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Table of Contents
Year : 2020  |  Volume : 63  |  Issue : 3  |  Page : 137-148

Effects of Cajanus cajan (L.) millsp. roots extracts on the antioxidant and anti-inflammatory activities

1 Department of Food Science and Biotechnology, College of Biotechnology and Bioresources, Da-Yeh University, Changhua, Taiwan
2 Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan
3 Department of Beauty Science and Graduate Institute of Beauty Science Technology, Chienkuo Technology University, Changhua, Taiwan
4 Undergraduate Program of Nutrition Science, School of Life Sciences, National Taiwan Normal University, Taipei, Taiwan, Taiwan

Date of Submission19-Nov-2019
Date of Decision09-Apr-2020
Date of Acceptance07-May-2020
Date of Web Publication23-Jun-2020

Correspondence Address:
Prof. Tuzz-Ying Song
Department of Food Science and Biotechnology, College of Biotechnology and Bioresources, Da-Yeh University, No.168, University Rd., Dacun, Changhua 51591
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/CJP.CJP_88_19

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Cajanus cajan (L.) Millsp., also named pigeon pea, is widely grown in the tropics and the subtropics. C. cajan roots (CR) and ribs stewed in hot water have been used as a traditional medicine in various cultures to treat diabetes. The purpose of this study was to determine the functional components of hot water (WCR) and 50%, 95% ethanol extracts (EECR50 and EECR95) from CR, then evaluating their antioxidant and anti-inflammatory effects. The results indicated that EECR95 had higher polyphenol, especially the isoflavones (e.x. daidzein, genistein, and cajanol) than those of the other extracts, and it also exhibited the most potent anti-oxidative activities by in vitro antioxidant assay. In the lipopolysaccharide-stimulated RAW 264.7 cells, we found that EECR95 significantly decreased intracellular reactive oxygen species and significantly enhanced the activities of superoxide dismutase and catalase. Mechanism studies showed that EECR95 mainly activated nuclear factor (NF) erythroid 2-related factor 2/antioxidant protein heme oxygenase-1 and inhibited nuclear factor kappa B (NF-κB) signaling pathway, and thus exhibited antioxidant and anti-inflammatory effects. Overall, this study suggests that CR may have the potential to be developed as a biomedical material and that genistein, which has relatively high uptakes (3.44% for the pure compound and 1.73% for endogenous genistein of EECR95) at 24 h of incubation with RAW 264.7 cells, could be the main active component of CR.

Keywords: Anti-inflammation, antioxidant, Cajanus cajan (L.) Millsp. root, uptake and genistein

How to cite this article:
Vo TLT, Yang NC, Yang SE, Chen CL, Wu CH, Song TY. Effects of Cajanus cajan (L.) millsp. roots extracts on the antioxidant and anti-inflammatory activities. Chin J Physiol 2020;63:137-48

How to cite this URL:
Vo TLT, Yang NC, Yang SE, Chen CL, Wu CH, Song TY. Effects of Cajanus cajan (L.) millsp. roots extracts on the antioxidant and anti-inflammatory activities. Chin J Physiol [serial online] 2020 [cited 2020 Jul 9];63:137-48. Available from: http://www.cjphysiology.org/text.asp?2020/63/3/137/287454

  Introduction Top

Inflammation is an important protective function of hosting against to infection of a foreign pathogen, such as physical or chemical stimuli or microbial toxins.[1] However, prolonged inflammation causes many diseases, such as arthritis, asthma, multiples sclerosis[2],[3] and increases the incidence of chronic diseases, including cancer[4],[5],[6],[7] and cardiovascular disease.[8] Oxidative stress commonly accompanies infection as a result of a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defenses.[9] Macrophages release many pro-inflammatory factors such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines, including interleukins-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α).[10] Furthermore, macrophages regulate the expression of genes such as nuclear factor kappa B (NF-κB), expressions of cyclooxygenase-2 (COX-2), and inducible NO synthase (iNOS), and consequently, the oxidative genes including heme oxygenase-1 (HO-1), nuclear factor-erythroid 2-related factor 2 (Nrf-2), and peroxisome proliferator-activated receptors (PPAR-γ).[11]

Cajanus cajan (L.) Millsp., known as pigeon pea in English, is widely grown in the tropics and the subtropics. Several studies have indicated that the leaf, root, and seed extracts of C. cajan (L.) Millsp. possess potent antioxidant, anti-inflammation, anticancer, hypoglycemic, anti-microbial, hypocholesterolemia, hepatoprotective and nephroprotective activities with potential applications in both medicine and the health food industry.[12],[13] In addition, the methanol extract of C. cajan (L) Millsp. roots (CR), was reported to inhibit plant pathogens[14] and have anti-cancer capabilities.[15] Zhang et al.[16] demonstrated that the major active compounds responsible for CR were polyphenols, especially isoflavonoids. Using a series of separation and purification from CR ethanol extract, Duker-Eshun et al.[17] identified the major components, which included betulinic acid, biochanin A, 5,2'-dihydroxy-7,4'-dimethoxyisoflavanone (cajanol), genistein, and 2'-hydroxygenistein. Genistein (4', 5, 7-trihydroxyisoflavone, C15H10O5), a flavonoid with phytoestrogen activity, is abundant in soy foods and possesses antioxidant[18],[19] anti-inflammatory[20],[21] and anti-cancer[22] activities.

