Diterpenes from the Aerial Parts of Salvia candelabrum and their Protective Effects against Lipid Peroxidation

• Abstract
• Materials and Methods
• References

Abstract

A methanolic extract of the aerial parts of Salvia candelabrum was subjected to multiple chromatographic separation under the guidance of anti-lipid peroxidation assay. From the most active fractions seven abietane and seco-abietane diterpenes were isolated by preparative TLC purification. Besides candesalvoquinone, candelabroquinone, 12-O-methylcandesalvone B, candesalvone B methyl ester and candelabrone (all reported earlier), the known candesalvone B and the new candesalvolactone were identified. The structures were established by means of mass spectroscopy and advanced 2D NMR methods. All the identified compounds were evaluated for antioxidant activity in enzyme-dependent (IC₅₀ values 3.49 - 10.42 μM) and enzyme-independent (IC₅₀ values 1.40 - 13.40 μM) systems of lipid peroxidation. All compounds displayed marked concentration-dependent effects in both tests as compared with those of authentic ascorbic, rosmarinic and caffeic acids. The differences in antioxidant capacities observed in the enzyme-independent system allowed conclusions concerning structure-activity relationships.

During the last years, many papers have dealt with antioxidants originating from plant sources. In this respect, special interest has focused on the Lamiaceae family [1], [2]. Analyses of Salvia officinalis L. and Rosmarinus officinalis L. have demonstrated that the antioxidant factors are primarily flavonoids, phenolic acids and diterpenoids [3], [4], [5].[]

Our previous screening of 11 European Salvia species revealed that the antioxidant activity of a 50 % methanolic extract of the leaves of S. candelabrum Boiss. is more potent than that of S. officinalis as concerns both the enzyme-dependent and the enzyme-independent systems of lipid peroxidation (LPO) [6].

The aim of the present work was to characterize the active constituents of S. candelabrum under antioxidant assay guidance. Five diterpenes, candesalvoquinone (1), candelabroquinone (2), 12-O-methylcandesalvone B (3), candesalvone B methyl ester (4), and candelabrone (5) were reported recently [7]. Investigation of the LPO-active fractions has now been continued in order to identify further compounds. The antioxidant potential of all isolated components was also studied.

Fractionation of the methanolic extract of S. candelabrum by polyamide CC led to the separation of seven fractions (P1-P7) [7] exhibiting concentration-dependent inhibitory activities comparable to those of ascorbic acid when tested against the autooxidation of a standard ox-brain homogenate (Fig. [1]). The most pronounced activities were demonstrated by the diterpenes containing fractions P2-P4. CC and preparative TLC separations of fractions P2 and P3 afforded compounds 6 and 7, besides the earlier-reported diterpenoids 1 - 5 [7]. Furthermore, the presence of RA and CA was detected in fractions P3 and P1, respectively.

Compound 6 as indicated by ¹H-NMR, JMOD (J-Modulated Spin Echo Experiment), ¹H-¹H COSY, HSQC and HMBC investigations was identical with candesalvone B, isolated previously from S. candelabrum [8].

Compound 7 was found to have the molecular formula C₂₀H₂₄O₆ by HR-ESI-MS. The ¹H-NMR spectrum contained signals indicative of two tertiary methyl groups, one isopropyl group and one chelating hydroxy group (δ_H = 13.54) (Table [1]). The JMOD spectrum exhibited 20 resonances, including those of one keto group (δ_C = 194.4), one ester carbonyl (δ_C = 172.8) and six aromatic quaternary carbons (δ_C = 111.7 - 160.2). Characteristic signals of an isopropenyl group were also observed suggesting an abietane diterpene with a seco rearranged ring A.

To ascertain the different carbon and hydrogen connectivities, a series of homo- and heteronuclear 2D NMR experiments was performed (Table [1]). Careful examination of the ¹H-¹H COSY, HSQC and HMBC spectra enabled us to elucidate the presence of 11,12,14-trihydroxy-7-oxo-3,4-seco-4(18),8,11,13-abietatetraene structure containing a lactone ring. This lactone ring is formed between C-3 and C-6 as demonstrated by the HMBC cross-peak between C-3 and H-6. The relative stereochemistry of 7 was determined on the basis of a NOESY experiment with respect to a Dreiding model. Starting from the β relative configuration of H-20, the Overhauser effects between H-20 and both H-1a and H-1b were indicative of H-1_equ/H-20_equ and H-1_ax/H-20_equ relationships. The NOESY correlation between H-1b_ax and H-5 indicated the axial orientation of H-5, which corresponds to its α position. NOE-enhanced signals between H-5 and H-6, and the coupling constant J _H-5/H-6 = 0 Hz provided evidence of β-oriented H-6_equ. On the basis of the above evidence the structure of this compound was elucidated as shown for 7, and it was assigned the trivial name candesalvolactone.

