In Vitro Antiprotozoal Activity of Abietane Diterpenoids Isolated from Plectranthus barbatus Andr.

Chromatographic separation of the n-hexane extract of the aerial part of Plectranthus barbatus led to the isolation of five abietane-type diterpenes: dehydroabietane (1); 5,6-didehydro-7-hydroxy-taxodone (2); taxodione (3); 20-deoxocarnosol (4) and 6α,11,12,-trihydroxy-7β,20-epoxy-8,11,13-abietatriene (5). The structures were determined using spectroscopic methods including one- and two-dimensional NMR methods. Compounds (1)–(3) and (5) are isolated here for the first time from the genus Plectranthus. The isolated abietane-type diterpenes tested in vitro for their antiprotozoal activity against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxicity was determined against fibroblast cell line MRC-5. Compound (2) 5,6-didehydro-7-hydroxy-taxodone showed remarkable activity with acceptable selectivity against P. falciparum (IC50 9.2 μM, SI 10.4) and T. brucei (IC50 1.9 μM, SI 50.5). Compounds (3)–(5) exhibited non-specific antiprotozoal activity due to high cytotoxicity. Compound (1) dehydroabietane showed no antiprotozoal potential.


Introduction
The genus Plectranthus (Labiatae) represents a large and widespread group of species with a diversity of traditional medicinal uses. The genus comprises a group of around 300 species, distributed in tropical and subtropical areas of Africa, Asia and Australia [1]. One of the most interesting species of this genus is Plectranthus barbatus Andr., which is well-known for the treatment of various ailments. A diversity of traditional medicinal uses of P. barbatus in India (Hindu and Ayurvedic medicine), East and Central Africa, China, and Brazil have been reported [2]. The majority of uses are for intestinal disturbances and liver fatigue, respiratory disorders, heart diseases and certain central nervous system disorders [2][3][4][5]. In previous work [6][7][8], we screened around 70 medicinal plants from the Arabian Peninsular region (Yemen and Saudi Arabia) for their antiplasmodial, antileishmanial and antitrypanosomal properties. Plectranthus barbatus represented one of the more interesting plants for its in vitro antiprotozoal effects [8]. The present study on P. barbatus specifically deals with bio-guided fractionation, isolation and structural elucidation of abietane diterpenoid constituents.

