Chemical Characterization of Novel Natural Products from the Roots of

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Chemical Characterization of Novel Natural Products from the Roots of Asian Rice (Oryza sativa) that Control Adipocyte and Osteoblast Differentiation Hee Rae Kang, Hyung Sik Yun, Tae Kyoung Lee, Seulah Lee, Seon-Hee Kim, Eunjung Moon, Ki-Moon Park, and Ki Hyun Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05030 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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Journal of Agricultural and Food Chemistry

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Chemical Characterization of Novel Natural Products from

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the Roots of Asian Rice (Oryza sativa) that Control Adipocyte

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and Osteoblast Differentiation

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Hee Rae Kang,†,+, Hyung Sik Yun,‡,+, Tae Kyoung Lee,† Seulah Lee,† Seon-Hee Kim,§ Eunjung

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Moon,∥ Ki-Moon Park,‡ Ki Hyun Kim†,*

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†

School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea

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‡

Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Republic

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of Korea

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§

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Sungkyun Biotech, Suwon 16419, Republic of Korea

∥

Charmzone R&D Center, Charmzone Co. LTD., Seoul 135-851, Republic of Korea

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+

These two authors contributed equally to this work.

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* Corresponding author:

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Ki Hyun Kim, Tel: +82-31-290-7700; Fax: +82-31-290-7730; E-mail: [email protected]

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ABSTRACT

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Oryza sativa L. is consumed globally as a staple food and its roots have been used as a Korean and

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Chinese medical supplement for protection of the stomach and lungs and for amelioration of vomiting

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and fever. In our continuing search for biologically effective metabolites from Korean natural materials,

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we found that an EtOH extract of O. sativa root reciprocally regulated adipocyte and osteoblast

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differentiation. Chemical analysis of the EtOH extract using a bioassay-guided fractionation protocol

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led to the isolation and determination of two novel lignans, oryzativols A and B, responsible for these

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regulatory activities. Using 1D and 2D nuclear magnetic resonance (NMR) spectroscopic analyses,

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high-resolution mass spectrometry (HRMS), and circular dichroism (CD) analysis, the structures of the

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novel compounds were elucidated. We examined their effects on the regulation of mesenchymal stem

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cell differentiation. Treatment with oryzativol A in the human mesenchymal cell line C3H10T1/2

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suppressed gene expression of peroxisome proliferator activated receptor γ (PPARγ), resulting in a

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reduction in adipogenesis. Oryzativol A also enhanced the expression of Runx2 and cellular

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differentiation into osteoblasts in the same mesenchymal stem cell line.

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Keywords: Oryza sativa, lignans, oryzativols A and B, osteoporosis, adipocyte and osteoblast

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differentiation

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INTRODUCTION

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Osteoporosis is a disease characterized by reduced bone mass and deterioration of bone tissue where

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decreased bone strength leads to high bone fragility and fracture.1 Currently, studies suggest that

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osteoporosis is related to defects in osteoblast progenitors (mesenchymal stem cells, MSCs) in the bone

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marrow.2 MSCs in bone marrow are pluripotent cells that are known to differentiate into osteocytes as

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well as adipocytes and are influenced by the microenvironment in bone marrow.2 The maintenance of

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bone homeostasis depends on appropriate determinations which affects MSC differentiation in the

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osteoblast lineage. The MSC differentiation, either into osteoblasts or adipocytes, is tightly regulated. A

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shift in the pattern of MSC differentiation to preferentially form adipocytes over osteoblasts has been

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reported in postmenopausal women.3 Alterations in the expression of Runx2 and PPARγ might disrupt

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the balance between osteoprogenitor and adipocyte progenitor cells in osteoporosis patients.4 Thus, a

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treatment to alter the expression of Runx2 and PPARγ in MSCs would be an excellent candidate for

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reducing or preventing postmenopausal osteoporosis.

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In our screening test, we found that an EtOH extract of Oryza sativa L. roots reciprocally regulated

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adipocyte and osteoblast differentiation. O. sativa (Gramineae), commonly known as Asian rice, is

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globally consumed as a staple food. The roots of O. sativa have been used as a Korean and Chinese

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medical supplement for protection of the stomach and lungs.5 Despite several trials investigating the

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chemical components of O. sativa roots, there have been few reports on the bioactive molecules present

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in the roots. On the basis of bioactivity as assessed by our screening test, phytochemical analysis of the

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EtOH extract of O. sativa root was carried out, which led to the isolation and determination of two novel

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lignans (1 and 2), oryzativols A and B (Figure 1) from the bioactive fraction. To the best of our

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knowledge, this is the first paper describing potential of the roots of O. sativa for regulation on

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adipocyte and osteoblast differentiation and identification of promising compounds for the treatment of

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osteoporosis.

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MATERIALS AND METHODS

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Sample Material. The roots of O. sativa were purchased at Kyungdong Market in Seoul, Korea, in

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October 2013 and the identity of the material was verified by one of the authors (K.M.P.). A voucher

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specimen (SKK-BBR-2014) was located in the herbarium of the School of Pharmacy, Sungkyunkwan

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University, Korea.

