PHYTOTHERAPY RESEARCH Phytother. Res. 29: 1600–1604 (2015) Published online 16 July 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5421
Antiamoebic and Antigiardial Activity of Clerodane Diterpenes from Mexican Salvia Species Used for the Treatment of Diarrhea Fernando Calzada,1* Elihú Bautista,2 Lilian Yépez-Mulia,3 Normand García-Hernandez4 and Alfredo Ortega2 1 Unidad de Investigación Médica en Farmacología, UMAE Hospital de Especialidades, 2° Piso CORCE, Centro Médico Nacional Siglo XXI, México D. F., México 2 Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, México D. F., México 3 Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, UMAE Hospital de Pediatría, 2° Piso, Centro Médico Nacional Siglo XXI, México City, México 4 Unidad de Investigación Médica en Genética Humana, UMAE Hospital de Pediatría, Centro Médico Nacional Siglo XXI, IMSS, I, México City, México
Terpenoids from Salvia species have been identified to possess biological properties as antiprotozoal agents. Here, we evaluated the antiamoebic and antigiardial activities of 14 known clerodane and modified clerodane-type diterpenes isolated from five Mexican Salvia species against Entamoeba histolytica and Giardia lamblia, and analyzed the effects of the functionalities in decalin ring or in the whole clerodane framework to visualize the structural requirements necessary to produce an antiprotozoal activity. Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 μM for E. histolytica and of 28.2 μM in the case of G. lamblia. In this context it may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: antiprotozoal activity; Entamoeba histolytica; Giardia lamblia; clerodane diterpenes; Salvia species.
INTRODUCTION Amoebiasis and giardiasis are parasitic infections caused by pathogenic unicellular protozoan, acquired through the ingestion of contaminated food or water and constitute one of the most widespread human health problems. These infections are prevalent particularly in developing countries and are associated with poor hygienic conditions (Singh et al., 2009; Mineno and Avery, 2003). In addition, both protozoa cause diarrhea, which is considered to be one of the five most common causes of death worldwide (1.6 millions of deaths each year) (Baldursson and Karanis, 2011). Amoebiasis is an endemic disease and a public health problem throughout Mexico, with incidence rates that vary among the geographic regions of the country. It the last six years, it has been a serious health problem, and it was the 10th cause of morbidity among all age groups. In the case of giardiasis, it currently accounts for an estimated nine millions of sicks each year, and it is the leading cause of gastrointestinal parasitosis of medical importance in children (DGE, 2013; Hernandez et al., 2015; GPC, 2012). Drugs, such as metronidazol, used to treat amoebic dysentery and giardiasis, have the disadvantage of causing many undesired effects. This situation highlights the * Correspondence to: Dr. Fernando Calzada Bermejo, Unidad de Investigación Médica en Farmacología. UMAE Hospital de Especialidades, 2° Piso CORCE, Centro Médico Nacional Siglo XXI, México D. F., México. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
need for discovery and development of new effective and safer antiprotozoal agents (Calzada et al., 2010) The name Salvia is derived from Latin ‘Salvare’ meaning ‘to heal or to be safe’ which was associated with the use of these species to treat multiple human ailments, for this reason they were considered ‘magical’ in ancient time (Yi-Bing et al., 2012). Salvia species belong to one of the largest genera of Lamiaceae family, which consists of approximately 1000 species. Plants of this genus have been organized in four sections: Salvia, Leonia, Clarea and Calosphace (Bentham, 1876). Subgenus Calosphace grouped 500 species which are endemic to North and South America; many of them constitute four largest