Diterpenoids from Rosmarinus officinalis L. and their nitric oxide inhibitory activity

Cite this paper: Vietnam J. Chem., 2021, 59(2), 229-234 Article DOI: 10.1002/vjch.202000161 229 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Diterpenoids from Rosmarinus officinalis L. and their nitric oxide inhibitory activity Le Thi Huyen * , Le Thi Oanh VNU University of Science, Vietnam National University, Hanoi, 19 Le Thanh Tong, Hoan Kiem, Hanoi 10000, Viet Nam Submitted September 17, 2020; Accepted February 4, 2021 Abstract One icetexane diterpenoid, demethylsalvicanol (1) and four abietane diterpenoids, sageone (2), 20-deoxocarnosol (3), 11,12,20-trihydroxy-abieta-8,11,13-triene (4), and 7α-ethoxyrosmanol (5) were isolated from the n-hexane layer of the leaves and twigs of Rosmarinus officinalis L. Chemical structures of compounds were identified by ESI-MS, 1D-, 2D-NMR spectra and by comparison of the spectral data in the literature. Compounds 1-5 were evaluated anti- inflammatory activity by inhibitory NO production, LPS stimulated on RAW 264.7 cells. At a concentration of 100 µM, compound 4 exhibited inhibitory percentage of 34.7±1.8 %, meanwhile compounds 1-3 and 5 showed cytotoxic effect. After dilution to concentration of 20 µM, except compound 1 and 2, compounds 3-5 did not show cytotoxic effect. Their NO inhibitory productions were ranging from 20.5±2.4 % to 26.7±1.9 %. Compounds 1, 3 and 4 have been reported for the first time from the Rosmarinus genus. Keywords. Rosmarinus officinalis, icetexane diterpenoid, abietane diterpenoid, nitric oxide inhibitor. 1. INTRODUCTION Rosmarinus officinalis L. (family Lamiaceae) is a shrub, woody, perennial herbs with fragrant evergreen needle-like leaves that found primarily in the Mediterranean region and widely spread in European, American, and Asian countries. [1] It has been used as a herbal medicine since ancient times for several diseases. It shows antioxidant, [2] anti- inflammator, [3] antimicrobial, [4,5] anticancer, [6-8] antidiabetic, [9] and hepatoprotective. [10] The main components have been identified as diterpenoids, triterpenoids, flavonoids, etc. [11-13] However, phytochemical study of this plant has not been studied yet in Vietnam. This paper reported the isolation, structure elucidation and anti- inflammatory activity of five diterpenoids from the n-hexane extract of the leaves and twigs of R. officinalis. 2. MATERIALS AND METHODS 2.1. Plant materials The leaves and twigs of R. officinalis were collected in Ha Giang, Vietnam in January 2019, and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology). A voucher specimen (RO.19.01) was stored at Faculty of Chemistry, VNU University of Science. 2.2. General experimental procedures The general experimental procedures and measurement techniques are as the same as described in Ref. [21]. 2.3. Extraction and separation The dried ground leaves and twigs of R. officinalis (2.0 kg) were grinded into fine powder and extracted with methanol (3 times  5 L) in ultrasonic extractor for 2 h each. After removal of the solvent under reduced pressure, the to get the residue was 160.0 g. Warm water (2 L, 50 o C) was added to this extract and then successively partitioned by solvents with increasing polarity n-hexane, EtOAc to obtain the n-hexane (ROH, 36.0 g), EtOAc (ROE, 85.0 g) and aqueous layer (ROW, 35.0 g) after evaporating solvents in vacuo under reduced pressure to dryness. Vietnam Journal of Chemistry Le Thi Huyen et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 230 Figure 1: Chemical structures of compounds 1-5 The ROH fraction was carried out by a silica gel column