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Extraction and Isolation. Dried O. sativa roots (0.6 kg) were extracted with 95% aqueous EtOH at

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60°C (1 day × 3) and then filtered. After evaporation of the filtrate in a laboratory freeze-dryer, 60 g of

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the resultant dried extract was obtained. The dried EtOH extract powder was resolved in sterile distilled

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water and fractionated with n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol

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(BuOH), and water (residue). Five fractions with increasing polarity, the n-hexane-soluble fraction (1.45

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g), DCM-soluble faction (3.23 g), EtOAc-soluble layer (0.56 g), n-BuOH-soluble fraction (4.33 g), and

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water residue (50.43 g), were produced. Powdered fractions were dissolved in dimethyl sulfoxide

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(DMSO) for the measurement of biological activity. After determining the DCM-soluble fraction as the

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active fraction responsible for controlling MSC differentiation, the DCM-soluble fraction (3.23 g) was

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then separated by silica gel column chromatography (200 g, 3 × 100 cm) into nine fractions (D1-D9)

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according to the solvent mixture ratio of chloroform/methanol [200:1 (D1), 100:1 (D2), 50:1 (D3), 20:1

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(D4), 10:1 (D5), 5:1 (D6), 2:1 (D7), 1:1 (D8), and 0:1 (D9)]. The most active subfraction, D6 (602.6

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mg), was applied to column chromatography with Sephadex LH-20 (100 g, 3 × 50 cm, GE Healthcare)

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by size-exclusion eluting methanol to yield six fractions (D61-D66). The next active fraction, D62 (431

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mg), was further separated under the same conditions using a Sephadex LH-20 column into six fractions

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(D621-D626). The next active fraction, D625 (242 mg), was isolated by preparative high performance

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liquid chromatography (HPLC) using a SunFire prep C18 column (10 × 250 mm i.d., 5 µm, Waters)

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with a flow rate of 1.0 mL/min using a mobile phase that consisted of 0.04% TFA in 5% methanol

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(solvent A) and methanol (solvent B) (the gradient and timing were as follows: initial, 10% B; 5 min,

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10% B; 80 min, 100% B; 100 min, 100% B; 105 min, 10% B; 120 min, 10% B) to yield eight fractions ACS Paragon Plus Environment

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(D6251-D6258). The next active fraction, D6255 (126 mg), was fractionated with column

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chromatography in a Sephadex LH-20 column (100 g, 1 × 55 cm) with methanol to give two main

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fractions (A1 and A2) according to TLC analysis. Fraction A1 (19.8 mg) was applied to semi-

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preparative HPLC analysis using a 250 × 10 mm i.d., 10 µm, Phenomenex Luna Phenyl-hexyl column

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with a flow rate of 2.0 mL/min [0-50 min linear gradient solvent of 72% methanol (+0.1% formic acid

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[FA]) to 76% methanol (+0.1% FA)], which revealed no valid peak in fraction A1. Fraction A2 (16.6

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mg) was separated by semi-preparative HPLC using the same column with 73% methanol (+0.1% FA)

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and a flow rate of 2.0 mL/min to yield oryzativol A (1.7 mg, tR = 30.1 min, yield: 0.00028%) and

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oryzativol B (1.5 mg, tR = 28.0 min, yield: 0.00025%).

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Oryzativol A (1). Yellowish gum. [α]25 D -12.3 (c 0.07, MeOH); IR (KBr) νmax: 3366, 2948, 2829, 1715,

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1647, 1610, 1515, 1337, 1032, 671 cm-1; CD (MeOH) λmax (∆ε) 244 (+4.9), 302 (-1.5) nm; UV (MeOH)

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λmax (log ε) 228 (3.5), 315 (1.9) nm; for 1H (700 MHz) and

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C (175 MHz) NMR data, see Table 1;

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ESIMS (positive-ion mode) m/z: 729.2 [M + H]+. HRESIMS (negative-ion mode) m/z: 727.2386 [M -

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H]- (calcd for C40H39O13, 727.2391).

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Oryzativol B (2). Yellowish gum. [α]25 D -6.7 (c 0.08, MeOH); IR (KBr) νmax: 3433, 2965, 2843, 1720,

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1646, 1613, 1517, 1397, 1033, 681 cm-1; CD (MeOH) λmax (∆ε) 243 (+4.6) nm; UV (MeOH) λmax (log ε)

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228 (3.5), 314 (1.9) nm; for 1H (700 MHz) and

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(positive-ion mode) m/z: 729.2 [M + H]+. HRESIMS (negative-ion mode) m/z: 727.2398 [M - H]- (calcd

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for C40H39O13, 727.2391).

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C (175 MHz) NMR data, see Table 1; ESIMS

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Cell Culture. C3H10T1/2 cells were cultured (see Supporting information), as described previously.6

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Cell Viability. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

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tetrazolium-bromide (MTT) assay.7-9 C3H10T1/2, 3T3-L1, and MC3T3-E1 cells were seeded at a

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concentration of 1.5×104 cells per well and were cultured until reaching confluence. The cells were then

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treated with 1 to 50 µM of oryzativol A. After 48 h, 5 mg/mL MTT was added, and the cells were


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incubated at 37°C for an additional 4 h. The resulting formazan crystals were dissolved in 200 µL

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DMSO, and using a microplate reader, the absorbance was measured at 520 nm.

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Oil Red O (ORO) Staining. A week after culture in adipogenic differentiation media, cells were fixed

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with 10% neutral buffered formalin (NBF) for 1 h and then stained with 0.5% Oil Red O (Sigma) in a

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mixture of isopropanol and distilled water at a 3:2 ratio for 1 h. Cells were washed with water three

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times to stop the reaction. To evaluate intracellular triglyceride content, stained cells were resolved with

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1 mL isopropanol and the absorbance was measured at 520 nm.

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Alkaline Phosphatase (ALP) Staining. After culture in osteogenic differentiation media for 7-9 days,

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the cells were rinsed with 2 mM MgCl2. The cells were incubated with AP buffer (100 mM Tris–HCl,

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pH 9.5, 100 mM NaCl, and 10 mM MgCl2) for 15 min. They were then incubated in AP buffer

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containing 0.4 mg/mL nitro-blue tetrazolium (NBT, Sigma) and 0.2 mg/mL 5-bromo-4-chloro-3-indolyl

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phosphate (BCIP, Sigma). The reaction was stopped with 5 mM EDTA (pH 8.0). The cells were fixed in

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10% NBF for 1 h.

