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Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Flavone glucosides from Artemisia juncea
Bakhodir S. Okhundedaev, Markus Bacher, Rimma F. Mukhamatkhanova,
Ildar J. Shamyanov, Gokhan Zengin, Stefan Böhmdorfer, Nilufar Z.
Mamadalieva & Thomas Rosenau
To cite this article: Bakhodir S. Okhundedaev, Markus Bacher, Rimma F. Mukhamatkhanova, Ildar J. Shamyanov, Gokhan Zengin, Stefan Böhmdorfer, Nilufar Z. Mamadalieva & Thomas Rosenau (2019) Flavone glucosides from Artemisia�juncea, Natural Product Research, 33:15, 2169-2175, DOI: 10.1080/14786419.2018.1490901
To link to this article: https://doi.org/10.1080/14786419.2018.1490901
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Published online: 13 Nov 2018.
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Flavone glucosides from
Artemisia juncea
Bakhodir S. Okhundedaeva, Markus Bacherb, Rimma F. Mukhamatkhanovaa, Ildar J. Shamyanova, Gokhan Zenginc, Stefan B€ohmdorferb,
Nilufar Z. Mamadalievaaand Thomas Rosenaub a
Institute of the Chemistry of Plant Substances of the Academy Sciences of Uzbekistan, Tashkent, Uzbekistan;bDivision of Chemistry of Renewables, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna (BOKU University), Tulln, Austria;cDepartment of Biology, Faculty of Science, University of Selcuk, Konya, Turkey
ABSTRACT
A new flavone glucoside, 40,5-dihydroxy-30,50 ,6-trimethoxyflavone-7-O-b-D-glucoside was obtained from aerial parts of Artemisia juncea, together with the known flavone eupatilin (5,7-dihydroxy-30,40,6-trimethoxyflavone). The compounds were comprehensively analytically characterized by IR, UV, NMR and HR-MS, and their chemical structures ascertained. The EtOAc fraction of A. juncea showed the strongest DPPH radical scavenging ability as well as reducing power (in CUPRAC and FRAP assays) and phosphomo-lybdenum activity. This fraction also exhibited the strongest inhibitory effects on tyrosinase. Additionally, the best antidiabetic effects were observed for eupatilin and the CHCl3fraction.
ARTICLE HISTORY
Received 31 January 2018 Accepted 16 June 2018
KEYWORDS
Artemisia juncea; flavones; flavonoid; 40,5-dihydroxy-30, 50 ,6-trimethoxyflavone-7-O-b-D-glucoside; eupatilin; antioxidant; enzyme inhibitor 1. Introduction
The genus Artemisia L. (Anthemideae tribe) is one of the largest and most widely distributed genera of the Asteraceae family. It consists of more than 500 species which are mainly found in Asia, Europe and North America (Abad et al. 2012; Ornano et al.
CONTACTThomas Rosenau thomas.rosenau@boku.ac.at
Supplemental data for this article can be accessed athttps://doi.org/10.1080/14786419.2018.1490901.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
2019, VOL. 33, NO. 15, 2169–2175
2016; Lv et al. 2018; Peron et al. 2017). 180 Artemisia species are present in Central Asia, of which 45 are endemic to this zone. From these taxa, 36 grow in Uzbekistan, some of them in desert or semi-desert zones, and others in mountain areas. The exist-ence of 19 Central Asian endemic species of the genus in Uzbekistan is remarkable. Sixteen Artemisia species form shrubland communities in the desert and mountain zones of Uzbekistan. Most widespread communities are dominated by species of the subgenus Seriphidium (Kapustina et al.2001).
Artemisia juncea Kar. et Kir. is a perennial shrub (up to 60 cm high) growing in Central Asia and China on gravelly, sandy plains and also on clayey, gravelly and dry stony slopes from the river valleys up to mid-belt of mountains (Vvedenskii 1962). In folk medicine, infusion of this species has been used to treat epilepsy, typhoid fever, kidney diseases, and also as an anti-inflammatory and anthelmintic agent. Oily infusion of herbs (in linseed or almond oil) have also been administered to treat asthma, oedema, and convulsions (Karomatov 2012). Previous studies and phytochemical analyses of this plant have focused on isolation and identification of compounds from essential oils, mono- and sesquiterpenoids, sesquiterpene lactones, alkaloids, resins and tannins (Kapustina et al.2001; Mukhamatkhanova et al.2018).