eluting with a n-hexane increasing amounts of EtOAc (100:0  0:1, v/v) to yield fractions H1– H7. H2 fraction was then separated on a silica gel column and eluted with a gradient mobile phase n- hexane-acetone (100:0  0:1, v/v) to give fractions, H2A – H2E. H2A fraction (776.9 mg) was chromatographed by an RP-18 column eluting with acetone-H2O (2.5:1, v/v) to give fractions, H3A- H3E. H3C was separated on an RP-18 column eluting with acetone- H2O (2.0:1, v/v) which afforded compound 2 (3.0 mg). Compounds 3 (10.0 mg) and 4 (17.6 mg) were obtained from H3D fraction using RP-18 column (solvent eluents of acetone-H2O, 2.0:1, v/v). H2C fraction (887.0 mg) was separated by an RP-18 column using acetone- H2O (2.0:1, v/v) as mobile phase to obtain fractions, H4A-H4C. Fraction H4C was further separated by an RP-18 column washing with acetone-H2O (1.5:1, v/v) giving 1 (21.0 mg). Fraction H2E (1.725 g) was loaded on an RP-18 column eluting with acetone- H2O (2.5:1, v/v) to give five fractions, H5A-H5E. H5C was further purified on RP-18 column, eluted with acetone-H2O (2.0:1, v/v), a compound labeled as 5 (27.2 mg) was obtained in pure state. Demethylsalvicanol (1): Colorless amorphous powder; ESI-MS m/z: 319.23 [M+H] + , C20H30O3; MW: 318; 1 H- and 13 C-NMR (CDCl3) data, see table 1. Sageone (2): Colorless amorphous powder; ESI- MS m/z: 301.18 [M+H] + , C19H24O3; MW: 300; 1 H- and 13 C-NMR (CDCl3) data, see table 1. 20-Deoxocarnosol (3): Colorless amorphous powder; ESI-MS m/z: 317.21 [M+H] + , C20H28O3; MW: 316; 1 H- and 13 C-NMR (CDCl3) data, see table 1. 11,12,20-Trihydroxy-abieta-8,11,13-triene (4): Colorless amorphous powder; ESI-MS m/z: 319.22 [M+H] + , C20H30O3; MW: 318; 1 H- and 13 C-NMR (CDCl3), data, see table 2. 7α-ethoxyrosmanol (5): Colorless amorphous powder; ESI-MS m/z: 375.23 [M+H] + , C22H30O5; MW: 374; 1 H- and 13 C-NMR (CDCl3) data, see table 2. 2.4. Inhibition of nitric oxide production Inhibition of NO production in LPS-stimulated RAW 264.7 cell assay, see reference [14]. 3. RESULTS AND DISCUSSION Compound 1 was appeared as a colorless amorphous powder. Its ESI-MS had a pseudo-molecular ion peak at m/z 319.23 [M+H] + , calculating to molecular formula of C20H30O3. The 1 H NMR and HSQC spectra of 1 indicated the signals of two tertiary methyl groups at δH 0.85 and 0.92 (each 3H, s), an isopropyl group at δH 1.21, 1.23 (each, 3H, d, J = 7.0 Vietnam Journal of Chemistry Diterpenoids from Rosmarinus officinalis L © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 231 Hz), and δH 3.18 (1H, m), an aromatic proton at δH 6.56 (1H, s), and a pair of doublets at δH 2.55 and 3.05 (each, 1H, d, J = 14.5 Hz). The 13 C NMR spectrum of 1 revealed signals of 20 carbons which were classified by DEPT spectrum as seven non- protonated carbons, three methines, six methylenes, and four methyl carbons. Of these, the signals at δC 21.5, 22.3 and 27.2 were assigned to the isopropyl group; six carbon signals ranging from δC 117.2 to 142.6 together with a singlet aromatic proton signal at δH 6.56 were assigned to a pentasubtituted aromatic ring. The above evidence leds suggestion that compound 1 was a diterpenoid type icetexane skeleton [15] (figure 1), which was further confimed by 2D-NMR spectra. The important HMBC correlations of 1 were shown in figure 2. Consequently, structure of 1 was established to be demethylsalvicanol, an icetexane diterpenoid previously isolated from Salvia broussonetii. [16] Compound 2 was obtained as a colorless amorphous powder. It had a molecular formula C20H28O3 which was suggested from a pseudo- molecular ion peak [M+H] + at m/z 317.18 in the ESI- MS and in consistent with 