In the present study, we measured the total phenolic (TPC) and the total flavonoid contents (TFC) in hot water (WCR) and ethanol extracts (EECR50, EECR95) of CR. We then analyzed their polyphenol composition by the reverse phase high-performance liquid chromatography (HPLC) technique. An extract (EECR95) with the highest antioxidant activity was chosen based on the non-cellular (1,1-diphenyl-2-picrylhydrazine [DPPH], ABTS+, and Ferric-reducing antioxidant power [FRAP]) and cellular antioxidant assays (ROS production, SOD and CAT activities in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages). EECR95 was then determined for its anti-inflammatory activities (cytokine release; IL-1, IL-6, TNF-α, and PGE2) in LPS-stimulated RAW 264.7 cells. Furthermore, the effect of EECR95 on inflammatory proteins (iNOS, COX-2, and NF-κB) and anti-inflammatory proteins (Nrf-2, PPAR-γ, and HO-1) were determined in LPS-stimulated RAW 264.7 cells. To elucidate the possible active compounds of EECR95, we investigated the cellular uptake of flavonoids (genistein, daidzein, and cajanol) in the macrophages. To the best of our knowledge, this is the first report on the antioxidant and anti-inflammation activities and the major active compounds in CR.

  Materials and Methods Top


Macrophage cells (RAW 264.7; BCRC Number: 6001) from Bioresource Collection and Research Center (Hsinchu, Taiwan). Lipopolysaccharide (LPS; Escherichia coli O26:B6) and bovine serum albumin were bought from Sigma-Aldrich Co. (St Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) from Gibco by Life Technologies (Frederick, MD, USA). Chemicals and reagents are of high quality. Tips, dishes, test tubes, etc. for cell culture were bought from Thermo Fisher Scientific (Waltham, MA, USA).

Preparation of extracts from Cajanus cajan (L.) Millsp. roots

Dried C. cajan (L.) Millsp. roots (CR) were collected from Taitung District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan. CR was washed and dried by sunlight for 3 h, then cut into about 8-cm segments for further lyophilization. The dried CR powder was obtained using grinder (Marconi, Piracicaba, SP, Brazil).

The dried CR powder were extracted by 20-fold (w/v) of hot water (98°C ± 2°C) for 2 h and different ethanol concentration (50% and 95%) for 48 h, then the extracts were filtered and concentrated at reduced pressure by using a rotary evaporator. Finally, the extracts were lyophilized to obtain WCR, EECR50, and EECR95, respectively. Then, they were stored at 4°C for following analysis.

Analysis of functional components in Cajanus cajan (L.) Millsp. roots

Total phenolic contents

TPC in each extract was determined applying the Folin-Ciocalteu (FC) method described by Do et al.[23] with minor modifications. TPC was expressed as milligram gallic acid equivalent (mg Ga/g) per gram of CR extract.

Total flavonoid contents

TFC of each extract was determined using the aluminum chloride colorimetric method described by Do et al.[23] with slight modifications. TFC was expressed as milligram quercetin equivalent per gram C. cajan (L.) Millsp. roots (mg Que/g).

Detect functional compounds by reverse phase high-performance liquid chromatography

The analysis was carried out using an Agilent 1200 reverse phase HPLC system (Hitachi, Chromaster 5430 Diode Array Detector) with a HIQ Sil C18W reversed-phase column (250 mm × 4.6 mm, i.e., 5 μm). The polyphenol compositions were measured as described by Chen et al.[24] with slight modifications. The mobile phase was: Solvent A: 0.25% formic acid and 2% methanol in distilled water, and solvent B: Acetonitrile and the flow rate was 1 mL/min. The gradients were: 0–20 min 85% (A), 20–30 min 85%-65% (A), 30–45 min 65% (A), 45–75 min 65%–35% (A), and 75–95 min 85% (A). The absorbance was measured at 290 nm. The polyphenols were identified and quantified using peak retention time, UV spectra, and the UV max absorbance bands and trough were compared with external commercial standards (Carbosynth (Berkshire, RG20 6NE, UK) and Sigma (St Louis, MO, USA)). The external standards (cajanol, cinnamic aicd, courmaric acid, ferulic acid, gallic acid, syringic acid, vanillic acid, flavonoid: Daidzein, genistein, rutin, and quercetin) were freshly prepared in 70% ethanol at a concentration of 1.0 mg/mL and applied onto the HPLC-DAD-UV-Vis system before the samples. The results were expressed as mg/g dry weight (dw). All solvents were of HPLC grade.

In vitro anti-oxidant activities of extracts of Cajanus cajan roots

1,1-diphenyl-2-picrylhydrazine radical scavenging activity

The radical scavenging activity was measured applying the method presented in Do et al.[23] with slight modifications. The IC50 value was half-maximal inhibit concentration as the amount of antioxidant required to decrease the initial DPPH concentration by 50%.

