Biol. Pharm. Bull. 38, 228–234 (2015)

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Vol. 38, No. 2

Regular Article

Antifibrotic Compounds from Liriodendron tulipifera Attenuating HSC-T6 Proliferation and TNF-α Production in RAW264.7 Cells Eun Ju Jeong,a Na-Hyun Kim,b Jeong-Doo Heo,b Ki Yong Lee,c Jung-Rae Rho,d Young Choong Kim,e and Sang Hyun Sung*,e a

 Department of Agronomy & Medicinal Plant Resources, College of Life Sciences and Natural Resources, Gyeongnam National University of Science and Technology; Jinju 660–758, Korea: b Gyeongnam Department of Environment & Toxicology, Korea Institute of Toxicology; 17 Jegok-gil, Munsan-eup, Gyeongnam 660–844, Korea: c  College of Pharmacy, Korea University; Sejong 339–700, Korea: d Department of Oceanography, Kunsan National University; Jeonbuk, 573–701, Korea: and e College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University; Seoul 151–742, Korea. Received August 12, 2014; accepted October 22, 2014 The inhibition of hepatic stellate cell (HSC) proliferation has been considered as an effective therapeutic target for the treatment of liver fibrosis. The methanolic extract of Liriodendron tulipifera showed significant inhibitory activity against the proliferation of HSCs. Bioactivity-guided isolation afforded twelve compounds including (−)-sesamin (1), (−)-syringaresinol (2), (+)-dihydrodehydrodiconiferyl alcohol (3), salvinal (4), (+)-guaiacylglycerol-8-O-4′-dihydroconiferyl ether (5), (±)-guaiacylglycerol-8-O-4′-sinapyl alcohol ether (6), tanegool (7), (+)-5,5′-dimethoxy-7-oxolariciresinol (8), 3-hydroxy-4-methoxyacetophenone (9), 4-acetoxymethylphenol (10), (−)-paramicholide (11), and blumenol A (12). Among the compounds isolated, 2, 3 and 4 significantly attenuated the proliferation of the activated HSC-T6 cells. The maximal dose of these compounds, however, showed no cytotoxicity in primary cultured rat hepatocytes. Collagen deposition in the activated HSC-T6 cells was reduced by 2, 3 and 4. Also, the increased production of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α induced by lipopolysaccharide was decreased by 3 and 4 in RAW264.7 macrophage cells. Collectively, (−)-syringaresinol (2), (+)-dihydrodehydrodiconiferyl alcohol (3), and salvinal (4) isolated from L. tulipifera leaves and twigs exhibited selective antifibrotic activities toward the activated HSCs and suppressed TNF-α production in RAW264.7 macrophages. These compounds may be useful candidates for developing therapeutic agents for the prevention and treatment of hepatic fibrosis. Key words

Liriodendron tulipifera; antifibrotic; hepatic stellate cell; HSC-T6; macrophage; RAW264.7

Hepatic stellate cells (HSCs), a non-parenchymal liver cell that resides in the space of Disse, mainly participate to regulate  sinusoidal  blood  flow  via contraction by releasing pro-inflammatory, and pro-fibrogenic cytokines.1,2) In a normal liver, HSCs exist in a quiescent state (qHSCs) that exhibit functions including  the  storage  of  vitamin  A  and  retinoid  as  well  as  the  suppression on extracellular matrix (ECM) formation. During the wound healing response to chronic liver injury from a  variety of causes including viral, autoimmune, drug induced, cholestatic and metabolic diseases, HSCs undergo a process called  “activation,”  by  which  qHSCs  are  trans-differentiated into  myofibroblast-like  activated  HSCs.  The  activated  HSCs  produce  the  excessive  ECM  which  leads  to  liver  dysfunction  and irreversible cirrhosis. Therefore, suppression of HSC activation and proliferation has been proposed as a therapeutic target against hepatic fibrosis.3) In  the  course  of  searching  for  antifibrotic  compounds  from  natural  resources  using  HSC-T6  cells,  an  immortalized  rat  hepatic stellate cell line, as an in vitro  assay  system,  it  was  found  that  a  methanolic  extract  of  the  leaves  and  twigs  of  Liriodendron tulipifera  inhibited  cell  proliferation  in  HSC-T6.  L. tulipifera,  also  well  known  as  the  tulip  tree  or  yellow  poplar, is the family of Magnoliaceae. Sesquiterpenes and aporphine alkaloids have been isolated from this plant as bioactive  constituents  with  anticancer,  antiplasmodial  and  antimicrobial  activities.4–8)  However,  there  has  been  no  report  related  to  the  antifibrotic  activity  of  this  plant.  Thus,  we  have  attempted  to  isolate  the  antifibrotic  constituents  from  the  leaves  and  twigs 

of L. tulipifera through bioactivity-guided fractionation.