complexes of medicinal plants, they are: Cantueso complex, Mirto complex, Manga– Paqui complex and Ñucchu complex. The first two complexes are endemic to Mexico, and it represents the major diversification center of Salvia species with approximately 275. In this region, some Mexican Salvia species are used in the traditional medicine to treat disorders of the central nervous system, obstetrical and gynecological, as well as gastrointestinal ailments (Jenks and Kim, 2013). Some salvias are used to treat particularly diarrhea, they are: Salvia adenophora, Salvia coccinea, Salvia divinorum, Salvia mexicana, Salvia microphylla, Salvia pinguifolia, Salvia polystachya and Salvia reptans (Aguilar et al., 1996). Of all these species, S. divinorum, known as ska María Pastora, ska Pastora, ska María or hierba de la Pastora, is also used as pyschotomimetic agent in magical rites by the Mazatec healers in Oaxaca (Valdes et al., 1983). Received 13 March 2015 Revised 01 July 2015 Accepted 02 July 2015
ANTIPROTOZOAL ACTIVITY OF CLERODANE DITERPENE FROM SALVIA SPECIES
Additional uses of this plant to stop diarrhea and relieve headache and rheumatism have been described (Prisinzano, 2009). Salvinorin A, a clerodane diterpene isolated previously from leaves of this plant, is the substance responsible of its hallucinogenic effect (Ortega et al., 1982; Roth et al., 2002). Recently, a study of salvinorin A described the potent antiinflammatory effect of this compound (Aviello et al., 2011). Importantly, the intestinal antisecretory and anti-motility effects of Salvinorin A and Salvia extract have been also documented in rodents (Capasso et al., 2008a; Capasso et al., 2008b; Fichna et al., 2009). However, the antiprotozoal properties, which can strengthen the traditional antidiarrheal use of Salvia divinorum, have been not reported to date. Among the Salvia species used in Mexico to relieve gastrointestinal disorders, only the antiprotozoal activity of the acetone soluble extract of the aerial parts of S. polystachya has been described. This study led to the isolation of linearolactone (14) (Fig. 1) as the active ingredient (Calzada et al., 2010). In addition, Salvia species are a rich source of terpenoid compounds, mainly diterpenes and triterpenes. This kind of secondary metabolites possesses a widespread variety of biological activities; one of them, recently investigated, is the antiprotozoal activity; this type of compounds has shown to be active against Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum and Trypanosoma brucei (Calzada et al., 2010; Ebrahimi et al., 2013; Moridi et al., 2011). This background prompted us to evaluate the antiprotozoal activity of fourteen clerodane and rearranged clerodane type diterpenoids isolated from Mexican Salvia species as a part of our search of new bioactive natural products.
Figure 1. Chemical structures of clerodane and rearranged clerodanetype diterpenes assayed. Copyright © 2015 John Wiley & Sons, Ltd.
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MATERIALS AND METHODS Chemicals. Metronidazole and emetine were used as positive controls and purchased from Sigma. 1(10)Dehydrosalviarin (1; 0.0105% dry weight), 1β,10βepoxysalviarin (2; 0.0006% dry weight), tehuanines D-F (3–5; 0.2288%, 0.0203% and 0.0145% dry weight respectively) and tehuanine H (6; 0.0012% dry weight) were isolated from S. herbacea (Bautista et al., 2012); salvinorin A (7; 0.0900% dry weight) was obtained from S. divinorum following the method previously described (Ortega et al., 1982); microphyllandiolide (8; 0.0510% dry weight), salvimicrophyllin B (9; 0.0160% dry weight) and salvimicrophyllin D (10; 0.0064% dry weight) were isolated from S. microphylla (Bautista et al., 2013; 2014); polystachyne E (13; 0.0064% dry weight) and linearolactone (14; 0.2221% dry weight) were obtained from S. polystachya using the method described (Maldonado and Ortega, 2000; Calzada et al., 2010).