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Measurement of mRNA Expression in C3H10T1/2 Cells. Total RNA was isolated from

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differentiated cells using Isol-RNA Lysis Reagent (5Prime, MD, USA). cDNA was synthesized from

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0.5 µg of total RNA using a ReverTra Ace qPCR RT Master Mix kit (TOYOBO, FSQ-201, Japan) with

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random primers. The synthesized cDNA was mixed with the amplification mixture containing

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THUNDERBIRD™ SYBRⓇ qPCR Mix (TOYOBO, QPS-201, Japan) and primers. cDNA was

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subjected to 40 amplification cycles of polymerase chain reaction (PCR) using a Thermal Cycler Dice

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(Takara, Shiga, Japan) normalized with 36B4 expression. The specific oligonucleotide primer sequences

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used for PCR were as follows: acidic ribosomal phosphoprotein P0 (36B4), 59 bp, forward 5′-

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AGATGCAGCAGATCCGCAT-3′ and reverse 5′-GTTCTTGCCCATCAGCACC-3′; peroxisome

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proliferator-activated receptor γ (PPARγ), 67 bp, forward 5′-CCATTCTGGCCCACCAAC-3′ and

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reverse 5′-AATGCGAGTGGTCTTCCATCA-3′; adipocyte binding protein 2 (aP2), 65 bp, forward 5′-

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CACCGCAGACGACAGGAAG-3′

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differentiation 36 (CD36), 129 bp, forward 5′-GGCCAAGCTATTGCGACAT-3′ and reverse 5′-

and

reverse

5′-GCACCTGCACCAGGGC-3′;

cluster

of


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CAGATCCGAACACAGCGTAGA-3′; runt-related transcription factor 2 (Runx2), 61 bp, forward 5′-

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AAGCTGCGGCAAGACAAG-3′ and reverse 5′-TCAAATCTGCAGCTTCAAGG-3′; osterix (OSX),

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AAGAGACCTGGCAAGAGG-3′;

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AAACCCAGAACACAAGCATTCC-3′ and reverse 5′-TCCACCAGCAAGAAGAAGCC-3′.

bp,

forward

5′-GCTAGAGATCTGAGCCGGGTA-3′ alkaline

phosphatase

(ALP),

and 218

reverse bp

forward

5′5′-

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Western Blots. Cells were harvested and lysed in RIPA buffer (1 % NP-40, 50 mM Tris-HCl, pH 7.4,

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150 mM NaCl, and 10 mM NaF) containing a protease inhibitor cocktail (Roche Diagnostics). Protein

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lysates were separated on SDS-PAGE. The protein-transferred membranes were blocked for 30 min

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with 5% non-fat dry milk and incubated overnight at 4°C with primary antibodies against PPAR-γ

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(1:1000, E-8 mouse monoclonal, Santa Cruz Biotechnology, CA), Runx2 (1:2000, M70 Rabbit

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polyclonal, Santa Cruz Biotechnology), or β-actin (1:2000, AC-15 mouse monoclonal, Sigma).

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Membranes were washed three times for 10 min in 0.1% TBST and incubated for 1 h at room

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temperature with HRP-conjugated secondary antibodies (1:10,000, Ab Frontier) in 5% non-fat dry milk.

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After washing unbound antibodies, the blot was developed using enhanced chemiluminescent Western

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blotting detection reagents (GE health care).

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RESULTS AND DISCUSSION

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Bioassay-guided Isolation of Active Compounds from O. sativa Roots. A 95% aqueous EtOH extract

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of O. sativa roots was resolved in sterile distilled water and sequentially fractionated with n-hexane,

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dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (BuOH), and water (residue), yielding five

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solvent-partitioned fractions, which were then filtered, concentrated, and freeze-dried. Powdered

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fractions were dissolved in DMSO for assessment of biological activity. C3H10T1/2 cells were treated

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with the five fractions at a concentration of 20 ng/mL, after which the degree of differentiation of these

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cells was measured. Adipocytes were detected using Oil Red O (ORO) staining (Figure S1). After a

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week in culture with each fraction, the cells were stained to measure adipogenesis. C3H10T1/2 cells ACS Paragon Plus Environment

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grown in medium treated with the DCM-soluble fraction demonstrated less differentiation into

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adipocytes than the cells treated with other fractions. In addition, after two weeks of osteogenic

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differentiation, cells were fixed and stained with an alkaline phosphatase (ALP) solution. Cells treated

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with the DCM-soluble fraction showed higher ALP staining than the other fractions or the control group.

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Our results showed that MSCs in cultures treated with the DCM-soluble fraction tended to effectively

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differentiate into osteoblasts while avoiding differentiation into adipocytes. These data required

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thorough investigation of the DCM-soluble fraction for the active substances that regulated MSC

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differentiation into adipocytes and osteoblasts. The DCM-soluble fraction was separated by successive

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column chromatography on silica gel and Sephadex LH-20, and semi-preparative HPLC purification

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according to the bioassay-guided fractionation for the biological activity of adipogenesis and

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osteogenesis (Figure 1A), which afforded compounds 1 and 2 as the main compounds of the final active

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fraction (Figure 1).

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Structural Determination of the Compounds. Compound 1 was isolated as a light yellowish gum.