Spectral Data
Compound (4) was revealed to have the molecular formula C 20 H 28 O 3 , by HR-ESIMS (m/z 316.9894) with 7 degree of unsaturation. The UV absorption at 320 and 282 nm indicated the presence of the benzene ring. In the IR spectrum, a hydroxyl group absorption was observed at 3400 cm −1 and absorptions at 1600 and 1510 cm −1 for the aromatic ring. Twenty carbon signals were observed in the 13 C-NMR and DEPT-experiment, six of them appeared in the aromatic area and indicated the presence of three double bonds at δ 143.5, 142.0, 134.7, 133.9, 129.6 and 112.9 assigned for carbons 12, 11, 8, 13, 9 and 14 respectively (Table 1). A singlet aromatic proton at δ H 6.60 in 1 H-NMR spectrum postulates the presence of a penta-substituted aromatic ring. In addition, it showed the presence of one hydroxymethylene group at δ H 4.31 and 3.01 (2H, d, J = 8.5 Hz), downfield oxymethine at δ H 4.67 (br t, J = 3.9 Hz) assigned for H7. The presence of an isopropyl group linked to a quaternary carbon was supported by the signals at δ H 3.25 (1H, m, H15), 1.20 (3H, br d, J = 6.0 Hz, CH 3 -16) and 1.21 (3H, br d, J = 6.0 Hz, CH 3 -17). Besides the isopropyl group, the presence of two tertiary methyl groups at δ H 0.87 (3H, s) and 1.15 (3H, s) in the 1 H-NMR and 20 carbon signal in the 13 C-NMR spectra (Table 1) revealed an abietane diterpenoid structure [9,10]. The absence of a tertiary methyl group (C20) at C10 frequently present in abietane-type diterpene and the presence of germinal hydroxymethelene protons at δ H 4.31 and 3.01 (2H, d, J = 8.5 Hz) suggests that methyl group 20 was replaced by hydroxymethylene. The measurement of long range HMBC experiment was useful in determining the final structure where two and three bond correlations were observed from the methylene protons (H 2 O) (δ H 4.31, 3.01) to C5, C7, C9 and C10, from H14 at δ H 6.60 and the carbons at δ C 72.7 (C7) and δ C 129.6 (C9); and between the proton resonance at δ H 4.67 (H7) and the carbons at δ C 44.6 (C5) and δ C 129.6 (C9). The above findings confirmed that Compound (4) is an abietane-type diterpene with an ether linkage between C20 and C7. The presence of such a type of ether linkage is uncommon in the plants belonging to family Lamiaceae. Comparing the above mentioned NMR data, MS and other spectral finding with those reported for 20-deoxocarnosol proved that both compounds were identical [9,10]. Compound (5) was obtained as colorless crystals. The HR-ESIMS gave a molecular ion peak at m/z 332.9893, corresponding to a molecular formula of C 20 H 28 O 4 with 16 mass units more than Compound (4). The IR spectrum showed absorption bands, like Compound (4), at 3400 (OH), and 1620 and 1500 cm −1 (aromatic). The presence of an aromatic ring was supported by the UV data (λ max 210 and 282, 320 nm). The 13 C-NMR and DEPT-experiment were in part similar to those of Compound (4), the main difference was the decrease number of methylene protons by one (3 in Compound (5) and 4 in Compound (4)) and the subsequent increase in the number of methine protons by one (5 in Compound (5) and 4 in Compound (4)). It showed, like Compound (4), 20 carbon signals, six of them appeared at δ C 144.2 (C12), 141.7 (C11), 134.8 (C13), 129.9 (C8), 129.6 (C9) and 116.6 (C14) ascribed for pentasubstutited aromatic ring ( Table 1). The major difference was the presence of an extra downfield signal at δ H 4.09 (br s, δ C 69.9) indicating a substitution with OH-group. The rest of 1 H-NMR spectral data was almost identical to Compound (4) and confirmed the presence of abietane-type diterpene with an aromatic ring and ether linkage between C20 and C7. The extra hydroxyl group was positioned at C6 through the observed long range cross peaks correlations in HMBC experiment between proton at δ 4.09 (br s, H6) and C4, C5 and C10; between hydroxymethylene group at δ H 4.11 and C5, C7, C9 and C10; between the multiple proton at δ 3.25 (H15) and C12, C13 and C14; between the proton at δ H 6.69 (H14) and C9 and C12; between the proton at δ 4.47 (H7, ether linkage proton) and C5, C6 and C9. The rest of HMBC correlations were closely similar to those of Compound (4). The orientation for hydroxyl group at C6 was confirmed to be α by comparing 13 C-NMR chemical shift for C5, C6 and C7 with those reported for 6α,11,12,-trihydroxy-7β,20-epoxy-8,11,13-abietatriene, recently isolated from Premna obtusifolia (Verbenaceae) [11]. It is worth noting that similar structural compounds have been isolated from Coleus eskirolii and Salvia aspera [12,13] and identified as esquirolin D and 6-epi-demethylesquirolin D. However in esquirolin D, the hydroxyl group at position 6 was β-orientated rather than α as in Compound (5) and in 6-epi-demethylesquirolin D.
The lipophilic nature of the abietane diterpenes enables easy transport across the parasitic membranes to accumulate in the parasitic food vacuole [20]. Comparing the IC 50 values of the isolated abietane diterpenes 1-5, some structure-activity relationships may be suggested. The results suggest that the antiprotozoal activity depends on oxygenated and dehydrogenated chromophoric systems through rings B and C since dehydroabietane (1), without oxygen functions in ring B and C as well as dehydrogenations in ring B, had no activity against all protozoal strains. The structural analysis of the diterpenes 2-5 allow us to conclude that the quinone-structure at C6, C7, C11 and C12 apparently increases the antiprotozoal activity as well as the antifungal and antibacterial activities of several abietane-type diterpene quinones [17]. Quinones should not be the only chemical group required for antiprotozoal activity. A comparison of Compounds (4) and (5) as well as Compounds (2) and (3) revealed that hydroxylation at C6, C7, C11 and C12 was translated into strong antiprotozoal and cytotoxic activity. Moreover, a comparison of Compounds (2) and (3) showed an en-ol-structure at C6 in Compound (2) instead of oxo-structure in Compound (3) which is apparently translated into more selectivity for 5,6-didehydro-7-hydroxy-taxodone (2). As most diterpenes are known to combine antiprotozoal activity with high cytotoxicity [26], 5.6-didehydro-7-hydroxy-taxodone (2) may be withheld as the better antiprotozoal agent in view of the more favorable selectivity indices.