Isolation of compounds 11 and 12 Plant material. The aerial parts of Salvia gesneriflora were collected in Huitzilac, Morelos, México near km 64 of México-Cuernavaca highway in November 2011. A voucher specimen (MEXU-1320390) was deposited at Nacional Herbarium, Instituto de Biología, Universidad Nacional Autónoma de México. Extraction and isolation. The aerial parts dried and powered (2.1 kg) were extracted by percolation with acetone (12 L) to obtain a gummy residue (96.3 g, 5.6% dry weight). The soluble acetone extract was dissolved in 0.5 L of a mixture of MeOH–H2O (4:1) and partitioned with hexanes (0.4 L × 10). The hydroalcoholic fraction was concentrated to one fifth of its volume and partitioned again with EtOAc (0.5 L × 3). The EtOAc fraction (34 g) was submitted to vacuum column chromatography (VCC) eluted with hexane–EtOAc, 4:1 (fraction A), hexane–EtOAc, 3:1 (fraction B), hexane– EtOAc, 7:3 (fraction C) and hexane:EtOAc, 3:2 (fraction D). Fraction A (2.86 g, 0.14%) was submitted to successive VCC eluted with CHCl3–EtOAc 97:3, hexane–EtOAc, 3:1 and hexane–EtOAc 7:3 to obtain by crystallization from EtOAc–hexane, 173 mg (8 × 10 3%) of compound 11. Fraction B (3.42 g, 0.16%) was subjected to successive VCC eluted with hexane–CHCl3–MeOH, 60:40:1 and hexane–EtOAc–MeOH, 80:20:1 to obtain by crystallization from EtOAc–hexane 87.6 mg (4 × 10 4%) of compound 12. Compounds 11 and 12 were identified by comparison of their 1H and 13C NMR spectra with those described for salvifulgenolide (Narukawa et al., 2006) and isosalvixalapadiene (Esquivel et al., 2005), respectively.
Evaluation of antiprotozoal activity. E.histolytica strain HM1-IMSS used in all experiments was grown axenically at 37 °C in TYI-S-33 medium supplemented with 10% heat inactivated bovine serum. In the case of G. lamblia, strain IMSS: 8909:1 was grown in TYIS-33 modified medium supplemented with 10% Phytother. Res. 29: 1600–1604 (2015)
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calf serum and bovine bile. The trophozoites were axenically maintained and for assays were employed in the log phase of growth. In vitro susceptibility tests were performed using a subculture method described previously (Calzada et al., 2010). Briefly, E. histolytica (6 × 103) or G. lamblia (5 × 104) trophozoites were incubated for 48 h at 37 °C in the presence of different concentrations (2.5–200 μg/mL) of pure compounds in dimethyl sulfoxide (DMSO). Each test included emetine and metronidazole as standard amoebicidal and giardicidal drugs, a control (culture medium plus trophozoites and DMSO) and a blank (culture medium). After incubation, the trophozoites were detached by chilling and 50-μL samples of each tube were subcultured in fresh medium for another 48 h, without antiprotozoal samples. The final number of parasites was determined with a hemocytometer and the percentages of trophozoites growth inhibition were calculated by comparison with the control culture. The results were confirmed by a colorimetric method: the trophozoites were washed and incubated for 45 min at 37 °C in phosphate buffer saline with MTT (3-[4, 5-dimethylhiazol-2-il]-2, 5-diphenyl tetrazolium bromide) and phenazine methosulfate. The dye produced (formazan) was extracted, and the absorbance was determined at 570 nm. The experiments were performed in duplicate for each protozoan and repeated at least three times. The in vitro results were classified as follows: if the samples displayed an IC50 less than 30 μM, the antiprotozoal activity was considered good, from 31 to 160 μM the antiprotozoal activity was considered moderate, from 161 to 200 μM the antiprotozoal activity was considered weak and over 200 μg/mL the samples were considered inactive (Calzada et al., 2010).
Statistical analysis. Data were analyzed using probit analysis. The percentage of trophozoites surviving was calculated by comparison with the growth in the control group. The plot of probit against log concentration was made; the best straight line was determined by regression analysis, and the 50% inhibitory concentration (IC50) values were calculated. The regression coefficient, its level of significance (P < 0.05 indicates significant difference between group) and correlation coefficient were calculated and 95% CI values determined (Finney, 1977; Keene et al., 1986; Calzada et al., 2010).
