Introduction

Ruellia tuberosa L. (Acanthaceae, Thai name: Toi-ting) is a perennial herb widely distributed in tropical areas of Asian countries. It has been externally used in Thai tradition medicine as an anti-inflammatory, an antiseptic as well as an antidote for detoxification of poisons. Previous phytochemical studies of this plant revealed the presence of steroids, terpenoids, long-chain aliphatic compounds and flavonoids [1–6]. In pharmacological studies, the aerial part extracts showed antioxidant, antinociceptive, and anti-inflammatory activities [7–9]. This present study deals with the isolation and structure determination of polar constituents including a new phenylethanoid (8) and two new flavone glycosides (11, 12) together with nine known compounds. Also, their antioxidant properties using DPPH and ORAC assays were evaluated.

Results and discussion

The methanolic extract of the aerial part of R. tuberosa was suspended in H2O and extracted with Et2O. The aqueous layer was applied to a column of Diaion HP-20, with H2O, MeOH and Me2CO as eluants, successively. The portion eluted with MeOH was separated by a combination of chromatographic procedures to provide 12 compounds (Fig. 1). Nine were identified as known compounds, including verbascoside (1), isoverbascoside (2), nuomioside (3), isonuomioside (4), forsythoside B (5), paucifloside (6), cassifolioside (7), hispidulin 7-O-β-d-glucuronopyranoside (6-O-methyl-scutellarein, 9) and comanthoside B (pectolinaringenin 7-O-β-d-glucuronopyranoside, 10) on the basis of spectroscopic evidence and comparison of the physical data with reported values [10–16].

Fig. 1
figure 1

The structures of compounds 1–12

Full size image

Compound 8 was isolated as a yellow amorphous powder, and its molecular formula was determined as C35H46O19 by high-resolution atmospheric pressure chemical ionization time-of-flight (HR-APCI-TOF) mass spectrometric analysis. The 1H- and 13C-NMR spectroscopic data (Table 1) indicated that compound 8 is an analogue of compounds 1–7 with three sugar moieties. The chemical shifts were related to those of isoverbascoside (2) except for a set of additional signals arising from an α-l-rhamnopyranosyl moiety, deduced from the chemical shifts of the carbon signals at δ C 102.9, 72.3, 72.3, 73.6, 70.1 and 18.0. This sugar moiety was suggested to be attached at C-2″ of the glucopyranosyl moiety due to the downfield shift of this carbon atom at δ C 80.1 as compared to isoverbascoside (2). The assignment was confirmed by means of COSY, HMQC and HMBC. In the HMBC spectrum, the significant correlations were observed from H-1″ (δ H 4.43) to C-8 (δ C 72.3), H-1″″ (δ H 4.98) to C-2″ (δ C 80.1), H-1′″ (δ H 4.93) to C-3″ (δ C 87.2), and H-6″ (δ H 4.50 and 4.34) to C-9′ (δ C 169.1) as illustrated in Fig. 2. In addition, acid hydrolysis provided d-glucose and l-rhamnose, identified by HPLC analysis using the optical rotation detector. Consequently, the structure was determined as shown. Since compound 8 was an isomer of crassifolioside (7), different on the location of the caffeoyl moiety at C-6″ instead of C-4″, therefore the name isocrassifolioside was proposed for this compound.

Table 1 1H-(400 MHz) and 13C-NMR (100 MHz) spectroscopic data of compound 8 (MeOH-d 4)
Full size table
Fig. 2
figure 2

HMBC correlations of compounds 8 and 11

Full size image

Compound 11 had the molecular formula C28H30O16 based on HR-APCI-TOF mass spectrometric analysis. The 1H- and 13C-NMR spectroscopic data (Table 2) were similar to those of hispidulin 7-O-β-d-glucuronopyranoside (9) except for the presence of an α-l-rhamnopyranosyl unit. This sugar moiety was assigned to be attached at C-2″ of the β-d-glucuronopyranosyl because the chemical shifts of C-1″ and C-2″ were significantly changed to 97.6 and 76.3 ppm, respectively, relative to compound 9. Moreover, HMBC correlation was observed between H-1′″ (δ H 5.23) and C-2″ (δ C 76.3), as shown in Fig. 2. The absolute configuration of two sugar units were identified as d-glucuronic acid and l-rhamnose by HPLC analysis. Consequently, the structure of this compound was elucidated to be hispidulin 7-O-α-l-rhamnopyranosyl-(1′″ → 2″)-O-β-d-glucuronopyranoside.

