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Abstract

Five flavonol O-glycosides and 4 C-glycosylflavones were isolated from the flowers of the Himalayan Megacodon stylophorus (Gentianaceae). They were characterized as quercetin 3-O-glucoside (1), quercetin 3-O-rutinoside (2), kaempferol 3-O-glucoside (3), isorhamnetin 3-O-glucoside (4) and kaempferol 3,7-di-O-glucoside (5) (flavonols), and isovitexin (6), isoorientin (7), isovitexin X″-O-arabinoside (8) and isovitexin 4′-O-glucoside (9) (C-glycosylflavones) by ultraviolet, liquid chromatography-mass spectrometry, acid hydrolysis, nuclear magnetic resonance, and/or high-performance liquid chromatography and thin-layer chromatography comparisons with authentic samples. On the other hand, 5 C-glycosylflavones were isolated from the leaves and identified as 6, 7, 9, vitexin (10), and orientin (11). Although many C-glycosylflavones and xanthones have been reported from Gentianaceae species, flavonols are minor occurrence in the family. Flavonoids were reported from the Megacodon species for the first time.

Keywords

flavonols flavonoids

The genus Megacodon consists of 2 species, M. stylophorus (C.B. Clark) H. Smith¹ and M. venosus (Hemsl.) H. Smith.² Recently, a new species, Megacodon lushuiensis J.C. Peng & Sun, was reported from China.³ Of their species, M. stylophorus is growing in high elevations between 3000 and 4400 m alt. in China, Bhutan, Nepal, and north-east India.³ As phenolic compounds in M. stylophorus, 4 xanthones, 1,5,8-trihydroxy-3-methoxyxanthone, 1,5,7-trihydroxy-3-methoxyxanthone, 1,7-dihydroxy-3,8-dimethoxyxanthone and 1,3,7,8-tetrahydroxyxanthone, and 5-formyl-2,3-dihydroxyiscoumarin, halenic acid, and ellagic acid have been reported, together with 4 triterpene derivatives.⁴ However, flavonoids are not isolated and identified from Megacodon species as far as we know. In this survey, flavonoids were isolated from the flowers and leaves from M. stylophorus and identified by ultraviolet (UV), liquid chromatography-mass spectrometry (LC-MS), acid hydrolysis, ¹H and ¹³C nuclear magnetic resonance (NMR), and/or high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) comparisons with authentic samples.

Results and Discussion

Five flavonol glycosides (1-5) and 4 C-glycosylflavones (6-9) were isolated from the flowers of M. stylophorus. Flavonols were identified as quercetin 3-O-glucoside (1), quercetin 3-O-rutinoside (2), kaempferol 3-O-glucoside (3), isorhamnetin 3-O-glucoside (4), and kaempferol 3,7-di-O-glucoside (5) (Figure 1) by UV spectral survey according to Mabry et al,⁵ LC-MS, acid hydrolysis, and HPLC and TLC comparisons with authentic samples. Of 4 C-glycosyflavones, 2 were identified as isovitexin (6) and isoorientin (7) (Figure 1) by UV, LC-MS, hot acid treatment, and HPLC and TLC comparisons with authentic samples. Compound 8 was characterized as isovitexin X″-O-arabinoside (Figure 1) by UV, LC-MS, and acid hydrolysis. Another C-glycosylflavone (9) was mainly identified as isovitexin 4′-O-β-d-glucopyranoside by ¹H and ¹³C NMR. That is, 4 aromatic proton signals corresponding to H-2′,6′, H-3′,5′, H-3, and H-8 appeared, together with C-glucosyl (δ_H 5.85, d, J = 9.0 Hz) and O-glucosyl (δ_H 5.75, d, J = 7.4 Hz) anomeric protons by ¹H NMR. The attachment of C-glucose to 6-position of apigenin was determined by heteronuclear multiple bond correlation (HMBC) between glucosyl anomeric proton at δ_H 5.85 and C-6 carbon signal of apigenin at δ_C 110.2. On the other hand, the attachment of another glucose to 4′-position of apigenin was shown by HMBC correlation between glucosyl anomeric proton at δ_H 5.75 and C-4′ carbon signal at δ_C 161.2.

