Chemical Constituents From the Ethyl Acetate Extract of Salvia hydrangea

Abstract

One new triterpenoid, 3β-O-(2-hydroxybenzoyl)-ursolic acid (1), and 11 known compounds, including 5 triterpenoids, 2 steroidal compounds, and 4 phenolic compounds were isolated from the ethyl acetate extract of the aerial parts of Salvia hydrangea. Their structures were elucidated based on the extensive spectroscopic data, including 1-dimensional and 2-dimensional nuclear magnetic resonance spectra, high resolution-electrospray ionization mass spectrometry, and by comparison with literature data. The antiprotozoal activity of compound 1 was evaluated against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, Plasmodium falciparum and showed no activities against all parasite tested.

Salvia is the largest genus of the family Lamiaceae, with over 900 species found throughout the world. Among them, circa 61 indigenous species grows wild in Iran. ¹ From ancient times, Salvia species have been used to treat more than 60 different ailments ranging from aches to epilepsy and mainly to treat colds, bronchitis, tuberculosis, hemorrhage, and menstrual disorders. ^2,3 Danshen, the dried root of Salvia miltiorrhiza, is a traditional Chinese medicine widely used for the treatment of cardiovascular diseases such as angina pectoris, myocardial infarction, and stroke, cerebrovascular disease, dysmenorrhea, chronic renal failure, and neuroasthenic insomnia. ^{4 -6} In Iranian folk medicine, the aerial parts of Salvia mirzayanii (locally known as Moor Talkh) are used as tonic and as a treatment for infectious and inflammatory diseases and stomach pains, ⁷ and Salvia sahendica as an antibacterial and antifungal agent. ⁸ The Salvia genus produce biologically active natural compounds that could be classified as terpenoids, steroids, flavonoids, and polyphenols.

In a project directed at structurally interesting bioactive metabolites from Iranian medicinal plants, ^{9 -14} we studied Salvia hydrangea. This species is locally known as “Gol-e Arooneh.” It grows widely in Iran, Anatolia, and Transcaucasia, ¹⁵ and the aerial parts are used in Iranian folk medicine as an anti-inflammatory and antispasmodic, and as a carminative. ¹⁶ In Pars province, infusions of the flowers are used to treat colds. In our previous works on the plant aerial parts, several isoprenoids with unique scaffolds, and potent antiprotozoal activity were isolated from the n-hexane extract. ^{17 -19} Thus, this findings prompted us to investigate the more polar extract of the plant. Examination of the ethyl acetate soluble fraction of the methanolic extract of the aerial parts by a combination of open column chromatography on silica gel and preparative thin-layer chromatography afforded one new triterpenoid (1), together with 11 known compounds including 5 triterpenoids, 2 steroids, and 4 phenolic compounds. Their structures were established on the basis of extensive spectroscopic analysis, including 1-dimensional and 2-dimensional nuclear magnetic resonance (NMR) and by comparison with literature data.

Compound 1 (Figure 1) was isolated as a white, amorphous solid. The molecular formula of C₃₇H₅₂O₅ was deduced from HR-ESIMS (m/z 577.3824 [M+H]⁺; calcd. 577.3819), accounting for 12 degrees of unsaturation (see supplementary material). The ¹³C-NMR spectrum revealed 37 carbon resonances, which were identified with the aid of HSQC and DEPTQ spectra as 7 methyls, 9 methylenes, 11 methines, and 10 quaternary carbons. The ¹H-NMR spectrum (see supplementary material) displayed the characteristic resonances of 5 methyl singlets (δ_H 0.62, 0.68, 0.78, 0.82, and 0.95), 2 methyl doublets (δ_H 0.71 [J = 6.7 Hz] and 0.83 [J = 6.8 Hz]), an oxygenated methine (δ_H 3.06, t, J = 7.9 Hz), and an olefinic proton (δ_H 5.11, brs). These structural features were reminiscent of an ursane-type triterpenoid. Comparison of the ¹H- and ¹³C-NMR spectroscopic data of the triterpenic part of the molecule with the literature data confirmed the backbone as ursolic acid. Furthermore, ¹H-NMR spectrum showed resonances at δ_H 7.73 (1H, d, J = 7.8 Hz), 6.73 (1H, t, J = 7.5 Hz), 7.28 (1H, t, J = 7.5 Hz), and 6.77 (1H, d, J = 7.2 Hz) indicating an ortho-substituted aromatic ring. Unambiguous assignment of all protons and their connectivities was achieved with the aid of ¹H-¹H COSY, HSQC, and HMBC experiments (Figure 1). HMBC correlations from H-7′ (δ 7.73) to C-1′ (δ 165.2), C-3′ (δ 161.1), and C-5′ (δ 134.9), and from H-4′ (δ 6.77) to C-6′ (δ 118.7) and C-2′ (δ 113.1), together with the 3J-COSY correlations between aromatic protons (Figure 1) corroborated the constitution of the aromatic ring as an ortho-hydroxybenzoyl moiety. Diagnostic HMBC correlation from H-3 (δ 3.06) to C-1′ confirmed the location of the benzoyl moiety at C-3. The relative configuration of 1 was deduced from the NOESY spectrum and was in agreement with that of ursolic acid (Figure 2). Therefore, the structure of compound 1 was elucidated to be 3β-O-(2-hydroxybenzoyl)-ursolic acid. Moreover, compound 1 was tested for its in vitro inhibitory activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani. None of the parasites were found to be sensitive to the tested compound.