13 C NMR data. 1 H NMR and HSQC spectra of 2 also showed the presence of two tertiary methyl groups at δH 0.85 and 1.14; each 3H, s), an isopropyl group at δH 1.24 (d, J = 7.0 Hz), 1.26 (d, J = 7.0 Hz) and 3.10 (1H, m), an aromatic proton signal at δH 6.61 (s), and a pair of doublets at δH 3.08 and 4.31 (d, J = 8.5 Hz) was due to H-20. The 13 C NMR spectrum of 2 showed 20 carbon resonances coresponding to seven non-protonated carbons, four methines, five methylenes, and four methyl carbons. Of these, three carbon signals at δC 21.5, 22.3 and 27.2 were assigned for an isopropyl group; six carbon signals ranging from δC 112.5 to 139.9 together with a singlet aromatic proton signal at δH 6.61 suggested of a pentasubtituted aromatic ring; two oxygen bearing carbons at δC 68.8 and 71.7. The NMR data of 2 were similar to the corresponding data of 20-deoxocarnosol [17] (Table 1). In addition, the HMBC correlations between H- 18 (δH 0.85) and C-3 (δC 41.3)/C-4 (δC 34.0)/C-5 (δC 43.2)/C-19 (δC 21.3) and between H-19 (δH 1.14) and C-3/C-4/C-5/C-18 (δC 33.0) confirmed both two angular methyl groups at C-4; the HMBC correlations from H-16 (δH 1.24)/H-17 (δH 1.26) to C-13 (δC 131.9)/C-15 (δC 27.3) and from H-15 (δH 3.10) to C-12 (δC 141.5)/C-13 (δC 132.8)/C-16 (δC 21.5)/C-17 (δC 22.3) indicated the location of isopropyl group at C-13 of the aromatic ring. The NMR data of ring C of 2 with those of 20- deoxocarnosol were found to match, identifying the two hydroxyl groups were at C-11 and C-12 , which were further confirmed by the observation of the HMBC correlations from H-14 (δH 6.56)/H-15 to C- 12 (δC 140.9). From the above data, compound 2 was identified to be 20-deoxocarnosol, an abietane diterpenoid previously reported from Coleus barbatus. [17] Compound 3 was isolated as a colorless amorphous powder. 1 H-NMR spectrum of 3 showed the presence of four tertiary groups at δH 1.24, 1.23, 1.27, 1.27 and one aromatic proton at δH 6.58 (s, H- 14). The 13 C-NMR and HSQC spectra displayed signals of 19 carbons, including a ketone group at δC 202.1 which appeared to be conjugated with a tetra- substituted olefin (δC 116.5 and 175.6). Therefore, the structure of 3 was determined to be sageone, a nor-diterpene compound isolated from Salvia officinalis. [18] Compound 4 was appeared as a white amorphous powder. Its ESI-MS had a pseudo- molecular ion peak at m/z 319.22 [M+H] + together with 13 C NMR analysis which was determined its molecular formula of C20H30O3. The 1 H and 13 C NMR spectra of 4 were quite similar to those of 2 except for the replacement of an oxygenated methine (δH 4.71/δC 71.1) in 2 instead of a methylene group (δH 2.85/δC 32.0) in 4. In addition, molecular formula of 4 (C20H30O3) had more two hydrogen than that of 2 (C20H28O3). These clear findings implied that the structure of 4 was similar to 2 except for the absence of ether bridge between C-7 and C-20 in compound 4. The remaining NMR signals of 4 were assigned by analysis of HSQC and HMBC spectra as mentioned in compound 2. From above spectral findings and comparison with the NMR data of 11,12,20-trihydroxy-abieta-8,11,13- triene - the compound previously isolated from Salvia mellifera, [19] compound 4 was identified as 11,12,20-trihydroxy-abieta-8,11,13-triene. Compound 5 was obtained as an yellow amorphous powder. It showed identical signals with those of compound 2 except the chemical shift of a carbonyl group at δC 129.3 (C-20) and the presence of ethoxy group in compound 5 (δC 66.2 and 15.8). Comparison of the NMR data of 5 with those published in the literatures, 5 was identified as 7α- ethoxyrosmanol. [20] To evaluate NO inhibitory