Antioxidant and Anti-Inflammatory Activities of Lindera glauca Extracts

Lindera glauca Blume (Siebold et Zucc.) is a deciduous shrub widely distributed in Asia and grows in the sun at the foot of a mountain [1, 2]. All parts of L. glauca are edible and used in traditional medicine. As a folk remedy, L. glauca’s fruit has been used as a traditional medicine to treat paralysis, including abdominal pain and speech disorders. In addition, roots have traditionally been used as a treatment for arthritis extrusion, bruising, and pain [3]. According to existing phytochemical studies, the physiological compounds such as alkaloids, butanolides, sesquiterpenoids, flavonoids, phenols, and steroids have been separated from L. glauca [4]. In Kim et al. [5] study, methanol extracts from L. glauca twig (LGT) showed excellent cytotoxicity in cancer cells such as A549, SK-OV-3, SK-MEL-2, and HCT-15. Even though several studies have been reported in the phytochemicals and pharmacological perspectives of L. glauca, no physiological activity study from the cosmetics perspective has been reported.

Reactive oxygen species is one of the causes of adult diseases such as cancer and aging. Hydrogen peroxide, superoxide ion, and hydroxyl radical occur naturally during human metabolism and protect against external substances. However, when overexpressed, they cause tissue damages like DNA and RNA damages and induce diseases [6]. In addition, reactive oxygen species is one of the causes of oxidative stress that accelerates skin aging. Then in the cosmetics industry, researches on single compounds and natural extracts that have antioxidant effects of removing oxidative stress have recently been increasing [7].

Inflammation usually occurs when infectious microorganisms such as bacteria, viruses, or fungi invade the body. The innate immune system is a defense mechanism against invading microorganisms, cancer cells, and macrophages [8]. Representative inflammatory mediators and cytokines involved in inflammation include nitric oxide (NO), PGE₂, inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) [9, 10]. When the inflammatory response begins, iNOS stimulated by an increase in inflammatory cytokines promotes NO. However, since excessive production of NO causes aggravation of diseases such as tissue damage, NO is used as a representative indicator of inflammation [11]. In addition, the expression of COX-2 has been reported to be associated with cell damage and oncogenesis and plays an essential role in the inflammatory response with iNOS. Therefore, a lot of studies are being conducted to search for substances that inhibit the secretion of these inflammatory mediators and cytokines from natural products [12].

In this study, we have investigated the antioxidant and anti-inflammatory effects in mouse macrophage cells for a cosmetic perspective. For these, the total polyphenol and flavonoid content, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, and ABTS⁺ radical scavenging assay were conducted to confirm the antioxidant effect of the extracts from LGT and L. glauca leaf/stem (LGLS). In addition, to evaluate the anti-inflammatory effect in the LPS-induced RAW 264.7 cells, protein and mRNA expression of major inflammatory factors were analyzed using Western blot analysis and RT-PCR. Moreover, it is intended to confirm the applicability as a natural material in the cosmetic and pharmaceutical fields and to provide primary data for future research.

The twigs, leaves, and stems of L. glauca used in this experiment were harvested from Geumseong Mountain, Gyeongsangbuk-do, Korea, in January 2022. First, the dried LGTs and LGLSs were pulverized into a fine powder using an electrical blender. Next, each powdered sample (60 g) was extracted with 300 mL of 70% ethanol at room temperature for 24 h, three times, and vacuum-filtered using Whatman no.2 filter paper. The extract was evaporated with rotary evaporator (EYELA N-1000; Tokyo Rikakikai Co., Ltd., Japan) at 40–45°C to remove excess ethanol. The remaining extracts are stored at −20°C until further used. Samples were labeled as LGT and LGLS for LGTs and LGLS extracts, respectively.

Total phenol content was measured by modifying the colorimetric quantification method of Folin-Denis [13]. Briefly, 80 μL of LGT and LGLS extracts diluted to 1 mg/mL was mixed with 80 μL 1N Folin-Ciocalteu reagent (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and placed at room temperature for 2 min. Then, 80 μL of 10% Na₂CO₃ solution was added to the mixture. After 1 h, absorbance was measured at 750 nm. A calibration curve was prepared using gallic acid (GA) as a reference material, and the results were expressed as GA mg/g.

The total flavonoid content of the sample was analyzed by modifying the method of Nieva-Moreno et al. [14]. Briefly, 100 μL of LGT and LGLS extracts diluted to 1 mg/mL was mixed with 1 mL of diethylene glycol and 100 μL of 1N NaOH. Absorbance was measured at 420 nm after incubating at 37°C for 1 h. A calibration curve was prepared using quercetin (QE) as a reference material, and the content of flavonoids in the LGT and LGLS was expressed as QE mg/g.

