Home | About PM | Editorial board | Search | Ahead of print | Current Issue | Archives | Instructions | Subscribe | Advertise | Contact us |  Login 
Pharmacognosy Magazine
Search Article 
  
Advanced search 
 


 
  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 56  |  Page : 461-464  

Peroxisome proliferator-activated receptor-γ agonistic effect of Chrysanthemum indicum capitulum and its active ingredients


1 Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
2 Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
3 Department of Health Chemistry, School of Pharmacy, Iwate Medical University, Morioka 020-0023, Japan

Date of Submission20-Dec-2017
Date of Acceptance10-Jan-2018
Date of Web Publication14-Aug-2018

Correspondence Address:
Toshiaki Makino
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603
Japan
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_607_17

Rights and Permissions
   Abstract 


Objective: The capitulum of Chrysanthemum indicum (Compositae) is the source of a crude drug used in Japanese traditional Kampo and traditional Chinese medicine. Previous research showed that C. indicum flowers promote adipocyte differentiation through the activation of peroxisome proliferator-activated receptor (PPAR)-γ that may be an important mechanism for controlling systemic insulin resistance or other biological functions. Materials and Methods: The capitulum of C. indicum was extracted with methanol, and its PPAR-γ agonistic activity was measured using luciferase assay. The active ingredients were isolated by the activity-guided fractionations. Results: We isolated (Z)-tonghaosu and (E)-tonghaosu, and (E)-tonghaosu had PPAR-γ agonistic constituents by the activity-guided fractionation from the capitulum of C. indicum. The isomer (Z)-tonghaosu did not show PPAR-γ-agonistic activity. Conclusion: (E)-tonghaosu is one of the active ingredients as PPAR-γ agonist in the capitulum of C. indicum.
Abbreviations used: ANOVA: One-way analysis of variance; DMEM: Dulbecco's modified minimum essential medium; FBS: Fetal bovine serum; HPLC: High-performance liquid chromatography; MCI: Methanol extract of the capitulum of Chrysanthemum indicum; NMR: Nuclear magnetic resonance; PPAR: Peroxisome proliferator-activated receptor.

Keywords: (E)-tonghaosu, (Z)-tonghaosu, activity-guided fractionation, Chrysanthemum indicum, diabetes, proliferator-activated receptor-γ


How to cite this article:
Zhang F, Iwaki A, Ishiuchi K, Sugiyama A, Ohsawa M, Makino T. Peroxisome proliferator-activated receptor-γ agonistic effect of Chrysanthemum indicum capitulum and its active ingredients. Phcog Mag 2018;14:461-4

How to cite this URL:
Zhang F, Iwaki A, Ishiuchi K, Sugiyama A, Ohsawa M, Makino T. Peroxisome proliferator-activated receptor-γ agonistic effect of Chrysanthemum indicum capitulum and its active ingredients. Phcog Mag [serial online] 2018 [cited 2021 Mar 2];14:461-4. Available from: http://www.phcog.com/text.asp?2018/14/56/461/238894





SUMMARY

  • We found (E)-tonghaosu from the capitulum of Chrysanthemum indicum as PPAR-γ-agonist.



   Introduction Top


Chrysanthemi Flos is a crude drug used in Japanese traditional Kampo medicine and traditional Chinese medicine. Japanese Pharmacopoeia 17th Edition (JPXVII)[1] defines that Chrysanthemi Flos is the capitulum of Chrysanthemum morifolium Ramatulle or C. indicum Linné (Compositae). On the other hand, Chinese Pharmacopoeia 2015 Edition[2] describes that the capitulum of these two plant species are recognized as different crude drug: the capitulum of the former plant species is defined as Chrysanthemi Flos, that is the same term as Japanese Pharmacopoeia, and that of the latter plant species is defined as Chrysanthemi Indici Flos. In traditional Chinese medicine, the capitulum of C. indicum is used to resolve fire toxicity,[3] and in Japanese traditional Kampo medicine, this crude drug is used as antipyretics, analgesics, and anti-inflammatory agents.[4]