MATERIALS AND METHODS Plant Material   The leaves and twigs of L. tulipifera were  collected from the Medicinal Plant Garden, Seoul National University,  Korea,  in  July  2008.  Plant  identification  was  authenticated  by  Dr.  Jong  Hee  Park,  Pusan  National  University,  Korea. A voucher specimen (CS-243) has been deposited in the Herbarium of the Medicinal plant Garden, College of Pharmacy, Seoul National University. Isolation of Compounds 1–12 from L. tulipifera Leaves and Twigs   The  air-dried  plant  material  (6.76 kg)  was  extracted  four  times  with  80%  MeOH  for  3 h  each  in  an  ultrasonic apparatus. Removal of the solvent in vacuo yielded a methanolic  extract  (482.5 g).  The  methanolic  extract  was  then  suspended  in  distilled  water  and  partitioned  successively  with  n-hexane (26.7 g), CHCl3 (34.8 g), EtOAc (21.5 g), and n-BuOH (70.0 g).  The  n-hexane and CHCl3  soluble  fractions  which  showed  anti-proliferative  activities  on  the  activated  HSC-T6  cells  were  used  to  elucidate  bioactive  compounds.  The  nhexane  fraction  was  subjected  to  silica  gel  column  using  mixtures of CHCl3–MeOH of increasing polarity as eluents to give  6  fractions  (HI–VI).  Compound  1  (9.8 mg)  was  obtained  from  HIV  by  subjecting  to  C18 RP column chromatography with  a  gradient  elution  of  MeOH–water  (30→100%  MeOH),  and  further  purification  on  Sephadex  LH-20  (MeOH).  The  CHCl3  fraction  was  subjected  to  silica  gel  column  chroma-

* To whom correspondence should be addressed.  e-mail: [email protected] © 2015 The Pharmaceutical Society of Japan

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tography  with  a  gradient  elution  of  n-hexane–EtOAc–MeOH (H : E=10 : 1→E : M=1 : 1→MeOH) to give 4 subfractions (CI– IV).  CII  was  subjected  to  C18 RP column chromatography with  a  gradient  elution  of  MeOH–water  (30→100%  MeOH).  Compound 9  (11.2 mg)  was  obtained  from  CII-2  by  additional  C18 RP HPLC (MeOH−H2O 10 : 90, 2.0 mL/min, 254 nm). CIII  was  subjected  to  silica  gel  column  chromatography  with  a  gradient elution of CHCl3–MeOH  (30 : 1→MeOH) to give 5 subfractions (CIII-1–5). Compound 10  (6.1 mg)  was  obtained  from CIII-5 by additional C18 RP HPLC (MeOH−H2O  25 : 75,  2.0 mL/min,  254 nm).  CIV  was  subjected  to  silica  gel  column  chromatography  with  a  gradient  elution  of  CHCl3–MeOH (50 : 1→MeOH) to give 9 subfractions (CIV-1–9). Fractions CIV-7,  CIV-8,  CIV-9  were  further  purified  by  recrystallization with MeOH to yield compounds 3 (23.7 mg), 5 (2.2 mg), 6 (2.2 mg), 7 (4.1 mg), 8 (8.0 mg), 11 (12.1 mg) and 12 (11.5 mg), respectively.  CIV-4  was  further  subjected  to  silica  gel  column  chromatography  with  a  gradient  elution  of  CHCl3–MeOH– water  (50 : 4 : 1→MeOH)  to  give  6  subfractions.  Compound  2 and 3  were  obtained  from  CIV-4-2  and  CIV-4-3  by  recrystallization with MeOH. Culture of HSC-T6 Hepatic Stellate Cells An immortalized  rat  hepatic  stellate  cell  line,  HSC-T6  was  kindly  provided  by  Prof.  SL  Friedman  (Columbia  University,  New  York,  U.S.A.).  HSC-T6  cells  were  maintained  in  Dulbecco’s  modified  Eagle’s  medinm  (DMEM)  supplemented  with  10%  heat-inactivated  fetal  bovine  serum,  100 IU/mL  penicillin  and  100 µg/mL  streptomycin  at  37°C  in  a  humidified  atmosphere  of 95% air–5% CO2. Assessment of Cell Viability For the assay, hepatocytes or  HSC-T6  cells  were  seeded  in  48-well  plates  at  a  density  of  5×104 cells/mL and incubated for 24 h. Cells were treated with  the sample to be tested at the concentration as indicated for 24 h or 48 h. Inhibitory effect on the proliferation was assessed  by  the  3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium  bromide  (MTT)  assay.  Cells  were  incubated  with  2 mg/mL  of  MTT  for  30 min–2 h.  Reduction  of  MTT  to  formazan  was  assessed  in  an  enzyme-linked  immunosorbent  assay  (ELISA)  plate  reader  at  450 nm.  Data  were  expressed  as  the  mean  of  three independent experiments. Measurement of Cell Proliferation Cell proliferation

