Despite progress in diagnosis and treatment, peptic ulcer disease remains a common cause of hospitalization and surgery [13]. The current medicinal treatment of peptic ulcer is generally based on the inhibition of gastric acid secretion as well as on acid-independent therapy. In recent years, there has been growing interest in alternative therapies and the use of natural products, especially those derived from plants. In traditional medicine, several plants and herbs have been used to treat gastrointestinal disorders [14]. In this regard, extracts of hexane, dichloromethane and methanol from the aerial parts of Hyptis suaveolens were evaluated, finding that the hexane extract is the most active (Table 1). Interestingly, this effect was not dose dependent. This extract presented a maximum gastroprotective effect (70.0 ± 2.8%) at 10 mg/kg, with similar values obtained at 30 and 100 mg/kg (69.5 ± 6.9% and 69.7 ± 8.3%, respectively).

Thus, 80 g of the hexane extract were separated over a silica gel column, giving four fractions (Table 2). Three of them had considerable activity, which suggests that H. suaveolens contains more than one active compound. F1 was found to be the most active, but the amount obtained of this fraction and F3 were minimal, and the compounds obtained when F1 was chromatographed on a silica gel column were unstable. Therefore, the decision was made to work with F2 in the present study. Accordingly, 35 g of F2 were separated on a silica gel column. Elution was performed with hexane and hexane/EtOAc mixtures, obtaining 50 fractions of 20 mL each. Fractions 45–50 (hexane/EtOAc, 8:2) yielded 2.5 g (7.1%) of a white solid (m.p. 182–183 °C). This solid was identified as suaveolol, an abietane diterpene (Figure 1), after a comparison between its spectral data (¹H and ¹³C-NMR) and that in the literature [6]. Although suaveolol was previously isolated from H. suaveolens, only its anti-inflammatory activity has been reported [6]. In recent years, some reports have been published on the gastroprotective activity of diterpenes, as well as that of its derivatives belonging to different structural skeletons [15,16]. In the case of abietane diterpenes, some compounds showed greater gastroprotective effect than lansoprazole [17].

In the current study, part of the gastroprotective activity of H. suaveolens was thus attributed to suaveolol, representing the first report of such activity by this compound. Suaveolol presented a gastroprotective effect (Figure 2A) similar to F2 (Table 2). Although there are apparently other compounds in H. suaveolens, apart from suaveolol, that contribute to its gastroprotective effect, they were not considered in this study because of the scarce quantity.

We found that suaveolol produced a dose dependent gastroprotective effect (Figure 2A), a condition not observed with the hexane extract (Table 1) from which suaveolol was obtained. This can be explained by the fact that the extract has more than one active compound, as shown by the fact three active fractions were obtained. The maximum gastroprotective effect of suaveolol was reached at a dose of 100 mg/kg (70.2 ± 7.1%), which was compared with the maximal gastroprotective effect induced by carbenoxolone (75.5 ± 5.5%), also at a dose of 100 mg/kg (Figure 2B).

Although the mechanism of action of diterpenes has not been well established, these compounds seem to protect the gastric mucosa mainly through mechanisms that enhance the defensive factors of the stomach [18]. Therefore, in the present study we investigated the mechanism of action of suaveolol in relation to some gastric mucosa protective factors, such as nitric oxide, prostagladins and sulfhydryl compounds.

Several studies have demonstrated the importance of endogenous NO in the protection of gastric mucosa. NO formed by endothelial NOS plays an important role in the modulation of gastric mucosal integrity by interacting with sensory neuropeptides and endogenous prostaglandins [19]. Considering that little information exists about the role of NO in the gastroprotection induced by abietane diterpenes, we decided to investigate the participation of NO in the mechanism of action of suaveolol, and for this purpose we used L-NAME (nitric oxide synthase inhibitor).

In the group treated with 70 mg/kg of L-NAME plus 100 mg/kg of suaveolol, the ulcer index was 128.3 ± 5.4 mm² (Table 3), and this value was greater than that obtained with saline solution (90.9 ± 10.2 mm²). Thus, in the presence of L-NAME the gastroprotective effect of suaveolol was inhibited, indicating that NO is implicated in the mechanism of action. This result suggests that NO could be important in the gastroprotection of abietane compounds. The result obtained with the reference drug carbenoxolone (Table 3) was the same as that reported in the literature [4].

Gastric mucosal integrity requires the continuous generation of prostaglandin E₂ (PGE₂) and prostacyclin (PGI₂). Inhibition of the synthesis of these molecules results in the reduction of blood flow and gastric mucosal damage [20]. Additionally, the gastroprotective activity of diterpenes and their derivatives has been explained by mechanisms that include stimulation of prostaglandin synthesis [17,18]. Therefore, in order to determine whether prostaglandins are involved in the mechanism of action of suaveolol, the animals were pretreated with indomethacin (10 mg/kg).

