PHCOG MAG.

ORIGINAL ARTICLE

Inhibition of inducible nitric oxide synthase and osteoclastic differentiation by Atractylodis Rhizoma Alba extract Sung‑Ho Choi, Sung‑Jin Kim Department of Pharmacology and Toxicology, School of Dentistry, Kyung Hee University, Seoul 130‑701, Republic of Korea Submitted: 08‑01‑2014

Revised: 27‑03‑2014

Published: ****

ABSTRACT

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Background: Atractylodis Rhizoma Alba (ARA) has been used in Korean folk medicine for constipation, dizziness, and anticancer agent. In the present study, we performed to test whether the methanolic extract of ARA has antioxidant and antiosteoclastogenesis activity in RAW 264.7 macrophage cells. Materials and Methods: Antioxidant capacities were tested by measuring free radical scavenging activity, nitric oxide (NO) levels, reducing power, and inducible nitric oxide synthase (iNOS) expression in response to lipopolysaccharides (LPS). Antiosteoclastogenesis activity was evaluated by performing tartrate‑resistant acid phosphatase assay in RAW 264.7 macrophage cells. Results: The extract exerted significant 1,1‑diphenyl‑2‑picrylhydrazyl and NO radical scavenging activity, and it exerted dramatic reducing power. Induction of iNOS and NO by LPS in RAW 264.7 cells was significantly inhibited by the extract, suggesting that the ARA extract inhibits NO production by suppressing iNOS expression. Strikingly, the ARA extracts substantially inhibited the receptor activator of NF‑κB ligand‑induced osteclastic differentiation of LPS‑activated RAW 264.7 cells. The ARA extract contains a significant amount of antioxidant components, including phenolics, flavonoids and anthocyanins. Conclusion: These results suggest that the methanolic extract of ARA exerts significant antioxidant activities potentially via inhibiting free radicals and iNOS induction, thereby leading to the inhibition of osteoclastogenesis.

Website: www.phcog.com DOI: 10.4103/0973-1296.139780 Quick Response Code:

Key words: Antioxidant, Atractylodis Rhizoma Alba, inducible nitric oxide synthase, nitric oxide, osteolastogenesis, receptor activator of NF‑κB ligand

INTRODUCTION Atractylodis Rhizoma Alba (ARA) is the dried rhizome of Atractylodes macrocephala Koidzumi, Atractylodes japonica Koidzumi ex Kitam and Atractylodes ovata Koidzumi, and belonging to the Composite family. It is widely distributed in Asian countries such as Korea, China and Japan,[1] and many medicinal prescriptions contain it as the principle drug.[2] It has been used in the oriental medicine for constipation, dizziness and anticancer agent.[3] In addition, it has been reported that ARA has antiulcer action and adipogenic differentiating activity.[4,5] Phytochemical investigations have revealed a number of chemical components such as sesquiterpenes, sesquiterpene glycosides, polyacetylenes, monoterpene glycosides, aromatic glycosides, sucrose esters, Address for correspondence: Prof. Sung‑Jin Kim, Department of Pharmacology and Toxicology, School of Dentistry, Kyung Hee University, 26, Kyunghee‑daero, Dongdaemun‑gu, Seoul 130‑701, Republic of Korea. E‑mail: [email protected] S494

and steroids were isolated.[6‑9] The main constituents from ARA are atractylon, atractylodin, and atractylenolides.[10,11] Many detrimental diseases are associated with the increase of reactive oxygen and nitrogen species (RONS), leading to imposing oxidative stress in the body, which can initiate tissue damages. Overproduction of free radicals more than their scavenging capacity of the antioxidative defense system in the cellular components plays a pivotal role in the damaging lipid, protein, carbohydrate and DNA, ultimately causing cell death.[12] Reactive oxygen species (ROS) contains superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (OH•), and they are produced during various cellular aerobic metabolism and as by‑products of many enzyme reactions. [12,13] Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is associated with the production of reactive nitrogen species,[14] which are responsible for the excessive production of lipid and protein oxidation processes.[12] Functional antioxidant system and sufficient supply of antioxidant molecules are critical in scavenging Pharmacognosy Magazine | Vol 10 | Issue 39 (Supplement 3)

Choi and Kim: Inhibition of Osteoclastogenesis by Atractylodis Rhizoma Alba

RONS. Novel nutraceuticals/drugs with anti‑iNOS activity will be of great value for the management of the oxidative stress, inflammation, bone metabolism and vascular tone.[15] Multinucleated osteoclasts are responsible for bone resorption and excessive bone resorption by osteoclasts could cause an imbalance in bone remodeling, leading to the bone lytic diseases and cancer metastasis to the bone.[16,17] Thus, understanding the molecular processes of osteoclast formation and identifying potential pharmacological interventions are critical for the prevention and treatment of abnormal bone loss.[18] In recent times, many herbal plants with antioxidant and antiinflammatory activity have been shown to suppress osteoclast differentiation through inhibition of specific signaling pathways.[18] The pharmacological action of ARA on the osteoclast formation has not yet been fully determined.[19] In this study, we sought to determine if 70% methanol extracts of ARA have antioxidative activities associated iNOS system. It has been also tested whether the ARA extracts could exert any effect on the osteoclastogenesis using a receptor activator of NF‑κB ligand (RANKL)/lipopolysaccharide  (LPS)‑induced bone resorption model in RAW 264.7 macrophage cells.