In this experiment, to determine the effective concentration of L. glauca extracts against RAW 264.7 cells, cell viability was measured by MTT assay using Mosmann’s colorimetric assay [17]. RAW 264.7 cells maintained in Dulbecco’s modified eagle medium with 1% penicillin-streptomycin and 5% fetal bovine serum were seeded onto a 96-well plate at 4 × 10 cells/well and incubated for 24 h. Cells were incubated with LGT and LGLS extracts at 25, 50, 100, or 125 μg/mL. After 24 h, 10 μL of MTT (5 mg/mL) solution was added to each well. After incubating for 3 h, absorbance was measured at 570 nm using a microplate reader (EZ Read 400; Biochrom Ltd., Cambridge, England).

NO production was measured to confirm the anti-inflammatory ability of LGT and LGLS extracts. RAW 264.7 cells were seeded at 4 × 10 cells/well in a 96-well plate and cultured for 24 h. The cells were stimulated with LPS (1 μg/mL) and co-treated with LGT and LGLS extracts (25, 50, 100, and 125 μg/mL), and then incubated for 24 h. The quantity of nitrite in the culture supernatant was measured using Griess reagent (Sigma-Aldrich, USA) for 10 min. The absorbance was read with a microplate reader (EZ Read 400; Biochrom Ltd., Cambridge, England) at 540 nm.

Western blot analysis was used to confirm the activity of iNOS and COX-2 protein factors. RAW 264.7 cells were seeded at 5 × 10 cells/well on a 6-well plate and then cultured for 24 h to stabilize the cells. After that, the medium was removed, LPS (1 μg/mL) was treated for 1 h, and the LGT and LGLS extracts were treated for indicated concentrations (25, 50, 100, and 125 μg/mL) and incubated for 24 h. The supernatant was removed and washed twice with phosphate-buffered saline. Cells suspended in 100 μL of RIPA buffer (150 mM NaCl, 1% non-ionic detergent, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail) then centrifuged at 13,200 rpm for 10 min at 4°C. Overall, 30–40 μg of protein was electroblotted onto polyvinylidene difluoride membranes following separation on an 8–10% SDS-polyacrylamide gel electrophoresis. The polyvinylidene difluoride membrane was blocked with blocking solution (5% skim milk; TBS, 0.1% Tween 20, TBS-T) for 1 h at room temperature, followed by incubation overnight with a primary antibody at 4°C. Membranes were washed three times with TBS-T and incubated with an HRP-conjugated secondary antibody (1:1,500 dilution) for 1 h at room temperature. Membranes were washed with TBS-T three times, and proteins were detected using ECL reagents (iNtRON Biotechnology Inc., Korea).

PCR was performed to investigate the mRNA expression of iNOS, COX-2, IL-6, and TNF-α. After seeding the RAW 264.7 cells at 5 × 10⁵ cells/well in a 12-well plate, it was incubated for 24 h and treated with LPS (1 μg/mL) for 1 h, and each extract was treated by concentration and cultured for 5 h. Total RNA was isolated with TRIzol reagent (Thermo Fisher Scientific, MA, USA). 2X green GoTaq master mix, template DNA, forward primer, reverse primer, and DNase-RNase-free water were added to the PCR tube. PCR was performed under the conditions of 5 min at 94°C, 30 s at 94°C, 30 s at 56°C, 1 min at 72°C, and 7 min at 72°C (30 cycles). The mRNA synthesized with PCR was electrophoresis of 1.2–1.5% agarose gel added with 0.002% red safe for 30 min, and then, the band was confirmed and quantified using gel document system (Doc-Print; Vilber Co., France). Sequences of oligonucleotide primers are listed in Table 1.

All the data were expressed as the mean ± SD derived at least three times. Statistical differences were determined by one-way ANOVA for multi-group comparisons followed by a Tukey post hoc test using GraphPad Prism version 9.3.1 (San Diego, CA, USA); p < 0.05 was considered statistically significant.

Before the investigation of antioxidant and anti-inflammatory activities of L. glauca, total polyphenol and flavonoid contents from twigs, leaves, and stems of L. glauca were measured since phenolic compounds and flavonoids are reported to have several physiological activities [18]. When expressed as an equivalent value of GA per g of dry sample in the total polyphenol content in LGT and LGLS extracts, LGLS extract showed higher polyphenol content whereas LGLS was 115.75 ± 7.87 mg (Table 2). Total flavonoid content was measured to confirm the antioxidant activity of LGT and LGLS extracts. When the flavonoid content of each extract was expressed as an equivalent value of QE, LGT was 66 ± 2.89, and LGLS was 74.33 ± 2.89 mg. As with the result of polyphenol, more flavonoids were measured in LGLS (Table 2).