The diterpenes 1 - 7 isolated from the aerial parts of S. candelabrum exhibited pronounced antioxidant effects in both enzyme-dependent and enzyme-independent anti-LPO systems (Fig. [2]). Excluding 3, the effectiveness observed against enzyme-independent LPO was similar to or higher than those of the positive controls RA and CA, the main antioxidative principles of sage and rosemary [3]. In the enzyme-dependent assay nearly equal activities were measured for diterpenes 1 and 2 and 4 - 7, indicating their similar enzyme inhibitory activities, while 3 showed only weak effect. As regards the enzyme-independent assay, remarkable differences were noted, which allowed some conclusions concerning structure-activity relationships. The IC₅₀ values of compounds 1 and 4, or 2 and 5, clearly showed that the phenoloid structure was more advantageous than the para-quinonoid ring C against the autooxidation of a standard ox-brain homogenate. Comparing the structurally related pairs 3 and 6, it is obvious that the methylation of a phenolic OH group causes a significant decrease in the antioxidant activity. However, methyl esterification at C-3 increases the antioxidant potency, most probably in consequence of the enhanced lipophilicity of the molecule, as demonstrated by 4 and 6.

Materials and Methods

Mass spectroscopy was carried out on a Perkin Elmer Q-STAR Pulsar Q-TOF spectrometer. Parameters of UV and NMR spectroscopies, optical rotations measurements, CC, TLC were described in ref. [7]. The presence of RA and CA was detected by TLC, using authentic samples (ICN Pharmaceuticals and Roth) according to ref. [6]. Ascorbic acid was purchased from Sigma.

The plant material, extraction procedure, polyamide CC and the isolation of compounds 1 - 5 from fraction P2 were described in ref. [7]. Fraction P3 (1.22 g) was fractionated by CC over silica gel (49 g), eluted with gradient systems of n-hexane-chloroform (2 : 8 and 1 : 9) and chloroform-methanol (100 : 0, 97 : 3, 90 : 10, 80 : 20, 1 : 1 and 0 : 100) using 100 mL of each eluent. Four subfractions (P3S1 - P3S4) were obtained, one of them P3S3 (350 mL) was further purified by preparative TLC on silica gel, with chloroform-ethyl formate-ethyl acetate-formic acid (7 : 1.5 : 0.5 : 1). The final preparative TLC separation was carried out on silica gel with chloroform-MeOH (19 : 1), to yield compounds 6 (R_f 0.48, 6.1 mg) and 7 (R_f 0.29, 10.7 mg).

Candesalvone B (6): yellow amorphous solid; [α]_D ³⁸: + 130 (c 0.1 MeOH); ¹H-NMR (CDCl₃, 500 MHz): δ = 1.34 (6H, d, J = 7.0 Hz, Me-16, 17), 1.42 (3H, s, Me-20), 1.77 (3H, s, Me-19), 2.13 (2H, m, H-1b, H-2b), 2.40 (1H, t, J = 9.0 Hz, H-2a), 2.56 (1H, d, J = 17.0 Hz, H-6b), 2.71 (2H, m, H-1a, H-5), 2.86 (1H, d, J = 17.0, H-6a), 3.47 (1H, sept, J = 7.0, H-15), 4.81 and 5.03 (each 1H, s, H-18), 13.68 (1H, s, 14-OH); ¹³C-NMR (CDCl₃, 125 MHz): δ = 20.2 and 20.3 (C-16 and C-17), 21.4 (C-20), 22.4 (C-19), 24.4 (C-15), 29.4 (C-2), 32.5 (C-1), 39.5 (C-6), 41.8 (C-10), 47.6 (C-5), 109.8 (C-8), 116.5 (C-18), 119.2 (C-13), 132.9 (C-9), 132.9 (C-11), 144.0 (C-4), 153.2 (C-12), 159.9 (C-14), 179.7 (C-3), 202.6 (C-7). Copies of the original spectra are obtainable from the author of correspondence.

Candesalvolactone (7): yellow amorphous solid; [α]_D ³⁸: + 85 (c 0.1 MeOH); UV (MeOH): λ_max (log ε) = 220 (2.85), 291 (2.44), 368 (2.68); ¹H- and ¹³C NMR see Table [1]; HR-ESI-MS: m/z = 361.1647 [M + H]⁺ (calcd for C₂₀H₂₅O₆: m/z 361.1651, Δ -1.1 ppm).

