A new glucosidic iridoid from Isodon rubescens

ABSTRACT

One new glucosidic iridoid, 6-O-veratroylbarlerin, was isolated from the chloroform/methanol extract of Isodon rubescens (Hemsl.) H.Hara, Lamiaceae aerial parts, along with the known compounds apigenin and caffeic acid. The structure of the new compound was elucidated on the basis of 1D and 2D NMR experiments and ESI-MS technique.

Keywords:
Rabdosia; Lamiaceae; Iridoid; NMR

Introduction

Isodon (formerly named Rabdosia), an important genus of Lamiaceae family, comprised roughly 150 species worldwide, mainly distributed throughout the tropical and subtropical Asia and southwestern China (Sun et al., 2006). Several species of the genus Isodon have been used in traditional Chinese medicine for the treatment of different diseases (Park, 2011). Among them, Isodon rubescens (Hemsl.) H.Hara, a perennial herb widely used in China against inflammation, bacterial infections, respiratory and gastrointestinal diseases and cancer (Ding et al., 2013), is the most popular species. Previous phytochemical investigations of this plant resulted in the isolation of several ent-kaurane and ent-abietane diterpenoids, that attracted considerable attention due to their diverse structures and interesting biological properties (Gao et al., 2011; Luo et al., 2017; Zhang et al., 2017), together with alkaloids, diterpenes (Liu et al., 2015), and phenolic compounds (Du et al., 2010a). To the best of our knowledge, while there have been numerous reports focused on the presence of diterpenoids in the Isodon genus, there are no papers concerning iridoids. The present study reports for the first time the isolation and the structure elucidation of a glucosidic iridoid (1) from I. rubescens aerial parts, along with three known compounds.

Materials and methods

General experimental procedure

Briefly, NMR experiments were recorded on a Bruker DRX-600 spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe, acquiring the spectra in methanol-d₄ (Milella et al., 2016). ESI-MS (positive mode) were obtained from a Finningan LC-Q Advantage Termoquest spectrometer (ThermoFinnigan, USA). Thin Layer Chromatographies (TLC) were performed on precoated Kieselgel 60 F₂₅₄ plates (Merck, Darmstadt, Germany) and compounds were detected by cerium disulfate/sulfuric acid (Sigma-Aldrich, Milan, Italy). Column chromatographies were performed over Sephadex LH-20 (40–70 µm, Amersham Pharmacia Biotech AB, Uppsala, Sweden) and over silica gel 60 (Merck, Darmstadt, Germany), followed by reverse phase-high performance liquid chromatography (RP-HPLC) performed on Shimadzu LC-8A series pumping system with Shimadzu RID-10A refractive index detector, C₁₈ µ-Bondapak column (30 cm × 7.8 mm, 10 µm, Waters, Milford, MA, USA), using mixtures of methanol/water at flow 2.0 ml/min) (Bisio et al., 2017). All solvents used for extraction and separation processes were purchased from Sigma-Aldrich (Milan, Italy).

Plant material

Dried aerial parts of Isodon rubescens (Hemsl.) H.Hara, Lamiaceae, were purchased in November 2008 from Yee Po International Company (Hong Kong) and authenticated by Dr. Fabiano Camangi (Scuola Superiore Sant’Anna, Pisa, Italy). A sample number was YP08-139.

Extraction and isolation

Dried aerial parts (700 g) were extracted using increasing polarity solvent hexane, chloroform, chloroform/methanol (9:1, v/v) and methanol by extensive maceration (3 times × 2 l) (De Leo et al., 2017). The solvent was evaporated under vacuum system obtaining the following yields 8.10, 17.29, 5.67 and 27.73 g, respectively. Briefly, part of the chloroform/methanol (9:1) extract (2.8 g) was separated by Sephadex LH- 20 with methanol as eluent. Fractions of 10 ml were collected, analyzed by TLC and grouped into seventeen fractions (A–Q) (Bisio et al., 2016). Fractions L and O were isolated as pure apigenin (39.5 mg) and caffeic acid (4 mg), respectively. Fractions C, D, E, and F (1548 mg) were regrouped and separated by silica gel CC eluting with chloroform followed by increasing concentrations of methanol (between 1% and 100%). Fractions of 5 ml were collected, analyzed by TLC and grouped into 21 fractions (A1-U1). Fraction J1 was isolated as pure oridonin (31.7 mg). Fraction H1 (100.1 mg) was subjected to RP-HPLC with methanol/water (35:65) as eluent and regrouped in fifteen fractions (A2-P2). Fractions C3 was isolated as a pure caffeic acid (1.5 mg, t_R 6 min). Fraction N2 (70.5 mg) was separated by RP-HPLC with methanol/water (45:55) as eluent to give pure compound 1 (1 mg, t_R 40 min).