Figure 1

Chemical structures of flavonols and C-glycosylflavones from the flowers and leaves of Megacodon stylophorus.

Five C-glycosylflavones (6, 7, 9-11) were isolated from the leaves. Of their flavonoids, 6, 7, and 9 were identified as isovitexin, isoorientin, and isovitexin 4′-O-glucoside which were found in the flowers. Other 2 compounds (10 and 11) were identified as vitexin and orientin (Figure 1), respectively, by UV, LC-MS, hot acid treatment, and HPLC and TLC comparisons with authentic samples.

In this survey, 5 flavonols and 6 C-glycosylflavones were found in the flowers and leaves of M. stylophorus for the first time. Many C-glycosylflavones have been reported from many Gentianaceae species, for example, Gentiana,^6,7 Gentianopsis,⁸ Gentianella,⁸ Lomatogonium,⁹ Swertia,^10,11 together with xanthones. However, flavonols which are popular flavonoids in plants have been found in a few Gentianaceae species, that is, santin from Gentiana algida,¹² quercetin from Lomatogonium carinthiacum,¹³ quercetin 3-O-glucoside from Gentiana pyrenaica,¹⁴ quercetin 3-O-rutinoside from Gentiana flavo-maculata,¹⁵ quercetin 3-O-robinobioside-7-O-rhamnoside and 3-O-[rhamnosyl-(1→6)-(4″-E-p-coumaroylgalactoside)]-7-O-rhamnoside from Coutoubea spicata,¹⁶ 3-O-rhamnosylgalactoside-7-O-rhamnoside of kaempferol, isorhamnetin, and myricetin, and 3-O-rhamnosylglucoside-7-O-rhamnoside of kaempferol and isorhamnetin from Eustoma grandiflorum,¹⁷ and kaempferol and isorhamnetin 3-O-rhamnosyl-(1→2)-galactosides from Blackstonia perfoliata.¹⁸ It was shown by this survey that 5 flavonols, quercetin 3-O-glucoside (1), quercetin 3-O-rutinoside (2), kaempferol 3-O-glucoside (3), isorhamnetin 3-O-glucoside (4), and kaempferol 3,7-di-O-glucoside (5) are contained in M. stylophorus. Of their flavonols, although 1 and 2 have been reported from Gentiana pyrenaica ¹⁴ and G. flavo-maculata,¹⁵ respectively, 3-5 were found in Gentianaceae species for the first time. Kaouadji¹⁸ described that the occurrence of flavonol glycosides in Blackstonia as well as in 3 other genera of Centaurium, Coutoubea, and Eustoma, is in agreement with the grouping of these 4 genera in the subtribe Chlorae of the Gentianeae. However, in this survey, flavonol glycosides were found in Megacodon which belongs to the subtribe Swertiinae of the Gentianeae.³

Materials and Methods

Plant Materials

Megacodon stylophorus (C.B. Clark) H. Smith was collected in the east of Lake Jyanetsch, ca. 3900 m alt., Paro, Bhutan on July 13, 2014. Voucher specimen was deposited in the herbarium of National Biodiversity Center (THIM), Bhutan.