MATERIALS AND METHODS Preparation of plant extracts

Authentic samples of ARA were purchased from Kyung‑Dong Oriental Market in Seoul. They were authenticated by Emeritus Professor Chang‑Soo Yook, Department of Oriental Pharmacy, Kyung Hee University, Seoul, the Republic of Korea. A  voucher specimen (No. 00-14) was deposited in the herbarium of the Department of Pharmacology, School of Dentistry, Kyung Hee University, Seoul, the Republic of Korea. They were collected during the early autumn season. ARA  (100  g) was cut into small pieces and extracted 3 times with 70% methanol (300 ml) for 3 h. The resulting methanol extracts was concentrated by rotary evaporator and dried by a freeze‑dryer. Reagents and materials

Scavenging of 1,1‑diphenyl‑2‑picrylhydrazyl radicals

1,1‑diphenyl‑2‑picrylhydrazyl (DPPH) radical scavenging activity was measured by evaluating the ability to remove DPPH under the principle of reduction reactions of DPPH radical solutions in the presence of hydrogen‑donating antioxidants.[20] Briefly, ARA extracts dissolved in 1 ml of MeOH was mixed with 1 ml of DPPH• solution at 23°C and an optical density were measured in 30 min at 515 nm. Scavenging of nitric oxide radicals

Nitric oxide generated from sodium nitroprusside (SNP) was measured by using the Griess reagent.[21] The extract was added to 0.2  ml of SNP  (10 mM) and 1.8  ml of phosphate buffer  (pH  7.4). The reaction mixture was allowed to incubate at 37°C for 3 h. A volume of 1 ml aliquots of the reaction mixture were mixed with 0.5 ml of Griess reagent and subjected to measure absorbance at 540 nm by using a spectrophotometer. Measurement of oxidation of deoxyribose

The experiment was carried out as described by Halliwell and Gutteridge. [22] The reaction mixture  (1.4  ml) containing extracts  (0.2  ml), deoxyribose  (6 mM), H2O2 (3 mM), FeCl3 (400 µM), ethylenediaminetetraacetic acid (400 µM), and ascorbic acid (400 µM) in phosphate buffer (20 mM, pH 7.4) was incubated at 37°C for 1 h. The extent of deoxyribose degradation was measured using the thiobarbituric acid (TBA) method. One milliliter of TBA  (1%, w/v) and 1.0  ml of trichloroacetic acid (2.8%, w/v) were added to the mixture, and allowed to be heated in a water bath at 90°C for 20 min. The absorbance of the mixture was read spectrophotometrically at 532 nm. Determination of reducing power

The reducing power of ARA extract was measured using the Oyaizu’s method.[23] 2.5 ml of ARA extract in 0.2 M phosphate buffer (pH 6.6) was added to 2.5 ml of potassium ferricyanide (10 mg/ml) solution and made to react for 15  min at 30°C. 2.5  ml of trichloroacetic acid (100 mg/ml) was put into the reactant and mixed up, and 2.5 ml of the mixture was again mixed with 2.5 ml of distilled water and 0.5 ml of ferric chloride (1.0 mg/ml) and the optical density was measured at 700 nm.

The iNOS and glyceraldehyde 3‑phosphate dehydrogenase (GAPDH) antibodies were purchased from Cell Signaling and Santa Cruz Biotechnology Co., respectively. The ECL kit was bought from Amersham Co. Other reagents were bought from Sigma Co. All cell culture media were purchased from Gibco Co.

Cell culture

Atractylenolide II was kindly provided by Prof. Jinwoong Kim at College of Pharmacy, Seoul National University, the Republic of Korea.