ABTS+ scavenging effects assay

The antioxidant capacity of the films was determined by applying spectrophotometric method described in Jeannineand Paulo.[25] Trolox was used as a standard antioxidant. The ABTS+ value was expressed as the mass of the extract which produces the same percentage of absorbance reduction as 1 μmole Trolox solution.

Ferric reducing antioxidant power assay

The method described in Chu et al.[26] would be applied in this work to determine the reducing power of freeze-dried extract. This reducing power was investigated by observing transformation of Fe3+ to Fe2+ and its absorbance was measured at 700 nm.

Antioxidant activities of EECR95 in lipopolysaccharide-induced RAW 264.7 cells

Cell culture

RAW cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 3.7 g sodium bicarbonate. Medium for cells used in the experiments was changed every 2 days, incubated at 37°C and 5% CO2.

Cell viability assay

Cell viability was determined by the MTT assay. Briefly, RAW 264.7 cells seeded into 96 well plates at a destiny of 1 × 104 cells per well were pretreated with various concentrations (0–100 μg/mL) of EECR95 for 2 h and then with LPS (2 μg/mL) for 24 h, after which 0.5 mg/mL of MTT solution was added. After addition of 100 μL DMSO and incubation for 3 h, the supernatant was removed, and cell viability was detected at 570 nm using a ELISA reader (Synergy HTX, BioTek, USA).

Measurement of superoxide dismutase and catalase activity

SOD activity assay was performed by pyrogallol autoxidation method as described in Marklund and Marklund[27] Catalase (CAT) assay was carried out according to a previous protocol with modifications Acuna et al.[28] RAW 264.7 cells were pretreated with various concentration of EECR95 for 2 h and were stimulated with LPS (2 μg/mL) for 24 h. Cell pellets were harvested, and lysed with lysis buffer containing protease inhibitor following by centrifuge 12,000×g for 30 min at 4°C. Determined protein concentration in the cell lysates by using detergent Bio-Rad protein assay (Hercules, CA, USA). One unit of SOD activity was defined as the amount required for inhibiting pyrogallol autoxidation by 50% per min. CAT activity was calculated based on the rate of decomposition of H2O2, which was proportional to the reduction in the absorbance. CAT activity was expressed as units of CAT per μg of protein (U/μg protein).

Measurement of intracellular reactive oxygen species

Intracellular ROS levels were measured using the fluorescent probe 2′, 7′-dichlorodihydrofluorescein diacetate (H2 DCFDA), as previously described in Lautraite et al.[29] DCFDA readily diffuses through the cell membrane and is enzymatically hydrolyzed by intercellular esterases to form nonfluorecent DCFH, which is then rapidly oxidized to form highly fluorescent DCF in the presence of ROS. RAW 264.7 cells (5 × 105 cells/mL) seeding into a 10 cm dish and incubated with different concentration of EECR95 for 2 h and incubated with LPS (2 μg/mL) for 24 h. After being treated, the cells were harvested, and stained by 30 μM of H2 DCFDA at 37°C for 1 h in dark. Removed staining by centrifugation at 4°C, 900×g for 10 min. The cell pellet was collected, 0.5 mL of 1×PBS was added, mixed and meshed before being transferred to a flow cytometer tube for measurement of ROS level using flow cytometry (BD FACSCanto II, USA).

Anti-inflammatory activities of EECR95 in lipopolysaccharide-induced RAW 264.7 cells

Measurement of nitrite production

RAW 264.7 cells (5 × 105 cells/well) were seeded in 24 well plates. LPS (2 μg/mL) was added to each well and incubated for 24 h. After collecting the supernatant, nitrite, an oxidation product of NO, was assayed by using spectrophotometry. Nitrite was determined with Griess reaction by mixing 100 μL of culture supernatant and 100 μL of Griess reagent (1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid and 0.1% (w/v) N-(1-naphthyl)-ethylenediamine-dihydrochloride) solution. After incubation for 10 min, absorbance was measured at 540 nm, and the nitrite concentration was calculated by using sodium nitrite as a standard.[30]

Measurement of prostaglandin E2 level

In brief, RAW 264.7 cells seeded in a 10-cm dish were treated with various concentration of the sample for 2 h followed by incubation with LPS (2 μg/mL) for 24 h. Then, cells were harvested, lysed, and the protein concentration was determined in the cell lysates using detergent Bio-Rad protein assay. The concentration of PGE2 pg/mL was calculated based on the standard concentrations using PGE2 ELISA kit according to the manufacturer's instructions from Life Technologies Corp.(Frederick, MD, USA).

Measurement of pro-inflammatory cytokines level

RAW 264.7 cells (5 × 105 cells/well) seeded into 24-well plates were treated with LPS (2 μg/mL) for 24 h. Next, the culture medium was collected, and the supernatant was measured for cytokines (involve IL-1, IL-6 and TNF-α) using ELISA kits according to the instructions of manufacturer from Invitrogen Co. (Camarillo, CA, USA).