The capitulum of C. indicum has been used as a tea beverage in the East Asian countries and as an edible garnish with raw fish served in Japan. C. indicum flowers are reported to have biological activities, including anti-inflammatory,[5],[6] hepatoprotective,[7] analgesic,[8] and prophylactic for photoaging,[9] atopic dermatitis,[10] cadmium-induced toxicity,[11] and cisplatin-induced nephrotoxicity.[12] The capitulum of C. morifolium, a crude drug with the same genus origin, is reported to have the ability to promote adipocyte differentiation through peroxisome proliferator-activated receptor (PPAR)-γ activation.[13] In the present study, we evaluated the agonistic effect of methanol extract of the capitulum of C. indicum and isolated its active ingredients by the activity-guided fractionation.


   Materials and Methods Top


Materials and its fractionation

The dried capitulum of C. indicum collected in Zhejian, China, with the grade of Japanese Pharmacopoeia seventeenth edition[1] (Lot number 8G08) is purchased from Daiko Shoyaku (Nagoya, Japan). Voucher specimens are deposited in the Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, and Nagoya City University. The dried capitulum of C. indicum (148 g) was powdered and mixed with 1.5 l of methanol and stirred overnight at room temperature. The solution was filtered with paper. The procedure was repeated twice with the resulting residue. The extracted solution was evaporated at 42°C under reduced pressure and then lyophilized to yield 36.5 g total methanol extract (methanol extract of the capitulum of Chrysanthemum indicum [MCI]). This extract (36 g) was suspended in water and serially partitioned with n-hexane, ethyl acetate, and water-saturated butanol to yield a hexane layer (5.7 g), an ethyl acetate layer (8.6 g), a butanol layer (8.0 g), and a water layer (14.0 g).

The hexane layer (100 mg) was spotted onto silica gel 60G F254 chromatography glass plate (2 mm, 20 cm × 20 cm, Merck Millipore, Billerica, MA, USA) and expanded with hexane/ethyl acetate (9:1) to yield fractions A—P. Because fraction F (Rf = 0.33) was the highest activity, hexane layer (5.5 g) was applied onto a silica gel open column chromatography (35 mm × 350 mm) and eluted with hexane/ethyl acetate (9:1) to yield 0.24 g of fraction F. Fraction F was further analyzed using silica gel open column chromatography (20 mm × 400 mm) eluted with chloroform to yield fractions F1—F14. Fraction F3 (27.3 mg) showed that the highest activity was analyzed with high-performance liquid chromatography (HPLC) (Cosmosil 5C18-AR-II, 10 mm × 150 mm, Nacalai Tesque, Kyoto, Japan), eluted with water/acetonitrile 1:1, and analyzed with ultraviolet absorbance at 238 nm to yield peak 1 (retention time, 15 min), 2 (21 min), and 3 (27 min). From 10 mg of F3, 1.0 mg of peak 1 and 2 and 1.4 mg of peak 3 were collected. The spectra of 1H- and 13C-nuclear magnetic resonance (NMR) of peaks 2 and 3 were analyzed, and their purities were 95% and 99%, respectively. Then, they were named compound 2 and 3, respectively, and NMR, electron impact ionization mass spectrometry (EI-MS) spectrum, and optical rotations data were measured as follows:

