Alkaloids and Phenolic Compounds from Sida rhombifolia L. (Malvaceae) and Vasorelaxant Activity of Two Indoquinoline Alkaloids

The follow-up of phytochemical and pharmacological studies of Sida rhombifolia L. (Malvaceae) aims to strengthen the chemosystematics and pharmacology of Sida genera and support the ethnopharmacological use of this species as hypotensive herb. The present work reports phytoconstituents isolated and identified from aerial parts of S. rhombifolia by using chromatographic and spectroscopic methods. The study led to the isolation of scopoletin (1), scoporone (2), ethoxy-ferulate (3), kaempferol (4), kaempferol-3-O-β-d-glycosyl-6′′-α-d-rhamnose (5), quindolinone (6), 11-methoxy-quindoline (7), quindoline (8), and the cryptolepine salt (9). The alkaloids quindolinone (6) and cryptolepine salt (9) showed vasorelaxant activity in rodent isolated mesenteric arteries.


Introduction
The Malvaceae family Juss. has predominantly pantropical distribution, including about 250 genera and 4200 species [1]. In Brazil, 70 genera are found widely spread, from which nine are considered endemic [2]. The Sida genus is considered native to Brazil and presents 95 species [3]. Many Sida species are used in American, African, and Asian countries, showing good efficacy in health disorders [4].
In order to strengthen chemosystematics and pharmacology of Sida genera, as well as to support the popular medicinal use of this plant, the phytochemical study of S. rhombifolia (Malvaceae) was continued. The study led to the isolation of nine secondary metabolites from the aerial parts of S. rhombifolia. Additionally, the vasorelaxant activity of two isolated alkaloids was demonstrated, justifying the popular use of the species against arterial hypertension.

Identification of Isolated Compounds
The isolated and identified compounds from aerial parts of S. rhombifolia are showed in Figure 1. The NMR data of compound 7 ( 1 H, 13 C, Heteronuclear Single Quantum Correlation-HSQC, Heteronuclear Multiple Bond Correlation-HMBC, and Nuclear Overhauser Spectroscopy-NOESY) are presented in Table 1. Spectral data from the other compounds are also presented. Additionally, the vasorelaxant activity of two isolated alkaloids was demonstrated, justifying the popular use of the species against arterial hypertension.

Identification of Isolated Compounds
The isolated and identified compounds from aerial parts of S. rhombifolia are showed in Figure 1. The NMR data of compound 7 ( 1 H, 13 C, Heteronuclear Single Quantum Correlation-HSQC, Heteronuclear Multiple Bond Correlation-HMBC, and Nuclear Overhauser Spectroscopy-NOESY) are presented in Table 1. Spectral data from the other compounds are also presented.        In agreement with literature [11].

Pharmacological Data
The vasorelaxant activity of compounds 6 (quindolinone) and 9 (salt of cryptolepine) were evaluated by increasing cumulative addition of the compound (10 −12 -10 −3 M) using rodent mesenteric arteries pre-contracted with phenylephrine (PHE) (1 µM). The experiments were carried out using arteries with functional endothelium and without functional endothelium (Figures 2 and 3). Data were expressed as maximum effect (Emax) and the potency of the drug was expressed as pD 2 , which is the log of the 50% of the effective concentration (EC 50 ).

Pharmacological Data
The vasorelaxant activity of compounds 6 (quindolinone) and 9 (salt of cryptolepine) were evaluated by increasing cumulative addition of the compound (10 −12 -10 −3 M) using rodent mesenteric arteries pre-contracted with phenylephrine (PHE) (1 μM). The experiments were carried out using arteries with functional endothelium and without functional endothelium (Figures 2 and 3). Data were expressed as maximum effect (Emax) and the potency of the drug was expressed as pD2, which is the log of the 50% of the effective concentration (EC50).

Discussion
The 1 H-NMR spectra of compound 7 showed signals with a pattern of substitution indicating an indoquinoline skeleton [5], aromatic rings ortho di-substituted with the presence of four broad doublets :

Pharmacological Data
The vasorelaxant activity of compounds 6 (quindolinone) and 9 (salt of cryptolepine) were evaluated by increasing cumulative addition of the compound (10 −12 -10 −3 M) using rodent mesenteric arteries pre-contracted with phenylephrine (PHE) (1 μM). The experiments were carried out using arteries with functional endothelium and without functional endothelium (Figures 2 and 3). Data were expressed as maximum effect (Emax) and the potency of the drug was expressed as pD2, which is the log of the 50% of the effective concentration (EC50).

