Adenophora triphylla (Thunb.) A. DC. var. triphylla (Family: Campanulaceae) is distributed in Japan, Korea, and China. It is locally known as “Saiyousyajin” in Japan and the roots are used in traditional medicine to treat chronic bronchitis and whooping cough, and also as anti-inflammatory and anti-tussive agents. Till now, there is no report on the chemical constituents of aerial parts. Thus, the main aim of this study was to isolate and identify major chemical constituents of aerial parts of A. triphylla var. triphylla, and to evaluate their free radical scavenging activity. The 70% methanol extract of the aerial parts was subjected to repeated column chromatography using MCI gel CHP-20P, Sephadex LH-20, ODS and silica gel columns to isolate the five phenolic components (1-5). Free radical scavenging activity of the extract and compounds was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity method. The structures of the isolated compounds were elucidated as luteolin (1), luteolin 4’-O-b-glucopyranoside (2), luteolin 7-O-b-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) based on their nuclear magnetic resonance (NMR) spectral data and comparison with literature values. All these compounds were isolated for the first time from A. triphylla var. triphylla. Extract showed weak free radical scavenging activity. Among isolated compounds, luteolin (1), luteolin 7-O-b-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) showed potent free radical scavenging activity. Results from this study suggest that the aerial parts of A. triphylla var. triphylla might be a potential plant source for the development of functional foods, however further detailed research is necessary

Devkota, Hari Prasad

Hori, Kengo

English

Nepal Journal of Biotechnology

Nepal J Biotechnol. 2 0 2 0  Ju l y ; 8(1):12-16 DOI: https://doi.org/10.3126/njb.v8i1.30205Research article ©NJB, BSN 12 Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity Kengo Hori1, Hari Prasad Devkota 1,2   1Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan  2Program for Leading Graduate Schools, Health life Sciences: Interdisciplinary and Glocal Oriented (HIGO) Program, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan Article history:-Received: 17 May 2020; Revised: 19 Jun 2020; Accepted: 25 Jun 2020; Published online: 31 Jul 2020 Abstract Adenophora triphylla (Thunb.) A. DC. var. triphylla (Family: Campanulaceae) is distributed in Japan, Korea, and China. It is locally known as “Saiyousyajin” in Japan and the roots are used in traditional medicine to treat chronic bronchitis and whooping cough, and also as anti-inflammatory and antitussive agents. Till now, there is no report on the chemical constituents of aerial parts. Thus, the main aim of this study was to isolate and identify major chemical constituents of aerial parts of A. triphylla var. triphylla, and to evaluate their free radical scavenging activity. The 70% methanol extract of the aerial parts was subjected to repeated column chromatography using MCI gel CHP-20P, Sephadex LH-20, ODS and silica gel columns to isolate the five phenolic components (1-5). Free radical scavenging activity of the extract and compounds was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity method. The structures of the isolated compounds were elucidated as luteolin (1), luteolin 4’-O-β-glucopyranoside (2), luteolin 7-O-β-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) based on their nuclear magnetic resonance (NMR) spectral data and comparison with literature values. All these compounds were isolated for the first time from A. triphylla var. triphylla. The extract showed weak free radical scavenging activity. Among isolated compounds, luteolin (1), luteolin 7-O-β-glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) showed potent free radical scavenging