Compound 2: colorless amorphous solid; [a]D20-1.4 (c 0.4, CHCl3); 1H-NMR (CDCl3, 500MHz) 6.23 (1H, d, J = 6.0 Hz, H-8), 6.15 (1H, d, J = 6.0 Hz, H-9), 4.60 (1H, brs, H-6), 4.24 (1H, ddd, J = 8.0, 8.0, 4.0 Hz, H-13a), 3.99 (1H, ddd, J = 8.0, 8.0, 8.0 Hz, H-13b), 2.33 (1H, m, H-12a), 2.23 (1H, m, H-11a), 2.06 (1H, m, H-12b), 2.05 (1H, m, H-11b), and 1.99 (3H, brs, H-1); and 13C-NMR (CDCl3, 125 MHz) 167.1 (C-7), 135.2 (C-9), 127.5 (C-8), 121.0 (C-10), 80.6 (C-2), 78.9 (C-4), 78.8 (C-6), 70.7 (C-5), 69.7 (C-13), 65.2 (C-3), 35.6 (C-11), 24.5 (C-12), and 4.8 (C-1). EIMS m/z 200 [M]+; HREIMS m/z 200.0828 (M; calcd for C13H12O2, 200.0837).

Compound 3: colorless amorphous solid; [a]D22 0.1 (c 0.6, CHCl3); 1H-NMR (CDCl3, 500MHz) 6.69 (1H, d, J = 6.0 Hz, H-8), 6.20 (1H, dd, J = 6.0, 2.0 Hz, H-9), 4.93 (1H, brs, H-6), 4.18 (1H, ddd, J = 8.0, 8.0, 3.0 Hz, H-13a), 3.99 (1H, ddd, J = 8.0, 8.0, 8.0 Hz, H-13b), 2.24 (1H, m, H-12a), 2.15 (1H, m, H-11a), 2.07 (1H, m, H-12b), 2.05 (1H, m, H-11b), and 1.98 (3H, d, J = 1.0 Hz, H-1); and 13C-NMR (CDCl3, 125 MHz) 168.8 (C-7), 135.7 (C-9), 126.0 (C-8), 120.8 (C-10), 79.9 (C-6), 79.7 (C-2), 76.3 (C-4), 71.5 (C-5), 69.7 (C-13), 64.9 (C-3), 35.5 (C-11), 24.5 (C-12), and 4.7 (C-1). EIMS m/z 200 [M]+; HREIMS m/z 200.0844 (M; calcd for C13H12O2, 200.0837).

Based on these spectra, compounds 2 and 3 were identified as (Z)- and (E)-tonghaosu, respectively.

Measurement of (Z)- and (E)-tonghaosu in the samples of chrysanthemi flos

Aliquot of the dried capitulum of C. indicum (5.0 g) was extracted using 100 ml of methanol with sonication for 30 min at room temperature, and the solution was filtered with paper. The procedure was repeated twice with the resulting residue. The solution was then evaporated at 42°C under reduced pressure and lyophilized to yield the extract. Three replicate extracts were prepared from one sample. Each methanol extract (50 μg) or (E)-tonghaosu (20 ng, 0.10, 0.50, and 1.0 μg) was injected to HPLC column (Cosmosil 5C18-AR-II, 4.6 × 150 mm, Nacalai) and eluted with water/acetonitrile 1:1 (1.0 ml/min) to measure the peak areas of (Z)- and (E)-tonghaosu (retention times, 8.8 and 11.3 min, respectively) detected with ultraviolet absorbance at 238 nm. A linear regression line with r2 value > 0.999 was plotted within the range from 20 ng to 1.0 μg of (Z)- and (E)-tonghaosu, and the detection limit was approximately 0.5 ng.