Discussion
The 1 H-NMR spectra of compound 7 showed signals with a pattern of substitution indicating an indoquinoline skeleton [5], aromatic rings ortho di-substituted with the presence of four broad doublets :  Table 1).
A singlet at δ H 11.50 (1H) was detected indicating the N-H of indol (NH-10) found in this group of alkaloids [5]. The hydrogens at δ H 4.39 (s, 3H) were attributed to a methoxy group bonded to C-11, since the chemical shift at δ H 9.02 characteristic of H-11 was not found [12].
The 13 C-NMR data supported the proposal of indoquinoline nucleous for compound 7 by showing signals for 15 carbons. The chemical shift at δ C 60.7 indicated a methoxy group as proposed by 1 H-NMR, being confirmed by the HMQC ( Table 1).
The 13 C-NMR data supported the proposal of indoquinoline nucleous for compound 7 by showing signals for 15 carbons. The chemical shift at δC 60.7 indicated a methoxy group as proposed by 1 H-NMR, being confirmed by the HMQC ( Table 1).
PHE is an agonist of α1-adrenergic receptors [13,14], whose are coupled to Gq/11 protein. Once activated, the receptors trigger the hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) by the action of phospholipase C-β (PLCβ). Then, the IP3 receptors at the sarcoplasmic reticulum are activated, releasing Ca 2+ into the cytosol. DAG and the elevation of intracellular Ca 2+ activate kinase proteins, including protein kinase C (PKC) in smooth muscle. It results in phosphorylation and inactivation of enzymes related to the contractile process, such as myosin light chain phosphatase (MLCP) [15,16].
Thus, the vasorelaxant activity of tested compounds seems to involve endothelium-derived substances, since the effect was decreased after the endothelium removal. However, the compounds The NOESY spectra of compound 7 showed correlations between neighbour hydrogens, as well as confirmed that the methoxy is bonded to C-11, by showing correlation between δ H 4.39(O-CH 3 ) with δ H 11.50 (NH-10) and δ H 8.30 (d, H-1).
PHE is an agonist of α1adrenergic receptors [13,14], whose are coupled to Gq/11 protein. Once activated, the receptors trigger the hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP 2 ) into inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG) by the action of phospholipase C-β (PLCβ). Then, the IP3 receptors at the sarcoplasmic reticulum are activated, releasing Ca 2+ into the cytosol. DAG and the elevation of intracellular Ca 2+ activate kinase proteins, including protein kinase C (PKC) in smooth muscle. It results in phosphorylation and inactivation of enzymes related to the contractile process, such as myosin light chain phosphatase (MLCP) [15,16].
Thus, the vasorelaxant activity of tested compounds seems to involve endothelium-derived substances, since the effect was decreased after the endothelium removal. However, the compounds also seem to act in smooth muscle cells, since in endothelium-free preparations it was possible to observe some vasorelaxant effect. The vasorelaxant effect of cryptolepinone isolated from S. rhombifolia has been previously reported [5].

Collection, Extraction, and Isolation
Sida rhombifolia was collected in Santa Rita city (PB, Brazil) in September 2008 and identified by Dr. Maria de Fátima Agra. A voucher specimen (Agra 7045) is kept at the herbarium Professor Lauro Pires Xavier (CCEN/UFPB). The dried and powdered aerial parts of the plant (5.5 kg) were extracted with 95% EtOH (10 L, room temperature) for 72 h. The EtOH extract was concentrated under reduced pressure at 40 • C, providing 570 g of crude ethanol extract (CEE).
The CEE of S. rhombifolia showed positive results when tested with Dragendorff, Bouchadart, and Mayer reagents for detection of alkaloids. Thus, the classic alkaloid extraction was performed using 100 g of the CEE.
Part of the CEE (200 g) was subjected to filtration under reduced pressure (VLC) using silica gel 60 as stationary phase and eluted with hexane (Hex.), ethyl acetate (EtOAc), and methanol (MeOH) alone or in binary mixtures increasing polarity.

Biological Assay
The experiments were approved by the Federal University of Paraíba Ethical Committee for Animal Use under the protocol CEPA#305/09. After animal euthanasia by decapitation using a guillotine, the cranial mesenteric artery was isolated, placed in Tyrode's solution and dissected in order to make it free of adhering tissue. In endothelium-denuded experiments, endothelium was removed by rubbing the intimal surface of the vessels. Rings with 1-2 mm were obtained and placed in physiological Tyrode's solution, maintained to 37 • C, gassed with carbogenic mixture (95% O 2 and 5% CO 2 ), and kept at pH 7.4. All preparations were stabilised under a resting tension of 0.75 g for 1 h. The solution was replaced every 15 min in order to prevent the accumulation of metabolites. The force of contraction was isometrically recorded by a force transducer (Miobath-4, WPI, Sarasota, FL, USA) coupled to an amplifier-recorder (Miobath-4) and to a computer equipped with an analog-to-digital converter board as described earlier. The presence of functional endothelium was assessed by the ability of acetylcholine (10 µM) to induce more than 80% relaxation of vessels pre-contracted with phenylephrine (10 µM). Less than 10% of relaxation to acetylcholine was taken as evidence that the vessel segments were functionally denuded of endothelium [5]. The preparations were exposed to cumulatively concentrations of tested compounds (10 −12 -10 −3 M) added to the preparations until a maximum response was observed as indicated by a plateau response (approximately 3-5 min).

Statistical Analysis
The results were expressed as mean ± standard error of the mean (SEM). Differences between means were considered significant when p < 0.05. Statistical comparisons between two variables were performed by using Student's t-test unpaired. The results were analyzed and plotted using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).
The quindolinone (6) and the salt of cryptolepine (9) induced vasorelaxation dependent on the vascular endothelium, justifying the use of the species in popular medicine in India. The vasorelaxation effect may be related to indoquinoline core, but more protocols are required to elucidate their complete mechanism of action.