2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical generally used to assess antioxidant activity. The distinct purple color of the DPPH radical solution disappeared as it reacted with antioxidants which prevent the radical chain reaction by providing hydrogen, and showed strong absorption at 517 nm. In this study, the DPPH RSA of LGT and LGLS was measured using l-ascorbic acid, well known as an antioxidant, as a positive control group. Both LGT and LGLS showed a concentration-dependently increase in scavenging activity. Furthermore, at 500 μg/mL, LGT was confirmed to have 88.56%, and LGLS had 87.86% DPPH RSA (shown in Fig. 1). The IC₅₀ values of the LGT and LGLS extracts were 102.29 and 105.07 μg/mL, respectively, where that of ascorbic acid was 43.71 μg/mL.

ABTS⁺ radical scavenging assay is based on decolorization that occurs when the radical ABTS⁺ is reduced. At a concentration of 50–500 μg/mL, both LGT and LGLS extracts showed a significant increase in antioxidant activity in a concentration-dependent manner. Moreover, at the highest concentration of 500 μg/mL, LGT showed excellent scavenging activity of 93.49% and LGLS of 99.23%, suggesting the potential as an antioxidant material like DPPH RSA (shown in Fig. 2). The IC₅₀ values of the LGT and LGLS extracts were 132.27 and 128.83 μg/mL, respectively, where that of ascorbic acid was 47.82 μg/mL.

Ahead of the NO production assay, MTT assay was performed to evaluate the cell safety of the L. glauca extracts. RAW 264.7 cells treated LGT or LGLS extracts at indicated concentrations (25, 50, 100, 125 μg/mL). When compared to the untreated group, LGT and LGLS showed 111% and 96% of viability, respectively, indicating that the effects of LGT and LGLS on cell viability were neglectable (shown in Fig. 3).

The changes of NO production were measured in LPS-induced RAW 264.7 cells to determine whether LGT and LGLS extracts are involved in the inflammatory response. The LPS (1 μg/mL)-treated group increased NO production compared to the untreated group. At a concentration of 125 μg/mL, LGT and LGLS showed 63.56% and 35.36%, respectively, indicating that they were effective in suppressing inflammation. LGT showed more potent NO production inhibition than LGLS (shown in Fig. 4).

Western blot analysis was used to measure the effect of inhibiting the expression of iNOS and COX-2 proteins, which are pro-inflammatory factors. As a positive control, β-actin, a housekeeping protein with no difference in expression even under various cell conditions, was used. It was confirmed that the expression level of the positive control was increased compared to the negative control due to stimulation by LPS (1 μg/mL), and the expression level of iNOS and COX-2 proteins was significantly inhibited in both of the extracts (shown in Fig. 5). At the concentration of 125 μg/mL, iNOS and COX-2 proteins were inhibited 97.7% and 79.3% by LGT extract, and 89.6% and 56.9% by LGLS extract, respectively.

RT-PCR was used to measure the inhibitory effect of iNOS and COX-2 mRNA expression, which are inflammatory factors. GAPDH, a housekeeping gene with little difference in expression even under various cell conditions, was used as a positive control. It was confirmed that the expression levels of iNOS and COX-2 mRNA, increased by LPS (1 μg/mL), were decreased by treating LGT and LGLS extracts (shown in Fig. 6).

Additionally, the expression of TNF-α and IL-6 in the inflammatory model of RAW 264.7 induced by LPS was confirmed to investigate the effect of LGT and LGLS extracts on inflammatory factor production. Both LGT and LGLS extracts significantly inhibited the production of IL-6 and showed insignificant inhibition against TNF-α (shown in Fig. 7). In addition, at the maximum concentration of 125 μg/mL, LGLS extract inhibited IL-6 by 87.7%, showing a higher effect than LGT.

This study evaluated the potentials of extracts of LGT and LGLS as a cosmetic substance through antioxidant and anti-inflammatory tests. Several studies on the efficacy of L. glauca have been reported [1‒5]. Among them, Kim et al. [1] used the stem extract, while Yu et al. [2] and Kim et al. [5] used twigs of L. glauca to assess the bioactivities of L. glauca. Over 90 bioactive chemical constituents have been isolated and reported [4]. Therefore, not only twigs but also leaves/stems of L. glauca were applied to the evaluation. Phenolic compounds are widely distributed in the Plantae and have various structures and molecular weights as one of the secondary metabolites. In addition, they have a phenolic hydroxyl group, which binds to macromolecules such as proteins and has physiological activities such as antioxidant and antimicrobial activity [18]. Furthermore, flavonoids widely distributed in plants are polyphenols known to have anti-inflammatory, antiviral, and anticancer effects by reducing oxidative stress and removing active oxygen [18]. The total polyphenol content of LGT and LGLS extracts was 88.42 and 115.75 GA mg/g, and flavonoid content was 66 and 74.33 QE mg/g, similar to or higher than those of other natural products. According to an antioxidant study on Lindera obtusiloba leaf and twig/stem extracts of the same genus, they showed similar or higher scavenging activity at lower concentrations compared to 84% (1,000 μg/mL) and 87% (500 μg/mL), respectively [19]. In contrast, LGT and LGLS extracts showed similar or high scavenging activity at a lower concentration, suggesting that LGT and LGLS extracts have a high potential as antioxidant materials.