Assay for peroxisome proliferator-activated receptor-γ agonistic activity

HEK293 (human embryonic kidney) cells were maintained in Dulbecco's modified minimum essential medium (DMEM, Nacalai) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich, St. Louis, MO, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Nacalai) under 5% CO2 atmosphere at 37°C. To prepare defatted FBS, 2 g of Cab-O-sil® M-5 (Sigma) was added in 25 ml of FBS and stirred at 4°C overnight. The FBS was then centrifuged (1.5 × 103 ×g) at 4°C for 1 h and sterilized using membrane filtration. The cells (1.5 × 105 cells/well) were seeded in 24-well culture plates and incubated in DMEM containing 10% defatted FBS at 37°C overnight. The cells were dual transfected with 0.5 μg each of pCMV-SPORT-PPAR-γ vector (Open Biosystems of Thermo Scientific, Waltham, MA, USA) and pGL4.26 PPRE5 × 48 vector, that was constructed by inserting five repeats of PPAR-γ response sequence into HindIII site of pGL4.26 luciferase reporter vector (Promega, Madison, WI, USA), by lipofection using X-tremeGENE transfection reagents (Roche Applied Science, Penzberg, Upper Bavaria, Germany) according to the manufacturer's protocol. After transfection, the medium was exchanged with fresh medium containing 10% defatted FBS with or without test samples. After 24 h, the cells were harvested to determine the luciferase activities. Pioglitazone (Wako Pure Chemicals, Osaka) was used as a positive control. Luciferase activity was determined as the ratio of the sample to the chemiluminescence of control group. Experiments were conducted in triplicate or quadruplicate.

Statistics

To compare results among groups, we used a one-way analysis of variance (ANOVA), followed by Bonferroni/Dunnett's multiple t-test (PASQ Statistics version 18, SPSS, IBM, Armonk, NY, USA). Differences were considered statistically significant at values of P < 0.05.


   Results Top


MCI showed concentration-dependent PPAR-γ agonistic activity [Figure 1]. We partitioned n-hexane, ethyl acetate, and water-saturated butanol, and water layer from MCI, and both n-hexane and water layers showed a significant PPAR-γ agonistic activity at the concentration related to 100 μg/ml of the original CFM [Figure 2]a. Further, chromatography showed that fraction F from n-hexane layer exhibited a significant PPAR-γ agonistic activity at 20 μg/ml [Figure 2]b. Fraction F was further separated into 14 fractions, and one of these (F3) exhibited a significant PPAR-γ agonistic activity at 20 μg/ml (data not shown).
Figure 1: Peroxisome proliferator-activated receptor-γ agonistic activity of methanol extract of the capitulum of Chrysanthemum indicum. HEK293 cells were transiently transfected with pGL PPRE-luciferase reporter plasmid and pCMV-peroxisome proliferator-activated receptor-γ plasmid and incubated with the medium containing defatted fetal bovine serum for 24 h. methanol extract of the capitulum of Chrysanthemum indicum or pioglitazone (Pio) (30 μM) was dissolved in the media and further incubated for 24 h. Then, cells were lysed and the substrates for luciferase were added, and chemiluminescences were measured. Data are shown as the ratio of the control (without any treatment with methanol extract of the capitulum of Chrysanthemum indicum or pioglitazone) and expressed as mean ± standard error (n = 4). **P < 0.01, ***P < 0.001 versus control group evaluated by Bonferroni–Dunnett's multiple t-test

Click here to view
Figure 2: Peroxisome proliferator-activated receptor -γ agonistic activity of the fractions of methanol extract of the capitulum of Chrysanthemum indicum. (a) methanol extract of the capitulum of Chrysanthemum indicum was partitioned according to the descriptions of Materials and Methods, and the cells was treated with the medium containing each layer (Hex, hexane layer; AcOEt, ethyl acetate layer; BuOH, buthanol layer, H2O, water layer; Pio, pioglitazone) at the concentration related to the original methanol extract of the capitulum of Chrysanthemum indicum (100 μg/ml). (b) Fractionated hexane layer of methanol extract of the capitulum of Chrysanthemum indicum shown as Fr. A– P. The cells were treated with the medium containing each fraction (20 μg/ml). Co, Control; Pio, pioglitazone. Data are shown as the ratio of the control and expressed as mean ± standard error (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control group evaluated by Bonferroni–Dunnett's multiple t-test

