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Adv Biomed Res 2021,  10:23

Isolation and characterization of methylated flavones from Artemisia kermanensis

Department of Pharmacognosy, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Submission02-Jan-2021
Date of Acceptance21-Jun-2021
Date of Web Publication29-Sep-2021

Correspondence Address:
Dr. Zeinab Yazdiniapour
Department of Pharmacognosy, Isfahan University of Medical Sciences, Isfahan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/abr.abr_1_21

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Background: Artemisia kermanensis Podl. is a green aromatic perennial shrub that belongs to the family Asteraceae and it grows widely in central deserts and south-eastern mountains of Iran such as Taftan Mountain in Sistan and Baluchestan Province. Artemisia species have been used traditionally as a remedy for various feverous diseases, including malaria, treatment of colds, infections, parasites, inflammations of the liver, as well as dyspepsia, diabetes, hypertension, and so many other conditions. Materials and Methods: Air-dried A. kermanensis extraction from all parts of the plant was done using different organic solvents. The methanolic extract was selected for isolation of flavonoids, using thin-layer chromatography. The chemical structures of the isolated compounds were determined based on analysis of mass and nuclear magnetic resonance spectra. Results: Two flavone aglycones were isolated and identified for the first time from this plant's methanolic extract, including 5,7-dihydroxy-3',4',6-trimethoxyflavone (eupatilin) and 5,7,3'-Trihydroxy-6,4',5'-trimethoxyflavone. Conclusions: Eupatilin is known for its anticancer, antioxidant, and anti-inflammatory activities. In future researches on A. kermanensis, as a rich source of these flavone compounds, it is wise to investigate for the proven eupatilin's biological activities that have been mentioned.

Keywords: Asteraceae, eupatilin, flavonoids, flavones

How to cite this article:
Yazdiniapour Z, Yegdaneh A, Akbari S. Isolation and characterization of methylated flavones from Artemisia kermanensis. Adv Biomed Res 2021;10:23

How to cite this URL:
Yazdiniapour Z, Yegdaneh A, Akbari S. Isolation and characterization of methylated flavones from Artemisia kermanensis. Adv Biomed Res [serial online] 2021 [cited 2022 Sep 29];10:23. Available from:

  Introduction Top

Artemisia kermanensis Podl. is a green aromatic perennial plant that belongs to the family Asteraceae, and it grows widely in central deserts and south-eastern mountains of Iran such as Taftan mountain in Sistan and Baluchestan Province. So far, A. kermanensis has been reported for its antimicrobial and antioxidant effects.[1] In general, Artemisia species has been used traditionally as a remedy for various feverous diseases, including malaria, treatment of colds, infections, parasites, inflammations of the liver, dyspepsia, diabetes, hypertension, and other conditions,[2],[3],[4],[5] and in modern medicine, Grech-Baran and Pietrosiuk[6] reported the synthesis of two drugs, artemisinin and arglabin, first isolated from Artemisia species[7] using for the treatment of malaria and multiple tumor cell lines, respectively.[8]

A variety of secondary metabolites has been discovered in Artemisia genus so far, such as terpenoids, flavonoids, coumarins, caffeoylquinic acids, sterols, and acetylenes.[9],[10] The flavonoids include apigenin, luteolin, chrysoeriol, kaempferol, rhamnocitrin, quercetin, tamarixetin, mikanin, casticin, cirsineol, eupatin, mearnsetin, chrysosplenol E and flavonoid glycosides include kaempferol-3-O-glucoside and isorhamnetin 3-glucoside.[11]

In this study, the known natural pharmacologically active flavone, 5,7-dihydroxy-3',4',6-trimethoxyflavone (eupatilin), 1 and 5, 7, 3'-Trihydroxy-6,4',5'-trimethoxyflavanone, 2 has been isolated for the first time, in large amounts.

  Materials and Methods Top

General experimental procedures

1H and 13C NMR spectra were recorded by Bruker 400 (1H at 400 MHz and 13C at 100 MHz) spectrometers using the solvent signal for calibration (CDCl3: δΗ 7.26, δC 77.0; CD3OD: δH 3.31, δC 49.0). The multiplicities of 13C NMR resonances were determined by DEPT experiments. ESIMS spectrometer was done on LCQ mass spectrometer using MeOH as the solvent. Medium-pressure liquid chromatography (MPLC) was performed on a Büchi 861 apparatus using glass columns (26 mm × 460 mm i.d.) and millipore silica gel (15–40 μm) as the stationary phase. Thin-layer chromatography (TLC) was performed on silicon dioxide (SiO2) plates with hexane/acetone (7:3) as a mobile phase and cerium sulfate in 2 N sulfuric acid (H2SO4) as a reagent for visualizing the spots.