RESULTS AND DISCUSSION As a part of our search to obtain new antiprotozoal agents by medicinal plants several diterpenes isolated from Mexican Salvia species were tested for their antiprotozoal activity against E. histolytica and G. lamblia and also to understand the structural requirements needed for this effect. The following diterpenes: 1(10)-dehydrosalviarin (1), 1β,10β-epoxysalviarin (2), tehuanines D-F (3–5), tehuanine H (6), salvinorin A (7), microphyllandiolide (8), salvimicrophyllin B (9), salvimicrophyllin D (10), salvifulgenolide (11), isosalvixalapadiene (12), polystachyne E (13) and linearolactone (14) were tested (Fig. 1). Among these compounds there are conventional neo-clerodanes with vinilyc and conjugated double bonds: acetate, epoxy, hydroxyl and ketone groups in the ring A; and the rearranged clerodanes: 5, 10-seco-neo-clerodanes and 5, 6-seco-salvigenanes. Results of the antiprotozoal activity for these compounds are shown in Table 1.
Table 1. Antiprotozoal activity data of compounds 1–13 against E. histolytica and G. lamblia trophozoites IC50 μM (CI)a Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 Linearolactone (14)b Kaempferolb Emetine Metronidazole
E. histolytica
G. lamblia
210.4 (211.6–210.1)#$ 188.9 (189.8–188.4) #$ 191.8 (194.0–191.7) #$ 184.8 (185.7–183.5) #$ 204.5 (205.3–204.2) #$ 167.6 (168.1–166.2) #$ 49.0 (49.5–48.8) #$ 182.2 (182.8–180.2) #$ 172.9 (173.5–172.1) #$ 187.2 (187.8–186.4) #$ 183.5 (184.7–182.9) #$ 200.5 (201.4–198.7) #$ 76.6 (77.2–75.8) #$ 22.9 (23.5–22.4)$ 27.7 2.18 (2.2–2.14) 0.23 (0.58–0–17)
241.3 (242.7–240.9) * & 228.0 (228.6–227.4) * & 208.6 (209.2–208.4) * & 221.7 (222.5–220.8) * & 219.5 (220.3–219.0) * & 186.4 (187.5–185.6) * & 64.8 (65.3–64.3)* & 201.3(201.6–200.4) * & 161.4(162.5–160.3) * & 215.3 (215.6–213.6) * & 259.7 (260.9–259.1) * & 241.9 (242.2–240.1) * & 83.6 (84.2–83.1)* & 28.2 (28.5–27.9) 30.5 0.83 (0.87–0.82) 1.22 (1.57–0.81)
&
a
Results are expressed as mean (n = 6), CI = 95% confidence intervals. Calzada et al., 2010. # P < 0.05 compared to linearolactone. $ P < 0.05 compared to emetine and metronidazole. *P < 0.05 compared to linearolactone. & P < 0.05 compared to emetine and metronidazole. **Correlation coefficient >0.874. b
Copyright © 2015 John Wiley & Sons, Ltd.
Phytother. Res. 29: 1600–1604 (2015)
ANTIPROTOZOAL ACTIVITY OF CLERODANE DITERPENE FROM SALVIA SPECIES
Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 μM for E. histolytica and from 28.2 μg/ml in the case for G. lamblia. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. Compounds 7 (E. histolytica IC50 = 49.0 μM and G. lamblia IC50 = 64.8 μM) and 13(E. histolytica IC50 = 76.6 μM and G. lamblia IC50 = 83.6 μM) showed moderate activity against both protozoa (Table 1). Compound 7 possesses a trans-fused decalin ring system with a ketone carbonyl and an acetate group at C-1 and C-2, respectively. Only this compound possesses different oxygenated functionalities other than epoxy and hydroxyl groups, which could explain its antiprotozoal activity. Compound 13 is closely related to linearolactone (14), a neo-clerodane active against E. histolytica and G. lamblia (Calzada et al., 2010). Both compounds were isolated from S. polystachya as a mixture being 13 the minor component (Maldonado and Ortega, 2000). Comparison between the data of antiprotozoal activity and structural features of the neo-clerodanes tested suggested that the following functionalities in ring A of the framework are preferred: a 1,3-diene over a 2,10-diene, a C-3 conjugated double bond over a C-2 vinyl double bond, as well as an 1,2-epoxy group over an 1,10-epoxy group. The hydroxyl group at C-10 in compound 10 decreases the activity in comparison to 14 with hydrogen atom. The 5,10-seco-neo-clerodanes and 5,6-seco-salvigenanes tested showed weak antiprotozoal activity. In conclusion, all the diterpenes assayed were less active than linearolactone (14) and antiprotozoal drugs
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used as controls (emetine and metronidazole). Salvinorin A (7) showed moderate activity against both parasites and it could be the responsible to stop diarrhea caused by protozoa. So the above also could explain the use of S. divinorum in the Mexican traditional medicine to treat this kind of ailment. However, additional studies are necessary. Polystachyne E (13) a minor component from S. polystachya showed weak activity. This result suggested that the antiprotozoal effect of the acetone soluble extract is mainly because of 14 (IC50 values of 22.9 μM for E. histolytica and 28.2 μM for G. lamblia). Also, data obtained in this investigation, combined with our previous results (Calzada et al., 2010, Barbosa et al., 2007), confirm that linearolactone may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea.