Continuing the search for structurally unique and biologically active compounds, the aerial parts of A. juncea were investigated and as a result, one new flavone glucoside (1) (Figure 1), along with the already known flavonoid eupatilin (2), has been isolated and identified. Herein, we describe the isolation and structural elucidation of these compounds, as well as the results of evaluation of their antioxidant and enzyme inhibition activities.
2. Results and discussion
The ethyl acetate and chloroform fractions obtained from ethanolic extract from the aerial parts of A. juncea were subjected to column chromatography on silica gel nor-mal-phase and Sephadex LH-20, and provided flavone glucoside (1) and the previously described compound eupatilin (2), respectively.
Compound 1 was obtained as a yellow, amorphous solid. The IR spectrum of this compound showed strong absorptions indicating hydroxyl groups, carbonyl groups and aromatic C¼ C bonds. The absorption maxima in the UV spectra at 243, 274 and 341 nm were attributed to a flavone skeleton. Upon addition of NaOAc to a solution of com-pound 1 in ethanol no bathochromic UV shift (band II) was observed, which indicated the absence of a free 7-hydroxyl group (Markham 1982). With anhydrous aluminium Figure 1. 40,5-Dihydroxy-30,50,6-trimethoxyflavone-7-O-b-D-glucoside.
chloride being added to the ethanolic solution of compound 1, a 10 nm UV shift was observed in band II, confirming the presence of the phenolic hydroxyl at C-5 (Litvinenko and Maksyutina 1965). The HR-ESI-MS data indicated a molecular formula of C24H26O13
based on the [Mþ H]þion signal at 523.1436 (calcd. 523.1373)(Figures S1 and S2). The1H NMR spectra of 1 showed a singlet atd 7.34 (integral 2H) and an additional 6H singlet from two identical methoxyl groups atd 3.87 indicating the presence of a symmetric, trioxygenated B ring. A signal atd 12.93 was assigned to 5-OH due to its low-field shift which is characteristic for these hydrogen bonded protons in flavonoid systems. This hydroxyl proton showed long range crosspeaks in the HMBC spectra to three quaternary carbon signals at d 105.87 (C-4a), 132.69 (C-6), and 152.46 (C-5), respectively. The assignment of these carbon resonances is based on chemical shift values and additional observed long range crosspeaks: C-4a to H-3 (d 7.04, which was assigned due to correlation to the B-ring) and H-8 (d 7.09), C-6 to H-8. An additional methoxyl group (d 3.77) revealed also a long range crosspeak to C-6 proving position 6 as methoxylated. Moreover the 1H spectra in combination with 13C and 2D spectra showed the presence of one carbohydrate pyranose moiety with its anomeric signals at d 5.09 and 100.64, respectively. A long range crosspeak in the HMBC spectra from this anomeric proton and also from H-8 to a carbon signal atd 156.56 proved position C-7 and not C-40 as site of glycosylation. Due to partial signal overlap no coupling constants of the sugar protons could be extracted except that for the anomeric proton with J¼ 7.6 Hz indicating the presence of b-O-glycosidic moiety. The missing coupling constant information and because 13C chemical shifts of especially gluco- or galacto-pyranosides are very similar no clear distinction about the nature of the attached sugar could be done solely on basis of the NMR data(Figures S3–S11). Therefore acid hydrolysis and subsequent TLC analysis was performed confirming the presence of D-glucose (Rf¼0.31), and thus an 7-O-b-D-glucopyranosyl motif in compound 1.
The structure of compound 2 was also determined with the help of 1D and 2D NMR spectroscopy (Table S1) and subsequent comparison with literature data. The compound was thus identified as eupatilin (Liu and Mabry, 1981). Eupatalin has been previously isolated from Artemisia frigida, A. annua, A. princeps, A. leucotricha, A. leucodes, (Ivanescu et al.2016) and has been reported to possess antioxidant as well as potent anticancer properties (Lee et al.2008; Choi et al.2008; Kim et al.2010).