Western blotting analysis

The cytosolic proteins 50 μg were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a poly-vinylidene fluoride membrane. The membrane was detected using an ECL chemiluminescent detection kit. The relative density of protein expression was quantified using ImageJ software, developed by Wayne Rasband at the National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin, USA). Western blot analysis was performed to detect the protein expression of HO-1, Nrf-2, PARP-γ, iNOS, COX-2, NF-κB, and β-actin in the RAW 264.7 cells, as described in a previous report in Lee et al.[31] with minor modification.

Cellular uptake

RAW 264.7 cells (5 × 105 cells/mL) seeded in a 10-cm dish were treated with 20 μg/mL of EECR95 (containing 0.4, 0.08, and 0.06 μg/mL of genistein, cajanol, and daidzein, respectively) or 25 μM of either genistein, cajanol, or daidzein (6.8, 7.9, and 6.4 μg/mL, respectively) and incubated at 37°C for 3, 6, 12 and 24 h. Cells were collected, lysed and centrifuged at 12,000×g for 30 min. A portion of the supernatant was applied onto a HPLC-DAD/UV-Vis system (Hitachi, Chromaster 5430 DAD) as described in previous paragraph. The cellular uptake was expressed as ng/106 cells.

Statistical analysis

All statistical analyses were performed using SPSS for Windows, version 18 (SPSS Inc., Chicago, IL, USA). Data are expressed as means ± standard deviation and analyzed using one-way ANOVA followed by Duncan's multiple range test. P < 0.05 is considered statistically significant.

  Results Top

Yields, total phenolic content and total flavonoid content of Cajanus cajan roots extracts

[Table 1] shows the extraction yields, TPC and TFC of WCR, EECR50, and EECR95. The extraction yields of EECR95 (28.90 g/kg) display the lowest, as compared with WCR (57.71 g/kg) and EECR50 (43.11 g/kg). However, EECR95 had higher TPC (33.33 mg Ga/g) and TFC (23.54 mg Que/g) than those of EECR50 (P < 0.05).
Table 1: Yields and contents of total phenolics and flavonoids in different solvent extracts of Cajanus cajan roots

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Polyphenol composition of extracts of Cajanus cajan roots

The HPLC profile of the nine polyphenols standard compounds and CR extracts (WCR, EECR50, and EECR95) was detected at 290 nm with the HPLC-DAD-UV/Vis system. As the result shown in [Figure 1], no polyphenolic peak was detected in WCR; however, three kinds of isoflavones (genistein, daidzein and cajanol) were detected in both EECR50 and EECR95. [Table 2] indicates that the genistein content was the highest (19.47 mg/g dw) in EECR95 followed by cajanol (3.72 mg/g dw) and daidzein (2.68 mg/g dw). Furthermore, the genistein content of EECR95 was about almost twice as high as that of EECR50 (10.27 mg/g dw) (P < 0.05).
Figure 1: High performance liquid chromatography chromatograms of polyphenol composition of hot water content reduction, 50% ethanol (EECR50) and 95% ethanol extracts (EECR95) from C. cajan (L.) Millsp. roots. Peaks 1-9 are as follows: (1) vannilic acid, (2) syringic acid, (3) courmaric acid, (4) ferullic acid, (5) daidzein, (6) rutin, (7) genistein, (8) quercetin, and (9) cajanol

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Table 2: Composition of polyphenol in different extracts from Cajanus cajan roots

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In vitro antioxidant activity of extracts of Cajanus cajan roots (WCR, EECR50, and EECR95)

1,1-diphenyl-2-picrylhydrazine radical scavenging effects

[Figure 2]a shows that DPPH radical-scavenging activities of the CR extracts. The IC50 of EECR95 was 459.7 μg/mL, which had the most potent DPPH scavenging effect than that of EECR50 (IC50 =677.0 μg/mL) and WCR (IC50 =928 μg/mL) (P < 0.05).
Figure 2: The antioxidant activity obtained from 1,1-diphenyl-2-picrylhydrazine (a), ABTS+ radical scavenging (b), and ferric-reducing power (c) of hot water content reduction, 50% ethanol (EECR50), and 95% ethanol extracts (EECR95) from C. cajan (L.) Millsp. roots. The results were expressed as mean ± SD; n = 3.

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ABTS+ scavenging effects

[Figure 2]b shows the antioxidant activity determined by the ABTS+ assays in CR extracts. The results indicated that the ABTS+-scavenging effect of EECR95 (IC50 =100 μg/mL) was higher than that of EECR50 (IC50 =184.26 μg/mL) and WCR (IC50 =4771 μg/mL). Under the concentration of 50–250 μg/mL, the ABTS+-scavenging capabilities were 39.37%–63.27% and 42.48%–81.21% for EECR50 and EECR95, respectively, whereas WCR had little ABTS scavenging effect at the concentration of 50–250 μg/mL (P < 0.05).