Figure 1

Key ¹H-¹H COSY and HMBC connectivities.

Figure 2

Key NOESY correlations.

In addition to oleanolic acid, β-sitosterol and salvigenin, which have been previously isolated from the plant [9c], the triterpenoids 3-formyl-ursolic acid, ²⁰ 2α,3β,23-trihydroxyurs-12-en-28-oic acid (asiatic acid), ²¹ 3-O-β-galactopyranosyloleanolic acid, ²² betulinic acid, ²³ the steroidal compound daucosterol, ²⁴ the flavonoid apigenin-7-O-glucoside, ²⁵ the phenyl ethanoid 2-(4-hydroxyphenethyl) tetratriacontanoate, ²⁶ and the phenolic compound rosmarinic acid ²⁷ were isolated and are described here from S. hydrangea for the first time.

Experimental

General

¹H NMR and ¹³C NMR spectra were recorded on an Avance III spectrometer (Bruker) operating at 500.13 MHz for ¹H and 125.77 MHz for ¹³C. ¹H NMR, COSY, HSQC, HMBC, and NOESY spectra were measured at 18°C in a 1 mm TXI probe with a z-gradient. ¹³C NMR/DEPTQ spectra were recorded at 23°C in a 5 mm BBO probe with a z-gradient. Standard Bruker pulse sequences were applied. TopSpin 3.5 software was used for data processing. HRESIMS data were recorded in positive ion mode on an Agilent 1290 Infinity system with Agilent 6540 UHD Accurate-Mass Quadrupole Time-of-Flight detector (G6540A). TLC plates were visualized under UV light (254 and 366 nm) and by spraying with 5% phosphomolybdic acid in EtOH, followed by heating at 150°C.

Plant Material

The aerial parts of S. hydrangea DC. Ex Benth. were collected at full flowering stage from Koohin region in Qazvin province, Iran, in May 2012 and identified by Dr G. R. Amin, Tehran University of Medical Sciences, Tehran, Iran. A voucher specimen (6719-THE) has been deposited at the herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Extraction and Isolation

