Chemical Constituents from Salvia fruticosa libanotica

Boukhary, Aboul-Ela, Al-Hanbali, and El-Lakany: Chemical Constituents from Salvia fruticosa libanotica

Authors

INTRODUCTION

Salvia constitutes the largest genus of the Family Lamiaceae with about 900 species. Plants of genus Salvia are ever green shrubs present all over the world and some species largely reputed in folk medicine because they are used as spices in food industry and flavoring agents in cosmetics and aromatherapy.1,2 Pharmacological and phytochemical studies showed that salvia species are considered “cure-all” type plants.3,4 Salvia aegyptiaca L is used in cosmetics, gonorrhea, hemorrhoids, eye diseases and as antispasmodic.5,6 Salvia hispanica reduces blood sugar level and is a novel agent in the prevention and treatment of cardiovascular diseases.7 Salvia officinalis known as Sage is effective in Alzheimer disease.8 The essential oil infusion and tinctures of salvia plants are used worldwide in traditional medicine such anti-inflammatory, antioxidant, for oral cavity inflammation, for tonsillitis and for certain gastric diseases.9,10,11 In 2000, Wang reported that, Salvia was always as the top of the list of house hold remedies for the relief of itching and lowering of fevers and relief of nervous headache. The leaves and roots of salvia are very popular for their antioxidative, anti-inflammatory and hypoglycemic properties due to the radical scavenging activity of their polyphenolics contents such as carnosic acid, carnosol, rosmarinic acid and flavonoids.12,13,14 These phenolic compounds alleviated hyperalgesia in pain conditions in rats.15

S. fruticosa libanotica is an indigenous plant growing wild in Lebanon with an antioxidant potential.7,10 Therefore, this research aims to isolate the antioxidative phenolic constituents including flavonoids and abietane diterpenoids from both aerial and root parts that might have biological activities.

MATERIALS AND METHODS

General

The IR spectra were determined on Shimadzu IR spectrophotometer (FT/IR-8300) in KBr discs and the absorption bands were measured in cm-1. The 1HNMR and 13CNMR spectra were recorded on Bruker Avance 500MHZ apparatus. Column chromatography was performed over silica gel (70-230, mesh, Fluka) using petroleum ether, methylene chloride, ethyl acetate and methanol gradients as eluents. UV spectra were determined using Ciba-Corning Double-beam spectrophotometer (2800 spectroscan) and mass spectra were recorded on a AEIMS-50 spectrometer.

Plant material

Fresh Salvia libanotica fruticosa was collected at the flowering stage from the littoral of Beirut in March and April 2011. The plant was identified by prof. Dr. Georges Tohme former professor of Taxonomy. A dried specimen (No.PS.14.12) was kept in the Faculty of Pharmacy. The plant was dried under shade at 25°C and the dried aerial parts and roots were grinded separately with a blender.

Extraction and Isolation

Air-dried powdered aerial parts of Salvia fruticosa libanotica (4.5 kg) were separately extracted with 95% ethanol at room temperature for 3 weeks. The combined alcoholic extract was then concentrated under reduced pressure to complete dryness giving 105.6 g of dry extract. The residue was successively extracted with light petroleum ether, methylene chloride, ethyl acetate, n-butanol and methanol. The methylene chloride extract was evaporated in vacuum to give 29.5g of residue that was subjected to column chromatography (CC) on silica gel and eluted with chloroform-methanol as eluent with increasing methanol content to provide 62 fractions.

Fractions 19-25 (1.75g) were combined subjected to PTLC on fluorescent silica gel plates using solvent system Chloroform: Ethyl acetate (6:4) giving two zones. Both zones were scrapped off and eluted with methanol and the solvent was distilled off. Crystallization of the obtained residues from methanol yielded 16 mg of yellowish crystals, m.p.345°C, designated as material 1 and 18 mg of yellow crystals, m.p.320°C designated as material 2. Moreover, the ethyl acetate extract 15 g was dissolved in methanol with slight warming. A dark yellow residue was formed up on cooling. The deposit was separated and crystallized from methanol to give 250 mg of yellowish crystals Rf value 0.8 system Chloroform-Methanol (3:5) m.p.242°C named as material 3.

1.5 kg of air-dried powdered roots of Salvia fruticosa libanotica were extracted in a soxhelet apparatus with acetone. The combined acetone extract was evaporated under pressure giving 17 g dark brown residue that was subjected to (CC) on silica gel and eluted with Hexane-Ethyl acetate as eluent with increasing ethyl acetate content. A total of 47 fractions were collected and concentrated to dryness under reduced pressure. Fractions 20-26 gave 0.84 g of shiny white crystals named as material 4.While Fractions 33-39 showed one major spot with an Rf value 0.64 system Chloroform-Methanol (9:1) acquiring a yellow color when exposed to ammonia vapor. The residue left after evaporation of the solvent was purified by PTLC fluorescent silica gel using the same system. Then the dark zone was scrapped off after visualization and eluted with methanol. The solvent was distilled off. Repeated crystallization from methanol afforded 200mg of white crystalline powder m.p.172-175°C designated as material 5.

