Extractives of Cercidiphyllum japonicum twigs: isolation and structural elucidation of a new galloylflavonol glycoside, anomeric tannins and flavonoids

Introduction

Tremendous amounts of tree twigs are incinerated or considered as useless by-products and disposed of in landfills in the course of logging or pulping. Of course, landfills are problematic from an environmental point of view (Feng et al. 2013; Si et al. 2013). On the other hand, the extraneous components of twigs could be a source of value-added products as they may contain undetected compounds. It is also well known that extractives are also markers for chemotaxonomical differentiation of tree species (Si et al. 2009; Wang et al. 2016; Vidakovic et al. 2018). The biosynthesis routes of extractives are highly relevant from the scientific point of view (Tsao et al. 2016; Mangindaan et al. 2017). New extractives with various activities may also serve for the development of medicines (Si et al. 2016a, 2017; Belt et al. 2017).

Cercidiphyllum japonicum Sieb. et Zucc., also known as katsura, is a deciduous and broad-leaved tree, while Cercidiphyllum is the sole genus of the Cercidiphyllaceae family (Sarker et al. 1997; Kubo et al. 2010). This species is well represented in fossil records, with occurrence in the late Cretaceous and Tertiary of Europe and North America. “Cercidiphyllum japonicum” is distributed only in East Asian countries, such as China, Japan and Korea (Tada and Sakurai 1991; Sarker et al. 1997). The katsura trees grow in cool-temperate riparian forests, where they can reach up to 30 m in height and a trunk with ca. 2 m in diameter. The trees can survive 1000 years several hundred years up to 1000 years by shooting sprouts from the base. Cercidiphyllum japonicum is dioecious, blooms in early spring before the leaves shoot and is wind-pollinated (Teifel and Berger 1993; Isagi et al. 2005).

Plant extractives of C. japonicum have been widely used in traditional Asian medicines owing to their antiinflammatory, antimicrobial, antifungal and antioxidative activities. They are also used as hair growth promoting agents and for other activities (Takasugi and Katui 1986; Kasuga et al. 2008; Bae et al. 2017). The components of xylem extractives, such as kaempferol-7-O-β-glucopyroanside, quercetin-3-O-β-glucopyranoside, kaempferol-3-O-β-glucopyranoside and 8-methoxykaempferol-3-O-β-glucopyranoside exhibit an anti-ice nucleation effect to promote supercooling of water (Wang et al. 2012). Our previous investigations on C. japonicum leaves and barks resulted in the extraction and separation of several phenolic compounds, including flavonoids, tannins and phenolic acids (Lee and Bae 2015; Lee et al. 2015, 2016; Bae et al. 2017). However, to the best of our knowledge, screening of the twig extractives of C. japonicum has never been carried out.

Aimed at the search for wood extractives, which may be responsible for the medicinal or pharmaceutical effects of the tree, the focus of the present paper was the isolation, purification and structural elucidation of three anomeric tannins and four flavonoid type components from the twigs of C. japonicum.

Materials and methods

Extraction, isolation and separation:

Air-dried twig samples from C. japonicum (5.02 kg) were finely ground and then extracted with acetone:H₂O mixture (3:1, v/v) in a jar (25 l, 96 h each time) at room temperature for at least five times. As shown in Figure 1, after filtration and concentration in an evaporator under reduced pressure, the obtained raw extracts were suspended in H₂O and then subjected to a successive liquid-liquid fractionation with petroleum ether, n-hexane and chloroform in separation funnels. Then, the chloroform fraction was freeze-dried to obtain 56.92 g of CHCl₃ soluble fraction powders (labeled as TCJC) (yield 1.13%, w/w, on dry plant materials). As demonstrated in Figure 1, 45.66 g of fraction TCJC powders was subjected to VLC filled with macroporous adsorbent resin D101 using gradient washing solvents of MeOH:H₂O (49:1→19:1→4:1→1:4→1:19→1:49, v/v) to obtain five main portions (TCJC₁–TCJC₅). During the whole process, the eluents were monitored and grouped by TLC visualization. The portion TCJC₃ (15.36 g) was separated by silica gel OCC eluting with a gradient of decreasing CHCl₃ in EtOH (from 9:1 to 1:9, v/v) to yield five portions (TCJC₃₁–TCJC₃₅). The portion TCJC₃₂ (1.05 g) was purified by OCC filled with Sephadex LH-20 using MeOH:H₂O (4:1, 1:1, 1:3, v/v) as the washing solvent to obtain compound I (61.4 mg). The portion TCJC₃₄ (9.82 g) was successively loaded over silica gel and Sephadex LH-20 OCCs eluting with CHCl₃:MeOH (29:1→1:4, v/v) and MeOH:H₂O (3:1→1:7, v/v), respectively, to present 42.3 mg of compound II and 1.14 g of portion TCJC₃₄₂₂. Compound III (59.6 mg) was obtained from TCJC₃₄₂₂ by Sephadex LH-20 OCC washing with MeOH:H₂O (2:1→1:3, v/v). The main portion TCJC₄ (21.09) was also subjected to silica gel OCC using a step gradient of decreasing CHCl₃ in EtOH (from 19:1 to 1:4, v/v) to yield three portions (TCJC₄₁–TCJC₄₃). TCJC₄₂ (16.52 g) was also purified by silica gel OCC eluting with CHCl₃:MeOH (9:1→1:9, v/v) to give portion TCJC₄₂₁ and compound IV (70.6 mg). A total of 14.35 g of TCJC₄₂₁ was similarly applied to Sephadex LH-20 OCC with MeOH:H₂O (3:1, 1:1, 1:3, v/v) which acts as a mobile phase to yield compound V (47.2 mg) and 5.60 g of TCJC₄₂₁₃. TCJC₄₂₁₃ was further separated by Sephadex LH-20 OCC and sequentially eluted with MeOH:H₂O (4:1→1:5, v/v) and n-hexane:EtOH (2:1→1:4, v/v) to obtain compounds VI (54.0 mg) and VII (40.8 mg).

Figure 1:

Flowchart of extraction, fractionation and purification of extractives from the twigs of C. japonicum.

Results and discussion

Air-dried twigs of C. japonicum were extracted with 75% aqueous acetone to obtain the raw extracts. Successive liquid-liquid fractionations and repeated chromatographic purification of CHCl₃ soluble powders resulted in the isolation of a new galloylflavonol glycoside, elucidated as 8-methoxykaempferol-4′-O-galloyl-3-O-α-L-rhamnopyranoside (VII), as well as six known extractives, including three anomeric tannins (compounds I and III, two anomeric galloyltannins; compound II, as an anomeric ellagitannin) and three flavonoids (compounds IV, V and VI). Based on their spectroscopic values and physicochemical clues, as well as by careful comparisons of the data with those reported in the literature, the six known extractives were determined as 3,4,6-tri-O-galloyl-β-D-glucopyranoside (I) (Haddock et al. 1982), pedunculagin (II) (Tanaka et al. 1993), 2,2′,5-tri-O-galloyl-α/β-D-hamamelose (III) (Lee et al. 2016), kaempferol-3-O-α-L-rhamnopyranoside (IV) (Harbone and Marby 1982), kaempferol (V) (Okuyama et al. 1978) and 8-methoxykaempferol (VI) (Dauguet et al. 1993) (as shown in Figure 2).

Figure 2:

Structures of extractives I to VII isolated from the twigs of C. japonicum.

Among the six known compounds (I–VI), 2,2′,5-tri-O-galloyl-α/β-D-hamamelose (III) and kaempferol (V) were previously isolated from xylem and heartwood of C. japonicum, respectively (Lee et al. 2016). However, this was the first time that 3,4,6-tri-O-galloyl-β-D-glucopyranoside (I), pedunculagin (II), kaempferol-3-O-α-L-rhamnopyranoside (IV) and 8-methoxykaempferol (VI) were purified from the genus Cercidiphyllum.

Compound VII was obtained as a yellowish amorphous powder, with an mp of 189–191°C and optical rotation [α]D20 −36.2° (c 0.5, in MeOH). The molecular formula of compound VII was determined to be C₂₉H₂₆O₁₅, for its positive FAB-MS spectrum displayed quasimolecular ion peaks [M+H]⁺, [M+Na]⁺ and [M+K]⁺ at m/z 615, 637 and 653, respectively, coinciding well with its molecular weight 614.

In TLC chromogenic reactions, when spraying with 1% ethanolic FeCl₃ solvent followed by heating, compound VII gave a dark brown color, accounting for the presence of one or more phenolic hydroxyl groups in its molecular structure. (Si et al. 2008). In two-dimensional (2D) TLC experiments, R_f values of compound VII were 0.20 and 0.77, when solvents A and B were used as the developing reagents, respectively. For compound VII, its IR spectrum presented characteristic absorption bands at 3395 cm⁻¹ (OH), 1630 cm⁻¹ (α, β-unsaturated C=O), 1590, 1515, 1480 cm⁻¹ (aromatic C=C) and 1165, 1065, 1020 cm⁻¹ (C-O) (Park et al. 2010; Hu et al. 2017). The UV spectrum of VII in MeOH exhibited absorption bands at λ_max 223, 273, 322 and 357 nm, corresponding with a 3-O-glycoside of 8-substitution flavonol skeleton (Dauguet et al. 1993; Koch et al. 2006).