General

Analytical HPLC was performed with Shimadzu HPLC systems using Inertsil ODS-4 column (I.D. 6.0 × 150 mm, Chemical Evaluation and Research Institute, Tokyo) at a flow rate of 1.0 mL/min. Detection wavelength was 350 nm. Eluent was acetonitrile (MeCN)/water (H₂O)/phosphoric acid (20:80:0.2). LC-MS was performed with Shimadzu LC-MS systems using Inertsil ODS-4 column (I.D. 2.1 × 100 mm) at a flow rate of 0.2 mL/min, electrospray ionization (ESI⁺) 4.5 kV and ESI⁻ 3.5 kV, 250 °C. Eluent was MeCN/H₂O/HCOOH (15:80:5). NMR spectra (¹H and ¹³C NMR, ¹H-¹H correlation spectroscopy, ¹H-¹H total CSY, heteronuclear multiple quantum correlation, and HMBC) were recorded on a Bruker AV-600 in pyridine-d ₅ at 600 MHz (¹H NMR) and 150 MHz (¹³C NMR). Acid hydrolysis was performed in 12% hydrochloric acid, 100 °C, 30 minutes. After shaking with diethyl ether, aglycones of flavonols were migrated to the organic layer, and sugars and C-glycosylflavones were left in the aqueous layer. Preparative HPLC was performed with Shimadzu HPLC systems using Inertsil ODS-4 column (I.D. 10 × 250 mm), at a flow rate of 3.0 mL/min, detection wavelength of 350 nm, and the eluent was MeCN/H₂O/HCOOH (25:70:5). TLC was performed with Cellulose Plastic Plate (Merck, Germany) using solvent systems, BAW (butanol [n-BuOH]/acetic acid [HOAc]/H₂O = 4:1:5, upper phase), BEW (n-BuOH/ethanol/H₂O = 4:1:2.2), and 15% HOAc. Preparative paper chromatography (prep. PC) was performed with solvent systems, BAW, and 15% HOAc.

Extraction and Isolation

Dry flowers (2.2 g) and leaves (24.0 g) were extracted with methanol (MeOH). After concentration, the extracts were applied to prep. PC with solvent systems, BAW, and then 15% HOAc. Isolated flavonoids were purified by Sephadex LH-20 column chromatography using a solvent system, 70% MeOH. The mixtures were further separated with prep. HPLC. Compounds 6 (ca 30 mg), 7 (ca 10 mg), and 9 (ca 30 mg) were obtained as pale yellow powders and other flavonoids as MeOH solution.

Identification of Flavonoids

Flavonoids were identified by UV-vis spectral survey according to Mabry et al,⁵ LC-MS, characterization of acid hydrolysates, ¹H and ¹³C NMR, and HPLC and TLC comparisons with authentic samples. UV, LC-MS, acid hydrolysis, HPLC, TLC, and NMR data are shown in Online Supplementary Material 1. Origins of authentic flavonoids were as follows: quercetin 3-O-glucoside from the aerial parts of Osyris alba (Santalaceae),¹⁹ quercetin 3-O-rutinoside from the flowers of Chimonanthus praecox (Calycanthaceae),²⁰ kaempferol 3-O-glucoside from the fronds of Cyrtomium falcatum subsp. australe (Dryopteridaceae),²¹ isorhamnetin 3-O-glucoside from Extrasynthese (Genay), kaempferol 3,7-di-O-glucoside from the flowers of Clematis armandii (Ranunculaceae),²² vitexin and isovitexin from the flowers of Iris ensata (Iridaceae),²³ orientin from the leaves of Colocasia esculenta (Araceae),²⁴ and isoorientin from the leaves of Japonolirion osense (Liliaceae).²⁵

Supplemental Material

Supplementary Material 1 - Supplemental material for Flavonoids From the Flowers and Leaves of the Himalayan Megacodon stylophorus (Gentianaceae)

Supplemental material, Supplementary Material 1, for Flavonoids From the Flowers and Leaves of the Himalayan Megacodon stylophorus (Gentianaceae) by Tsukasa Iwashina, Rinchen Yangzom, Hari Prasad Devkota and Takayuki Mizuno in Natural Product Communications

Footnotes

Acknowledgments

Authors thank to Dr Yoshinori Murai (National Museum of Nature and Science, Japan) for coordination of Bhutan trip, and also to Kencho Dorji, Choki Wangmo, and Choki Gyeltshen (National Biodiversity Centre, Bhutan) for support of plant collection.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Discretionary expenses of Director General of the National Museum of Nature and Science, Japan.

ORCID ID

Tsukasa Iwashina

Supplemental Material

Supplemental material for this article is available online.

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