Ferric reducing antioxidant power effects

The FRAP effect of extracts was determined by monitoring the reduction of Fe3+ to Fe2+. This measurement follows the formation of Perl's Prussian blue absorbance at 700 nm. As shown in [Figure 2]c, EECR95 exhibited the strongest FRAP activity, as compared with those of EECR50 and WCR (P < 0.05).

Antioxidant activity of EECR95 in RAW 264.7 cells

Effect of EECR95 on lipopolysaccharide-induced cytotoxicity in RAW 264.7 macrophages

The possible cytotoxicity of EECR95 in RAW 264.7 macrophages was evaluated using the MTT assay. Cytotoxicity of EECR95 on RAW 264.7 cells was observed to be dose-dependent. When the concentration was <100 μg/mL, cell viability was >80% [Figure 3]. A reduction in cell viability to 80.56% was observed after incubation with LPS (2 μg/mL) for 24 h. EECR95 pre-treated cells restored the cell viability of LPS-induced cytotoxicity to 82.41%, 87.46%, 90.54%, and 92.86% after incubation with 1, 5, 10, and 20 μg/mL EECR95 for 24 h, respectively. The results indicated that pretreatment with 10 and 20 μg/mL of EECR95 could increase the cell viability induced by LPS (+LPS), and there was no significant difference between + LPS or -LPS group on the cell viability (P < 0.01). Thus, we chose the concentration <20 μg/mL of EECR95 for the following experiments.
Figure 3: Effect of EECR95 on lipopolysaccharide-induced cytotoxicity in RAW 264.7 macrophages. Cells were pretreated with EECR95 for 2 h and then incubated with lipopolysaccharide (2 μg/mL) for 24 h. The viability was measured by MTT assay. The results were expressed as mean ± standard deviation; n = 3. Asterisks indicate significant difference by comparison with the control (without lipopolysaccharide) as determined by ANOVA: *P < 0.01.

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Effect of EECR95 on lipopolysaccharide-induced reactive oxygen species level in RAW 264.7 macrophages

The DCFH-DA assay was employed to investigate the intracellular ROS level in LPS-stimulated RAW 264.7 cells. As shown in [Figure 4]a, LPS-induced an increase in ROS level from 3.85% to 129%. Treatment of LPS-induced RAW 264.7 cells with 1, 5 and 20 μg/mL EECR95 diminished the ROS level to 62%, 34.87%, and 13.71%, respectively. [Figure 4]b shows that the inhibitory effect of 20 μg/mL EECR95 was approximately equal to that of 25 μM of genistein (96.59%) (P < 0.05).
Figure 4: Effect of EECR95 on ROS production. RAW 264.7 cells treated with 1, 5, 20 μg/mL EECR95 or Gen (Genistein, 25 μM) for 2 h and then incubated of lipopolysaccharide (2 μg/mL) for 24 h. (a) Cells were collected and incubated with 30 μM DCFH-DA at 37°C for 1 h in dark, washed with PBS×1, and evaluated by flow cytometry. (b) Inhibition (%) of intracellular ROS production was calculated. Values (mean ± standard deviation; n = 3) not sharing a common letter are significantly different (P < 0.05).

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Effect of EECR95 on lipopolysaccharide-induced superoxide dismutase and catalase activities in RAW 264.7 macrophages

[Table 3] shows the effects of EECR95 on SOD and CAT activities in LPS-stimulated RAW 264.7 cells. Pretreatment with EECR95 (1, 5, and 20 μg/mL) significantly and concentration-dependently increased SOD activity (by 17.4%, 33.1%, and 135.6%, respectively), as compared with the LPS control group. The increment of CAT activity by EECR95 (1, 5, and 20 μg/mL) was even more remarkable (by 30.6%, 41.9%, and 338.6%, respectively) in LPS-stimulated RAW 264.7 cells. Pre-treatment with genistein (25 μM) also significantly increased the activity of SOD (85.7%) and CAT (150.1%) (P < 0.05).
Table 3: Effect of EECR95 on activity of superoxide dismutase and catalase in RAW 264.7 cells

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Anti-inflammatory activities of EECR95 in RAW 264.7 macrophages

Effect of EECR95 on lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages

Nitrite concentration in the medium was determined by using Griess reagent. [Figure 5]a indicates that RAW 264.7 macrophages with LPS alone (111.47 μmol/mL) resulted in extremely significant increases in NO production as compared to the control group (29.45 μmol/mL). The NO level was increased by 82.02% as compared to control group. However, pretreated with EECR95 (1, 5, 10, and 20 μg/mL) significantly inhibited the production of NO in 24 h after LPS stimulated. The NO level was decreased by 41.69%, 58.77%, 77.09%, and 86.05%, respectively [Figure 5]a (P < 0.05).
Figure 5: Effects of EECR95 on (a) NO production and (b) Prostaglandin E2 in lipopolysaccharide-induced RAW 264.7 cells. Cells were treated with EECR95 (1-20 μg/mL) and Gen (genistein; 25 μM) for 2 h and incubated of lipopolysaccharide (2 μg/mL) for 24 h. Values (mean ± standard deviation; n = 3) not sharing a common letter are significantly different (P < 0.05).