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The molecular formula was deduced to be C40H40O13 from the molecular ion peak [M - H]- at m/z

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727.2386 (calcd for C40H39O13, 727.2391) in the high-resolution (HR)-electrospray ionization (ESI)-MS

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negative-ion mode. The infrared (IR) spectrum showed absorptions for hydroxyl (3366 cm−1), α,β-

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unsaturated carbonyl ester (1715 cm−1), and aromatic groups (1610 and 1515 cm−1). The 1H NMR

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spectrum of 1 (Table 1) exhibited signals for the presence of two trans-substituted double bonds at δH

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7.44 (2H, d, J = 16.0 Hz) and 6.23 (2H, d, J = 16.0 Hz) and two typical 1,4-disubstituted aromatic rings

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at δH 7.38 (4H, d, J = 8.5 Hz) and 6.80 (4H, d, J = 8.5 Hz). The 1H–1H correlation spectroscopy (COSY)

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and heteronuclear multiple bond correlation (HMBC) analyses of these peaks suggested the existence of

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two trans-p-coumaroyl moieties in this molecule (Figure 1C).10,11 Furthermore, the signals for aromatic

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proton and carbon at δH 6.78 (4H, s) and δC 105.1, indicative of two 1,3,4,5-tetrasubstituted aromatic

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rings; two oxygenated methines at δH 5.06 (2H, d, J = 8.0 Hz) and δC 85.8; two methylenes at δH 4.45

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(2H, dd, J = 11.5, 4.5 Hz)/4.44 (2H, dd, J = 11.5, 5.5 Hz) and δC 64.8; two methines at δH 2.70 (2H, m) ACS Paragon Plus Environment

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and δC 51.9; and four methoxyl groups at δH 3.85 (12H, s) and δC 57.0 were observed in the NMR data

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of 1 (Table 1) with assistance of heteronuclear single quantum correlation (HSQC) experiment. Analysis

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of these NMR data suggested the presence of an icariol A2 moiety, a 2,5-diaryl tetrahydrofuran lignan in

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the molecule,12 which was confirmed by 2D NMR data analysis (HSQC, 1H-1H COSY, and HMBC)

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(Figure 1C). Finally, the complete gross structure of 1 was established through assignment of the

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connection of the trans-p-coumaroyl groups. The relatively down-field shifted resonances of H-9 and H-

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9' at δH 4.45 (2H, dd, J = 11.5, 4.5 Hz) and 4.44 (2H, dd, J = 11.5, 5.5 Hz) were comparable to those of

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an icariol A2 moiety12 and implied the location of the trans-p-coumaroyl groups on the 9- and 9'-

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hydroxy groups, which was confirmed by the HMBC cross-peaks of H-9/C-9'' (δC 169.0) and H-9'/C-9'''

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(δC 169.0) (Figure 1C). The relatively large coupling constants of H-7 (J7,8 = 8.0 Hz) and H-7' (J7',8' =

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8.0 Hz) indicated each hydroxymethylene group to be oriented in a trans relationship to the vicinal aryl

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group in the 2,5-diaryl tetrahydrofuran lignan system,12 which allowed us to conclude that the

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configuration of the lignan system was either the all-trans or meso form. Thus, the absolute

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configuration of 1 was determined by circular dichroism (CD) data, which showed a positive Cotton

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effect at 244 nm. While 7S,7'S,8R,8'R-icariol A2 showed a negative band around 240 nm, 7R,7'R,8S,8'S-

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icariol A2 displayed a positive Cotton effect at 244 nm.12,13 In accordance with the above evidence, the

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absolute configuration of 1 was elucidated as 7R,7'R,8S,8'S, and the structure was determined to be

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[(2R,3S,4S,5R)-tetrahydro-2,5-bis(4-hydroxy-3,5-dimethoxyphenyl)-3,4-furandiyl]bis(methylene) ester

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(E)-p-hydroxycinnamic acid as shown in Figure 1B. The compound was named oryzativol A.

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Compound 2 was obtained as a light yellowish gum with the molecular formula of C40H40O13, as

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determined by negative-ion mode HRESIMS data that showed a molecular ion peak [M - H]- at m/z

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727.2398 (calcd for C40H39O13, 727.2391). The 1H and 13C NMR data of 2 were quite similar to those of

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compound 1 except for the aromatic region (Table 1). Detailed inspection of the 1H and 13C NMR data

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of 2 revealed that one trans-p-coumaroyl moiety seen in 1 was replaced with a cis-p-coumaroyl moiety

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in 2. The coupling constant (12.5 Hz) between H-7''' and H-8''' and their characteristic chemical shifts ACS Paragon Plus Environment

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(δH 6.88 and 5.73) supported the presence of cis-olefinic protons.11,12,14,15 The presence of a cis-p-

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coumaroyl moiety was confirmed by 2D NMR data analysis (HSQC, 1H-1H COSY, and HMBC), and

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HMBC correlations from the proton signals of H-9' (δC 4.42 and 4.37) to C-9''' (δC 168.4) indicated that

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the cis-p-coumaroyl group was located at C-9'''. Similarity between the CD spectra of 1 and 2 suggested

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the same absolute configuration of 2 with that of 1, namely 7R,7'R,8S,8'S.12,13 The structure of 2 was

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thus determined to be an isomer of 1 (Figure 1B). This compound was named oryzativol B.

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Regulatory Effects of the Compounds on MSCs Differentiation into Adipocytes and Osteoblasts.