Acknowledgements E. Bautista also thanks Dirección General del Personal Académico (DGAPA) for the postdoctoral scholarship and M. Sc. María del Rosario García Peña for identifying the plant material.
Conflict of Interest The authors declare that there are no conflicts of interest.
REFERENCES Aguilar A, Camacho JR, Chino S, Jacquez PE, López ME. 1996. Plantas Medicinales del Herbario IMSS. Instituto Mexicano del Seguro Social: Mexico D. F. 107–110. Aviello G, Borelli F, Guida F, et al. 2011. Ultrapotent effects of salvinorin A, a hallucinogenic compound from Salvia divinorum, on LPS-stimulated murine macrophages and its anti-inflammatory action in vivo, J Mol Med 89: 891–902. Baldursson S, Karanis P. 2011. Waterborne transmission of protozoan parasites: review of worldwide outbreaks—an update 2004–2010. Water Res 45: 6603–6614. Barbosa E, Calzada F, Campos R. 2007. In vivo antigiardial activity of three flavonoids isolated of some medicinal plants used in Mexican traditional medicine for the treatment of diarrhea. J Ethnopharmacol 109: 552–554. Bautista E, Maldonado E, Ortega A. 2012. Neo-clerodane diterpenes from Salvia herbacea. J Nat Prod 75: 951–958 Bautista E, Toscano RA, Ortega A. 2013. Microphyllandiolide, a new diterpene with an unprecedented skeleton from Salvia microphylla. Org Lett 15: 3210–3213 Bautista E, Toscano RA, Ortega A. 2014. 5,10-seco-neo-clerodanes and neo-clerodanes from Salvia microphylla. J Nat Prod 77: 1088–1092. Bentham G 1876. Labiatae. In Genera Plantarum. vol. 2, Bentham G and Hoocker JD (eds). Revve& Co: London pp. 1160–1222. Calzada F, Yépez-Mulia L,Tapia-Contreras A, Bautista E, Maldonado E, Ortega A. 2010. Evaluation of the antiprotozoal activity of neo-clerodane type diterpenes from Salvia polystachya against Entamoeba histolytica and Giardia lamblia. Phytother Res 24: 662–665. Capasso R, Borrelli F, Zjawiony J, et al. 2008a. The hallucinogenic herb Salvia divinorum and its active ingredient salvinorin A reduce inflammation-induced hypermotility in mice. Neurogastroenterol Motil 20(2):142–148. Capasso R, Borrelli F, Cascio MG, et al. 2008b. Inhibitory effect of salvinorin A, from Salvia divinorum, on ileitis-induced hypermotility: cross-talk between kappa-opioid and cannabinoid CB(1) receptors. Br J Pharmacol 155(5):681–689. Copyright © 2015 John Wiley & Sons, Ltd.