production on RAW 264.7 cells, LPS-stimulated, all compounds were sceened for cytotoxic activity at concentrations of 100 and 20 µM. All compounds showed cell viability over than 80% at the concentration of 20 µM was further chosen for evaluating NO inhibitory production. As the results, compounds 1-5 showed weak inhibitory activity inhibitory rates ranging from 20.5±2.4 % to 51.6±1.5 %. L-NMMA was used as a positive control with NO inhibitory values Vietnam Journal of Chemistry Le Thi Huyen et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 232 of 82.2±2.5 % at the concentration of 20 µM. Table 1: NMR spectral data of 1-3 and reference compounds C 1 2 3 #δC ,a δC a,b δH a,c (mult., J in Hz) $δC ,a δC a,b δH a,c (mult., J in Hz) %δC ,a δC a,b δH a,c (mult., J in Hz) 1 41.6 41.3 1.50 (m)/1.80 (m) 202.1 202.1 - 30.1 31.1 2.59 (m) 2.12 (brd, 13.5) 2 18.7 18.6 1.41 (m)/1.80 (m) 35.1 35.1 2.69 (dd, 7.0, 12.0) 19.0 19.1 1.62 (m) 3 42.3 42.3 1.27 (m)/1.40 (m) 35.6 35.7 1.94 (t, 7.0) 41.2 41.3 1.28 (m)/1.56 (m) 4 34.4 34.4 - 37.2 37.2 - 33.9 34.0 - 5 58.1 58.2 1.32 (dd, 12.5, 2.5) 175.8 175.6 - 43.0 43.2 1.46 (ddd, 1.5, 5.5, 11.5) 6 24.3 24.3 1.15 (m)/2.00 (m) 27.4 27.4 2.53 (t, 7.5) 30.8 30.2 1.57 (m)/2.03 (m) 7 36.0 36.1 2.64 (dd, 12.0, 14.0) 2.73 (dd, 7.5, 14.0) 28.4 28.4 2.39 (t, 7.5) 71.1 71.1 4.71 (dd, 1.5, 3.0) 8 136.4 136.1 - 127.5 127.5 - 132.9 133.3 - 9 120.3 121.1 - 130.3 130.3 - 127.5 127.6 - 10 71.3 71.9 - 116.5 116.5 - 39.9 40.1 - 11 142.6 142.6 - 139.9 140.0 - 140.9 139.0 - 12 140.4 141.5 - 143.2 143.3 - 139.3 140.9 - 13 132.4 132.8 - 133.1 133.1 - 132.2 131.9 - 14 117.5 117.9 6.56 (s) 116.5 116.6 6.58 (s) 112.2 112.5 6.61 (s) 15 27.2 27.2 3.18 (m) 27.1 27.2 3.29 (m) 27.1 27.3 3.10 (m) 16 21.5 21.5 1.23 (d, 7.0) 22.4 22.4 1.24 (d, 6.5) 22.7 22.6 1.24 (d, 7.0) 17 22.3 22.3 1.21 (d, 7.0) 22.4 22.4 1.23 (d, 6.5) 22.7 22.7 1.26 (d, 7.0) 18 32.2 32.2 0.85 (s) 26.1 26.2 1.27 (s) 32.0 33.0 0.85 (s) 19 22.8 22.9 0.92 (s) 26.1 26.2 1.27 (s) 21.2 21.3 1.14 (s) 20 41.6 41.6 2.57 (s)/3.03 (s) 68.5 68.6 3.08 (d, 8.5)/4.31 (d, 8.5) Measured in a) CDCl3, b) 125 MHz, c) 500 MHz. #)δC of demethylsalvicanol, [16] $)δC of sageone [18] , %)δC of 20-deoxocarnosol. [17] Figure 2: The key HMBC correlations of compounds 1-5 Vietnam Journal of Chemistry Diterpenoids from Rosmarinus officinalis L © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 233 Table 2: The 1 H- and 13 C-NMR data of compounds 4-5 and reference compounds C 4 5 *δC a δC a,b δH a,c (mult., J in Hz) &δC a δC a,b δH a,c (mult., J in Hz) 1 31.4 31.3 1.22 (m)/3.21 (m) 27.4 27.2 1.97 (ddd, 1.5, 5.5, 11.5)/3.18 (brd, 14.0) 2 18.9 18.9 1.43 (m)/1.67 (m) 19.0 19.0 1.53 (m)/1.64 (m) 3 41.4 41.1 1.26 (m)/1.50 (m) 38.0 38.0 1.20 (m)/1.43 (m) 4 33.6 33.6 - 31.4 31.4 - 5 52.7 52.7 1.41 (m) 50.9 50.9 2.28 (s) 6 19.0 19.0 1.63 (m)/1.70 (m) 75.3 75.4 4.35 (d, 3.0) 7 32.0 32.0 2.85 (m) 75.7 75.8 4.66 (d, 3.0) 8 130.0 130.1 - 126.6 126.7 - 9 127.5 127.5 - 124.6 124.4 - 10 44.0 44.0 - 47.0 47.1 - 11 142.1 142.1 - 142.7 142.4 - 12 142.2 142.1 - 141.4 141.9 - 13 132.5 132.4 - 134.6 135.0 - 14 118.9 118.9 6.52 (s) 120.8 120.6 6.77 (s) 15 27.2 27.2 3.20 (m) 27.2 27.3 3.10 (m) 16 22.5 22.6 1.22 (d, 6.5) 22.2 22.2 1.20 (d, 7.0) 17 22.2 22.3 1.23 (d, 6.5) 22.3 22.4 1.21 (d, 7.0) 18 33.8 33.9 0.88 (s) 22.0 22.0 0.92 (s) 19 22.8 22.8 0.97 (s) 31.3 31.4 1.01 (s) 20 67.3 67.2 3.93 (d, 9.5) / 4.47 (d, 9.5) 179.1 179.3 - -CH2CH3 66.2 66.2 3.85 (m) -CH2CH3 15.8 15.8 1.32 (t, 6.5) Measured in a)CDCl3, b)125 MHz, c)500 MHz. *)δC of 11,12,20-trihydroxy-abieta-8,11,13-triene [19], &)δC of 7α-ethoxyrosmanol. 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