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To identify the regulatory effects of these compounds on the MSCs differentiation into adipocytes and

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osteoblasts, the C3H10T1/2 cell line was treated with various concentrations of oryzativol A or B during

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adipogenesis or osteogenesis. Oryzativols A and B dose-dependently inhibited lipid production to a

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similar degree in C3H10T1/2 cells (Figure 2A). ALP, which is bound to the membrane of osteoblasts,

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is a biomarker for osteoblast differentiation. ALP staining showed that oryzativols A and B accelerated

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osteogenesis, as measured by the deep purple color of ALP staining (Figure 2B). Although oryzativols

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A and B levels were similar in both suppression of adipogenesis and promotion of osteogenesis,

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oryzativol A was selected for testing in the following experiments due to its structural stability. A week

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after adipogenic differentiation, cells were stained with Oil Red O dye (Figure 2C). Cells showed low

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levels of staining in the presence of ≥1 µM of oryzativol A, suggesting a suppressive effect in

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adipogenesis. In contrast, C3H10T1/2 cells vigorously differentiated into osteoblasts at ≥1 µM

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oryzativol A, showing the purple color indicative of positive ALP staining after osteogenic

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differentiation (Figure 2C).

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Various concentrations of oryzativol A were added into the culture media of several undifferentiated

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cell lines, including the mouse MSC line C3H10T1/2, the mouse embryonic fibroblast clonal sub cell

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line NIH3T3-L1, and the aneuploid immortal keratinocyte cell line HACAT. Cells were treated for 48 h

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to analyze oryzativol A toxicity in vitro (Figure 3). At a 10 µM or lower, all of the cell lines showed

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100% viability as assessed by MTT assay. The LC50 of oryzativol A in HACAT cells was found to be ACS Paragon Plus Environment

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43.95 µM, 88 times higher than the actual working concentration (Figure 3A); however, the LC50 in the

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undifferentiated C3H10T1/2 and NIH3T3-L1 cells lines was 16.51 µM and 19.16 µM, respectively. In

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addition, C3H10T1/2 and NIH3T3-L1 cells were stimulated to differentiate into osteoblasts and/or

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adipocytes in the presence of various oryzativol A concentrations (Figure 3B). After 48 h exposure to

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the compound, the viabilities of osteoblast-differentiated C3H10T1/2, adipocyte-differentiated

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C3H10T1/2, and adipocyte-differentiated NIH3T3-L1 cells were measured using the MTT assay.

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Although all of the differentiated cells maintained approximately 100% viability at a concentration of 10

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µM or less, the viabilities significantly decreased at a concentration of 20 µM oryzativol A. The LC50

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value of oryzativol A was calculated to be in the range of 14.04~16.03 µM in the osteoblast- or

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adipocyte-differentiated C3H10T1/2 and NIH3T3-L1 cells. Thus, the differentiated cells were found to

248

be slightly more sensitive to the compound than the undifferentiated cells.

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The mRNA expression of various genes relating to the MSCs differentiation into adipocytes or

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osteoblasts was measured by quantitative PCR (Figure 4). Since peroxisome proliferator-activated

251

receptor γ (PPARγ) is the central regulator of adipogenesis,16,17 we first tested whether oryzativol A

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could act as a regulator of PPARγ expression (Figure 4A). Also, expression of the genes encoding the

253

adipogenic markers CD36 (Figure 4B) and aP2 (Figure 4C) was measured to further identify the

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regulatory effect of oryzativol A on adipogenic differentiation. Treatment with oryzativol A decreased

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gene expression of the genes encoding PPARγ, aP2, and CD36 in C3H10T1/2 cells, implicating that the

256

compound suppressed adipogenic gene expression and inhibited adipogenesis in MSCs. The IC50 of

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oryzativol A for the suppression of adipogenic gene expression ranged from 0.70~1.05 µM. Expression

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of the genes encoding PPARγ, aP2, and CD36 was hardly seen in the presence of 5 µM or more

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oryzativol A in culture media. The levels of adipogenic gene expression with oryzativol A were

260

compared with the levels of expression from resveratrol-treated cells (R in Figure 4A-C) since

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resveratrol, a phytoalexin, is known to have anti-adipogenic and pro-osteogenic effects.6,18 The

262

suppressive effect of 40 µM resveratrol on adipogenic gene expression was comparable to the ACS Paragon Plus Environment

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suppressive effect shown in cells treated with 5 µM oryzativol A. To define the reciprocal activity of

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oryzativol A on adipogenesis and osteogenesis in MSCs, we also examined the gene expression of the

265

osteogenic markers Runx2 (Figure 4D), osterix (Figure 4E), and ALP (Figure 4F). Treatment with 5

266

µM oryzativol A caused an up to 88-fold increase in mRNA expression of Runx2, an activator of

267

osteoblasts, compared to the untreated control group. The compound also stimulated a dose-dependent

268

increase in the mRNA expression of osterix. Oryzativol A at 5 µM increased ALP expression up to

269

34.5-fold compared to controls. The expression of PPARγ and Runx2 protein was examined in

270

differentiated MSCs (Figure 5). Oryzativol A regulated PPARγ expression (Figure 5) resulting in the

271

inhibition of adipogenesis in MSCs. The inhibitory effect of 5 µM oryzativol A on PPARγ expression

272

was comparable to 40 µM resveratrol. Protein expression of the osteogenic marker Runx2 (Figure 5)

273

was slightly increased, consistent with Runx2 mRNA level (Figure 4D), in the media containing

274

oryzativol A. Treatment with 5 µM oryzativol A caused an increase in Runx2 protein expression to a

275

level similar to treatment with 100 nM 17β-estradiol, an estrogen receptor agonist. Concentration of

276

oryzativol A under 1 µM hardly affected protein expression in differentiated MSC adipocytes or

277

osteoblasts. Taken together, these findings suggest that oryzativol A simultaneously regulates the

278

expression of key genes related to both adipogenesis and osteogenesis, playing a pivotal role in the

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reciprocal differentiation of MSCs into adipocytes and osteoblasts.