The air-dried, powdered aerial parts of S. hydrangea (7.5 kg) were crushed and extracted with MeOH (8 × 45 L) at room temperature for 7 days. Extracts were concentrated in vacuo, to afford a dark gummy residue (698 g). The residue was mixed with water (2.0 L) to form a suspension and partitioned successively with n-hexane and EtOAc to yield n-hexane (91.4 g), EtOAc (94.7 g), and water (511.9 g) soluble fractions. The EtOAc soluble fraction (94.7 g) was loaded on a silica gel column (700 g, 5 × 120 cm) and separated with a gradient of n-hexane-EtOAc (100/0 to 0/100) as eluent, followed by increasing concentrations of MeOH (up to 30%) in EtOAc. On the basis of TLC analysis, fractions with similar composition were combined to yield 55 main fractions (F₁-F₅₅). From fraction F₅ (100 mg, eluted with n-hexane/EtOAc [85:15]) an amorphous white powder was obtained and recrystallized from MeOH to afford phenyl ethanoid, 2-(4-hydroxyphenethyl) tetratriacontanoate (3 mg). Fraction F₈ (1.2 g, eluted with n-hexane/EtOAc [60:40]) was separated over a silica gel CC (50 g, 2 × 60 cm) with n-hexane/EtOAc (60:40) as eluent, to afford oleanolic acid (10 mg). Fraction F₉ [2 g, eluted with n-hexane/EtOAc (90:10)] was triturated with acetone and MeOH to give β-sitosterol (10 mg). Fraction F₁₂ (2.2 g, eluted with n-hexane/EtOAc [70:30]) was triturated with acetone, and the insoluble solid was recrystallized from acetone to afford salvigenin (100 mg). Fraction F₁₃ (100 mg, eluted with n-hexane/EtOAc [30:70]) was triturated with acetone to give betulinic acid (8.2 mg).Fraction F₃₃ (2.37 g, eluted with n-hexane/EtOAc [55:45]) was again subjected to repeated CC over silica gel (160 g, 2.5 × 70 cm) and eluted with chloroform/acetone/formic acid (88.23:9.80:1.97) to afford 6 subfractions (F_33a-F_33f). Subfraction F_33d was recrystallized from chloroform to give compound 1 (3 mg). From subfraction F_33c crude crystals were obtained which were recrystallized from acetone to afford 3-formyl-ursolic acid (2 mg). Fraction F₄₅ (3.87 g, eluted with n-hexane/EtOAc 20:80]) was subjected to silica gel CC (230 g, 3 × 85 cm), using a gradient of CHCl₃/MeOH (99:1-94:6) as mobile phase to afford subfractions (F_45a-F_45o). Subfraction F_45n (120 mg, eluted with CHCl₃/MeOH [95:5]) was separated on a silica gel CC (50 g, 1 × 40 cm), eluted with chloroform/isopropyl alcohol (90:10) to afford 2α,3β,23-trihydroxyurs-12-en-28-oic acid (asiatic acid) (0.9 mg). From fraction F₄₈ (100 mg, eluted with EtOAc/MeOH [90:10]) crude crystals were obtained and the insoluble solid was recrystallized from MeOH to yield apigenin-7-O-glucoside (5.56 mg). From fraction F₅₀ (eluted with EtOAc/MeOH [80:20]) an amorphous white powder of daucosterol (30 mg) was obtained. Fraction F₅₂ (1.25 g, eluted with EtOAc/MeOH [80:20]) was subjected to silica gel CC (100 g, 2 × 75 cm) with CHCl₃/MeOH (81:19) as eluent and subfractions (F_52a-F_52j) were obtained. Subfraction F_52d was further purified by preparative TLC (CHCl₃/MeOH [85/15]) to afford compound 3-O-β-galactopyranosyloleanolic acid (1.1 mg). Subfraction F_52b (213 mg) was subjected to silica gel CC (70 g, 1.5 × 40 cm), eluted with a gradient of CHCl₃/MeOH (75:25 to 50: 50) to give subfractions (F_52b-1-F5_2b-10). Subfraction F_52b-1 was further purified by preparative TLC [CHCl₃/MeOH (80:20)] to afford rosmarinic acid (4 mg).

3β-O-(2-Hydroxybenzoyl)-Urs-12-Ene-28-Oic Acid (1)

White amorphous powder (3 mg).

${\mathrm{}\left[\alpha \right]}_D^{25}$ +42.0 (c 0.11, CHCl₃).

¹H-NMR (CDCl₃ & CD₃OD, 500 MHz): δ 0.55(1H, d, J = 11.0 Hz, H-5), 0.62 (3H, s, H-23), 0.68 (3H, s, H-26), 0.71 (3H, d, J = 6.7 Hz, H-29), 0.78 (3H, s, H-25), 0.82 (3H, s, H-24), 0.83 (3H, d, J = 6.8 Hz, H-30), 0.95 (3H, s, H-27), 2.05 (1H, d, J = 11.2 Hz, H-18), 3.06 (1H, t, J = 7.9 Hz, H-3), 5.11 (1H, brs, H-12), 6.73 (1H, t, J = 7.5 Hz, H-6'), 6.77 (1H, d, J = 7.2 Hz, H-4'), 7.28 (1H, t, J = 7.5 Hz, H-5'), 7.73 (1H, d, J = 7.8 Hz, H-7').

¹³C-NMR (CDCl₃ & CD₃OD,125 MHz): δ 15.1 (C-25), 15.6 (C-23), 16.8 (C-26), 16.9 (C-29), 18.7 (C-6), 20.6 (C-30), 23.3 (C-27), 23.4 (C-11), 24.2 (C-16), 27.3 (C-15), 27.7 (C-2), 28.1 (C-24), 30.0 (C-21), 32.3 (C-7), 37.2 (C-22), 37.9 (C-10), 38.5 (C-1), 38.7 (C-4), 38.9 (C-20), 39.0 (C-19), 39.1 (C-8), 42.1 (C-14), 47.7 (C-17), 47.8 (C-9), 52.7 (C-18), 55.3 (C-5), 78.2 (C-3), 113.1 (C-2'), 116.6 (C-4'), 118.7 (C-6'), 125.4 (C-12), 130.2 (C-7'), 134.9 (C-5'), 138.7 (C-13), 161.0 (C-3'), 165.2 (C-1'), 181.0 (C-28).

HR-ESIMS: m/z 577.3824 [M+H]⁺ (calcd. for C₃₇H₅₂O₅, 577.3819).

In Vitro Antiprotozoal Assays

In vitro inhibitory activity against the protozoan parasites T. brucei rhodesiense (STIB900) trypomastigotes, T. cruzi (Tulahuen C4) amastigotes, L. donovani (MHOM-ET-67/L82) axenically grown amastigotes, and P. falciparum (NF54) IEF stage were determined as reported previously. ²⁸