Click here to view


Analysis of F3 by HPLC revealed three distinct peaks. Peak 2 and Peak 3 were designated as compounds 2 and 3, respectively. On the basis of MS, NMR, optical rotation analysis, and comparison with previous studies,[14] compounds 2 and 3 were identified as (Z)-tonghaosu and (E)-tonghaosu, respectively [Figure 3]. The contents of (Z)- and (E)-tonghaosu in MCI were 0.063 ± 0.019 (w/w) % and 0.12 ± 0.04 (w/w) %, respectively. (E)-tonghaosu showed a significant concentration-dependent PPAR-γ agonistic activity, but (Z)-tonghaosu did not at a concentration of 100 μg/ml [Figure 4].
Figure 3: Chemical structures of compound 2 and 3

Click here to view
Figure 4: Peroxisome proliferator-activated receptor - γ agonistic activity of (Z)and (E)tonghaosu. (Z)tonghaosu, (E)tonghaosu, or pioglitazone was dissolved in the media and further incubated for 24 h. Then, cells were lysed and the substrates for luciferase were added, and chemiluminescence was measured. Data are shown as the ratio to the control and expressed as mean ± standard error (n = 4). **P < 0.01 versus control group evaluated by Bonferroni–Dunnett's multiple t-test

Click here to view



   Discussion Top


PPAR-γ agonists have a wide spectrum of biological functions, including regulating metabolism, reducing inflammation, influencing the balance of immune cells, inhibiting apoptosis and oxidative stress, and improving endothelial function; moreover, they are widely prescribed to type II diabetes mellitus in Japan.[15]

In the present study, MCI at 100 μg/ml showed PPAR-γ agonistic activity approximately double of an experimental control. Previous study indicated that the hot water extract of C. morifolium edible flower (100 μg/ml) caused doubling of PPAR-γ mRNA expression in 3T3-L1 adipocyte cells.[16] Although Chrysanthemum species and the extracting methods are different from our study, the results are consistent with the previous report.[16]

We isolated the active constituents from CFM by activity-guided fractionation. Results show that PPAR-γ agonistic activities were distributed among several fractions of the extract, suggesting that Chrysanthemi Flos contained several active constituents. We isolated three peaks from the fraction exhibiting the highest activity and found that two of these (compounds 2and 3) had PPAR-γ agonistic activity. We further analyzed the chemical structures of compounds 2 and 3. Both compounds were geometric isomers of tonghaosu, and only (E)-isomer had PPAR-γ agonistic activity. The polyacetylene motif binding to carbon 6 of tonghaosu with different geometry would affect the binding of tonghaosu to the target site of PPAR-γ. Although conjugated linoleic acids have different anti-inflammatory activities among geometric isomers,[17] the geometric character of tonghaosu could affect the PPAR-γ agonistic activity. (E)-tonghaosu showed significant PPAR-γ agonistic activity at 3 μg/ml = about 15 μ M, suggesting that (E)-tonghaosu have about one-tenth activity of pioglitazone.


   Conclusion Top


We identified (E)-tonghaosu as PPAR-γ agonistic constituents in the capitulum of C. indicum. Considerable chemical modulation would be needed to derive a new PPAR-γ agonist using (E)-tonghaosu as the seed.

Acknowledgements

This study was partially supported by the A-STEP Program from Japan Science and Technology Agency, JST (2013).

Financial support and sponsorship

This study was partially supported by the A-STEP Program from Japan. Science and Technology Agency, JST (2013).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Pharmaceutical and Medical Device Regulatory Science Society of Japan. The Japanese Pharmacopoeia. 17th ed. Tokyo: Jiho; 2016.  Back to cited text no. 1
    
2.
Pharmacopoeia Commission of the Ministry of Health of the People's Republic of China. Pharmacopoeia of the People's Republic of China. Beijing: China Medical Science Press; 2015.  Back to cited text no. 2
    
3.
Bensky D, Clavey S, Stöger E. Chinese Herbal Medicine: Materia Medica. 3rd ed. Seattle: Eastland Press; 2004.  Back to cited text no. 3
    