280

In conclusion, we identified the novel lignans, oryzativols A and B from O. sativa roots using a

281

bioassay-guided isolation and this is the first report of lignans in the rice roots. We examined their

282

effects on MSC differentiation into adipocytes and osteoblasts, which determined that oryzativol A

283

reciprocally regulates adipocyte and osteoblast differentiation. To the best of our knowledge, this is the

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first paper describing the potential of O. sativa root components for the regulation of MSCs

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differentiation into adipocytes and osteoblasts and for the treatment of osteoporosis.

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ASSOCIATED CONTENT ACS Paragon Plus Environment

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*Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at

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AUTHOR INFORMATION

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Corresponding Authors

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Ki Hyun Kim: (Tel: +82-31-290-7700; Fax: +82-31-290-7730; E-mail: [email protected])

294 295

Author Contributions

296

S.H.K., K.M.P., and K.H.K. designed the experiments. H.R.K. and T.K.L. performed the phytochemical

297

experiments. H.S.Y. contributed to the biological studies. H.R.K., H.S.Y., S.L., S.H.K., E.M., K.M.P.,

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and K.H.K. analyzed the data. S.L., S.H.K., and K.H.K. wrote the main manuscript text including all

299

figures. All authors reviewed and approved the manuscript.

300 301

Funding

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This research was supported by the Basic Science Research Program through the National Research

303

Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning

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(2015R1C1A1A02037383). This research was supported by Basic Science Research Program through

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the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-

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2017R1D1A1B03030419)

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The authors declare that there are no conflicts of interests.

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References

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(1) Chen, Q.; Yang, L.; Zhang, G.; Wang, F. Bioactivity-guided isolation of antiosteoporotic

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compounds from Ligustrum lucidum. Phytother. Res. 2013, 27, 973-979.

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(2) Rodríguez, J. P.; Astudillo, P.; Ríos, S.; Pino, A. M. Involvement of adipogenic potential of human

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bone marrow mesenchymal stem cells (MSCs) in osteoporosis. Curr. Stem Cell Res. Ther. 2008, 3, 208-

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(3) Kirkland, J. L.; Tchkonia, T.; Pirtskhalava, T.; Han, J.; Karagiannides, I. Adipogenesis and aging:

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Does aging make fat go mad? Exp. Gerontol. 2002, 37, 757-767.

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(4) Raisz, L. G. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J. Clin. Invest. 2005,

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115, 3318–3325.

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(5) Kimura. T.; But. P. P. H.; Guo, J. X.; Sung, C. K. International Collation of Traditional and Folk

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Medicine: Northeast Asia part 1. World Scientific; 1996, p. 198-9.

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(6) Rayalam, S.; Della-Fera, M. A.; Baile, C. A. Synergism between resveratrol and other

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phytochemicals: Implications for obesity and osteoporosis. Mol. Nurt. Food Res. 2011, 55, 1177-1185.

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(7) Taher, M.; Aminuddin, A.; Susanti, D.; Aminudin, N. I.; On, S.; Ahmad, F.; Hamidon, H. Cytotoxic,

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anti-Inflammatory and adipogenic effects of inophyllum D, calanone, isocordato-oblongic acid, and

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morelloflavone on cell lines. Nat. Prod. Sci. 2016, 22, 122-128.

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(8) Lee, H.; Kim, J.; Park, J. Y.; Kang, K. S.; Park, J. H.; Hwang, G. S. Processed Panax ginseng, sun

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ginseng, inhibits the differentiation and proliferation of 3T3-L1 preadipocytes and fat accumulation in

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Caenorhabditis elegans. J. Ginseng Res. 2017, 41, 257-267.

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(9) Peng, Y.; Zhong, Y.; Li, G. Tubeimoside-1 suppresses breast cancer metastasis through

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downregulation of CXCR4 chemokine receptor expression. BMB Rep. 2016, 49, 502-507.


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(10) Kim, K. H.; Choi, S. U.; Lee, K. R. Bioactivity-guided isolation of cytotoxic triterpenoids from the

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trunk of Berberis koreana. Bioorg. Med. Chem. Lett. 2010, 20, 1944-7.

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(11) Eom, H. J.; Kang, H. R.; Kim, H. K.; Jung, E. B.; Park, H. B.; Kang, K. S.; Kim, K. H. Bioactivity-

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guided isolation of antioxidant triterpenoids from Betula platyphylla var. japonica bark. Bioorg. Chem.

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2016, 66, 97-101.

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(12) Kim, K. H.; Kim, H. K.; Choi, S. U.; Moon, E.; Kim, S. Y.; Lee, K. R. Bioactive lignans from the

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rhizomes of Acorus gramineus. J. Nat. Prod. 2011, 74, 2187-2192.

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(13) Yamauchi, H.; Kakuda, R.; Yaoita, Y.; Machida, K.; Kikuchi, M. Two new glycosides from the

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whole plants of Glechoma hederacea L. Chem. Pharm. Bull. 2007, 55, 346-347.

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(14) Kim, K. H.; Choi, S. U.; Son, M. W.; Lee, K. R. Two new phenolic amides from the seeds of

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Pharbitis nil. Chem. Pharm. Bull. 2010, 58, 1532-1535.

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(15) Kim, K. H.; Chang, S. W.; Lee, K. R. Feruloyl sucrose derivatives from Bistorta manshuriensis.

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Can. J. Chem. 2010, 88, 519-523.

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(16) Colaianni, G.; Brunetti, G.; Faienza, M. F.; Colucci, S.; Grano, M. Osteoporosis and obesity: Role

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of Wnt pathway in human and murine models. World J. Orthop. 2014, 5, 242-246.

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(17) Park, K. W.; Waki, H.; Choi, S. P.; Park, K. M.; Tontonoz, P. The small molecule phenamil is a

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modulator of adipocyte differentiation and PPARgamma expression. J. Lipid. Res. 2010, 51, 2775-2784.

