Flavonoids, cinnamic acid and phenyl propanoid from aerial parts of Scrophularia striata

Abstract

No phytochemical investigation regarding Scrophularia striata Boiss. (Scrophulariaceae) has been performed, although several reports about other Scrophularia species have been published. The inhibitory effects of aerial parts of S. striata on matrix metalloproteinase expression elaborate a new approach to treat variety of malignant and inflammatory disorders. Five known compounds, including cinnamic acid, three flavonoids (quercetine, isorhamnetin-3-O-rutinoside and nepitrin) and one phenyl propanoid glycoside (acteoside 1) were isolated from S. striata Boiss. by chromatographic techniques and the structures of compounds were characterized by spectroscopic methods. This is the first report regarding the isolation of these compounds from S. striata.

Keywords::

• Acteoside
• flavonoid
• nepitrin
• phenolic compounds
• Scrophularia striata

Introduction

The genus Scrophularia, consisting of about 300 species, is one of the most important genera belonging to Scrophulariaceae. Many species belonging to this genus have been used since ancient times as folk remedies for some medical treatments (scrophulas, scabies, tumours, eczema, psoriasis, inflammatory affections, etc.) (Heather & Henderson, 1994).

Scrophularia ningpoensis Hemsl. is found in China their ancient folkloric use includes anti-fever, and treatment of laryngitis, pharyngitis, neuritis and constipation swelling (Miyazawa et al., 1998). The roots of S. ningpoensis are used as a famous Chinese medicine named “Xuan shen” for the treatment of various inflammatory diseases (Li et al., 1999). Other species, such as Scrophularia grossheimi Schischk. and Scrophularia nodosa Linn. are used as diuretic agents (Akhmedov et al., 1969).

There are several reports that describe isolation, purification and structure elucidation of new compounds including iridoids, phenyl propanoids, phenolic acids, flavonoids and saponins from different species of Scrophularia (Guisalberti, 1998; Emam et al., 1997; Nishibe, 1994).

Scrophularia striata Boiss. is a small, many branched perennial herb that occurs abundantly in all parts of Iran and also grows spontaneously in Turkey and Azerbaijan. The dimensions of serrated leaves are about 7.5 × 2 cm and the length of their stems is about 30-90 cm (Rechinger et al., 1994).

There is one report on the inhibitory effect of aerial parts of Scrophularia striata on matrix metalloproteinases expression (Hajiaghaee et al., 2007), but no phytochemical investigation has been found in literature.

In this paper we report the isolation and structural elucidation of cinnamic acid, three flavonoids and a phenyl propanoid glycoside for the first time from this species.

Materials and methods

The sequential extracts were prepared from dried and powdered aerial parts of S. striata and one phenolic acid, three flavonoids and one phenyl propanoid glycoside were purified.

Plant material

The aerial parts of S. striata were collected from the northeastern part of Iran, in the Ruin region (1350 m above sea level) in May 2006, and were air dried at room temperature.

A sample was authenticated by F. Attar, and a voucher specimen was preserved in the Faculty of Sciences’ Herbarium at Tehran University, Tehran, Iran (TUH No: 36501).

Chemicals and instruments

The ¹H and ¹³C-NMR spectra of the isolated compounds were measured in CD₃OD at 500 and 125 MHz, respectively, using a Bruker AC 500 spectrophotometer (Dramstadt, Germany). MS spectra were taken on a Finnigan TSQ-Mat 70(70eV) spectrometer. UV spectra were obtained using a Shimadzu UV-240 spectrophotometer.

In the case of flavonoids, all shift reagents spectra were measured by adding the reagents separately to a solution of the investigated material in methanol (Harborne et al., 1975).

Silica gel (Merck), Sephadex LH-20 (Fluka) and Polyamide 6 (Fluka) were used as stationary phases in column chromatography fractionations. All solvents and reagents were purchased from Merck and were extra pure or reagent grade.

Extraction and isolation

Petroleum ether extract

Dried powdered aerial parts (1 kg) of the plant were extracted with petroleum ether (3 × 500 mL) by maceration for 36 h. The mixture was filtered and the solvent was evaporated under reduced pressure. The residue (6.6 g) was subjected to column chromatography (CC), (4 × 60 cm, 300 g silica gel), the mobile phase was petroleum ether: dichloromethane (50:50). Four fractions were yielded that were further purified by preparative TLC. Some alkanes and fatty acid compounds were distinguished from petroleum ether fraction.

