Phlomiside: A new steroid from Phlomis bracteosa

Riaz Ullah; Zain Ullah; Iqbal Hussain; Shabir Ahmad

doi:10.1016/j.arabjc.2013.03.014

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Original article

10 (

1_suppl

); S1303-S1305

doi:

10.1016/j.arabjc.2013.03.014

Phlomiside: A new steroid from Phlomis bracteosa

Riaz Ullah^{^a,⁎}, Zain Ullah^{^b}, Iqbal Hussain^{^c}, Shabir Ahmad^{^c}

a Department of Chemistry, Sarhad University of Science and Information Technology, Peshawar KPK, Pakistan

b Department of Chemistry, Gomal University, Dera Ismail Khan KPK, Pakistan

c Department of Chemistry, Kohat University of Science and Technology, Kohat KPK, Pakistan

⁎Corresponding author. afridiriaz@yahoo.com (Riaz Ullah)

Received: 2012-7-24, Accepted: 2013-3-15, Published: 2013-02-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

A new steroid named Phlomiside (1) has been isolated from the whole plant including roots from the ethylacetate fraction of Phlomis bracteosa along with four known compounds namely β-amyrin (2), β-sitosterol (3), stigmasterol (4) and oleanolic acid (5). The structure assignments are based primarily on two-dimensional (2D)-NMR techniques including correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) and nuclear overhauser effect spectroscopy (NOESY) experiments.

Keywords

Phlomis bracteosa

Labiatae

Phlomiside

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1 Introduction

The genus Phlomis (Labiatae) consists of about 100 species (Albaladejo et al., 2004; Kyriakopoulou et al., 2001), few species of them are used as tonics and stimulants in Anatolian folk medicine (Calis and Kırmızıbekmez, 2004). Phlomis species are explained by Dioscorides as herbal drugs, and are used ethno-pharmacologically in herbal medicine for respiratory tract diseases and for local treatment of wounds. Some Phlomis species are used in folk medicine for their analgesic and antidiarrheal properties, and for the treatment of ulcers and hemorrhoids. There are few reports about the pharmacological and biological effects of Phlomis. Some studies have shown various activities such as anti-inflammatory, immuno-suppressive, antimutagenic, anti-nociceptive, antifibriel, free radical scavenging, anti-malarial, and anti-microbial effects (Sarkhail et al., 2006). Different classes of glycosides comprising diterpenoids, iridoids, phenylpropanoids, phenylethanoids and flavonoids have been identified from the genus Phlomis. Many of these phenylpropanoids showed significant biological activities, such as cytotoxic, cytostatic, anti-inflammatory, immuno-suppressant and anti-microbial (Kamel et al., 2000). The present work has led to the isolation of one new steriods, trivially named Phlomiside (1) and four known compounds β-amyrin (2), β-sitosterol (3), stigmasterol (4), and oleanolic acid (5). These four compounds from Phlomis bracteosa are reported for the first time.

2 Experimental

2.1

2.1 General

Column chromatography (CC) utilized silica gel, 70–230 mesh. Flash chromatography was carried out using silica gel 230–400 mesh. Thin layer chromatography (TLC) was performed with pre-coated silica gel G-25-UV254 plates and detection was achieved at 254 nm, and by spraying with ceric sulfate in 10% H₂SO₄ solution. The IR and UV spectra were recorded on a Jasco-320-A and Hitachi-UV-240 spectrophotometers, respectively. Optical rotations were measured on a Jasco-DIP-360-digital polarimeter using a 10 cm cell-tube. Mass spectra (EI- and HR-EI–MS) were measured in an electron impact mode on either Finnigan MAT 12 or MAT 312 spectrophotometers; ions are presented in m/z (%). The ¹H- and ¹³C-NMR spectra were recorded on a Bruker AMX-400 spectrometer in CDCl₃. 2D-NMR spectra were obtained using a Bruker AMX-400 spectrometer. Chemical shifts, in parts per million (δ), relative to tetramethyl silane (TMS) as in ternal standard, and scalar couplings (J) are reported in Hertz.

2.2

2.2 Plant material

Whole parts of P. bracteosa were collected from Parachinar, NWFP, Pakistan in July 2005 and identified by Mr. Muhammad Naveed, Assistant Professor, Botany Department, University of Peshawar, NWFP, Pakistan. A voucher specimen (No. PB1) was deposited in the herbarium of the Botany Department Kohat University of Science and Technology Kohat.

2.3

2.3 Extraction and compound isolation

The whole plant of P. bracteosa was dried in the shade, chopped and ground to coarse powder. The powdered plant (3 kg) was initially extracted with methanol for 25 days at room temp. The methanolic extract was evaporated under reduced pressure to give 300 g residue. It was partitioned between n-hexane, chloroform, ethyl acetate, n-butanol and water. 60 g of ethyl acetate was chromatographed over a silica gel column. Elution was carried out with a gradient of increasing polarity of ethyl acetate in n-hexane up to 100% ethyl acetate, and then a gradient of methanol in ethyl acetate up to 100% methanol. Fraction A was obtained through elution with n-hexane/ethyl acetate (1:1) and was subjected to CC with n-hexane/ethyl acetate. This fraction afforded compounds 1–5 on elution with n-hexane/ethyl acetate at a polarity range from 20% to 50%.