The antioxidant properties of the EtOAc and CHCl3fractions, and of compounds 1
and 2 were comparatively examined by different assays, including DPPH free radical scavenging, CUPRAC, FRAP, phosphomolybdenum and metal chelation tests. The results are summarized in Table 1. The EtOAc fraction exhibited the strongest DPPH Table 1. Antioxidant properties of two extracted fractions, and pure compounds 1 and 2.
Sample
Phosphomolybdenum (mmol TE/g extract
or compound) DPPH (mg TE/g extract or compound) CUPRAC (mg TE/g extract or compound) FRAP (mg TE/g extract or compound) Metal chelating activity (mg EDTAE/g extract or compound) Compound 1 1.99 ± 0.11 53.51 ± 5.51 265.47 ± 0.97 128.00 ± 0.90 12.54 ± 0.55 Compound 2 0.83 ± 0.05 31.24 ± 5.19 188.63 ± 7.00 58.53 ± 2.53 13.42 ± 0.75 CHCl3fraction 2.53 ± 0.09 42.00 ± 1.78 157.90 ± 6.14 79.95 ± 1.01 10.44 ± 0.87 EtOAc fraction 3.09 ± 0.03 156.58 ± 4.63 668.03 ± 4.85 466.87 ± 12.90 5.29 ± 0.31 Values expressed are means ± S.D. of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA equivalent.
radical scavenging ability as well as cupric and ferric reducing power. In addition, the greatest activity was observed by the EtOAc fraction in the phosphomolybdenum assay, followed by CHCl3fraction and new flavone glucoside (1). The metal chelating
abilities can be ranked in the order: eupatilin (2)> new flavone glucoside (1) > CHCl3
fraction> EtOAc fraction.
Enzyme inhibition is considered one of the most useful therapeutic approaches for managing global health problems including Alzheimer’s disease and diabetes mellitus. In this context, we investigated the enzymatic inhibitory potentials of the components isolated from A. juncea (Table 2). The EtOAc fraction exhibited the strongest inhibitory effects on tyrosinase. The best inhibitory effect on amylase and glucosidase was observed in eupatilin (2) and the CHCl3 fraction, respectively. Eupatilin (2) exerted
significant BChE inhibition with 3.82 mgGALAEs/g compound, while other samples were inactive in the case of this enzyme. Also, the strongest AChE inhibitory effect was observed for eupatilin (2) in the tested samples.
3. Experimental
3.1. General experimental procedures
Ultraviolet (UV) spectra were recorded in ethanol on a SF-2000 spectrophotometer (ZAO OKB Spectrum, Russia) and IR spectra on a Perkin Elmer FT-IR spectrometer. NMR experiments were performed on a Bruker Avance II 400 spectrometer (resonance frequencies 400.13 MHz for 1H and 100.63 MHz for 13C, respectively) equipped with a 5 mm observe broadband probe head with z-gradients at room temperature with standard Bruker pulse programs. Chemical shifts are presented in parts per million (d/ppm) and referenced to residual solvent signals (DMSO-d6: 2.49 ppm for
1
H, 39.6 ppm for13C; pyridine-d5: 7.22 ppm for1H, 123.87 ppm for13C). Coupling constants
(J) are reported in Hz. HR-ESI-MS spectra were recorded on the Orbitrap HF mass spec-trometer coupled to a Vanquish HPLC (Thermo Fisher Scientific). Silica gel (100/200 mesh, Tianjin Sinomed Pharmaceutical, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used as the stationary media for column chromato-graphy. Thin layer chromatography (TLC) was performed on aluminum plates pre-coated with silica gel 60 F254 (Merck, Germany). The flavonoid spots were observed directly under UV light (at 254 nm and 366 nm) and/or visualized after treating with ammonia vapor, and/or 3% vanillin in ethanol/conc. hydrochloric acid (4:1, v/v). TLC Table 2. Enzyme inhibitory activities of the two extracted fractions and pure compounds 1 and 2. Sample AChE inhibition (mg GALAE/g extract or compound) BChE inhibition (mg GALAE/g extract or compound) Tyrosinase inhibition (mg KAE/g extract or compound) Amylase inhibition (mmol ACAE/g extract or compound) Glucosidase inhibition (mmol ACAE/g extract or compound) Compound 1 1.68 ± 0.16 n.a. 35.34 ± 1.45 0.14 ± 0.01 46.57 ± 0.10 Compound 2 2.68 ± 0.06 3.82 ± 0.15 24.41 ± 3.15 0.94 ± 0.01 46.40 ± 0.02 CHCl3fraction 1.88 ± 0.07 n.a. 26.98 ± 2.64 0.26 ± 0.05 46.71 ± 0.36
EtOAc fraction 2.32 ± 0.02 n.a. 38.56 ± 2.64 0.10 ± 0.01 46.14 ± 0.36 Values expressed are means ± S.D. of three parallel measurements. GALAE: Galanthamine equivalent; KAE: Kojic acid
equivalent; ACAE: Acarbose equivalent; n.a.: not active. 2172 B. S. OKHUNDEDAEV ET AL.