4.
Namba T, Tsuda Y. Outline of Pharmacognosy a Textbook. Tokyo: Nankodo; 1990.  Back to cited text no. 4
    
5.
Luyen BT, Tai BH, Thao NP, Cha JY, Lee HY, Lee YM, et al. Anti-inflammatory components of Chrysanthemum indicum flowers. Bioorg Med Chem Lett 2015;25:266-9.  Back to cited text no. 5
    
6.
Yang WS, Kim D, Yi YS, Kim JH, Jeong HY, Hwang K, et al. AKT-targeted anti-inflammatory activity of the methanol extract of Chrysanthemum indicum var. albescens. J Ethnopharmacol 2017;201:82-90.  Back to cited text no. 6
    
7.
Jeong SC, Kim SM, Jeong YT, Song CH. Hepatoprotective effect of water extract from Chrysanthemumindicum L. flower. Chin Med 2013;8:7.  Back to cited text no. 7
    
8.
Chen YF, Zhao MH, Yan M, Shi GB, Hou GX, Huang Y, et al. Analgesic activity of the aqueous fraction from the ethanolic extract of Chrysanthemumindicum in mice. Pharmazie 2011;66:538-42.  Back to cited text no. 8
    
9.
Sun S, Jiang P, Su W, Xiang Y, Li J, Zeng L, et al. Wild Chrysanthemum extract prevents UVB radiation-induced acute cell death and photoaging. Cytotechnology 2016;68:229-40.  Back to cited text no. 9
    
10.
Park S, Lee JB, Kang S. Topical application of Chrysanthemum indicum L. Attenuates the development of atopic dermatitis-like skin lesions by suppressing serum IgE levels, IFN-γ, and IL-4 in Nc/Nga mice. Evid Based Complement Alternat Med 2012;2012:821967.  Back to cited text no. 10
    
11.
Kim SJ, Myung NY, Shin BG, Lee JH, So HS, Park RK, et al. Protective effect of a Chrysanthemumindicum containing formulation in cadmium-induced ototoxicity. Am J Chin Med 2011;39:587-600.  Back to cited text no. 11
    
12.
Kim TW, Kim YJ, Park SR, Seo CS, Ha H, Shin HK, et al.Chrysanthemum indicum attenuates cisplatin-induced nephrotoxicity both in vivo and in vitro . Nat Prod Commun 2015;10:397-402.  Back to cited text no. 12
    
13.
Yamamoto J, Yamane T, Oishi Y, Shimizu M, Tadaishi M, Kobayashi-Hattori K, et al.Chrysanthemum promotes adipocyte differentiation, adiponectin secretion and glucose uptake. Am J Chin Med 2015;43:255-67.  Back to cited text no. 13
    
14.
Martínez V, Barberá O, Sánchez-Parareda J, Alberto Marco J. Phenolic and acetylenic metabolites from Artemisia assoana. Phytochemistry 1987;26:2619-24.  Back to cited text no. 14
    
15.
Ivanova EA, Parolari A, Myasoedova V, Melnichenko AA, Bobryshev YV, Orekhov AN, et al. Peroxisome proliferator-activated receptor (PPAR) gamma in cardiovascular disorders and cardiovascular surgery. J Cardiol 2015;66:271-8.  Back to cited text no. 15
    
16.
Yamamoto J, Tadaishi M, Yamane T, Oishi Y, Shimizu M, Kobayashi-Hattori K, et al. Hot water extracts of edible Chrysanthemummorifolium Ramat. Exert antidiabetic effects in obese diabetic KK-Ay mice. Biosci Biotechnol Biochem 2015;79:1147-54.  Back to cited text no. 16
    
17.
Reynolds CM, Roche HM. Conjugated linoleic acid and inflammatory cell signalling. Prostaglandins Leukot Essent Fatty Acids 2010;82:199-204.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

Top
   
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures

 Article Access Statistics
    Viewed1296    
    Printed32    
    Emailed0    
    PDF Downloaded92    
    Comments [Add]    

Recommend this journal