Table 2 1H-(400 MHz) and 13C-NMR (100 MHz) spectroscopic data of compounds 11 and 12 (DMSO-d 6)
Full size table

The molecular formula of compound 12 was determined as C29H32O16by HR-APCI-TOF mass spectrometric analysis. Inspection of the 1H- and 13C-NMR spectroscopic data (Table 2) revealed that this compound had the same aglycone as pectolinaringenin 7-O-β-d-glucuronopyranoside (10), while the sugar moieties were identical to those of compound 11. Accordingly, this compound was determined as pectolinaringenin 7-O-α-l-rhamnopyranosyl-(1′′′ → 2″)-O-β-d-glucuronopyranoside.

In this study, the isolated compounds 1–12 were evaluated for their radical scavenging activities using both DPPH and ORAC assays (Table 3) [17]. In DPPH assay, phenylethanoid glycosides (1–8) displayed relative scavenging activity in same range of the positive control, ascorbic acid, while flavonoid glycosides (9–12) were inactive. In the ORAC assay, the unit values of all isolated compounds were in the range of 2–6 folds more potent than the positive control, Trolox. The predominantly antioxidant activity of phenylethanoid glycosides (1–8) and was due to the presence of a caffeoyl and free two hydroxyl groups on phenyl ring. Position of a caffeoyl group locating on C-4″ or C-6″ of sugar moiety seemed to be no effect on the activity. In addition, flavonoid glycosides having free hydroxyl group on phenyl ring (9, 11) were active about 2 folds more potent than methoxy analogues (10, 12).

Table 3 Radical scavenging activities of isolated compounds 1–12
Full size table

Experimental section

General procedures

1H- and 13C-NMR spectra were recorded using a Bruker AV-400 and a Brucker AVANCE III ultrashield 300 MHz spectrometers. Mass spectra were obtained on a Bruker Micro TOF-LC mass spectrometer. Optical rotations were measured with a Jasco P-1020 digital polarimeter. For column chromatography, Diaion HP-20 (Mitsubishi Chemical Industries Co. Ltd.), silica gel 60 (70–230 mesh, Merck), and RP-18 (50 μm, YMC) were used. HPLC (Jasco PU-980 pump) was carried out on ODS column (21.2 × 250 mm i.d., Vertisep™ AQS, 8 mL/min) with a Jasco MD-2010 detector at 220 nm. The spraying reagent used for TLC was 10% H2SO4 in 50% EtOH.

Plant material

The aerial portions of Ruellia tuberosa L. were collected in March 2010, Bangkok, Thailand. The identification of the plant was done by Mr. Nopporn Nontapa of Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher specimen (TK-PSKKU-0069) is on file in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University.