General Experimental Procedures
The UV and IR spectra were recorded on UV-1601-PC (Shimadzo, Koyoto, Japan) and JASCO 320-A spectrometers (JASCO Germany GmbH, Gross-Umstadt, Germany). The 1 H-, 13 C-NMR and 2D-NMR spectra were recorded on a Bruker AMX-500 spectrometer (Bruker, Faellanden, Switzerland) with tetramethylsilane (TMS) as internal standard. Chemical shifts are given in ppm (δ) relative to tetramethylsilane internal standard and scalar coupling constants (J) are reported in Hertz. Jeol JMS-700 High Resolution Mass Spectrophotometer (JEOL (Germany) GmbH, Muenchen, Germany) was used for the mass determination. Electron Impact mode of ionization was used, keeping ionization energy of 70 eV. Resolution was set up to 10 k. A direct probe was used with temperature ramp setting, initial temperature 50 °C rise with rate of 32 °C per minute and final temperature set up to 350 °C. Thin layer chromatography (TLC) was performed on precoated silica gel F 254 plates (E. Merck, Darmstadt, Germany); detection was done at 254 nm and by spraying with p-anisaldehyde/H 2 SO 4 reagent. All chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA).

Plant Materials
Aerial part (leaves, stems and flowers) of P. barbatus was collected from Wadi Gama in Taif province of Saudi Arabia in March 2010 and identified at the Pharmacognosy Department, College of Pharmacy, King Saud University. A voucher specimen (Voucher # P-15451) was deposited at the Pharmacognosy Department, College of Pharmacy, King Saud University.

Extraction and Isolation
The air-dried and powdered aerial part of P. barbatus (1 kg) was extracted by maceration with 70% ethanol (5 × 2 L) at room temperature. The combined obtained ethanolic extract was filtered and concentrated under reduced pressure at 40 °C using a rotary evaporator. The dried ethanolic extract (65 g) was subsequently redissolved in 30% ethanol (200 mL) and partitioned successively for several times with n-hexane (3 × 200 mL), chloroform (3 × 200 mL) and n-butanol (3 × 200 mL) to provide the corresponding extracts. Each extract was tested for its antiprotozoal activity. Consequently, it was shown that both activities resided predominantly in the hexane and chloroform extracts. Hence, the n-hexane extract (26 g) was subjected to column chromatography on a pre-packed silica gel column (35 mm i.d. × 350 mm) to give 15 fractions. The elution was performed with a gradient of hexane:acetone (10:1) to pure acetone. TLC analysis of the fractions with anisaldehyde/sulfuric acid and heating at 100 °C allowed the constitution of 15 fractions. Fraction 3 (4.5 g) was separated on a RP18 column with methanol:acetonitile (1:9) to afford 485 mg of colorless oil (Compound (1)). Fraction 14 (350 mg) was subjected to a silica gel column chromatography using dichloromethane:methanol (100:1) as a solvent to afford two subfractions (Fraction 14a and 14b) Fraction 14a afforded Compound (2) which required a further separation on the chromatotron (centrifugal TLC) (silica gel 60, 0.04-0.06 mm, 1 mm and methanol:DCM, 0.5:99.5) to give 36 mg of yellow crystalline powder (Compound (2)). The purification of fraction 14b on a silica gel column with acetone:hexane (1:20) as eluent gave an orange viscous Compound (3) (24 mg). Fraction 15 (2.8 g) was separated on a silica gel column with acetone:hexane (1:20) as eluent to afford a subfraction containing Compound (4) and Compound (5). Both compounds required a further purification on a chromatotron (Silica gel, 0.04-0.06 mm, 1 mm, EtoAc:dichloromethane, (2:8) to give two colorless crystalline powders, namely Compound (4) (395 mg) and Compound (5) (42 mg).