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(18) Baur, J. A.; Pearson, K. J.; Price, N. L.; Jamieson, H. A.; Lerin, C.; Kalra, A.; Prabhu, V. V.; Allard,

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J. S.; Lopez-Lluch, G.; Lewis, K.; Pistell, P. J.; Poosala, S.; Becker, K. G.; Boss, O.; Gwinn, D.; Wang,

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M.; Ramaswamy, S.; Fishbein, K. W.; Spencer, R. G.; Lakatta, E. G.; Le Couteur, D.; Shaw, R. J.;

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Navas, P.; Puigserver, P.; Ingram, D. K.; de Cabo, R.; Sinclair, D. A. Resveratrol improves health and

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survival of mice of high calorie diet. Nature 2006, 444, 337-342.


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357


Table 1. 1H (700 MHz) and 13C (175 MHz) NMR data of compounds 1 and 2 in CD3ODa Position 1 2 3 4 5 6 7 8 9

δC 133.7 105.1 149.5 136.6 149.5 105.1 85.8 51.9 64.8

1' 2' 3' 4' 5' 6' 7' 8' 9'

133.7 105.1 149.5 136.6 149.5 105.1 85.8 51.9 64.8

1 δH (J in Hz) 6.78, s

6.78, s 5.06, d (8.0) 2.70, m 4.45, dd (11.5, 4.5) 4.44, dd (11.5, 5.5) 6.78, s

6.78, s 5.06, d (8.0) 2.70, m 4.45, dd (11.5, 4.5) 4.44, dd (11.5, 5.5)

1'' 127.1 2'' 131.5 7.38, d (8.5) 3'' 117.0 6.80, d (8.5) 4'' 161.6 5'' 117.0 6.80, d (8.5) 6'' 131.5 7.38, d (8.5) 7'' 147.2 7.44, d (16.0) 8'' 114.9 6.23, d (16.0) 9'' 169.0 1''' 127.1 2''' 131.5 7.38, d (8.5) 3''' 117.0 6.80, d (8.5) 4''' 161.6 5''' 117.0 6.80, d (8.5) 6''' 131.5 7.38, d (8.5) 7''' 147.2 7.44, d (16.0) 8''' 114.9 6.23, d (16.0) 9''' 169.0 3-OMe 57.0 3.85, s 5-OMe 57.0 3.85, s 3'-OMe 57.0 3.85, s 5'-OMe 57.0 3.85, s a Coupling constants (in Hz) are given in parentheses.

δC 133.8 105.2 149.6 136.6 149.6 105.2 85.8 51.9 64.3 133.8 105.0 149.6 136.6 149.6 105.0 85.2 51.9 64.6 127.2 131.5 117.1 161.6 117.1 131.5 147.2 115.0 169.1 127.8 133.8 116.1 160.4 116.1 133.8 145.8 116.5 168.4 57.1 57.1 57.1 57.1

2 δH (J in Hz) 6.73, s

6.73, s 4.99, d (8.0) 2.60, m 4.36, dd (11.5, 4.5) 4.40, dd (11.5, 5.5) 6.72, s

6.72, s 5.00, d (8.0) 2.60, m 4.37, dd (11.5, 4.5) 4.42, dd (11.5, 5.5) 7.38, d (8.5) 6.78, d (8.5) 6.78, d (8.5) 7.38, d (8.5) 7.42, d (16.0) 6.21, d (16.0)

7.59, d (8.5) 6.73, d (8.5) 6.73, d (8.5) 7.59, d (8.5) 6.88, d (12.5) 5.73, d (12.5) 3.85, s 3.85, s 3.84, s 3.84, s

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Figure captions

361 362

Figure 1. (A) Bioassay-guided isolation of the active DCM-soluble fraction from O. sativa extracts. The

363

extract was separated according to effectiveness in regulating the differentiation of the MSC

364

C3H10T1/2 cell line into adipocytes or osteoblasts. (B) Chemical structures of oryzativols A (1) and B

365

(2). (C) The 1H-1H COSY correlations (blue bond) and key HMBC correlations (H→C) of oryzativol A

366

(1).

367

Figure 2. Reciprocal effects of oryzativols A and B on the differentiation of MSCs. (A) Inhibitory effect

368

of oryzativols A and B on adipogenesis. Lipid drops in adipogenically differentiated C3H10T1/2 cells

369

were stained with ORO, and the isopropanol-dissolved intracellular triglyceride content was measured

370

at 520 nm with a spectrophotometer. (B) Stimulatory effects of oryzativols A and B on osteogenesis.

371

Staining intensity in C3H10T1/2 cells stained for ALP was measured with a program using the b value

372

of LAB in color. (C) Visualization of adipogeneic and osteogenic differentiation of MSCs. After

373

differentiation of MSCs in the presence of oryzativols A and B, cells were stained with ORO or ALP

374

and photographed.

375

Figure 3. Cell viability in the presence of oryzativol A. Cells were cultured in the presence of oryzativol

376

A at various concentrations. (A) MTT assay with undifferentiated cells in the presence of oryzativol A.

377

Undifferentiated C3H10T1/2 (mouse MSC line, CN ●), 3T3-L1 (mouse embryonic fibroblast clonal

378

sub-cell line, LN▲), and HACAT (aneuploid immortal keratinocyte cell line HN □) cells were exposed

379

to various concentrations of oryzativol A for 2 days prior to the MTT assay. (B) MTT assay with

380

differentiated cells in the presence of oryzativol A. C3H10T1/2 cells were cultured in osteoblast

381

differentiation media (CO ●) or adipocyte differentiation media (CA ○) for 9 or 7 days, respectively.

382

3T3-L1 cells were cultured in adipocyte differentiation media (LA ▲) for 7 days. All differentiated

383

cells were exposed to oryzativol A for 2 days prior to the MTT assay.