Extraction and isolation

The air-dried aerial portions of R. tuberosa (3.2 kg) were macerated with MeOH three times (12 L for each extraction) at room temperature. The MeOH extract was concentrated in vacuo to dryness. This residue (318.1 g) was suspended in H2O, and partitioned with Et2O (each 1.0 L, 3 times). The aqueous soluble fraction (255.0 g) was subjected to a Diaion HP-20 column, and eluted with H2O, MeOH and (CH3)2CO, successively. The fraction eluted with MeOH (41.5 g) was subjected to a silica gel column using solvent systems EtOAc–MeOH (9:1, 5.0 L), EtOAc–MeOH–H2O (40:10:1, 4.0 L), EtOAc–MeOH–H2O (70:30:3, 5.0 L) and EtOAc–MeOH–H2O (6:4:1, 10.0 L), respectively, to afford seven fractions. Fraction 3 (6.6 g) was applied to a RP-18 column using a gradient solvent system H2O–MeOH (90:10 → 20:80, v/v) to provide eight fractions. Fraction 3–4 (2.4 g) was purified by preparative HPLC-ODS using solvent system H2O–MeCN (80:20, v/v) to give compounds 1 (792.3 mg), 2 (171.8 mg), 3 (19.4 mg) and 4 (17.7 mg). Fraction 5 (9.7 g) was subjected to a RP-18 column using solvent system H2O–MeOH (90:10 → 20:80, v/v) to provide eleven fractions. Fraction 5–6 (3.4 g) was purified by preparative HPLC-ODS with solvent system H2O–MeCN (80:20, v/v) to provide compounds 5 (332.5 mg), 6 (32.6 mg), 7 (27.4 mg) and 8 (66.2 mg). Fraction 6 (5.8 g) was applied to a RP-18 column using solvent system H2O–MeOH (90:10 → 20:80, v/v) to afford six fractions. Fraction 6–2 (2.1 g) was purified by preparative HPLC-ODS with solvent system H2O–MeCN (85:15, v/v) to obtain compounds 9 (87.8 mg), 10 (59.9 mg), 11 (89.0 mg) and 12 (90.1 mg).

Isocassifolioside (8)

Yellow amorphous powder; [α] 27D -54.5 (MeOH, c 1.05); IR spectrum: ν max = 3386, 2936, 1693, 1604, 1278, 1040 cm−1; negative HRMS (APCI-TOF): [M−H]−, found 769.2545. C35H45O19 requires 769.2561. 1H- and 13C-NMR data: see Table 1.

Hispidulin 7-O-α-l-rhamnopyranosyl-(1′″ → 2″)-O-β-d-glucuronopyranoside (11)

Yellow amorphous powder; [α] 26D -44.2 (DMSO, c 1.00); IR spectrum: ν max = 3266, 2926, 1604, 1458, 1351, 1286, 1023 cm−1; negative HRMS (APCI-TOF): [M−H]−, found 621.1444. C28H29O16 requires 621.1461. 1H- and 13C-NMR data: see Table 2.

Pectolinaringenin 7-O-α-l-rhamnopyranosyl-(1′″ → 2″)-O-β-d-glucuronopyranoside (12)

Yellow amorphous powder; [α] 26D -69.4 (DMSO, c 1.03); IR spectrum: νmax = 3320, 2926, 1603, 1459, 1354, 1250, 1022 cm−1; negative HRMS (APCI-TOF): [M−H]−, found 635.1604. C29H31O16 requires 635.1617. 1H- and 13C-NMR data: see Table 2.

Determination of the absolute configurations of sugars

Monosaccharide subunits of new compounds (8, 11 and 12) were obtained by acid hydrolysis. Each sample (ca. 5 mg) in 2 N HCl-dioxane (6:1, 3.5 ml) was heated at 80 °C for 6 h. After cooling, each reaction was diluted with H2O and extracted with EtOAc. Each aqueous layer was concentrated to dryness providing the sugar fraction. Each of these was dissolved in H2O (1 mL) and analysed by HPLC (Jasco OR-2090 plus chiral detector; column A: Vertisep™ sugar LMP or column B: Vertisep™ OA, 7.8 × 300 mm i.d.; mobile phase A: water or B: 0.003 M H2SO4 aq.; flow rate 0.4 ml/min; temperature 40 or 80 °C) and comparison of their retention times and optical rotations with authentic samples.

Hydrolysis of compound 8 gave peaks corresponding to d-glucose at 19.0 min and l-rhamnose at 23.0 min (both positive optical rotation) (column A, mobile phase A, 80 °C).

Hydrolysis of compound 11 gave peaks of d-glucuronic acid at 12.6 min and l-rhamnose at 15.2 min (both positive optical rotations) (column B, mobile phase B, 40 °C).

Hydrolysis of compound 12 gave peaks of d-glucuronic acid at 12.6 min and l-rhamnose at 15.2 min (both positive optical rotations) (column B, mobile phase B, 40 °C).