Measurement of antioxidant activity: The assays were carried out as described in ref. [6]. The fractions and the compounds (1 - 7) were studied at concentrations of 10 ^{- 5}, 10 ^{- 4}, 10 ^{- 3}, 10 ^{- 2}, 10 ^{- 1} and 1 mg/mL. The purity of the isolates was investigated by TLC and ¹H-NMR spectra (purity over 95 %).

References

• 1 Lamaison J L, Petitjean-Freytet C, Duke J A, Walker J. Hydroxycinnamic derivative levels and antioxidant activity in North American Lamiaceae.  Plant Med Phytother. 1993;  26 143-8
  PubMedSearch in Google Scholar
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  Thieme ConnectPubMedSearch in Google Scholar
• 3 Santos-Gomes P C, Seabra R M, Andrade P B, Fernandes-Ferreira M. Phenolic antioxidant compounds produced by in vitro shoots of sage (Salvia officinalis L.)  Plant Sci. 2002;  162 981-7
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  PubMedSearch in Google Scholar
• 5 Masuda T, Inaba Y, Takeda Y. Antioxidant mechanism of carnosolic acid: structural identification of two oxidation products.  J Agric Food Chem. 2001;  49 5560-5
  CrossrefPubMedSearch in Google Scholar
• 6 Zupkó I, Hohmann J, Rédei D, Falkay G y, Janicsák G, Máthé I. Antioxidant activity of leaves of Salvia species in enzyme-dependent and enzyme-independent systems of lipid peroxidation and their phenolic constituents.  Planta Med. 2001;  67 366-8
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Hohmann J, Janicsák G, Forgo P, Rédei D, Máthé I, Bartók T. New diterpenoids from the aerial parts of Salvia candelabrum .  Planta Med. 2003;  69 254-7
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Mendes E, Marco J L, Rodríguez B, Jimeno M L, Lobo A M, Prabhakar S. Diterpenoids from Salvia candelabrum .  Phytochemistry. 1989;  28 1685-90
  CrossrefPubMedSearch in Google Scholar

Dr. Gábor Janicsák

Institute of Ecology and Botany

Hungarian Academy of Sciences

2163 Vácrátót

Hungary

Fax: +36-28-360110

Email: janicsak@botanika.hu

References

• 1 Lamaison J L, Petitjean-Freytet C, Duke J A, Walker J. Hydroxycinnamic derivative levels and antioxidant activity in North American Lamiaceae.  Plant Med Phytother. 1993;  26 143-8
  PubMedSearch in Google Scholar
• 2 Hohmann J, Zupkó I, Rédei D, Csányi M, Falkay G y, Máthé I, Janicsák G. Protective effects of the aerial parts of Salvia officinalis, Melissa officinalis and Lavandula angustifolia and their constituents against enzyme-dependent and enzyme-independent lipid peroxidation.  Planta Med. 1999;  65 576-8
  Thieme ConnectPubMedSearch in Google Scholar
• 3 Santos-Gomes P C, Seabra R M, Andrade P B, Fernandes-Ferreira M. Phenolic antioxidant compounds produced by in vitro shoots of sage (Salvia officinalis L.)  Plant Sci. 2002;  162 981-7
  CrossrefPubMedSearch in Google Scholar
• 4 Ho C T, Wang M F, Wei G J, Huang T C, Huang M T. Chemistry and antioxidative factors in rosemary and sage.  Biofactors. 2000;  13 161-6
  PubMedSearch in Google Scholar
• 5 Masuda T, Inaba Y, Takeda Y. Antioxidant mechanism of carnosolic acid: structural identification of two oxidation products.  J Agric Food Chem. 2001;  49 5560-5
  CrossrefPubMedSearch in Google Scholar
• 6 Zupkó I, Hohmann J, Rédei D, Falkay G y, Janicsák G, Máthé I. Antioxidant activity of leaves of Salvia species in enzyme-dependent and enzyme-independent systems of lipid peroxidation and their phenolic constituents.  Planta Med. 2001;  67 366-8
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Hohmann J, Janicsák G, Forgo P, Rédei D, Máthé I, Bartók T. New diterpenoids from the aerial parts of Salvia candelabrum .  Planta Med. 2003;  69 254-7
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Mendes E, Marco J L, Rodríguez B, Jimeno M L, Lobo A M, Prabhakar S. Diterpenoids from Salvia candelabrum .  Phytochemistry. 1989;  28 1685-90
  CrossrefPubMedSearch in Google Scholar

Dr. Gábor Janicsák

Institute of Ecology and Botany

Hungarian Academy of Sciences

2163 Vácrátót

Hungary

Fax: +36-28-360110

Email: janicsak@botanika.hu