chromatograms were developed using following solvent systems: benzene-ethanol (9:1, v/v); chloroform-methanol-acetic acid (4:1:0.3, v/v/v); chloroform-methanol-acetic acid-water (9:3:1:0.5 and 7:3:0.5:0.5, v/v/v/v).
3.2. Plant materials
The aerial parts of A. juncea were collected at the township Churuh of the Bukhara region in Uzbekistan during flowering in September 2016. The taxonomic authentica-tion was accomplished at the Institute of Botany, Academy Sciences of Uzbekistan. The voucher specimen of A. juncea was deposited at the Central Herbarium of Uzbekistan under the code #310.
3.3. Extraction and isolation
The air-dried aerial parts of the plant (2 kg) were extracted five times with 93% aqueous ethanol at room temperature. After removal of the solvent, the crude extract (507 g) was subject to silica gel column chromatography (CC) with petroleum ether (PE, 3000 ml), chloroform (3000 ml) and EtOAc (3000 ml) as the mobile phases to afford the PE (18 g), CHCl3(22.8 g), and EtOAc (38 g) fractions, respectively.
The EtOAc fraction (38 g) was chromatographed on silica gel (l¼ 190 cm, d ¼ 4.5 cm), using a gradient system of CHCl3:MeOH (from 90:10 to 70:30, v/v) providing 3
subfrac-tions EA1-EA3. Subfraction EA1 (10 g) was further separated by Sephadex LH-20 in a mix-ture of EtOH: water (80:20, v/v) as the solvent system, to obtain compound 1 (358.5 mg). 40,5-Dihydroxy-30,50,6-trimethoxyflavone-7-O-b-D-glucoside (1). Yellow amorphous compound. C24H26O13, m.p. 233–235, Rf¼ 0.62 (CHCl3:MeOH:acetic acid¼ 4:1:0.3, v/v/v).
UV (EtOH,kmax, nm): 243, 274, 341 nm. IR (KBr,mmax, cm1): 3368 (), 2928 (3),
1662 (¼), 1613, 1527, 1503 (Ar), 1040, 1015 (C-O). HR-ESI-MS: m/z 523.1436 [Mþ H]þ (Dppm ¼ 1.944) and 521.12964 [M-H]- (Dppm ¼ 0.814) (C24H26O13 calcd.). 1
NMR (400 MHz, DMSO-d6, d/ppm, J/Hz): 3.17 (m, 1H, Glc-H4), 3.31 (m, 1H, Glc -H3),
3.35 (m, 1H, Glc-H2), 3.45 (m, 1H, Glc-H5), 3.46 (m, 1H, Glc-H6a), 3.74 (m, 1H, Glc-H6b), 3.77 (s, 3H, 6-OCH3), 3.87 (s, 6H, 30,50-OCH3), 4.64 (t, J¼ 5.4, Glc-6-OH), 5.06 (d, J ¼ 5.2,
Glc-4-OH), 5.09 (d, J¼ 7.6, Glc-H1), 5.41 (d, J ¼ 4.9, Glc-2-OH), 7.04 (s, 1H, H-3), 7.09 (s, 1H, H-8), 7.34 (s, 2H, H-20, H-60), 12.93 (s, 1H, 5-OH).