Biological Assays
The integrated panel of microbial screens and standard screening methodologies were adopted as previously described [27]. All assays were performed in triplicate, at the Laboratory of Microbiology, Parasitology and Hygiene at the University of Antwerp, Belgium. Plant extracts were tested at 5 concentrations (64, 16, 4, 1 and 0.25 µg/mL) to establish a full dose-titration and determination of the IC 50 (inhibitory concentration 50%). The concentration of DMSO did not exceed 0.5%. The selectivity antiprotozoal potential was assessed by simultaneous evaluation of cytotoxicity on a fibroblast (MRC-5) cell line. The criterion for activity was an IC 50 <10 µg/mL (<5 µg/mL for T. brucei) and a selectivity index of ≥4.

Antileishmanial Activity
L.infantum MHOM/MA(BE)/67 amastigotes were collected from the spleen of an infected donor hamster and used to infect primary peritoneal mouse macrophages. To determine in vitro antileishmanial activity, 3 × 10 4 macrophages were seeded in each well of a 96-well plate. After 2 days outgrowth, 5 × 10 5 amastigotes/well were added and incubated for 2 h at 37 °C. Pre-diluted plant extracts were subsequently added and the plates were further incubated for 5 days at 37 °C and 5% CO 2 . Parasite burdens (mean number of amastigotes/macrophage) were microscopically assessed after Giemsa staining, and expressed as a percentage of the blank controls without plant extract.

Antitrypanosomal Activity
Trypanosoma brucei Squib-427 strain (suramin-sensitive) was cultured at 37 °C and 5% CO 2 in Hirumi-9 medium [29], supplemented with 10% fetal calf serum (FCS). About 1.5 × 10 4 trypomastigotes/well were added to each well and parasite growth was assessed after 72 h at 37 °C by adding resazurin [30]. For Chagas disease, T. cruzi Tulahuen CL2 (benznidazole-sensitive) was maintained on MRC-5 cells in minimal essential medium (MEM) supplemented with 20 mM L-glutamine, 16.5 mM sodium hydrogen carbonate and 5% FCS. In the assay, 4 × 10 3 MRC-5 cells and 4 × 10 4 parasites were added to each well and after incubation at 37 °C for 7 days, parasite growth was assessed by adding the β-galactosidase substrate chlorophenol red β-D-galactopyranoside [31]. The color reaction was read at 540 nm after 4 h and absorbance values were expressed as a percentage of the blank controls.

Cytotoxicity Assay
MRC-5 SV2 cells were cultivated in MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate and 5% FCS. For the assay, 10 4 MRC-5 cells/well were seeded onto the test plates containing the pre-diluted sample and incubated at 37 °C and 5% CO 2 for 72 h. Cell viability was assessed fluorimetrically after 4 h of addition of resazurin. Fluorescence was measured (excitation 550 nm, emission 590 nm) and the results were expressed as % reduction in cell viability compared to control.

Conclusions
In conclusion, five Compounds (1)-(5) belonging to abietane-type diterpenes were isolated from the aerial part of Plectranthus barbatus. Compounds (1)-(3) and (5) were isolated here for the first time from the genus Plectranthus. The antiprotozoal activity against P. falciparum, L. infantum, T. brucei and T. cruzi is being reported for the first time for four of the isolated diterpenes. Compound (2) 5,6-didehydro-7-hydroxy taxodone showed moderate activity against P. falciparum and T. brucei with acceptable selectivity. Compounds (3)-(5) exhibited a high antiprotozoal activity but this was due to high cytotoxicity. Compound (1) dehydroabietane showed no antiprotozoal potential. These findings stress the importance of structure-activity relationships for biological and toxicological properties of isolated plant constituents whereby potency and selectivity are combined in vitro endpoint parameters.