Chloroform extract

To the filtered part (from previous step) was added 500 mL of chloroform (three times) and shaken successively for 24 h. The mixture was filtered and combined chloroform extract was concentrated in vacuum. The residue (11.4 g) was subjected to CC on silica gel eluted repeatly with CHCl₃-ethylacetate (90:10) with an increasing percentage of EtOAc to obtain five main parts (a-e). Fraction c was further purified by second column chromatography with CHCl₃-EtOAc as eluent to obtain 23 mg of compound 1.

Ethyl acetate extract

To the filtered part (from previous step) was added 500 mL of EtOAc (three times) and shaken successively for 24 h. The mixture was filtered and combined EtOAc extract was concentrated under reduced pressure. The residue (12.8g) was subjected to CC on silica gel eluted with 500 ml of CHCl3-ethylacetate (90:10) and then with an increasing percentage of ETOAc to reach 100%. Compound 2 (20 mg) and compound 3 (23 mg) were obtained as pure compounds.

Figure 1.  Chemical structures of cinnamic acid (1), quercetin (2), isorhamnetin 3-O-rutinoside (3), nepitrin (4) and acteoside 1 (5) from Scrophularia striata.

80% Methanolic extract

To the filtered part (from the previous step) was added 1 L (twice) methanol solution (80%) and shaken successively for 24 h. The mixture was filtered and combined, the solvent was concentrated under reduced pressure. The residue (16.4 g) was subjected to a polyamide column chromatography and eluted by H₂O:methanol (100:0) with an increasing percentage of methanol to 100%. Eight main parts were obtained (a-h). Fractions b and c were combined and rechromatographed on Sephadex LH-20 with methanol to afford 32 mg of compound 4 as pure compound. Fractions e and f were combined and rechromatographed on Sephadex LH-20 with methanol to afford 28 mg of compound 5 as pure compound.

Results and discussion

The EIMS spectrum of compound 3 showed a [M- (Glu+Rh)]⁺ at m/z: 316. The ¹H NMR was used to confirm the structure of compound 3. The methoxy (δ_H = 3.9) and two aromatic spin systems, δ_H 7.6 ( J = 8.4 and 2.4 Hz), 6.9( J = 8.4 Hz) and 7. 85 ( J = 2.4 Hz) corresponded to H-6′, H-5′ and H-2′, respectively, and δ_H 6.3 ( J = 2.4 Hz) and 6.1 (J = 2.4 Hz) to H-8 and H-6, respectively, was observed (Table 1). The ¹H NMR spectrum of compound 3 indicated the presence of one rhamnosyl and one glucosyl moieties with characteristic signals at 4.5 ppm (H-1′′′, d, J = 7.5 Hz) and 1.1 ppm ( H-6′′′, d, J = 6 Hz) for rhamnose and the anomeric proton of β-glucose at 5.2 ppm (H-1′′, d, J = 7.3 Hz). The rhamnose moiety should be attached to glucosyl, as the glucose ¹³C signals equivalent to those seen in data available from the literature (Tulyaganov et al., 2001; Wenkert & Gottlieb, 1977). Thus, compound 3 is isorhamnetin 3-O-rutinoside.

Table 1.  ¹H NMR and ¹³C NMR spectroscopic data of isorhamnetin 3-O-rutinoside (3) and nepitrin (4) in CD₃OD.

Position	3		4
	δ ^1H	δ ^13C	δ ^1H	δ ^13C
2		158.4		166.2
3		135.5	6.6 s	105.2
4		179.2		184.0
5		162.9	6.5 s	152.1
6	6.1 d (2.4)	100.1		130.6
7		166.2		155.5
8	6.3 d (2.4)	95.0		96.0
9		158.8		153.8
10		105.6		106.7
1′		122.9		123.7
2′	7.8 d (2.4)	114.6	7.5 d (2.1)	110.8
3′		148.3		149.5
4′		150.8		151.3
5′	6.9 d (8.4)	116.1	6.9 d (8.5)	116.8
6′	7.6 dd (8.4, 2.4)	124.0	7.4 dd (8.5, 2.1)	121.7
OCH3	3.9 s	56.8	3.9 s	56.7
glucose
1′′	5.2 d (7.3)	104.5	5.1 d (7.3)	103.7
2′′	3.01-3.37 m	75.9	3.01-3.7 m	75.4
3′′	3.01-3.37 m	77.3	3.01-3.7 m	77.8
4′′	3.01-3.37 m	71.6	3.01-3.7 m	70.9
5′′	3.01-3.37 m	78.2	3.01-3.7 m	78.7
6′′	3.01-3.37 m	68.5	3.01-3.7 m	62.2
rhamnose
1′′′	4.5 d (1.5)	102.5
2′′′	3.01-3.37 m	72.0
3′′′	3.01-3.37 m	72.3
4′′′	3.01-3.37 m	73.8
5′′′	3.01-3.37 m	69.7
6′′′	1.1 d (6.0)	17.8