2.4

2.4 Phlomiside (1)

Amorphous solid. – ¹H and ¹³CNMR (CDCl₃) data see Table 1. – ${[α]}_{D}^{23} = + 46.28$ (c = 0.15, CHCl₃). – IR: ν = (CHCl₃) = 3600–3400, 1590, 1460, 1750 cm⁻¹. – EIMS: (C₅₁H₈₈O₇) m/z = 812, 57 (100), 97 (57), 135 (30), 125 (15), 417 (87), 395 (40).

Table 1 NMR spectral data of compound 1 (δ in ppm).

C. No.	Multiplicity (DEPT)	¹³C NMR (δ)	δ H	C. No.	Multiplicity (DEPT)	¹³C NMR (δ)	δ H
3	CH	78.7	3.47, m	21	CH₃	22.8	0.91, d
4	CH₂	38.6	–	22	CH	138.4	5.31, dd
5	C	130.8	–	23	CH	128.6	5.03, dd
6	CH	118.7	5.16, t	24	CH	48.5	–
7	CH₂	33.9	–	25	CH	26.7	–
8	CH	36.8	–	26	CH₃	19.8	0.85, d
9	CH	48.1	–	27	CH₃	19.6	0.65, d
10	C	38.2	–	28	CH₂	22.5	–
11	CH₂	21.3	–	29	CH₃	22.8	0.83, t
12	CH₂	39.7	–	1′	CH	106.9	4.50, d
13	C	39.3	–	2′	CH	78.7	–
14	CH	55.1	–	3′	CH	77.2	–
15	CH₂	26.0	–	4′	CH	68.8	–
16	CH₂	27.8	–	5′	CH	68.1	–
17	CH	56.1	–	6′	CH₂	62.0	4.16, m
18	CH₃	13.8	0.67, s	C2″-15″	(CH₂) n	34.4–22.6	0.86–2.29
19	CH₃	21.3	0.97, s	1″	C⚌O	153.5	–
20	CH	36.1	–	16″	CH₃	14.5	0.86, t

3 Results and discussion

Phlomiside 1 (Fig. 1) was isolated from the ethylacetate soluble portion of P. bracteosa. The mass fragmentation pattern of 1 observed in EIMS clearly indicated the presence of stigma sterol type skeleton, which was confirmed by comparing ¹H and ¹³C NMR data with those reported for sterol (Matsui et al., 1996). The IR spectrum showed an absorption band at 3600–3400 cm⁻¹ indicating the presence of hydroxyl group, a strong absorption band at 1590, 1460 cm⁻¹ indicating (C⚌C) and at 1750 cm⁻¹ indicating (C⚌O).

The ¹H NMR spectrum of 1 (Table 1) showed three olefinic signals at δ = 5.31 (m, H-22), 5.03 (m, H-23) and 5.16 (t, J = 6.834 Hz, H-6). The same spectrum showed signals for H-3 at δ = 3.47 (m). The seven methyls indicated by the ¹³C NMR spectrum of 1 (Table 1) appeared in the ¹H NMR spectrum at δ = 0.67 (s, Me-18), .97 (s, Me-19), 0.91 (d, J = 6.2 Hz, Me-21), 0.85 (d, J = 6.3 Hz, Me-26), 0.65 (d, J = 6.4 Hz, Me-27) and δ = 0.83 (t, J = 7.0 Hz, Me-29) and long chain fatty acid methyl showed a signal at δ = 0.86 (t, J = 6.50 Hz). This methyl resonated in the ¹³C NMR spectrum at δ = 14.5 (Table 1). This information revealed that methylene 6 proton of glucose unit was coupled with fatty acid. The presence of sugar moiety in the molecule was confirmed due to the presence of an anomeric proton at δ = 4.50 (d, J = 7.0 Hz), methylene 6 protons at δ = 4.16 (m), and an anomeric carbon at δ = 106.9 in the NMR spectrum. In addition to the signal due to stigma sterol and sugar moiety, additional signals appeared in the proton NMR spectrum at δ = 1.23 showing δ range of all protons of the long chain fatty acid. The ¹³C NMR also showed extra carbon at δ = 153.5 confirming the presence of carbonyl in the molecule. Chemical shifts of (¹H and ¹³C) of sugar unit were exactly matched with the reported data (Jensen et al., 1981). The EIMS of 1 showed a peak at m/z = 414 after the loss of sugar moiety. Therefore, in order to get the complete mass of 1, the FAB-MS in the negative mode of the sample was scanned, which showed the pseudo molecular ion peak. The ¹H and ¹³C NMR data for compound 1 are presented in Table 1. All chemical shifts and attachment of different groups were confirmed through ¹H-NMR, ¹³C-NMR, HMBC (Fig. 2), HMQC and COSY techniques and comparison with the reported data of related compounds and trivially named Phlomiside. Along with Phlomiside four known compounds β-amyrin (2) (Heupel, 1985), β-sitosterol (3) (Habib et al., 2007), stigmasterol (4) (Habib et al., 2007), oleanolic acid (5) (Akuta and Itokawa, 1988) were isolated for the first time from P. bracteosa.

4 Conclusion

A new steroid named Phlomiside (1) has been isolated from the whole plant including roots from the ethylacetate fraction of P. bracteosa along with four known compounds namely β-amyrin (2), β-sitosterol (3), stigmasterol (4) and oleanolic acid (5) for the first time.

Acknowledgement

The authors wish to thank the Pakistan Science Foundation, Government of Pakistan, for providing financial support for the current study.

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