Measurement of nitric oxide

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Murine RAW 264.7 macrophage cells were cultured in Dulbecco’s modified Eagle’s Medium  (DMEM; Gibco BRL, Grand Island, NY) with 10% heat‑inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, NY) in 5% humidified CO2 atmosphere at 37°C. RAW 264.7  cells were cultured with DMEM and 10% FBS. NO was measured by measuring the amount in the S495

Choi and Kim: Inhibition of Osteoclastogenesis by Atractylodis Rhizoma Alba

cell supernatant as nitrite and nitrate. The safest form of nitrite after being reduced to nitrate was measured using the Griess reagent  (Sigma, USA). The cells  (2  ×  106) were seeded into 6 well plate and washed 2  times with phosphate buffered saline (PBS) when the confluence was approximately 80% and then cultured for at least 24 h, and the ARA extract were made into the final concentrations of 10, 1.0 and 0.1  mg/ml for experiments. Four hours later, LPS of the final concentration 10 µg/ml was added into all wells except for the well for the control group to stimulate the samples. The amounts of NO generated were measured by gathering the supernatant around 18 h later, having them react with the Greiss reagent for 10 min in darkness and then measuring the optical density at 540 nm. Measurement of inducible nitric oxide synthase expression by western blotting

When the cells reached confluence, the DMEM culture medium was removed and replaced by the serum‑free DMEM medium, and subsequently, the cells were treated with ARA extracts and cultured for 24  h. The cells were washed 2  times with PBS and scraped into a buffer containing 10 mM Tris‑HCl  (pH  7.4), 50 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 30 mM NaF, 0.1 mM Na3VO4, 1% Triton X-100, 0.5% NP‑40, 1 µg/ml leupetin, 1 µg/ml aprotinin after that, the cells were disrupted by passing them through a 1 ml tuberculin syringe 5 times. The cell lyaste was subjected to centrifugation at 10,000 ×g for 10 min, and the supernatant was used for Western blot analysis. The protein content of the soluble fraction was assessed by the method of Bradford.[24] Protein (50 µg/lane) was electrophoretically separated in 10% polyacrylamide gels containing sodium dodecyl sulfate and transferred to nitrocellulose membranes (Schleicher and Schuell) for 1 h at 100 V (constant) as described by Towbin et al.[25] The membranes were blocked for 1 h at 23°C with PBS containing 0.1% Tween 20 and 5% skim milk and washed with PBS containing 0.1% Tween 20  3  times for 10  min each. Followed by, the blots were probed with primary antibody directed against iNOS (1:1000) and GAPDH  (1:1000) for 2  h at room temperature or overnight at 4°C diluted in blocking buffer. The blots were then incubated with horseradish peroxidase‑conjugated antirabbit IgG (1:1000 for iNOS and GAPDH) for 1 h at room temperature and washed with PBS containing Tween 20 3 times for 10 min each. The detection of immobilized specific antigens was carried out by ECL. The images were analyzed using ImageJ software (NIH, USA). Determination of osteoclastic differentiation

RAW 264.7 cells (2 × 103) were seeded into 6 well plate and washed 2 times with PBS when the confluence was 80% and then cultured for at least 24  h and the ARA S496

extract were made into the final concentrations of 10, 1.0 and 0.1  mg/ml for experiments. Four hours later, RANKL (50 ng/ml) was put into all wells for 72 h and LPS of the final concentration 10 µg/ml was added to stimulate osteoclastic differentiation. After 6~7  days, tartrate‑resistant acid phosphatase  (TRAP) assay was performed according to the manufacturer’s instruction to count multinucleated osteoclastic cell numbers (Sigma Cat #387A). Component analysis (anthocyanin, phenolics, flavonoids) Measurement of total phenolics

The total phenolic content was measured using the Folin–Ciocalteu procedure at 725 nm.[26] Gallic acid was used as a standard for phenolic compounds and the phenolic concentration was calculated by using a gallic acid standard calibration curve. The total phenolics content was expressed as the gallic acid equivalent (mg gallic acid/g extract). Measurement of total flavonoids

The total flavonoids was measured using the method of Miliauskas et  al.[27] and was expressed as the rutin equivalent (mg rutin acid/g extract) using rutin as a standard flavonoid. 1.0 ml of ARA extract was mixed with aluminum thiochloride in ethanol (20 mg/ml) and diluted to 25 ml. After incubation for 40 min at 20°C, the optical density was measured at 415 nm. Measurement of total anthocyanin

The total anthocyanin was measured using color reactions.[28] ARA extracts were dissolved in 1.0 ml of acetate buffer (25 mM, pH 4.5), and an optical density was measured at 520  nm. The content of anthocyanin was expressed as kuromanin equivalent (mg Kuromanin/g extract). High‑performance liquid chromatography analysis

Atractylodis Rhizoma Alba extract was analyzed by high‑performance liquid chromatography (HPLC) (Nova‑Pak® C18, 60Å 4uM, 3.9 × 150 mm). A mobile phase system (50:50 of Acetonitrile: Water) was applied at a flow rate of 1 ml/min for 35 min, and the absorption peaks were detected at 236 nm and compared with a standard marker molecule, Atractylenolide II. The HPLC profile was quantified using its integrated area [Figure 1]. Statistical analysis

All data were expressed as mean ± standard error of the mean. Statistical analysis was performed using the  GraphPad Prism 4 (GraphPad software, Inc., La Jolla, CA, USA) 4 with one‑way ANOVA followed by Tukey’s multiple comparison test and P