activity. Results from this study suggest that the aerial parts of A. triphylla var. triphylla might be a potential plant source for the development of functional foods, however further detailed research is necessary. Keywords: Adenophora triphylla; Saiyousyajin; Phenolic compounds; Free radical scavenging  Corresponding author, email: devkotah@kumamoto-u.ac.jp Introduction Medicinal plants and their phytochemicals have played a vital role in human healthcare as an important source of traditional medicines, drug discovery and development of nutritional and functional foods [1–3]. However, many plant species are yet to be explored for their chemical constituents and potential biological activities and health-promoting effects. The genus Adenophora belonging to belonging to Campanulaceae family consists of about 62 species distributed in East Asia and Europe, among which, 12 species are distributed in Japan [4]. Adenophora triphylla (Thunb.) A. DC. var. triphylla (“Saiyousyajin” in Japanese) and A. triphylla (Thunb.) A. DC. var. japonica (Regel) H. Hara (“Tsuriganeninjin” in Japanese) are among many varieties of plant A. triphylla (Thunb.) A. DC. and are distributed in Japan, Korea, and China [4, 5]. Roots of both of these plants are used in traditional medicine to treat chronic bronchitis and whooping cough and as an anti-inflammatory and antitussive agents [5–8]. There have been many studies on the chemical constituents/biological activities of roots and leaves of A. triphylla var. japonica [5, 7, 9, 10]. Kim et al. [5] reported the high phenolic and flavonoid contents and potent free radical scavenging activities of leaves of A. triphylla var. japonica, however, no  Nepal Journal of Biotechnology Publisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313  Journal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 Nepal J Biotechnol. Ju l y  2 0 2 0 ;8(1):12-16  Hori and Devkota ©NJB, BSN 13 such studies are reported on the aerial parts of A. triphylla var. triphylla for the best of our knowledge. Thus, in this study, the main aim was to isolate and identify major chemical constituents of aerial parts of A. triphylla var. triphylla and to evaluate their free radical scavenging activity. Materials and Methods General Experimental Procedure 1H- and 13C- NMR spectra were measured on BRUKER AVANCE 600 NMR Spectrometer (Bruker, Billerica, MA, USA) (1H-NMR: 600 Hz and 13C-NMR: 150 Hz). Chemical shift values (δH and δC) are given in ppm with reference to tetramethyl silane (TMS). Column chromatography (CC) was carried out with MCI gel CHP20P (75 ~ 150 μm, Mitsubishi Chemical Industries Co. Ltd., Tokyo, Japan), Sephadex LH-20 (Amersham Pharmacia Biotech, Tokyo, Japan) and silica gel 60 (0.040-0.063 mm, Merck KGaA, Darmstadt, Germany). Thin layer chromatography (TLC) was performed on a pre-coated silica gel 60 F254 (Aluminum sheet, Merck KGaA, Darmstadt, Germany).  Plant Material The aerial parts (stems and leaves) of A. triphylla var. triphylla were collected from Mt. Tawarayama Kumamoto, Japan in August 2015 and shade dried for two weeks. Plant material was identified by Mr. Masato Watanabe, Technical Officer, School of Pharmacy, Kumamoto University.  Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Aldrich, Co. (Tokyo, Japan). 6–Hydroxy- 2, 5, 7, 8– tetramethylchroman–2-carboxylic acid (Trolox) was from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and 2-morpholinoethanesulfonic acid monohydrate (MES) was purchased from Dojindo Chemical Research (Kumamoto, Japan). Extraction and Isolation The dried aerial parts (2600 g) were then extracted twice with 70% MeOH (18 L). The combined extract was evaporated under reduced pressure to give 414.0 g of extract. The extract was suspended in water and subjected on MCI gel CHP20P column chromatography (CC) and eluted successively with water, 40%, 70% and 100% MeOH to give eight fractions (1~8). Fraction 2 (109.4 g, H2O eluate) was subjected on Sephadex LH-20 CC (H2O) to obtain four subfractions (2-1~2-4). Subfraction 2-3 was subjected on MCI gel CC (H2O) to afford compound 5 (153.8 mg). Fraction 7 (3.05 g, 80% MeOH eluate) was subjected on Sephadex LH-20 CC (MeOH) to afford compound 1 (174.4 mg). Fraction 6 (4.0 g, 60% MeOH eluate) was subjected to Sephadex LH-20 CC (H2O-MeOH; 1:1) to afford compound 2 (211.4 mg).  