DPPH radical and ABTS⁺ radical scavenging assays are widely used to assess the antioxidant ability of natural products [20]. It is reported that cation radical scavenging is an important attribute of antioxidants, and ABTS radical cation (ABTS⁺) has characteristic absorbance at 734 nm, which decreases with the scavenging of cation radicals [16, 21]. As a result of DPPH scavenging assay, at 100 μg/mL, the activity was lower than that of the reference material, l-ascorbic acid, which was 93.5%. However, at the highest concentration of 500 μg/mL, LGT showed 88.56% and LGLS was 87.86%. The IC₅₀ values of the LGT and LGLS extracts were determined as 102.29 and 105.07 μg/mL, respectively, which are about double of ascorbic acid. In the ABTS⁺ radical scavenging assay, at 100 μg/mL, QE, the reference substance, showed 91.59%, superior to LGT and LGLS. However, LGT and LGLS showed 93.49% and 99.23% at 500 μg/mL, respectively. The IC₅₀ values of the LGT and LGLS extracts were 132.27 and 128.83 μg/mL, respectively. The effects of ascorbic acid have been studied in diverse ways such as antioxidant, anti-inflammatory effects [22, 23]. Therefore, l-ascorbic acid was used as a positive control to compare the DPPH and ABTS⁺ RSA in this study.

It was confirmed that the antioxidant activity was significantly increased in a concentration-dependent manner of the LGT and LGLS. In order to confirm the anti-inflammatory effect of LGT and LGLS, NO production activity in RAW 264.7 cells at a concentration that does not show cytotoxicity was measured through an MTT assay. NO production showed a concentration-dependent decrease in both LGT and LGLS extracts, and at the highest concentration of 125 μg/mL, LGT was 63.56%, and LGLS was 35.36%, showing stronger NO production inhibition in the LGT. NO is a representative inflammatory factor synthesized from iNOS and is known to cause several inflammatory diseases. Excessive NO production has been reported to lead to inflammation intensification and tissue damage [11, 24].

Western blot and RT-PCR were used to evaluate the anti-inflammatory effects of LGT and LGLS on protein and mRNA. The expressions of mRNA and the protein productions of iNOS and COX-2 showed a tendency to decrease in a concentration-dependent manner by LGT and LGLS. iNOS protein of LGT and LGLS showed 97.7% and 89.6% inhibitory ability, and 79.3% and 56.9% inhibitory activity, respectively, against COX-2 protein at a maximum concentration of 125 μg/mL. In a study on the anti-inflammatory effects of compounds isolated from L. glauca roots, root fractions were reported to have concentration-dependent inhibition of the expression of LPS-induced iNOS and COX-2 [25]. LPS is recognized as a potent activator of inflammation [26]. In addition to iNOS and COX-2 mRNA measured using RT-PCR, iNOS of LGT and LGLS showed a decrease of 98.7% and 44.5%, and COX-2 showed a decrease of 62.1% and 13.4%, respectively. Furthermore, it was confirmed that the expression decreased in the IL-6 and TNF-α mRNA experiment. Infectious cytokines such as TNF-α and interleukin have been reported to cause cell aging and disease by promoting inflammation in the immune system and engaging in the upregulation of inflammatory responses [9]. The inhibition of the expression of IL-6 mRNA was significantly higher than that of TNF-α. Conclusively, it could be considered that the L. glauca extract has an antioxidant effect and suppressed inflammation, and has a high potential as a constituent in the functional cosmetics and pharmaceutical industry.

Ethical approval is not required for this study in accordance with local or national guidelines.

The authors have no conflicts of interest to declare.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A3072936).

Professor Seung-Ho Ohk has contributed to this study by organizing the concept, establishing the methodology, and writing the manuscript as a project manager. Professor Hyun Ju Lee has contributed to this study by supervising the whole process of this study concept, establishing the methodology, and validating and curating the data. Ms. Yi-Na Park has contributed to this study by conducting the whole procedures and formal analyses of this study as a first author of this manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.