : non-significant; * p<0,05; ** p<0,001; *** p<0,0001).
The concentrations of total flavonoids in Ethanol extraxct of PS (EEPS), Ethyl acetat of PS (EAPS), and n-hexane of PS (nhPS) were quantified, with values ranging from 5.1 to 25.4 mg/g flavonoids. According to the data presented in Table
The effect of EEPS and EAPSin RAW 264.7 macrophage cells in the presence of LPS on tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and IL-12. Cells were treated with various concentrations of EEPS and EAPS (40, and 50 μg/mL) for 24 h followed by LPS (1 µg/mL. (ns : non-significant; * p<0,05; ** p<0,001; *** p<0,0001).
To examine the cytotoxic effects of EEPS and EAPS derivatives on RAW 264.7 cells, the viability of the cells was assessed by the utilization of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The cells were subjected to treatment with various concentrations of PE (0, 10, 20, 30, 40, and 50 μg/mL) and LPS (1 μg/mL), followed by incubation for a duration of 24 hours to assess absorbance levels. Consequently, no notable distinctions were observed between the untreated control group and the group treated with PE in RAW 264.7 cells. Consequently, it was determined that the treated doses of PE exhibited no harmful effects. Subsequently, more investigations were carried out employing the concentrations of 100, 300, 500, and 700 μM. To investigate the impact of PE on the production of nitric oxide (NO), cellular samples were subjected to pre-treatment with varying concentrations (0, 10, 20, 30, 40, and 50 μg/mL) of each PE for a duration of 1 hour. Following this pre-treatment, the cells were subsequently stimulated with lipopolysaccharide (LPS) at a concentration of 1 μg/mL for a period of 24 hours. The cells that were solely treated with LPS exhibited a significant increase in the generation of nitric oxide (NO) in comparison to the cells that were not treated and served as the control group. Nevertheless, the application of PE resulted in a concentration-dependent reduction in NO generation.
The RAW 264.7 cell line consists of macrophages, which are essential in the modulation of inflammatory conditions. In this study, we examined the potential of EEPS and EAPS to modulate the inflammatory response of macrophages upon exposure to LPS stimulation. The ELISA technique was employed to assess the quantity of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and IL-12 in the supernatants obtained from cells that were subjected to different doses of AL extract and stimulated with LPS for 24 hours. The findings of this study demonstrated that treatment with EEPS and EAPS extract effectively suppressed the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and IL-12 in RAW264.7 cells stimulated with LPS. The reported inhibitory effects of the EEPS and EAPS extract were found to be depending on the concentration. The findings of this study indicate that EEPS and EAPS, which is rich in quercetin, may have the potential to inhibit the synthesis and release of inflammatory mediators and cytokines from RAW 264.7 cells that have been activated with LPS.
Polyscias scutellaria (PS) is increasingly gaining recognition as a natural source of antioxidants and anti-inflammatory compounds that can significantly impact human health and wellness. In this study, we explored various parameters to comprehensively assess the potential health benefits of PS, including its DPPH scavenging activity, total phenol content, and total flavonoid content in different extracts (EEPS, EAPS, and nhPS). Additionally, we delved into the effects of EEPS and EAPS on cell viability, nitric oxide (NO) production, and the secretion of critical pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-12) using the RAW 264.7 cell line. The remarkable DPPH scavenging activity observed in PS extracts highlights its inherent ability to neutralize free radicals and oxidative stress. Among the extracts tested, EEPS, EAPS, and nhPS demonstrated significant antioxidant potential, with EEPS exhibiting the highest scavenging activity. This finding suggests that PS could serve as an effective natural defense against oxidative damage, which is implicated in various chronic diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders (
The author thank to sasniwatihasibuan for assisting the research experiment.