One new royleanumoate from Teucrium royleanum Wall. ex Benth.

One new royleanumoate, a benzene ester (1), has been isolated from T. royleanum Wall. ex Benth along with two known compounds, namely, 3,4-dihydroxymethyl benzoate (2) and oleanolic acid (3). The structure elucidation of the isolated compounds was established on two-dimensional (2D) NMR techniques including heteronuclear multiple bond correlation (HMBC), heteronuclear multiple quantum Coherence (HMQC), and correlation spectroscopy (COSY) experiment.

1. Introduction

The family Lamiaceae is a large family of order Lamiales [1]. It contains about 170 genera and 300 species of worldwide distribution, growing under great variety of soils and climates but more abundant in Mediterranean and mountainous region [2]. Several genera of the family Lamiaceae contain biologically active compounds [3]. Teucrium is one of the important genera of this family [4]. The genus Teucrium comprises mainly herbaceous plants. It contains about 7,000 species in temperate regions, only four species of which are reported in Pakistan, namely, Teucrium stocksianum, Teucrium scordium, Teucrium royleanum, and Teucrium quadrifarium [5]. Many biological activitieshave been attributed to the genus Teucrium. Some of the species have been used as medicinal plants since time immemorial and are still being used in folk medicine as antispasmodics, tonics, antipyretics, and antiseptics [6]. The literature survey reveals that the terpenoids in these plants have also shown insect antifeedant activity [6-8]. These medicinal properties prompted us to carry out phytochemical investigation on T. royleanum in continuation to our ongoing research on this species [9-11]. Our current study has led to the isolation of one new royleanumoate, abenzene ester 1. In addition to the new compound 1, some known compounds 3,4-dihydroxymethyl benzoate 2 and oleanolic acid 3 have been isolated for the first time from this species (see Figures 2, 3, and 4).

Figure 2

Figure 3

Figure 4

2. Material and Methods

2.1. Plant Materials

The aerial parts of T. royleanum were collected from Swat (Pakistan) in June 2003 and identified by Professor Dr. Abdul Rashid, Plant Taxonomist, Department of Botany, University of Peshawar, Peshawar, Pakistan, where a voucher specimen (number Shabir 2651979 (PUP)) is deposited.

2.2. Methods for Purification

The powdered air-dried aerial parts of T. royleanum (10 kg) were soaked in MeOH (3 × 45 L) at room temperature for about 24 hours. The combined methanolic extract was then concentrated via rotavapour to get a thick gummy extract (850 g). The resultant concentrated extract was then dissolved in water and was subjected to solvent-solvent extraction process using n-hexane, chloroform, and n-butanol.

The fraction Tb-SA1 was eluted on a silica gel column loaded with initial chloroform-hexane (1 : 1) which on further column chromatography in chloroform-hexane (6.5 : 3.5) provided compound 1 as amorphous solid (7 mg).

2.3. Physical and Spectral Data of Royleanumoate (1)

IRν max⁡ (KBr) cm−1 3440, 1735, 1617

ElMS m/z: 121 (100), 107 (6), 71 (11), and 57  (40)

FAB+MS m/z: 333.3713 (caled. for C21H34O3)

1H-NMR 13C-NMR (C5D5N, 400 MHz and 100 MHz): Table 1.

Table 1

1H-NMR and 13C-NMR (C5D5N, 400 MHz, C5D5N, 100 MHz), chemical shifts, and multiplicities of (1).

C. number	Multiplicity (DEPT)	13C-NMR (δ)	1H-NMR (δ)	1 J HH (Hz)
C-1	C	173.80	—	—
C-2-C-11	CH2	29.25	1.23	brs
C-12	CH3	14.11	0.88	t, J = 6.4
C-1′	C	130.15	—	—
C-3′-5′	CH	115.38	6.75	d, J = 10.0
C-2′-6′	CH	130.04	7.05	d, J = 10.0
C-4′	C	153.84	—	—
C-1′′	CH2	34.38	2.81	t, J = 2.8
C-2′′	CH2	64.87	2.24	t, J = 2.3

3. Results and Discussion

Compound 1 was isolated from the VLC fraction of the chloroform soluble part obtained from the methanol extract of T. royleanum Wall. ex Benth as amorphous solid. The Fab +ve of 1 showed the [M+1]+ at m/z 333.3713 in agreement with the molecular formula C21H34O3 indicating five degrees of unsaturation. Other prominent mass fragments at m/z 121 (100), 107 (6), 71 (11), and 57 (40) were also observed in the mass spectrum as shown in Scheme 1. The IR spectrum of compound 1 exhibited absorption bands at 1735 (ester C=O), 3430 for (OH), and 1617 for (aryl).

Scheme 1

The mass spectral fragmentation pattern for royleanumoate (1).

The 1H-NMR spectrum corroborated the presence of one methyl, thirteen methylene, and aromatic groups in the high-field region. In the downfield region of the spectrum two doublets at δ 6.75 and 7.05 each of two protons integration were assigned to C-2′, C-6′ and C-3′, C-5′ aromatic protons. The methyl group attached at the terminal position of the aliphatic chain appeared as a triplet at δ 0.88 with a J = 6.36. Similarly, methylene protons at C-1′′ and C-2′′ at δ 2.81 and 2.24 show two triplets each of 2 H integration with a J value of 2.81 Hz and 2.25 Hz.

The 13C-NMR spectrum (BB, DEPT) (Table 1) showed twenty-one signals, including one methyl, thirteen methylene, four methine, and three quaternary carbons. In the downfield region signals appeared at δ 130.2, 115.4, 130.04, and 153.8 which were assigned to the C-1′, C-2′, C-6′, C-3′, C-5′, and C-4′ of aromatic carbons, while a signal at δ 173.7 indicated the presence of a carbonyl carbon in the form of ester in the molecule.

Similarly, two signals at δ 34.4 and 64.9 were assigned to the methylene carbons present in between ether oxygen and aromatic ring, while in the upfield region a signal at δ 14.1 was assigned to the methyl carbon attached at terminal position of the aliphatic chain. The long-range 1H-13C connectivities were established through HMBC technique.

In the HMBC spectrum (Figure 1), the C-1′′ methylene protons (δ 2.81, t) showed correlations with C-2′′ (δ 34.38) and another correlations of C-2′′ (δ 130.04) and C-1′ (δ 130.15), thus supporting the attachment of –CH2–CH2– to the phenol ring at paraposition. Similarly the two orthoprotons (C-2′, C-6′) also showed correlations with C-3′ and C-5′, respectively.

Figure 1

Important HMBC correlations of royleanumoate (1).

On the basis of all the above spectral data and comparison with the analogous structures in the literature [12] the compound 1 was named as royleanumoate. 3,4-Dihydroxymethyl benzoate 2 and oleanolic acid 3 were also isolated for the first time from thechloroform soluble fraction of the crude extract of T. royleanum and identified by comparison with the literature data [13].

4. Conclusion

One new compound (benzene ester 1) and two known compounds (3,4-dihydroxymethyl benzoate 2 and oleanolic acid 3) have been isolated from T. royleanum Wall. ex Benth. The isolated compounds were confirmed by two-dimensional NMR technique, IR, and mass spectra.