Compound 1: brownish amorphous solid; [α]²⁵_D: −7 (c 0.1, MeOH); ¹H and ¹³C NMR (CD₃OD, 600 MHz), see Table 1; ESIMS m/z 613 [M+H]⁺, 451 [M+H-162]⁺; HRESIMS m/z 613.2120 [M+H]⁺ (calcd 613.2132 for C₂₈H₃₇O₁₅).

Table 1
¹H- and ¹³C-NMR data of compound 1 in CD₃OD, δ ppm, J (Hz).^a

Position	1
	δ H	δ C
1	5.95 d (2.8)	95.2
3	7.56 s	153.4
4		107.0
5	3.45 m	39.7
6	5.52 br d (5.0)	79.0
7a	2.26 dd (14.0, 5.0)	44.5
7b	2.50 br d (14.0)
8		89.0
9	3.11 dd (3.5, 8.5)	50.2
10	1.62 s	21.4
11		168.0
12	3.69 s	51.6
Glc-1'	4.71 d (8.0)	100.1
2'	3.23 dd (8.0, 9.0)	74.2
3'	3.37 t (9.0)	78.0
4'	3.30 t (9.0)	71.3
5'	3.39 m	77.7
6'	3.70 o	62.8
	3.54 dd (12.0, 3.5)
Vrt-7'		167.0
1"	7.58 d (1.5)	123.5
2"		113.3
3"	7.09 d (8.3)	149.9
4"	7.70 dd (8.3, 1.5)	154.6
5"	3.93 s	111.6
6"	3.91 s	124.9
OCH3-3"		56.2
OCH3-4"	1.91 s	56.0
OCOCH3		172.3
OCOCH3		21.2

• ^a
  Data assignments were confirmed by DQF-COSY, HSQC and HMBC experiments.
  Glc, glucose; Vrt, veratroyl; o, overlapped.

Results and discussion

The phytochemical study of the chloroform/methanol (9:1) extract from I. rubescens aerial parts, by means of different chromatographic techniques, led to the isolation of four compounds, of which a new iridoid glucoside (1).