Apigenin (1): yellowish crystals, m.p.345°C. UV (λmax, MEOH ): 350, 265 nm. Degradation takes place with all used shifts reagents. EIMS m/z (rel.abund. %): 270 (14) [M+, C15H10O5], 253 (80), 259 (49), 249 (38). IR (KBr, νmax, cm-1) 3640, 3400,1710,1663 cm-1. 1HNMR (DMSO, 500 MHz); d 6.7 (1H, S, H-3), 6.19 (1H, d, J=2 Hz, H-6 ), 6.25 (1H, d, J=2 Hz, H-8), 7.9 (1H, dd, J=8, 2 Hz, H-2’), 6.93 (1H, dd, J=8, 2Hz, H-3’), 6.93 (1H, dd, J=8, 2Hz, H-5’), 7.9 (1H, dd, J =8, 2Hz, H-6’).

Luteolin (2): Dark yellow crystals, m.p.320°C. UV (λmax, MeOH): 350, 250 nm, (MeOH + NaOMe): 390, 273 nm, (MeOH + AlCl3): 388, 270 nm , (MeOH + AlCl3 + HCl ): 388, 270 nm, (MeOH + NaOAc) : 385, 275 nm. EIMS m/z (rel.abund.%): 286 (98) [M+,C15H11O6], 259 (40), 241 (17), 153 (97), 135 (23). IR (KBr, νmax, cm-1) 3375,1657,1610,1125. 1HNMR (DMSO, 500 MHz), integrated for 11 protons: d 6.6 (1H, s, H-3), 6.23 (1H, br, s, H-6), 6.48 (1H, br, s, H-8), 7.4 (1H, d, J=2.1 Hz, H-2’), 6.85 (1H, d, J=8.2 Hz, H-5’) and 7.45 (1H, dd, J=8.2 Hz, H-6’).

Rutin (3): yellow crystals, m.p. 242°C. UV( λmax, MEOH ): 215, 250, 350 nm, (MeOH + NaOMe): 395, 270 nm, (MeOH + AlCl3): 355, 290, (MeOH + AlCl3 + HCl ): 355, 291, (MeOH + NaOAc): 374, 260. IR (KBr, νmax, cm-1) 3400, 1650, 1450,1050. EIMS m/z (rel.abund.%): 611(54) [(M++1), C27H30O16]; 465 (15); 303 (20). 1HNMR (DMSO, 500 MHz); 6.25 (1H, d, J=2Hz, H-6), 6.4 (1H, d, J=2Hz, H-8), 7.6 (1H, s, H-2’), 6.82 (1H, d, J=8Hz, H-5’), 7.52 (1H, dd, J=8.2Hz,2Hz, H-6’), 5.15 (1H, d, J=2Hz,1H-sugar), 4.54 (4H, d, J=8Hz, H-1-Gluc) and 3.82 (1H, d, J=1Hz,1H-Rhamn).13CNMR (DMSO,500 MHz); 158.5 (C-2), 134 (C-3), 178 (C-4), 162.5 (C-5), 101.5 (C-6), 164 (C-7), 93.8 (C-8), 123.1 (C-1’), 117.5 (C-2’), 144 (C-3’), 148.5 (C-4’), 116.5 (C-5’) and 123.5 (C-6’).

Carnosol (4): white crystals, m.p.210-220°C. UV (λmax, MEOH ): 310, 382 nm. IR (KBr, νmax, cm-1) 3320,1700,1600,1060. EIMS m/z (rel.abund.%): 330 (100) [M+, C20H26O4], 301 (17), 285 (53), 259 (7). IR (KBr, νm, cm-1) 3320,1700,1600, 1060.1HNMR (DMSO, 500 MHz); d 2.65 (2H, triplet, J=7Hz, CH2 -1), 2.09 (2H, quintet, J=7Hz, CH2 -2), 1.25 (2H, m, J=7Hz, CH2 -3), 6.67 (1H, s, H-14), 5.47 (1H, d, J=2. Hz, H-7), 3.2 (1H, heptet, J=7Hz, H-15), 1.14 (3H, d, J=7Hz, CH3 -16, CH3-17). 13CNMR (DMSO, 500 MHz); 29.7 (C-1), 19.9 (C-2), 41 (C-3), 31.5 (C-4), 45.4 (C-5), 29.6 (C-6), 77.4 (C-7), 132.5 (C-8), 122.3 (C-9), 48.3 (C-10), 143.5 (C-11), 144 (C-12), 134 (C-13), 116 (C-14), 26.6 (C-15), 23.1 (C-16), 23.2 (C-17), 19.7 (C-18), 31.7 (C-19) and 175.95 (C-20).