Indomethacin inhibited the gastroprotective action elicited by suaveolol (127.7 ± 9.2 mm²), evidenced by the fact that its ulcer index value was greater than that of the respective control using the vehicle (78.9 ± 6.3 mm²). Hence, prostaglandins also participate in the mechanism of action of suaveolol (Table 4), thus confirming the involvement of prostaglandins in the gastroprotection of the abietane diterpenes. Similarly, the gastroprotective effect of carbenoxolone was attenuated (77.3 ± 7.5 mm²) by pretreatment with indomethacin (Table 4), also indicating a role played by prostaglandins.

It is well known that reduced glutathione GSH protects the gastric mucosa submitted to an ulcerative challenge. It has been demonstrated that the development of ethanol induced gastric damage is accompanied by a decrease in mucosal sulfhydryl compounds, due to the fact that these compounds are neutralized when they bind to the free radicals produced with tissue injury by noxious agents [21]. Aiming to investigate the possible role of endogenous sulfhydryl compounds in the gastroprotective effect promoted by suaveolol, the animals were pretreated with NEM, a blocker of sulfhydryl compounds. This pretreatment produced a reduction in the gastroprotection exerted on ethanol-induced gastric lesions by the administration of suaveolol, evidenced by an increase in the value (176.5 ± 10.7 mm²) of the ulcer index (Table 5). A significant decrease in gastric GSH following ethanol administration indicated massive generation of free radicals [22]. In this context, the antiulcerogenic activity of suaveolol (100 mg/kg) may depend on mucosal GSH levels. Accordingly, it is likely that an increase of endogenous sulfhydryl compounds plays an important role in the gastroprotective properties of suaveolol, as is the case with other diterpenes, including the abietanes [15,17]. Regarding carbenoxolone, the results obtained with NEM are in agreement with the literature [4].

The ¹H and ¹³C-NMR spectra were recorded in a CDCl₃ solution on a VARIAN VXR-3005 spectrometer (Palo Alto, CA, USA), working at 400 and 100 MHz, respectively.

The leaves of Hyptis suaveolens were collected in the Municipality of Copainala, the State of Chiapas, Mexico during August of 2009. The plant was identified and registered by Francisco Hernández Najarro working in the Flora Department of the Chip Herbarium, which is part of the Botanical Garden of the Secretary of Environmental Protection, Housing and Natural History of the State of Chiapas, Mexico. A specimen of the original collection can be found with the voucher number 27939.

The leaves of H. suaveolens were dried at room temperature (22 ± 2 °C) in the shade. After grinding 3 kg of leaves, compounds were successively extracted by maceration at room temperature (22 ± 2 °C) for 3 days, first with hexane (28 L × 3), then dichloromethane (28 L × 3) and finally methanol (28 L × 3). Evaporation of the solvents in vacuum yielded 77, 72, and 170 g of syrupy residues, respectively. The hexane extract obtained from the leaves of H. suaveolens showed the most active gastroprotective effect (Table 1). Thus 70 g of this extract were subjected to percolation over a silica gel column (particle size: 0.063–0.200 mm, 500 g) by using a step gradient of hexane (1.7 L, F1) hexane/EtOAc (9:1, 1.5 L, F2), hexane/EtOAc (7:3, 1.5 L, F3) and hexane/EtOAc (1:1, 1.5 L, F4). 10 g of the most active fraction (F1), were chromatographed on a silica gel column (200 g). However, the compounds obtained were unstable. Therefore, it was decided to work with fraction 2 (F2). 29 g of fraction 2 (F2) were chromatographed on a silica gel column (290 g) by using a step gradient of hexane, hexane/EtOAc and EtOAc. From this procedure, we obtained 50 fractions of 20 mL each. Fractions 15 to 25 (Hexane/EtOAc, 9:1) yielded a white solid (2.5 g, m.p. 182–83 °C), which was identified as suaveolol by comparing its spectral data (¹H and ¹³C-NMR) with that of the literature [6].