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[Figure 5]b also indicates that RAW 264.7 macrophages incubated with LPS alone (9004.21 pg/mL) resulted in marked increases in PGE2 production, as compared with the control group (4304.59 pg/mL). However, macrophages pretreated with EECR95 (1, 5, 10 and 20 μg/mL) for 24 h significantly inhibited the PGE2 levels in the LPS-stimulated cells (by 21.63%, 34.84%, 47.59%, and 61.26%, respectively) [Figure 5]b. EECR95 also significantly attenuated NO and PGE2 levels in LPS-stimulated RAW 264.7 in a dose-dependent manner (P < 0.05). The inhibition of NO and PGE2 by EECR95 at 5 and 20 μg/mL in LPS-stimulated cells was approximately equivalent to that of 25 μM genistein (about 60%) (P < 0.05).

Effect of EECR95 on pro-inflammatory cytokines in lipopolysaccharide-induced RAW 264.7 macrophages

To investigate the effect of Gen and EECR95 on the inflammatory cytokines, IL-1, IL-6, and TNF-α, the levels of cytokines in the medium of RAW 264.7 cells stimulated with LPS (2 μg/mL) were measured using an ELISA kit. As shown in [Figure 6], LPS markedly increased IL-1, IL-6 and TNF-α levels, as compared with untreated controls; however, all of these cytokines were significantly decreased by EECR95 (1–20 μg/mL) in a concentration-dependent manner. The IL-1, IL-6 and TNF-α level in RAW 264.7 cells treated with 20 μg/mL of EECR95 were decreased by 32.91%, 42.02%, and 56.61%, (P < 0.05), respectively. Pre-treatment with genistein (25 μM) also significantly decreased the level of IL-1 (34.07%), IL-6 (42.92%), and TNF-α (67.76%) (P < 0.05).
Figure 6: Effects of EECR95 on cytokines levels in RAW 264.7 cells stimulated with lipopolysaccharide. Cells were treated with EECR95 (1-20 μg/mL), Gen (genistein; 25 μM) for 2 h and then incubated with lipopolysaccharide (2 μg/mL) for 24 h. Interleukins-1 (a), interleukins-6 (b) and tumor necrosis factor-α (c) levels were determined by ELISA kits. Values (mean ± standard deviation; n = 3) not sharing a common letter are significantly different (P < 0.05).

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Effect of EECR95 on expression of anti-inflammatory protein in lipopolysaccharide-induced RAW 264.7 cells

As shown in [Figure 7]a, EECR95 at concentration of 1–20 μg/mL led to a significant increase (P < 0.05) in the expression of HO-1, Nrf-2 and PPAR-γ compared to control group treated with LPS only. Especially, 20 μg/mL EECR95 increased the level of expression by 2 folds in HO-1 [Figure 7]b, by 3 folds in Nrf-2 [Figure 7]c, and by 5 folds in PPAR-γ [Figure 7]d. Cells incubated with 25 μM of Gen also showed increased expression levels of anti-inflammatory factors approximately equivalent to that of 20 μg/mL EECR95 (HO-1 and Nrf-2) and 1 μg/mL EECR95 (PPAR-γ).
Figure 7: Effects of EECR95 on the expression levels of antioxidant factors in lipopolysaccharide-induced RAW 264.7 cells. Cells were treated with EECR95 (1–20 μg/mL), Gen (genistein; 25 μM) for 2 h and incubated lipopolysaccharide (2 μg/mL) for 24 h. (a) protein expression of heme oxygenase-1, nuclear related factor 2, peroxisome proliferator-activated receptors-γ and β-actin were detected in the cytoplasm by Western blotting assay. The quantitative of (b) heme oxygenase-1, (c) nuclear related factor 2 and (d) peroxisome proliferator-activated receptors-γ were analysed by ImageJ software. Values (mean ± standard deviation; n = 3) not sharing a common letter are significantly different (P < 0.05).

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Effect of EECR95 on expression of inflammatory protein in lipopolysaccharide-induced RAW 264.7 cells

As shown in [Figure 8]a, EECR95 at concentration of 1–20 μg/mL significantly (P < 0.05) reduced the expression of iNOS, COX-2 and NF-ĸB compared to control group treated with LPS only. Expression of the iNOS [Figure 8]b, COX-2 [Figure 8]c and NF-ĸB [Figure 8]d protein was significantly decreased by more than 2 folds in a dose-dependent manner within the concentration of 20 μg/mL EECR95. Cells incubated with 25 μM of Gen also decreased the expression levels of pro-inflammation factors in LPS-induced RAW 264.7 cells approximately equivalent to that of 1 μg/mL EECR95 (iNOS and NF-κB) and 20 μg/mL EECR95 (COX-2).
Figure 8: Effects of EECR95 on the expression levels of pro-inflammation factors in lipopolysaccharide-induced RAW 264.7 cells. Cells were treated with EECR95 (1–20 μg/mL), Gen (genistein; 25 μM) for 2 h and incubated with lipopolysaccharide (2 μg/mL) for 24 h. (a) protein expression of inducible nitric oxide synthase, COX-2, NF-κB and β-actin were detected in the cytoplasm by Western blotting assay. The quantitative of (b) inducible nitric oxide synthase, (c) COX-2, and (d) NF-κB were analysed by ImageJ software. Values (mean ± standard deviation; n = 3) not sharing a common letter are significantly different (P < 0.05).