Compound 1 was isolated as brownish amorphous solid and was assigned the molecular formula C₂₈H₃₆O₁₅ as deduced from the HRESIMS (m/z 613.2120 [M+H]⁺) and ¹³C NMR analyses. Spectral data indicated the presence of 11 degree of unsaturation. The ESIMS/MS fragmentation pattern showed an ion fragment at m/z 451 due to the loss of a hexose unit [M+H−162]⁺. Analysis of ¹H NMR data (Table 1) evidenced the presence of a double bond at δ 7.56 (1H, s), characteristic for a 4-substituted enol-ether system, typical of iridoid skeleton. According to an iridoid structure, one acetal proton (δ 5.95, d, J = 2.8 Hz), one hydroxylated methine (δ 5.52, br d, J = 5.0 Hz), two methines (δ 3.45, m; δ 3.11, dd, J = 3.5, 8.5 Hz), one methylene (δ 2.26, dd, J = 14.0, 5.0 Hz); δ 2.50, br d, J = 14.0 Hz), and one methyl (δ 1.62, s) were observed. Furthermore, protons ascribable to a monosaccharide portion were present, with anomeric proton at δ 4.71 (1H, d, J = 8.0). In addition, the ¹H NMR spectrum of 1 exhibited characteristic signals of the veratroyl group at δ 7.09 (1H, d, J = 8.3 Hz), 7.58 (1H, d, J = 1.5 Hz), and 7.70 (1H, dd, J = 8.3, 1.5 Hz). The ¹³C NMR spectrum confirmed ¹H-NMR data, showing 28 signals (Table 1), of which ten attributable to an iridoidal aglycon moiety, while the remaining eighteen signals were ascribable to a hexose residue, a veratroyl group, and an acetyl function. Thus, the aglycon was identified as shanzhigenin methyl ester (Guo et al., 2001). The downfield shift of 10.0 ppm for C-8 (δ 89.0), compared with a hydroxy-substituted carbon, led to establish the location of the acetyl group at the same carbon (Damtoft et al., 1981). The location of the veratroyl substituent at C-6 was deduced from the downfield shift of 1.47 ppm for H-6 (δ 5.52) (Kato et al., 2012). The monosaccharide portion was established to be a glucose on the basis of literature data (Kato et al., 2012). HMBC correlations between H-1'−C-1 and H-1−C-1' confirmed the position at C-1 of the glucopyranosyl portion. The other substituent sites were derived from the HSQC and HMBC correlations which also allowed the assignments of all the resonances of ¹³C NMR spectrum. The correlations between H-3−C-1, H-3−C-4, H-3−C-5, and H-3−C-11 substantiated the location of the double bond. The presence of methoxy group at C-11 was confirmed by HMBC correlation between H-12−C-11, while the position of methyl group at C-10 was deduced by correlations between H-10−C-7, H-10−C-8, and H-10−C-9. Finally, correlations between H-7−C-5, H-7−C-6, H-7−C-8, H-7−C-9, and H-7−C-10 were consistent with the established aglycon structure. Thus, compound 1 was identified as 6-O-veratroylbarlerin.

Together with the new iridoid glucoside, oridonin (Lu et al., 2006), caffeic acid (Chang et al., 2009), and apigenin (Alwahsh et al., 2015) were isolated and characterized by comparison of ¹H and ¹³C NMR spectra and MS data with those reported in the literature. I. rubescens is known as one of the best source of oridonin (Harris et al., 2012). The occurrence of caffeic acid and apigenin with its derivatives in Isodon/Rabdosia genera is illustrated in Table 2. In conclusion, this is the first report about the isolation of a glucosidic iridoid from Isodon genus.

Table 2
Occurrence of caffeic acid and apigenin/apigenin derivatives in the Isodon/Rabdosia genera.

Plant species	Caffeic acid	Apigenin	Apigenin glycosides	References
I. eriocalyx var. laxiflora	+			Niu et al. (2003)
I. japonicus	+			Hong et al. (2009)
I. lophanthoides	+			Feng et al. (2013)
I. lophanthoides var. gerardianus	+		+	Tang et al. (2015); Feng et al. (2016)
I. lophathoides var. graciliflorus	+		+	Zhou et al. (2014); Tang et al. (2015)
I. nervosa	+			Du et al. (2010b)
I. oresbius		+		Hao et al. (1996)
I. rubescens	+			Du et al. (2010a)
I. sculponeata	+			Jiang et al. (2002)
I. serra	+		+	Liu et al. (2010); Tang et al. (2015)
I. sorra	+		+	Chen et al. (2013); Tang et al. (2015)
I. striatus	+		+	Tang et al. (2015)
I. ternifolius		+		Na et al. (2002)
I. xerophilus	+			Hou et al. (2011)
R. excisa	+			Tang et al. (2014)
R. japonica		+		Shen et al. (2009); Liu et al. (2013)
R. japonica var. glaucocalyx	+	+	+	Zhang et al. (2006); Yao et al. (2013)
R. lophanthoides	+		+	Kuang et al. (2014); Lu (2015)
R. lophanthoides var gerardiana	+			Lu et al. (2013)
R. rubescens	+			Tang et al. (2011); Guo et al. (2017)
R. flexicaulis	+			Li et al. (2015)
R. serra	+			Zhu et al. (2013)

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Publication Dates

• Publication in this collection
  May-Jun 2018

History

• Received
  26 Jan 2018
• Accepted
  7 Mar 2018