Plant material

A. kermanensis was authenticated and collected by Mohammad Amir Heidari in the September of 2018 from Taftan Mountain in south-east of Iran. A voucher specimen (4001) was deposited in the Herbarium of Pharmacy Faculty of Medical Sciences University of Isfahan. The collected plan was air dried away from the direct sunlight.

Extraction and isolation

5.0 kg of all parts of A. kermanensis was air-dried and extracted several times by maceration method. The solvent combination was two parts of dichloromethane, one part acetone. The extract was filtered and then evaporated under reduced pressure to get crude extract. The crude extract was coated on reverse silica gel and treated with methanol: water (7:3) to remove chlorophyll from the extract. The methanolic extract was filtered and the solvent evaporated and a waxy extract was left.

The methanolic crude extract (288.2 g) was pulverated by normal silica gel and was subjected to fractionation using MPLC. The pulverated extract was loaded on a previously packed MPLC silica gel column, fractionated using a gradient solvent system from n-hexane 100% to EtOAc 100%. The fractions were collected in 250 cc volumes, riched to 185 fractions. They were analyzed by TLC and combined into 23 fractions that were concentrated and kept for later use. Before the combination, the fractions were given a few days in room temperature in case of possible crystallization. Fortunately, considerable amounts of yellow crystals were observed and collected from the middle polarity zone tubes. TLC analysis showed three compounds that were isolated one after another. They were isolated from 19 tubes, and the first crystal was pure. The second compound isolated by preparative TLC and extra fractionation by column chromatography to the isolation of the third compound was failed [Figure 1].
Figure 1: Chemical structures of compounds 1 and 2

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  Results Top

Two methylated flavones [Figure 1] were isolated from the methanol soluble part of the acetone/DCM extract of A. kermanensis by silica gel MPLC and preparative TLC methods. Their structure elucidation was obtained by extensive spectroscopic analysis, including NMR and ESIMS experiments.

Compound 1 was obtained as yellow crystals. The ESIMS (negative ions) of 1 showed a molecular ion peak at m/z 343.175 [M-H]- and m/z 345.237 [M + H]+ at ESIMS (positive ions), which following 13C NMR data suggested the molecular formula C18H16O7 and it was assigned to 1 [Table 1]. Its structural assignment was obtained by extensive use of NMR spectroscopy and in comparison, with literature data.[12] Preliminary inspection of the 1H NMR spectrum of 1 [Table 1] suggested the presence of a flavone skeleton methylated with three methoxy groups (3H singlets at δ3.95, 3.96, 4.02). The 1H NMR spectrum showed signals attributed to two hydroxyl groups 1H signals at 6.53 (bs) and 13.05 (s). Other signals in the 1H NMR spectrum at δ6.59 (s, H-3) indicated the presence of typical flavone aglycone type of flavonoids.[13] The proton spectrum also showed that four aromatic protons at 6.56 (s, H-8), 6.96 (d, J = 8.4 Hz, H-5'), 7.31 (d, J = 2 Hz, H-2'), 7.50 (dd, J = 8.4-2 Hz, H-6') were ascribable to protons on sp2 carbon on A and B ring of highly oxygenated flavones. Two of them appeared as doublets (J = 2 Hz) at δ 7.31 and 7.50, suggesting meta coupling, a doublet (J = 8.4) at 6.96 corresponding to ortho coupling and one as a singlet at δ 6.56.
Table 1: 1H and 13C nuclear magnetic resonance data of compounds 1 and 2 at 400 and 100 MHz

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13C NMR and DEPT experiments showed that the skeleton is composed of 15 carbons: five methines and ten quaternary carbons between δ 93.6 and 164.3, a carbonyl carbon at δ 183.1. Signals at δ 56.3, 56.3, and 61.1 indicate the presence of three methoxyl groups.

This evidence pointed to flavone skeleton for 1 with oxygenated carbons at positions 5, 6, 7, 3', and 4' as a dihydroxy-trimethoxyflavone. Comparison of NMR data with analog compounds reported in the literature pointed to the 5,7-Dihydroxy-3',4',6-trimethoxyflavone (eupatilin) structure.[14],[15]