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384

Figure 4. Gene expression of adipogenic or osteogenic markers in C3H10T1/2 cells. Cells differentiated

385

into adipocytes or into osteocytes in the presence of various oryzativol A concentrations. mRNA

386

expression of marker genes for adipogenesis and osteogenesis was measured by real-time quantitative

387

PCR. Adipogenic markers are (A) PPARγ, (B) CD36, and (C) aP2. Resveratrol (40 µM, R) was used as

388

a positive control for adipogenic differentiation. The osteogenic markers include (D) Runx2, (E) Osterix,

389

and (F) ALP. * indicates difference from non-treated control (p < 0.05).

390

Figure 5. Protein expression of PPARγ, an adipogenic marker (A) and Runx2, an osteogenic marker (B)

391

in C3H10T1/2 cells. Cells differentiated into adipocytes or into osteocytes in the presence of various

392

doses of oryzativol A. Protein expression of adipogenesis and osteogenesis marker genes was measured

393

by Western blot. Resveratrol (40 µM, R) and 17β-estradiol (100 nM, E2) were used as positive controls

394

in adipogenic- (A) and osteogenic- (B) differentiation, respectively.

395


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(A)

397 398 399 400 401 402 403 404 405 406 HO

407

(B) 3''

2'' 7''

HO

408

5''

8'' 9''

6''

H 3CO

409

O

3

O 2

9 8

1 7

O

4

HO 5

OH

OCH3

1' 1

O

O

O

H 3CO

OCH 3 O

HO

OH OCH 3

OH

H3CO

OCH 3 2

1

410 (C)

O

O

O

O

OH

HO

412 H3 CO

413

O HO

O

6

H3CO

411

1'''

O

1''

4''

OCH 3 O

HO H3 CO

OH OCH 3

414 ACS Paragon Plus Environment

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415

Figure 1. (A) Bioassay-guided isolation of the active DCM-soluble fraction from O. sativa extracts. The

416

extract was separated according to effectiveness in regulating the differentiation of the MSC

417

C3H10T1/2 cell line into adipocytes or osteoblasts. (B) Chemical structures of oryzativols A (1) and B

418

(2). (C) The 1H-1H COSY correlations (blue bond) and key HMBC correlations (H→C) of oryzativol A

419

(1).

420


21


Page 22 of 27

421 422

Figure 2. Reciprocal effects of oryzativols A and B on the differentiation of MSCs. (A) Inhibitory effect

423

of oryzativols A and B on adipogenesis. Lipid drops in adipogenically differentiated C3H10T1/2 cells

424

were stained with ORO, and the isopropanol-dissolved intracellular triglyceride content was measured

425

at 520 nm with a spectrophotometer. (B) Stimulatory effects of oryzativols A and B on osteogenesis.

426

Staining intensity in C3H10T1/2 cells stained for ALP was measured with a program using the b value

427

of LAB in color. (C) Visualization of adipogeneic and osteogenic differentiation of MSCs. After

428

differentiation of MSCs in the presence of oryzativols A and B, cells were stained with ORO or ALP

429

and photographed.

430


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431 432

Figure 3. Cell viability in the presence of oryzativol A. Cells were cultured in the presence of oryzativol

433

A at various concentrations. (A) MTT assay with undifferentiated cells in the presence of oryzativol A.

434

Undifferentiated C3H10T1/2 (mouse MSC line, CN ●), 3T3-L1 (mouse embryonic fibroblast clonal

435

sub-cell line, LN▲), and HACAT (aneuploid immortal keratinocyte cell line HN □) cells were exposed

436

to various concentrations of oryzativol A for 2 days prior to the MTT assay. (B) MTT assay with

437

differentiated cells in the presence of oryzativol A. C3H10T1/2 cells were cultured in osteoblast

438

differentiation media (CO ●) or adipocyte differentiation media (CA ○) for 9 or 7 days, respectively. ACS Paragon Plus Environment

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439

3T3-L1 cells were cultured in adipocyte differentiation media (LA ▲) for 7 days. All differentiated

440

cells were exposed to oryzativol A for 2 days prior to the MTT assay.

441


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442 443

Figure 4. Gene expression of adipogenic or osteogenic markers in C3H10T1/2 cells. Cells differentiated

444

into adipocytes or into osteocytes in the presence of various oryzativol A concentrations. mRNA

445

expression of marker genes for adipogenesis and osteogenesis was measured by real-time quantitative

446

PCR. Adipogenic markers are (A) PPARγ, (B) CD36, and (C) aP2. Resveratrol (40 µM, R) was used as

447

a positive control for adipogenic differentiation. The osteogenic markers include (D) Runx2, (E) Osterix,

448

and (F) ALP. * indicates difference from non-treated control (p < 0.05).


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449 450

Figure 5. Protein expression of PPARγ, an adipogenic marker (A) and Runx2, an osteogenic marker (B)

451

in C3H10T1/2 cells. Cells differentiated into adipocytes or into osteocytes in the presence of various

452

doses of oryzativol A. Protein expression of adipogenesis and osteogenesis marker genes was measured

453

by Western blot. Resveratrol (40 µM, R) and 17β-estradiol (100 nM, E2) were used as positive controls

454

in adipogenic- (A) and osteogenic- (B) differentiation, respectively.

455


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456


TOC graphic HO

457 458

O

O

OH

HO O H 3CO

O O

OCH3

O

H 3CO

O HO

O

HO

O

OH

OCH3 O

HO H3CO

OH

OCH 3 H 3 CO Oryzativol A

OCH 3 Oryzativol B


27

Chemical Characterization of Novel Natural Products from the Roots of

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