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Cellular uptake of genistein or EECR95

The increase of culture time, the detected amounts of genistein standard and genistein in EECR95 uptake by macrophage cells were increased, and the elusion time was 6 h and 12 h, respectively. As shown in [Table 4], the genistein uptake level of RAW 264.7 cells treated by 25 μM genistein (6.8 μg/mL) was about 425 ng/106 cells, and the cellular uptake was from 1.33% to 3.44% after 6–24 h incubation. Relatively, the genistein uptake level of RAW 264.7 cells treated by 20 μg/mL of EECR95 (containing 0.4 μg/mL genistein) was only 13 ng/106 cells, and the cellular uptake was from 0.39% to 1.73% after 12–24 h incubation. We also analyzed the cajanol and daidzein uptake level of RAW 264.7 cells treated by 20 μg/mL of EECR95. However, neither cajanol nor daidzein was detected in macrophages cells (P < 0.05).
Table 4: Determination of cellular uptake of genistein in RAW 264.7 macrophages

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  Discussion Top

Currently available studies on C. cajan have focused on determination of flavonoids in extracts of leaves and seeds of C. cajan, and few studies have explored the bioactivities and active components of CR extracts. Thus, the main purpose of the present study was to evaluate the antioxidant and anti-inflammatory effects of CR extracts (WCR, EECR50, and EECR95), their major active compounds, and possible mechanisms of action. The results indicated that EECR95 not only had the most potent antioxidant potential but also had the highest polyphenol and isoflavone contents (genistein, daidzein, and cajanol). Among the isoflavones identified, genistein was most abundant in EECR95, with a content of 19.47 mg/g dw, which is in consistency with that (18.77 mg/g dw in CR) reported by Shuang et al.[32] Interestingly, the contents of genistein in CR are higher than those of soybean (4.6–18.2 μg/g dry weight), tofu (94.8–137.7 μg/g dw), and soy nut products (200.6-968.1 μg/g dw).[33] Genistein is a natural phytoestrogen with low toxicity and multiple bio-functions such as antioxidant,[18],[19] anti-inflammatory[20],[21] and anti-cancer activities.[22] The present study confirms and strengthens the antioxidant effect of EECR95 by its free radical-scavenging activities and its attenuation of cellular ROS, as all these assays exhibit similar concentration-dependent actions.

Then, we explored the cellular antioxidant and anti-inflammatory mechanisms of EECR95 in RAW 264.7 cells as well as the major active compounds of EECR95 that could account for its antioxidant and anti-inflammatory capacities. To examine the protective ability of EECR against inflammation in the macrophages, we first tested the effects of different concentration (0–5 μg/mL) of LPS on cell viability using MTT method. Then, we chose the concentration of 2 μg/mL of LPS to stimulate moderate cytotoxicity (>80%) in RAW 264.7 cells. EECR95 at the concentration <100 μg/mL was found to have low cytotoxicity in RAW 264.7 cells, and the effective doses of EECR95 for the antioxidant and anti-inflammation activities were <20 μg/mL. Thus, EECR95 may have a substantial “therapeutic window.” Although LPS inhibited the viability of RAW 264.7 cells in a significant manner, EECR95 was able to protect against the LPS-induced cytotoxicity.

LPS-induced toxicity in RAW 264.7 cells is attributed to the high NO2+/NO3 levels that induce inflammatory responses (NO production, cytokine (IL-1 β, IL-6, and TNF-α) secretion, and PGE2 synthesis) and the decrease of cell viability.[34],[35] In addition, these inflammatory responses play important roles in the pathogenesis of various acute and chronic inflammatory diseases.[36] Many studies have demonstrated that phytochemicals or plant extracts possess anti-inflammatory effects with a decrease in NO and PGE2 production and cytokine secretion.[37],[38] EECR95 has the ability to inhibit NO and PGE2 synthesis and cytokine secretion (IL-1, IL-6, and TNF-α) in LPS-stimulated macrophages without inducing cytotoxicity, and hence, the anti-inflammatory ability of EECR95 is apparent.

iNOS and COX-2 responsible for synthesizing NO and PGE2, respectively, are important enzymes that mediate inflammatory processes. iNOS and COX-2 are up-regulated mainly by NF-ĸB activation in macrophages with LPS-stimulation.[39] Much evidence shows the inhibition of LPS-induced NO, PGE2, iNOS, COX-2, TNF-α, and IL-6 through the inactivation of NF-ĸB pathway in RAW 264.7 cells.[39] NF-ĸB signaling pathway plays a key role in inflammation, immune response, and protection against apoptosis. Downregulation of NF-ĸB is regarded as a potential therapy in many diseases, such as cancer, inflammation, and autoimmune disease.[36] Our results revealed that treatment of EECR95 inhibited the expression of NF-ĸB, iNOS, and COX-2. The results suggest that EECR95 blocks the NF-ĸB signaling pathway, thereby down-regulating the expression levels of iNOS and COX-2. This is common to other flavonoids-derived compounds (e.g., curcumin, quercetin, and resveratrol) with known anti-inflammatory effects.[40]