13
NMR (100 MHz, DMSO-d6, d/ppm): 56.49 (30-, 50-OCH3), 60.37 (6-OCH3), 60.76 (Glc
-C6), 69.81 (Glc -C4), 73.29 (Glc -C2), 76.88 (Glc -C3), 77.68 (Glc -C5), 94.93 (C-8), 100.64 (Glc -C1), 103.45 (C-3), 104.63 (C-20, C-60), 105.87 (C-4a), 120.29 (C-10), 132.69 (C-6), 140.22 (C-40), 148.30 (C-30, C-50), 152.11 (C-8a), 152.46 (C-5), 156.56 (C-7), 164.22 (C-2), 182.41 (C-4).
The chloroform fraction (20 g) was chromatographed on silica gel (l¼ 165 cm, d¼ 4.5 cm), using a gradient system with increasing polarity of PE: EtOAc (from 90:10 to 10:90, v/v) to yield subfraction C1-C5. Subfraction C2 (2.38 g) was re-chromoto-graphed on Sephadex LH-20 using gradient mixture of EtOH:water (95:5, v/v) to give 106 mg of compound 2, which was identified as eupatilin (5,7-dihydroxy-30,40 ,6-tri-methoxyflavone) by comparison of its spectral data with those reported in the litera-ture (Belenovskaya and Korobkov,2005).
Eupatilin (5,7-dihydroxy-30,40,6-trimethoxyflavone) (2). Yellow compound, 18167, m.p. 232–234 , Rf ¼ 0.53 (benzene-ethanol, 9:1, v/v). UV (EtOH, kmax,
nm): 272, 350. IR (KBr, mmax, cm1): 3429 (), 2941 (–3), 1650 (¼), 1620, 1587,
1510, 1463 (Ar), 1148, 1109, 1024, (C-O).1 and13 NMR data seeTable S1.
3.4. Identification of sugar moiety of compound 1 by TLC
The sugar moiety in the new flavonoid 1 was determined after complete acid hydroly-sis of compound 1 in H2SO4 (3 ml, 2 N) after 8 hours at 100C. The hydrolyzate was
neutralized with BaCO3, deionized over cation exchanger KU-2 (Hþ) and analyzed by
paper chromatography (PC) (Filtrak No.12). The solvent system n-butanol-pyridine-water (6:4:3, v/v/v) was used as a mobile phase. The hydrolysis products were compared by TLC with authentic samples (D-galactose, D-glucose, L-arabinose, D-xylose and L-rhamnose). The chromatogram was then visualized with acidic aniline phthalate followed by heating for 3–5 minutes at 90–100C. PC of the hydrolyzate detected 40,5,7-trihydroxy-30,50,6-trimethoxyflavone (Martinez-Vazquez et al.1993) and D-glucose.
3.5. Antioxidant assays
The antioxidant activity was evaluated by free radical scavenging (DPPH), reducing power (CUPRAC and FRAP), phosphomolybdenum, and metal chelating assays. The results of these assays were expressed as trolox equivalents (TE/g extract or TE/g compound). Metal chelating activity was evaluated as EDTA equivalent (mgEDTA/g extract or mgEDTA/g com-pound). The experimental procedures were as previously described (Zengin et al.,2015).
3.6. Enzyme inhibition assays
Enzyme inhibition effects were investigated against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, a-amylase, and a-glucosidase as previously described (Zengin2016). The inhibitory effects were expressed as standard compound equivalents. Briefly, galanthamine was used for AChE and BChE; kojic acid for tyrosin-ase, and acarbose fora-amylase and a-glucosidase.
4. Conclusions
A new flavone glycoside, besides the known compound eupatilin, was isolated from the aerial parts of Artemisia juncea. The structure of the new compound was identified based on a combination of analytical techniques (UV, FT-IR, HR-ESI-MS, 1D and 2D NMR spectroscopy). The EtOAc fraction extracted from the sample material exhibited the strongest antioxidant effects. We also demonstrated the capability of the EtOAc fraction to inhibit AChE and tyrosinase activity, which suggests that it may be effective against Alzheimer’s disease and hyperpigmentation. The best antidiabetic effect was observed from pure eupatilin.
Acknowledgements
The project was funded through a grant from the Republic of Uzbekistan State Foundation for Basic Research (grant number M/UZB-KNR-25-2015 and TA-FA-F7-008). We gratefully
acknowledge Dr. Roland Hellinger (Center for Analytical Chemistry, Department for Agrobiotechnology, BOKU) for measuring the HR-MS spectra.
Disclosure statement
No potential conflict of interest was reported by the authors.
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