Analysis of NMR and MS spectral data of the second yellow crystals obtained from 12 fractions of A. kermanensis by preparative TLC method showed the flavone aglycone skeleton. The molecular formula of C18H1608, determined by ESIMS analysis and 13C NMR data. 1H NMR spectrum of 2 exhibited a typical flavone skeleton with the signals of three distinct methyl groups (3H singlets at 3.94, 3.87, and 3.86). Aromatic protons showed two singlets and two doublets at δ 6.40 (s, H-8), 6.54 (s, H-3), 7.06 (d, J = 2 Hz, H-6'), and 7.10 (d, J = 2 Hz, H-2'). The characteristic small germinal values (2 Hz) indicated the presence of meta-aromatic protons. 13C NMR data were closely related to compound 1 and agreed with the molecular formula, thus showing 18 carbon signals. The carbon flavone skeleton, including five methines and ten quaternary carbons, was appeared between δ 97.3 and 164.8 and carbonyl carbon at δ 183.5. Methoxyl group carbons signals were exhibited at δ 56.8, 61.3, and 64.5. A comparative NMR and MS analysis of compound 2 with compound 1 indicated compound 2 to be the derivative of compound 1 with oxygenated carbons at positions 5, 6, 7, 3',4', and 5'.

Compound 2 was found identical in all the characteristics, including NMR and MS data, with 5, 7, 3'-Trihydroxy-6,4',5'-trimethoxyflavone previously isolated from Artemisia ludoviciana[12] and Artemisia frigida.[13]

  Discussion Top

Artemisia is a diversified genus encompassing about 500 species in the temperate regions of Europe, Asia, and North America. It has been reported as a rich source of flavonoids, including eupatilin.[16],[17] In general, flavonoids of Artemisia species have been demonstrated to have cytotoxic, antioxidant, antimalarial, antihemolytic, and estrogenic activities.[7],[18],[19],[20] As we discussed, there are two pharmacologically active flavone compounds, eupatilin, and its hydroxylated form, in A. kermanensis with their wide range of biological activity. Eupatilin is known for its anticancer,[17],[18],[21],[22],[23],[24],[25] anti-inflammatory,[26],[27] anti-oxidant,[28] neuroprotective,[29] and antiallergic[30] activities. It is speculated that eupatilin could be subjected to structural optimization to synthesize derivative analogs to reinforce its efficacy, minimize toxicity, and optimize absorption profiles, ultimately leading to potent drug candidates.

  Conclusions Top

In future researches on A. kermanensis, as a source of these flavone compounds, it is wise to investigate for the proven eupatilin's biological activities that have been mentioned earlier.