Table 1. Proton NMR spectroscopic data of compound 1 - 4 (H, mult. (J in Hz)) Position 1a 2 b 3 a 4 b 3 6.65, s 6.56, s 6.74, s 6.57, s 6 6.17, d (2.1) 6.18, d (2.0) 6.43, d (2.1) 6.38, d (2.1) 8 6.43, d (2.1) 6.41, d (2.0) 6.78, d (2.1) 6.73, d (2.1) 2’ 7.40, d (2.3) 7.41, d (2.3) 7.40, d (2.1) 7.38, d (2.5) 5’ 6.87, d (8.2) 7.28, d (8.5) )6.89, d (8.4) )6.89, d (8.5) )6’ 7.38, dd (8.2, 2.3) 7.39, dd (8.5, 2.3) 7.43, dd (8.4, 2.1) 7.40, dd (8.5, 2.5) Glc-1 4.93, d (7.5) 5.05, d (7.5) 5.18, d (6.7) Glc-2 3.40-4.00 3.40-4.00 3.40-4.00 Glc-3 3.40-4.00 3.40-4.00 3.40-4.00 Glc-4 3.40-4.00 3.40-4.00 3.40-4.00 Glc-5 3.40-4.00 3.40-4.00 3.40-4.00 Glc-6 3.40-4.00 3.40-4.00 3.40-4.00 Rha-1 5.28, d (1.2) Rha-2 3.40-4.00 Rha-3 3.40-4.00 Rha-4 3.40-4.00 Rha-5 3.40-4.00 Rha-6 1.33, d (6.2) a in DMSO-d6, b in CD3OD Nepal J Biotechnol. Ju l y  2 0 2 0 ;8(1):12-16  Hori and Devkota ©NJB, BSN 14 Fraction 3 (14.3 g) and fraction 4 (2.5 g) were combined and subjected to Sephadex LH-20 CC (50% MeOH) followed by ODS CC (20%, 30%, 40%  MeOH) and silica gel CC (CH2Cl2: MeOH: H2O =8:2:0.1) to afford compound 3 (1610.7 mg) and 4 (7.0 mg).  Measurement of DPPH Free Radical Scavenging Activity The antioxidant potential was determined using the DPPH free radical scavenging method as described previously [3].  Results and Discussion  The 70% methanol extract of aerial parts of A. triphylla var. triphylla was subjected to various column chromatographic methods including MCI gel CHP20P, Sephadex LH-20, ODS and silica gel to afford five compounds. The structures of these compounds were elucidated as luteolin (1) [11], luteolin 4’-O- β -glucopyranoside (2) [12], luteolin 7-O- β -glucopyranoside (3) [11, 12], luteolin 7-O-neohesperidoside (4) [13] and chlorogenic acid (5) [14] (Figure 1) based on their NMR spectral data and comparison with literature values. Proton and 13C NMR data for compounds 1-4 are provided in Table 1 and Table 2, respectively. All of these compounds were isolated for the first time from A. triphylla var. triphylla. Previously, Hashiba et al. [10] had reported flavonoids including luteolin (1), luteolin 7-O- β -glucopyranoside (3), luteolin 4’7-di-O-β-glucopyranoside, quercetin, quercetin 3-O-β-glucopyranoside from the leaves of A. triphylla var. japonica. Characterization of similar flavonoids in the aerial parts of A. triphylla var. triphylla in this study suggests their chemotaxonomic similarity and these compounds can be used as the chemotaxonomic markers for these varieties. Further studies on other varieties of A. triphylla or other species of Adenophora may help explore their further similarity. The 70% extract and all isolated compounds were evaluated for their DPPH free radical scavenging activity (Table 3). The extract showed weak free Table 2. 