¹H-NMR (CDCl₃): δ = 0.76 (3H, s, H19), 0.81 (3H, d, J = 8 Hz, H16), 0.95 (3H, d, J = 8 Hz, H17), 1.00 (3H, s, H20), 1.04 (1H, m, H1b), 1.09 (1H, m, H6b), 1.20 (1H, m, H13), 1.25 (1H, m, H3b), 1.40 (1H, m, H3a), 1.47 (1H, m, H12b or H5), 1.50 (1H, m, H5 or H12b), 1.56 (1H, m, H2b), 1.62 (1H, m, H2a), 1.65 (1H, m, H12a), 1.68 (1H, m, H6a), 1.72 (1H, m, H1a), 1.83 (1H, m, H7b or H11b), 1.88 (1H, m, H11b or H7b), 1.97 (1H, m, H15), 2.04 (1H, m, H7a), 2.20 (1H, s br, OH), 2.42 ( 1H, m, H11a), 2.68 (1H, s br, OH), 3.03 (1H, d, J = 12 Hz, H18b), 3.45 (1H, d, J = 12 Hz, H18a), 3.8 (1H, d, J = 8 Hz, H14). ¹³C-NMR (CDCl₃): δ = 17.21 (C16), 17.74 (C19), 18.35 (C12 or C2), 18.40 (C2 or C12), 19.67 (C20), 21.46 (C17), 21.82 (C6), 24.52 (C7), 27.24 (C15), 28.05 (C11), 34.90 (C3), 35.85 (C1), 37.75 (C10 or C4), 37.78 (C4 or C10), 44.19 (C5), 48.98 (C13), 71.40 (C18), 73.38 (C14), 128.70 (C8), 142.82 (C9). These data match those in the literature [6].

All the experiments were performed with male Wistar rats, weighing 180–220 g, obtained from the animal house of the Universidad Autónoma Metropolitana Campus Xochimilco, in Mexico City, Mexico. Procedures involving animals and their care were conducted in conformity with the Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999), and in compliance with international rules on care and use of laboratory animals. Unless otherwise specified, the rats were placed in single cages with wire-net floors and deprived of food 24 h before experimentation. Animals were allowed free access to tap water throughout the experimental procedures. All experiments were carried out using 8–10 animals per group.

Carbenoxolone (Sigma-Aldrich Co. St. Louis, MO, USA) was used as the gastroprotective reference drug. This drug was prepared freshly for each use, suspended in 0.5% Tween 80 and administered by the intragastric route. Control rats received the vehicle (0.5% Tween 80) in the same volume (0.5 mL/100 g) and by the same route. N^G-nitro-L-arginine methyl ester (L-NAME), N-ethylmaleimide (NEM) and indomethacin (IND) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

A gastric ulcer was induced by administering absolute ethanol orally (1 mL) [4]. The extracts or drugs were administered to different groups 30 min before ethanol administration. Two hours after ethanol administration, the animals were sacrificed in a CO₂ chamber. The stomach and duodenum were dissected, inflated with formalin (10 mL), and then placed in 2% formalin for 5 min to fix both the inner and outer layers. The duodenum was opened along its anti-mesenteric side and the stomach along the greater curvature. The damaged area (mm²) was measured under a dissection microscope (×10) with an ocular micrometer. The ulcer index was calculated as the sum of all the lesions (area in mm²) in the stomach of each animal. Gastroprotection (%) was calculated according to: % gastroprotection = (UIC − UIT) × 100/UIC, where UIC is the ulcer index in control and UIT is the test animal index [4].

To investigate the involvement of endogenous NO in the gastroprotective effects of the compounds, L-NAME (70 mg/kg dissolved in saline solution) was intraperitoneally injected into 3 experimental groups 30 min before the administration of either the vehicle, suaveolol (100 mg/kg) or carbenoxolone (100 mg/kg) by the oral route [4]. Absolute ethanol was given to each rat in these groups 30 min later, and the animals were sacrificed 2 h after the administration of ethanol to measure the ulcer index. Two control groups (L-NAME-treated and non-L-NAME-treated) were included in this evaluation.

To investigate the involvement of endogenous prostaglandins in the gastroprotective effect of the compounds, a control group received a subcutaneous injection of 5 mM NaHCO₃ in saline solution and another group an injection of indomethacin (10 mg/kg dissolved in NaHCO₃ at 5 mM) by the same route [4]. After 75 min, the animals in each of these two groups received one of three oral treatments (saline solution, 100 mg/kg suaveolol or 100 mg/kg carbenoxolone). Absolute ethanol was given to each rat 30 min after suaveolol or carbenoxolone administration and the rats were sacrificed 2 h later in a CO₂ chamber. The stomachs were subsequently removed to measure the ulcer index, as aforementioned.

To investigate the involvement of the endogenous sulfhydryls in the protective effects of suaveolol and carbenoxolone, NEM (dissolved in saline solution) was subcutaneously injected (10 mg/kg) in 3 groups of animals 30 min before the oral administration of either the vehicle, suaveolol (100 mg/kg) or carbenoxolone (100 mg/kg) [4]. Absolute ethanol was given to each rat 30 min later and rats were sacrificed 2 h after the administration of ethanol to measure the intensity of the gastric ulcer. Two control groups (NEM-treated and non-NEM-treated) were included in this experiment.

Data are presented as the mean ± SEM from 8–10 rats per group. Statistical significance between the treatments was evaluated by the Kruskal-Wallis test, followed by Dunn’s multiple comparison tests, with p< 0.05 considered as significant.