Fig. 1.

was  assessed  by  bromodeoxyuridine  (BrdU)  incorporation  using a colorimetric ELISA Kit (Roche Diagnostics, GmbH, Mannheim,  Germany).  HSC-T6  cells  were  plated  and  treated  as described for MTT assay. At the end of each treatment period,  the  medium  was  discarded  and  the  cell  pellet  was  used  for assay according to the manufacturer’s instructions. Measurement of Intracellular Collagen Content The collagen  content  was  quantified  by  the  Sirius  Red-based  colorimetric assay.9)  Briefly,  after  treatment,  the  cultured  HSC-T6  cells  were  washed  with  phosphate  buffered  saline  (PBS),  followed by fixation with Bouin’s fluid for 1 h. After fixation, the  fixation fluid was removed and the culture dishes were washed  by  immersion  in  running  tap  water  for  15 min.  The  culture  dishes  were  air  dried  and  stained  by  Sirius  Red  dye  reagent  for 1 h on the rocker with the speed of about 5 rpm. Thereafter,  the  solution  was  removed  and  the  cultures  were  washed  with  0.01 N HCl to remove non-bound dye. The stained material was  dissolved  in  0.1 N  NaOH  and  the  absorbance  was  measured at 550 nm against 0.1 N NaOH as a blank. Determination of Tumor Necrosis Factor (TNF)-α Production   R AW264.7  cells  were  plated  overnight  in  12  well  plates at a density of 5×105  cells/mL.  The  cells  were  treated  with the samples to be tested for 1 h before exposure to 100 ng/ mL of lipopolysaccharide (LPS). After incubation for 24 h, the supernatants  were  collected  and  used  for  cytokine  measurement. The concentration of TNF-α in the culture medium was  determined  by  a  mouse  TNF  enzyme-linked  immunosorbent  assay (ELISA) kit (BD Biosciences, San Jose, CA, U.S.A.).

RESULTS Isolation of Anti-proliferative Compounds from L. tulipifera The methanolic extract of L. tulipifera leaves and twigs  (6.76 kg)  was  successively  fractionated  into  n-hexane (26.7 g),  chloroform  (34.8 g),  EtOAc  (21.5 g),  n-BuOH  (70 g),  and H2O fractions. The n-hexane and chloroform soluble fractions  showed  significant  antiproliferative  activity,  which  suppressed the proliferation of HSC-T6 cells to 7.4% and 9.6%  of control, respectively, at a concentration of 100 µM (Fig. 1).  These  two  fractions  were  subjected  to  repeated  column  chromatography  to  yield  twelve  compounds.  The  isolated 

The Inhibitory Effects of Total Extract and Subfractions of L. tulipifera Leaves and Twigs on the Proliferation in HSC-T6 Cells

HSC-T6  cells  were  incubated  with  total  methanolic  extract  or  each  fraction  at  the  concentration  of  10,  50  and  100 µg/mL  for  48 h.  Cell  viability  was  measured  by  the  MTT assay. The percent of cell viability (%) was calculated as 100×{(Absorbance of compound-treated)/(Absorbance of non-treated control)}. Results are expressed as the  mean±S.D. of three independent experiments, each performed using triplicate wells. * p