Dehydro-abietic acid (5): white crystals, m.p.172-175°C. UV (λ max, MEOH ): 568, 425nm. EIMS m/z (rel.abund.%): [M++1]; 301, 279 (25); 252 (78); 211 (39). IR (KBr. νmax, cm-1) 3330, 1705,1610. 1HNMR (DMSO, 500 MHz) ; 2.18 (2H, m, CH2 -1 ), 1.67 (2H, quartet, J=7 Hz, CH2 -2), 2 (2H, m, CH2 -3), 1.4 (1H, triplet, J=7Hz, H-5), 1.67 (2H, quartet, J=7Hz, CH2 -6), 2.37 (2H, triplet, J=7Hz, CH2 -7), 7.17 (1H, d, J=9Hz, H-11) ,7 (1H, d, J=9Hz, H-12) and 1.17 (6H,d, H-16, H-17). 13CNMR (DMSO, 500 MHz); 40.57 (C-1), 18.6 (C-2), 36.7 (C-3), 45.1 (C-4), 46.8 (C-5), 21.58 (C-6), 29.9 (C-7), 134.5 (C-8), 147.2 (C-9), 38.2 (C-10), 124.5 (C-11), 124.1 (C-12), 145.5 (C-13), 126.9 (C-14), 33.3 (C-15), 24.4 (C-16 and C-17), 16.8 (C-19) and 25.2 (C-20).

RESULTS AND DISCUSSION

Compounds 1, 2 and 3 were found to be 5-hydroxyflavonol derivatives. Compound 1 was deduced to be C15H11O5 and its UV spectra in AlCl3 and AlCl3/HCl showed the absence of 3’-OH. EIMS showed its molecular ion peak at m/z=270.1HNMR was integrated for 11 protons. Accordingly, compound 1 was identified as apigenin. It was confirmed by co-chromatography with apigenin standard. The molecular formula of compound 2 was deduced to be C15H11O6 based on different spectral evidence. EIMS showed its molecular ion peak at m/z=286. 1HNMR was integrated for 11 protons. UV absorption at 213, 250, and 350 indicated that it is highly conjugated. Comparison of the different spectral data of compound 2 with published data showed that it is Luteolin.16,17 Acid hydrolysis of compound 3 afforded glucose as a sugar part and quercetin (Figure 1).

Figure 1

The isolated compounds

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Table 1

1H-NMR spectral data of Compound 4

PositionMaterial S6 [DMSO] δ1H (Hz)
CH2-12.65 (2H, ddd, J=7)
CH2-22.09 (2H, quintet, J=7 Hz)
CH2-31.25 (2H, m)
H-51.75 (lH,dd)
H-62.45 (IK m)
H-75.47 (1H; d)
H-11-
H-12-
H-146.67 (lH S)
H-153.2 (1H, heptet. J=7 Hz)
CH3-161.14 (3H, d, J=7Hz)
CH3-17 
CH3-180.82 (3H, S)
CH3-190.84 (3H, S)
Table 2

1H-NMR spectral data of Compound 5

PositionMaterial S7 [DMSO] δ1H (Hz)
CH2-12.18 (2H, m)
CH2-22.8 (2H, quintet, J=7 Hz)
CH2-32 (2H, m)
CH-51.4 (1H, triplet)
CH2-61.67 (2H, quartet, J=7Hz)
CH2-72.37 (2H, triplet, J=7 Hz)
CH-117.17 (1H, d, J=9.1 Hz)
CH-127(1H, d, J=9 Hz)
CH-146.85 (1H, S)
CH-152.79 (2H, heptet. J=7Hz)
CH3-161.17 (3H, d)
CH3-171.17 (3H, d)
CH3-191.24 (3H, S)
CH3-201.13 (3H, S)

Compound 3 was isolated as yellow powder and was determined to be C27H30O16 and was obtained on the basis of 1HNMR,13CNMR and MS analysis. UV spectra of compound 3 in different shift reagents indicated the presence of 5-,7-,3’- and 4’- hydroxyl groups and its UV spectra in AlCl3 and AlCl3/HCl showed the absence of 3’-OH. 1HNMR was integrated for 11 protons and 13CNMR for 27 carbon atoms Moreover, 1HNMR confirmed the presence of protons at positions 2’, 5’ and 6’. It also showed a doublet a d 6.4 assigned for H-8. The identity of the sugar moiety in “3’’was determined as rhamnoglucosyl, as showed by the signals in the 1H-NMR at d 5.15 (1H, d, J=2Hz) and 3.82 (1H, d, J=8Hz) and their corresponding carbon signals at 100 and 103, respectively in the 13C-NMR spectra. The signals appearing in the 1H-NMR at 3.33 -3.64 (m,12 H of sugar moieties) proved that too. Its 1H-NMR spectrum displayed the presence of one doublet at d 7.52 (1H, d, J=2Hz) and one singlet at 7.6 (1H, S) for protons at positions 6’ and 2’. 13C-NMR showed 27 carbon atoms of which one quaternary carbon atom appeared at d 178 due to the carbonyl group at position C-4. These spectral data were identical to those reported to rutin “Quercetin 3-O-rutinoside”18 (Figure 1).