The ¹H NMR spectrum of compound VII displayed a singlet at δ_H 12.29 which was attributable to a free hydroxyl group at C-5 of the A-ring. Another singlet observed at δ_H 6.32 was assigned to H-6 (Rayyan et al. 2005). A set of AA′BB′ coupling proton signals constituted by two doublets (J=8.9 Hz) at δ_H 8.12 and 7.09 was assignable to H-2′, 6′ and H-3′, 5′ due to a para-substituted B-ring (Okoye et al. 2015). A methoxy group was recognized by its NMR evidence [δ_H 3.84 (3H, s), δ_C 60.9], and its linkage to C-8 was confirmed by ¹H detected HMBC experiment for ¹H→¹³C coupling correlation was observed between δ_H 3.84 (3H, s) of the OCH₃ and C-8 (δ_C 128.1) of the A-ring, as presented in Figure 3. In ¹³C NMR spectrum of compound VII, characteristic flavonol signals resonated at δ_C 156.6 (C-2), 133.9 (C-3) and 178.0 (C-4). Thus, the aglycone was determined as a 8-methoxykaempferol (Dauguet et al. 1993).

Figure 3:

Key HMBC correlations observed in compound VII.

The presence of a galloyl moiety in compound VII was easily recognized from its ¹H NMR spectrum for a pair of symmetric protons H-2″ and H-6″ diagonally irradiating at δ_H 7.08 (1H each, s) (Si et al. 2016b). The HMBC spectroscopic correlations of VII, as demonstrated in Figure 3, presented interlinks between the aglycone B-ring proton H-3′/5′ (δ_H 7.09) and the quaternary carbon C-7″ (δ_C 165.7), which evidenced that the galloyl moiety was connected to C-4′ of the aglycone.

In the ¹H NMR spectrum of compound VII, the α-configuration of L-rhamnose was confirmed from a secondary methyl group characteristically irradiated at δ_H 1.01 (3H, d, J=6.1 Hz, H-6′″) and an anomeric proton typically resonated at δ_H 4.63 (1H, d, J=1.5) (Liu et al. 2017). In the ¹³C NMR spectrum, the down-field shift of the C-2 signal (approximately Δ-10 ppm) in compound VII compared with kaempferol (V) indicated that the glycosylation happened at 3-hydroxy of the aglycone (Dauguet et al. 1993). Moreover, the connection of α-L-rhamnosyl unit at C-3 of the 8-methoxykaempferol was also supported by the HMBC experiment of compound VII, in which the long-range coupling correlation was observed from H-1′″ (δ_H 4.63) of the rhamnosyl residue to C-3 (δ_C 133.9) (Okoye et al. 2015).

The combination of DEPT and ¹³C NMR can distinguish the signals of different types of carbons. The detailed assignments for compound VII are illustrated in Table 1, where 29 carbon signals are sorted into 2 methyl, 12 methene, 12 methine and 15 tertiary carbons, while each carbon agrees well with the analyses presented above.

Consequently, compound VII was determined to be a galloylflavonol glycoside, and its chemical structure was elucidated as 8-methoxykaempferol-4′-O-galloyl-3-O-α-L-rhamnopyranoside, which is a new compound and has never previously been isolated from any other natural source.

Conclusions

The raw extracts from C. japonicum twigs are widely used in medicines to cure a variety of diseases or disorders. In this work, from the chloroform extracts of C. japonicum twigs, three anomeric tannins [3,4,6-tri-O-galloyl-β-D-glucopyranoside (I), pedunculagin (II), 2,2′,5-tri-O-galloyl-α/β-D-hamamelose (III)] and four flavonoids [kaempferol-3-O-α-L-rhamnopyranoside (IV), kaempferol (V), 8-methoxykaempferol (VI), and 8-methoxykaempferol-4′-O-galloyl-3-O-α-L-rhamnopyranoside (VII)] were purified and identified. The elucidation of the chemical structure of the compounds I–VII was achieved by their spectroscopic and chemical data. To date, I, II, IV and VI have not been isolated from the species of the Cercidiphyllum genus. Compound VII is a new galloylflavonol glycoside and its structure was elucidated for the first time in this work.