This study is the thesis of Soudeh Akbari submitted for fulfillment of the degree of Pharm D in Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran. NMR spectra were recorded at Central Laboratory, University of Isfahan, Iran.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Kazemi M, Dakhili M, Dadkhah A, Yasrebifar Z, Larijani K. J Med Plant Res 2011;5:18:4481-86.  Back to cited text no. 1
Patil G, Dass S, Chandra RJ. Artemisia afra and modern diseases. J Pharmacogenomics Pharmacoproteomics 2011;2:1-22.  Back to cited text no. 2
Suresh J, Reddy V, Rajan D, Ihsanullah M, Khan MJ. Antimicrobial activity of Artemisia abrotanum and Artemisia pallens. J Pharmacogn Phytochem Res 2010;3:18-21.  Back to cited text no. 3
Abad MJ, Bedoya LM, Apaza L, Bermejo P. The Artemisia L. genus: A review of bioactive essential oils. J Mol 2012;17:2542-66.  Back to cited text no. 4
Mighri H, Hajlaoui H, Akrout A, Najjaa H, Neffati M. Antimicrobial and antioxidant activities of Artemisia herba-alba essential oil cultivated in Tunisian arid zone. J C R Chim 2010;13:380-6.  Back to cited text no. 5
Grech-Baran M, Pietrosiuk A. Artemisia species in vitro cultures for production of biologically active secondary metabolites. BioTechnologia 2012;93:371-80.  Back to cited text no. 6
Ferreira JF, Luthria DL, Sasaki T, Heyerick A. Flavonoids from Artemisia annua L. as antioxidants and their potential synergism with artemisinin against malaria and cancer. Molecules 2010;15:3135-70.  Back to cited text no. 7
Lone SH, Bhat KA, Khuroo MA. Arglabin: From isolation to antitumor evaluation. Chem Biol Interact 2015;240:180-98.  Back to cited text no. 8
Koul B, Taak P, Kumar A. The Artemisia genus: A review on traditional uses, phytochemical constituents, pharmacological properties and germplasm conservation. J Glycomics Lipidomics 2017;7 (1):142.  Back to cited text no. 9
Brown GD, Liang GY, Sy LK. Terpenoids from the seeds of Artemisia annua. Phytochemistry 2003;64:303-23.  Back to cited text no. 10
Lee JY, Chang EJ, Kim HJ, Park JH, Choi SW. Antioxidative flavonoids from leaves of carthamus tinctorius. Arch Pharm Res 2002;25:313.  Back to cited text no. 11
Kim AR, Ko HJ, Chowdhury MA, Chang YS, Woo ER. Chemical constituents on the aerial parts of Artemisia selengensis and their IL-6 inhibitory activity. Arch Pharm Res 2015;38:1059-65.  Back to cited text no. 12
Mabry TJ, Markham KR, Thomas MB. The Systematic Identification of Flavonoids. New York etc., Springer. 1970.  Back to cited text no. 13
Liu YL, Mabry T. Flavonoids from Artemisia ludoviciana var. ludoviciana. Phytochemistry 1982;21:209-14.  Back to cited text no. 14
Liu YL, Mabry T. Two methylated flavones from Artemisia frigida. Phytochemistry 1981;20:309-11.  Back to cited text no. 15
Bora KS, Sharma A. The genus Artemisia: A comprehensive review. Pharm Biol 2011;49:101-9.  Back to cited text no. 16
Son JE, Lee E, Seo SG, Lee J, Kim JE, Kim J, et al. Eupatilin, a major flavonoid of Artemisia, attenuates aortic smooth muscle cell proliferation and migration by inhibiting PI3K, MKK3/6, and MKK4 activities. Planta Med 2013;79:1009-16.  Back to cited text no. 17
Yuan H, Lu X, Ma Q, Li D, Xu G, Piao G. Flavonoids from Artemisia sacrorum Ledeb. and their cytotoxic activities against human cancer cell lines. Exp Ther Med 2016;12:1873-8.  Back to cited text no. 18
Naqinezhad A, Nabavi SM, Nabavi SF, Ebrahimzadeh MA. Antioxidant and antihemolytic activities of flavonoid rich fractions of Artemisia tschernieviana Besser. Eur Rev Med Pharmacol Sci 2012;16 Suppl 3:88-94.  Back to cited text no. 19
Lee SJ, Chung HY, Maier CG, Wood AR, Dixon RA, Mabry TJ. Estrogenic flavonoids from Artemisia vulgaris L. J Agric Food Chem 1998;46:3325-9.  Back to cited text no. 20
Kim JS, Lee SG, Min K, Kwon TK, Kim HJ, Nam JO. Eupatilin inhibits adipogenesis through suppression of PPARγ activity in 3T3-L1 cells. Biomed Pharmacother 2018;103:135-9.  Back to cited text no. 21
Lee JY, Bae H, Yang C, Park S, Youn BS, Kim HS, et al. Eupatilin promotes cell death by calcium influx through ER-mitochondria axis with SERPINB11 inhibition in epithelial ovarian cancer. Cancers (Basel) 2020;12 (6):1459.  Back to cited text no. 22
Zhong WF, Wang XH, Pan B, Li F, Kuang L, Su ZX. Eupatilin induces human renal cancer cell apoptosis via ROS-mediated MAPK and PI3K/AKT signaling pathways. Oncol Lett 2016;12:2894-9.  Back to cited text no. 23
Wang X, Zhu Y, Zhu L, Chen X, Xu Y, Zhao Y, et al. Eupatilin inhibits the proliferation of human esophageal cancer TE1 cells by targeting the Akt-GSK3 β and MAPK/ERK signaling cascades. Oncol Rep 2018;39:2942-50.  Back to cited text no. 24
Li YY, Wu H, Dong YG, Lin BO, Xu G, Ma YB. Application of eupatilin in the treatment of osteosarcoma. Oncol Lett 2015;10:2505-10.  Back to cited text no. 25
Kim J, Kim Y, Yi H, Jung H, Rim YA, Park N, et al. Eupatilin ameliorates collagen induced arthritis. J Korean Med Sci 2015;30:233-9.  Back to cited text no. 26
Liu H, Hao J, Wu C, Liu G, Wang X, Yu J, et al. Eupatilin Alleviates lipopolysaccharide-induced acute lung injury by inhibiting inflammation and oxidative stress. Med Sci Monit 2019;25:8289-96.  Back to cited text no. 27
Du L, Chen J, Xing YQ. Eupatilin prevents H2O2-induced oxidative stress and apoptosis in human retinal pigment epithelial cells. Biomed Pharmacother 2017;85:136-40.  Back to cited text no. 28
Nageen B, Sarfraz I, Rasul A, Hussain G, Rukhsar F, Irshad S, et al. Eupatilin: A natural pharmacologically active flavone compound with its wide range applications. J Asian Nat Prod Res 2020;22:1-6.  Back to cited text no. 29
Song EH, Chung KS, Kang YM, Lee JH, Lee M, An HJ. Eupatilin suppresses the allergic inflammatory response in vitro and in vivo. Phytomedicine 2018;42:1-8.  Back to cited text no. 30


  [Figure 1]

  [Table 1]

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