Compound 4 was isolated from 80% methanol extract as yellow needles. The UV spectra of 4 suggested the presence of free 5-3′-4′-hydroxyl and substituted 7-hydroxyl groups via treatment with NaOMe, AlCl₃ and NaOAC (Mabry et al., 1970). The EIMS spectrum showed a [M – (Glu)] of m/z: 315.

The presence of 1-glycosyl and 6-methoxyl groups was indicated by comparison of C-6 (δ 130.6) and C-8 (δ 96.0) ¹³C NMR signals of 4 with those of 6-methoxyl luteolin-7-glucoside (Weng & Wang, 2000). The absence of 3-hydroxy was indicated by C-4 (δ 189.0) and H-3 (δ 6.6, S) that corresponding with flavone structures. The ¹H NMR spectrum of compound 4 indicated the presence of glucosyl moieties with characteristic signal at 5.1 ppm (H 1′′, d, J = 7.3 Hz), the anomeric proton of β-glucose. Therefore, compound 4 was identified as nepitrin. The chemical shifts of the compound 4 are shown in Table 1.

The characteristics of compound 5 were as follows. Amorphous powder, UV ( MeOH) λ_max (log ϵ): 333 (4.28), 291 (4.10), 246 (3.98), 219 (4.23) nm; ¹HNMR (CD₃OD): 1.07 (3H, d, J = 6 Hz, H-6′′′), 2.7 (2H, m, H-7), 4.3 (1H, d, J = 8.0 Hz, H-1′′), 4.9 (1H, t, J = 10.0 Hz, H-4′′), 5.1 (1H, S, H-1′′′), 6.2 (1H, d, J = 16.5 Hz, H-8′), 6.5 (1H, dd, J = 7.5 and 1.5 Hz, H-6), 6.60 (1H, d, J = 7.5 Hz, H-5), 6.68 (1H, d, J = 1.5 Hz, H-2), 6.7 (1H, d, J = 8.5 Hz, H-5′), 6.9 (1H, d, J = 8.5 Hz, H-6′), 7.0 (1H, S, H-2′), 7.5 (1H, d, J = 16.5 Hz, H-7); ¹³C NMR (CD₃OD) δ: 18.1 (C-6′′′), 36.5 (C-7), 62.4 (C-6′′), 70.4 (C-5′′′), 70.6 (C-4′′), 72.0 (C-3′′′), 72.2 (C-8), 72.3 (C-2′′′), 73.8 (C-4′′′), 76.0 (C-5′′′), 76.2 (C-2′′), 81.6 (C-3′′), 102.9 (C-1′′′), 104.2 (C-1′′), 114.5 (C-8′), 115.2 (C-2′), 116.4 (C-5), 116.8 (C-5′), 117.2 (C-2), 121.3 (C-6), 123.6 (C-6), 127.5 (C-1′), 131.6 (C-1), 144.6 (C-4), 146.1 (C-3), 147.0 (C-3′), 148.1 (C-7′), 150.2 (C-4′), 168.4 (C-9′), acteoside 1 ¹H and ¹³C NMR data were consistent with previously published literature (Kobayashi et al., 1987). Compounds 1 and 2 were identified as cinnamic acid and quercetin by comparing their EIMS, ¹H NMR and ¹³C NMR with published data (Bourn et al., 1963; Markham & Geiger, 1994).

Acknowledgement

This research was supported by a grant from the Vice Chancellor for Research, Medical Sciences, University of Tehran, Iran.

Declaration of interest

The authors thank from Chancellor for Research, Medical Sciences, Tehran university of Medical Sciences for financial supporting of this research.