Compound 4 was isolated from the acetone extract of the roots of Salvia libanotica fruticosa in the form of a white crystalline powder, m.p.210-220°C and was confirmed to be carnosol through different spectral data. The molecular formula of material 4 was determined as C20H26O4 through M.S spectra that showed a molecular peak at m/z=330 along with 13C-NMR that revealed the presence of 20 carbon atoms.1H-NMR spectrum of S6 showed a low field signal at d 6.67 due to proton at position 14. The spectrum also displayed the presence of two singlet signals, each integrated for three protons at 0.82 and 0.84 due to the two angular methyls at positions 18 and 19, respectively. In addition, the spectrum exhibited two proton signals characteristic for an isopropyl group at d 1.14 (6H, d, J=7Hz) and at d 3.2 (1H, hept, J=7Hz). 13C-NMR showed nineteen resolved signals representing 20 carbons. It displayed the presence of two oxygenated aromatic protons at d 143.5 and 144 due to carbons at 11 and 12 position. In addition, it showed a peak at d 175.95, characteristic for a carbonyl of an ester. Comparing the obtained spectral data with those reported for carnosol, indicated that they are almost identical19 (Figure 1 and Table 1).

Compound 5 was also isolated from the acetone extract of the roots of Salvia libanotica fruticosa in the form of a white crystalline powder m.p.172-175°C. In the Mass spectrum, the appearance of a peak at 301(M++1) established the molecular formula to be C20H28O2. 13C-NMR showed the presence of six aromatic signals, three of which are quaternary and three are olefinics. In 1H-NMR, the three olefinic protons appeared at d 6.85 (s), 7.00 (d, J=9Hz), and at 7.17 (d, J=9Hz) due to protons at positions 14,11 and 12, respectively. The presence of an isopropyl group was evident through the appearance of a one – proton heptet at d 2.79 (J=7Hz) and a six-proton doublet (J=7Hz) at d 1.17. 13C-NMR spectrum revealed the presence of two methyl signals at d 16.8 and 25.2 due to carbons at positions 19 and 20 respectively. Referring to literature, it was found that, all the observed spectral data are similar to those reported for dehydro-abietic acid.20,21 Also, it is isolated for the first time from Salvia fruticosa libanotica (Figure 1 and Table 2).

CONCLUSION

In this work we performed a phytochemical determination of aerial and root parts of Salvia libanotica fruticosa. We isolated the Phenolic diterpenes Carnosol and Dehydro-abietic acid from for the first time from the roots of salvia fruticosa libanotica growing widely in Lebanon and fully assigned for their protons and carbons for the first time too. In addition the flavonoids, luteolin, rutin and apigenin and phenolic acids gallic, rosmarinic acid and ferulic acids were obtained also from aerial parts . All the previously mentioned compounds were responsible for the antioxidant activity of Salvia.22

GRAPHICAL ABSTRACT

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SUMMARY

Isolation of eight phenolic materials from Salvia fruticosa libanotica providing a proof about the wide use of this plant in folk medicine as antidiabetic, anti-oxidant and in the cure of many illness.

ACKNOWLEDGMENT

The authors are grateful to Miss F. Mostafa (Department of Pharmaceutical Sciences, Faculty of Pharmacy, AL-Zaytoonah University, Amman, Jordan ) for NMR spectra, MS, IR and UV generated for this study. This work was supported by the Faculty of Pharmacy, Beirut Arab University, Lebanon.

Notes

[1] Conflicts of interest CONFLICT OF INTEREST We declare that we have no conflict of interest.

ABBREVIATIONS USED

NMR

Nuclear magnetic resonance

Ms

Mass spectroscopy

IR

Infra red spectroscopy

UV

Ultra violet

G

Grams

Kg

Kilograms

MHZ

Mega hertz

13C-NMR

Carbon nuclear magnetic resonance

1H-NMR

Proton nuclear magnetic resonance

CC

Column chromatography

PTLC

Preparative thin layer chromatography

Rf

Rate of flow

MEOH

Methanol

NaOMe

Sodium methoxide

DMSO-d6

Deuterated.dimethyl.sulphoxide

HZ

Hertz

m.p

Melting point

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