13C NMR spectroscopic data of compound 1 - 4 Position 1a 2 b 3 a 4 b 2 146.8 165.4 164.5 166.9 3 135.7 105.1 103.2 105.6 4 175.8 183.8 182.0 184.0 5 160.7 163.2 161.1 163.0 6 98.1 100.3 99.6 102.5 7 163.8 166.1 163.0 164.4 8 93.3 95.1 94.8 97.3 9 156.1 159.4 156.7 159.0 10 102.9 105.5 105.4 107.1 1’ 121.9 127.2 121.4 123.5 2’ 115.4 118.0 113.6 114.3 3’ 145.0 148.6 145.8 147.1 4’ 147.6 150.0 150.1 151.2 5’ 115.5 114.9 116.0 117.0 6’ 119.9 120.0 119.2 120.6 Glc-1 103.3 99.9 99.6 Glc-2 74.8 73.1 78.3 Glc-3 77.5 76.4 79.0 Glc-4 71.3 69.6 71.4 Glc-5 78.5 77.2 79.1 Glc-6 62.3 60.7 62.5 Rha-1 101.0 Rha-2 72.2 Rha-3 72.2 Rha-4 74.0 Rha-5 70.0 Rha-6 18.3 a in DMSO-d6,  b in CD3OD. Figure 1. Chemicalstructures of compounds isolated from A. triphylla var. triphylla Nepal J Biotechnol. Ju l y  2 0 2 0 ;8(1):12-16  Hori and Devkota ©NJB, BSN 15 radical scavenging activity with IC50 value of 248.3 µg/mL. Compounds 1, 3, 4, and 5 showed potent free radical scavenging activity with IC50 values of 10.6, 14.3, 20.4 and 19.3 µg/mL, respectively as compared to positive control Trolox (IC50=12.8 µg/mL). Table 3. IC50 (µg/mL) values of extract and isolated compounds of A. triphylla var. triphylla for DPPH free radical scavenging activity Compounds IC50 value (µg/mL) 70% Methanol Extract 248.3±2.4 Luteolin (1) 10.6±0.7 Luteolin 4’-O- β -glucopyranoside (2) 62.9±0.9 Luteolin 7-O- β -glucopyranoside (3) 14.3±1.5 Luteolin 7-O-neohesperidoside (4) 20.4±2.4 Chlorogenic acid (5) 19.3±2.1 Trolox (positive control) 12.8± 1.1 In recent years, there is growing attention on the plant-based functional foods. Previously, Kim et al. [5] reported the high phenolic and flavonoid contents and free radical scavenging activities of leaves of A. triphylla var. japonica but the active compounds were not reported. In our study, the extract of aerial parts of A. triphylla var. triphylla showed weak activity but the isolated compounds showed strong free radical scavenging activity. Hashiba et al. [10] had also reported similar flavonoids from A. triphylla var. japonica, hence, luteolin derivatives can be regarded as the active antioxidant compounds in both of these verities. Flavonoids including luteolin derivatives are well reported as strong antioxidant phytochemicals with various health-promoting and disease prevention activities [15–20]. Further detailed research on these plants may help in the development of functional foods. Conclusion Five bioactive phenolic compounds; luteolin (1), luteolin 4’-O- β -glucopyranoside (2), luteolin 7-O- β -glucopyranoside (3), luteolin 7-O-neohesperidoside (4) and chlorogenic acid (5) were isolated for the first time from the leaves A. triphylla var. triphylla. Some of the isolated compounds showed potent free radical scavenging activity. The aerial parts of A. triphylla var. triphylla might be an important plant source for the development of functional foods. However, detailed research related to pharmacological activity and safety are necessary in future. Author’s Contribution HPD conceived the idea and designed the study. Both authors contributed to experiments and analysis, and wrote the manuscript. Competing Interest No competing interests were disclosed. Funding This work was supported partially by Program for Leading Graduate Schools, Health life Sciences: Interdisciplinary and Glocal Oriented (HIGO) Program, MEXT, Japan Acknowledgments We are grateful to Ms. Teruo Tanaka of Institute of Resource Development and Analysis, Kumamoto University for the measurement of NMR.  Ethical Approval and Consent Not applicable. References 1. Belwal T, Devkota HP, Hassan HA, Ahluwalia S, RamadanMF, Mocan A, Atanasov AG. Phytopharmacology of Acerola(Malpighia spp.) and its potential as functional food. Trends inFood Science & Technology. 2018 Apr 1;74:99-106. https://doi.org/10.1016/j.tifs.2018.01.0142. Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, LinderT, Wawrosch C, Uhrin P, Temml V, Wang L, Schwaiger S,Heiss EH, Rollinger JM. Discovery and resupply ofpharmacologically active plant-derived natural products: Areview. Biotechnology advances. 2015 Dec 1;33(8):1582-614. https://doi.org /10.1016/j.biotechadv.2015.08.0013. Dirar AI, Alsaadi DH, Wada M, Mohamed MA, Watanabe T,Devkota HP. Effects of extraction solvents on total phenolicand flavonoid contents and biological activities of extracts from Sudanese medicinal plants. South African Journal ofBotany. 