HO-1 can protect cells from oxidative stress and inflammatory responses through its ability of inhibiting the expression of inflammatory cytokines and can direct the differentiation of immune cells toward anti-inflammatory phenotypes.[41] In activated of macrophages, HO-1 has been regarded as an adaptive protective mechanism to preserve homeostasis in response to oxidative stress. Besides, Nrf-2 is responsible for the transcription of several antioxidant response element (ARE)-related cytoprotective genes, including HO-1.[42] Nrf-2 and NF-κB are the two key transcription factors that regulate cellular responses to oxidative stress and inflammation, respectively. Wardyn et al.[43] pointed out that the absence of Nrf-2 can exacerbate NF-κB activity leading to increased cytokine production. In this study, we found that EECR95 inhibited the expressions of inflammatory proteins (COX-2 and iNOS) and pro-inflammatory cytokines (IL-1, IL-6, and TNF-α) accompanied by the increase of anti-inflammatory protein HO-1. These results suggest that EECR95 may activate Nrf-2 pathway to exert its anti-inflammatory effects.

Choi et al.[44] showed that the inhibitory effect of genistein on NF-κB activation induced by LPS in RAW 264.7 cells is related with the ameliorated intracellular oxidative stress in the signaling pathway of LPS to NF-κB activation. The effect of genistein (at 50 μM and 100 μM) on NF-κB activation is consistent with attenuated NO generation and elevated anti-oxidative enzyme activities.[44] In the present study, we demonstrate that genistein is the major flavonoid in EECR95 (19.47 mg/g).

Recently, Jia et al.[45] reported that flavonoids from Rhynchosia minima root (a species of Papilionoideae in the Leguminosae family commonly known as snout-bean) exerts anti-inflammatory activity in LPS-stimulated RAW 264.7 cells via MAPK/NF-κB signaling pathway. Our results also showed that daidzein, genistein, and 5,2'-dihydroxy-7,4'-dimethoxyisoflavanone (cajanol) were the major flavonoids in EECR95 and that genistein, a major flavonoids in EECR95, exert antioxidant and anti-inflammatory activities, has a high cellular uptake in RAW 264.7 cells. EECR95 directly diminished LPS-induced ROS production in macrophages, and increased the expression of ARE (Nrf-2 and HO-1) to provide robust protection against oxidative challenge. Inflammation and oxidative stress go hand in hand, since ROS lead to the production of inflammatory cytokines.[46] Thus, EECR95 decreases the expression of iNOS, COX-2 and pro-inflammatory cytokines through NF-κB pathway through antioxidant activities. [Figure 9] schematically summarizes the effect of EECR95 in LPS-stimulated RAW 264.7 cells.
Figure 9: Schematic summaries the effect of EECR95 on lipopolysaccharide.stimulated RAW 264.7 cells.

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Coldham and Sauer[34] found that after genistein ingestion by rats, the highest content of genistein was in the intestine (18.5 μg/g), followed by the liver (0.98 μg/g), serum (0.79 μg/g), and reproductive tissues (in the range of 0.12–0.28 μg/g). Genistein can remain in the body for a long time owing to its good protein binding ability. In the present study, we found that the maximal genistein uptake was achieved in RAW 264.7 cells within 24 h incubation. Liu et al.[47] reported that the cellular uptake of quercetin (2.28%) in macrophage cells after 24 h incubation. Interestingly, we also found that the cellular uptake of pure genistein added at 25 μM (6.8 μg genistein/mL) was 3.44% [Table 3]. However, the cellular uptake of genistein originally present in EECR95 (added at 20 μg/mL containing 0.4 μg genistein/mL) was only 1.73%. This difference in cellular uptake of genistein by RAW 264.7 cells suggests that the co-existence of other polyphenols, especially isoflavones, in EECR95 may compete for cellular uptake of genistein.

  Conclusions Top

The present study indicates that CR extracts are abundant in polyphenols, especially the isoflavones genistein, daidzein, and cajanol. In addition, EECR95, the 95% ethanol extracts of CR, possesses potent antioxidant and anti-inflammatory activities. Mechanistically, EECR95 activates the Nrf-2/HO-1 system and inhibits the NF-ĸB signaling pathway in RAW 264.7 cells. In addition to the abundance of genistein in CR, the relatively high cellular uptake of genistein at 24 h of incubation (3.44% for added genistein and 1.73% for genistein contained in EECR95) suggests that genistein is a major active component of CR.In vivo studies are warranted for elucidating the potential of EECR95 for future biomedical applications.

Financial support and sponsorship

This study was financially supported by the Ministry of Science and Technology of the Republic of China, Taiwan (MOST 106-2320-B-212-00).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]

  [Table 1], [Table 2], [Table 3], [Table 4]


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