2019 Jan 1;120:261-7. https://doi.org/ 10.1016/j.sajb.2018.07.0034. Ohashi H, Monda Y, Murata J, Yonekura K, Kihara H. WildFlowers of Japan. Tokyo: Heibonsha. 2017. 5. Kim JH, Hong JY, Shin SR, Yoon KY. Comparison ofantioxidant activity in wild plant (Adenophora triphylla) leaves and roots as a potential source of functional foods.International journal of food sciences and nutrition. 2009 Jan 1;60(sup2):150-61. https://doi.org/10.1080/096374809029565946. Suzuki H. Encyclopedia of Traditional Oriental Medicine.Ishiyaku Publishers. 2005. 7. Ahn EK, Oh JS. Lupenone isolated from Adenophora triphyllavar. japonica extract inhibits adipogenic differentiationthrough the downregulation of PPARγ in 3T3‐ L1 cells.Phytotherapy Research. 2013 May;2 7(5):7 61-6. https://doi.org/10.1002/ptr.4779Nepal J Biotechnol. Ju l y  2 0 2 0 ;8(1):12-16  Hori and Devkota ©NJB, BSN 16 8. Watanabe M, Devkota HP, Sugimura K, Watanabe T. AGuidebook of Medicinal Plant park. Kumamoto:School ofPharmacy, Kumamoto University. 2018. 9. Konno C, Saito T, Oshima Y, Hikino H, Kabuto C. Structureof methyl adenophorate and triphyllol, triterpenoids ofAdenophora triphylla var. japonica roots. Planta medica. 1981Jul;42(07):268-74. https://doi.org/10.1055/s-2007-97163910. Hashiba K, Iwashina T, Matsumoto S. Variation in the qualityand quantity of flavonoids in the leaves of coastal and inlandCampanula punctata. Biochemical systematics and ecology.2006 Dec 1;34(12):854-61. https://doi.org/10.1016/j.bse.2006.04.012 11.  Harborne JB, Mabry TJ. The flavonoids. Boston, MA: SpringerUS. 1982. https://doi.org/10.1007/978-1-4899-2915-0 12.  Lee MH, Son YK, Han YN. Tissue factor inhibitory flavonoidsfrom the fruits of Chaenomeles sinensis. Archives of pharmacalresearch. 2002 Dec 1;25(6):842. https://doi.org/10.1007/BF0297700213. Li YL, Li J, Wang NL, Yao XS. Flavonoids and a newpolyacetylene from Bidens parviflora Willd. Molecules. 2008Aug;13(8):1931-41. https://doi.org/10.3390/molecules1308193114. Devkota HP, Joshi KR, Ozasa T, Fukunaga S, Yoshida N,Yahara S. Steroidal glycosides from the fruits, aerial parts andtubers of potato (Solanum tuberosum). J. Pharmacognosy &Phytochemistry. 2015;3:252-5. 15.  Khan H, Sureda A, Belwal T, Çetinkaya S, Süntar İ, Tejada S,Devkota HP, Ullah H, Aschner M. Polyphenols in thetreatment of autoimmune diseases. Autoimmunity reviews.2019 Jul 1;18(7):647-57. https://doi.org/10.1016/j.autrev.2019.05.001 16. Rice-Evans C. Flavonoids and isoflavones: absorption,metabolism, and bioactivity. Free Radical Biology andMedicine. 2004;7(36):827-8. https://doi.org/10.1016/j.freeradbiomed.2003.12.01217. Raffa D, Maggio B, Raimondi MV, Plescia F, Daidone G.Recent discoveries of anticancer flavonoids. European journalof medicinal chemistry. 2017 Dec 15 ; 142 :2 13-28. https://doi.org /10.1016/j.ejmech.2017.07.03418. Rodriguez-Mateos A, Vauzour D, Krueger CG,Shanmuganayagam D, Reed J, Calani L, Mena P, Del Rio D,Crozier A. Bioavailability, bioactivity and impact on health of dietary flavonoids and related compounds: an update.Archives of toxicology. 2014 Oct 1;88(10):1803-53. https://doi.org /10.1007/s00204-014-1330-719. López-Lázaro M. Distribution and biological activities of theflavonoid luteolin. Mini reviews in medicinal chemistry. 2009Jan 1;9(1):31-59. https://doi.org/10.2174/13895570978700171220. Lin Y, Shi R, Wang X, Shen HM. Luteolin, a flavonoid withpotential for cancer prevention and therapy. Current cancerdrug targets. 2008 Nov 1;8(7):634-46. https://doi.org/10.2174/156800908786241050

Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity

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Phenolic Compounds from the Aerial Parts of Adenophora triphylla (Thunb.) A. DC. var. triphylla and their Free Radical Scavenging Activity

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