Dirección General de Epidemiología (DGE). 2013. Anuarios de morbilidad 2008–2013. www.epidemiologia.salud.gob.mx/ anuario/html/anuarios.html. Ebrahimi SN, Zimmermann S, Zaugg J, Smiesko M, Reto B, Hamburger M. 2013. Abietane diterpennoids from Salvia sahendica—antiprotozoal activity and determination of their absolute configurations. Planta Med 79:150–156. Esquivel B, Tello R, Sánchez AA. 2005. Unsaturated diterpenoids with a novel carbocyclic skeleton from Salvia xalapensis. J Nat Prod 68: 787–790. Fichna J, Schicho R, Andrews CN, et al. 2009. Salvinorin A inhibits colonic transit and neurogenic ion transport in mice by activating kappa-opioid and cannabinoid receptors. Neurogastroenterol Motil 21(12):1326-e128. doi: 10.1111/j.1365-2982.2009.01369.x. Finney DL. 1977. Probit Analysis. Cambridge University Press: New York. Hernandez EG, Granados J, Partida-Rodriguez O, et al. 2015. Prevalent HLA class II alleles in Mexico City appear to confer resistance to the development of amebic liver abscess. Plos One May 4;10(5): e0126195. doi: 10.1371/journal. pone.0126195.eCollection. Jenks AA, Kim SC. 2013. Medicinal plant complexes of Salvia subgenus Calosphace: an ethnobotanical study of new world sages. J Ethnopharmacol 146: 214–224. Keene AT, Harris A, Phillipson JD, Warhurst DC. 1986. In vitro amoebicidal testing of natural products; Part I. Methodology. Planta Med 4: 278–285. Maldonado E, Ortega A. 2000. Polystachynes A–E, five cis-neo-clerodane diterpenoids from Salvia polystachya. Phytochemistry 53: 103–109. Mineno T, Avery MA. 2003. Giardiasis: recent progress in chemotherapy and drug development. Curr Pharm Des 9: 841–855. Moridi FM, Bahadori MB, Taheri S, Ebrahimi SN, Zimmermann S, Brun R, Amin G, Hamburger M. 2011. Triterpenoids with rare carbon skeletons from Salvia hydrangea: antiprotozoal activity and absolute configurations. J Nat Prod 74: 2200–2205. Narukawa Y, Fukui M, Hatano K, Takeda T. 2006. Four new diterpenoids from Salvia fulgens Cav. J Nat Med 60: 58–63. Phytother. Res. 29: 1600–1604 (2015)
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Ortega A, Blount JF, Manchand PS. 1982. Salvinorin, a new transneoclerodane diterpene from Salvia divinorum (Labiatae). J Chem Soc Perkin Trans 1: 2505–2508. Prisinzano TE. 2009. Natural products as tools for neuroscience: discovery and development of novel agents to treat drug abuse. J Nat Prod 72: 581–587. Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P, Rothman RB. 2002. Salvinorin A: a potent naturally occurring non-nitro genousκ opioid selective agonist. Proc Natl Acad Sci U S A 99: 11,934–11,939 Singh S, Bharti N, Mohapatra PP. 2009. Chemistry and biology of synthetic and naturally occurring antiamoebic agents. Chem Rev 109: 1900–1947.
Copyright © 2015 John Wiley & Sons, Ltd.
Guía práctica clínica (GPC). 2012. Prevención, diagnóstico y tratamiento farmacológico de la giardiasis en niños y adolescentes de 1 a 18 años en el primer y segundo nivel de atención. Evidencias y recomendaciones. Publicado por CENETEC. ISSSTE-252-12. www.cenetec.salud.gob.mx/interior/gpc.html. Valdes III JL, Díaz JL, Paul AG. 1983. Ethnopharmacology of ska María Pastora (Salvia divinorum, Epling and Jativa M.). J Ethnopharmacol 7: 287–312. Yi-Bing W, Zhi-Yu N, Qing-When S, et al. 2012. Constituents from Salvia species and their biological activities. Chem Rev 112: 5967–6026.
Phytother. Res. 29: 1600–1604 (2015)
Antiamoebic and Antigiardial Activity of Clerodane Diterpenes from Mexican Salvia Species Used for the Treatment of Diarrhea Fernando Calzada,1* Elihú Bautista,2 Lilian Yépez-Mulia,3 Normand García-Hernandez4 and Alfredo Ortega2 1 Unidad de Investigación Médica en Farmacología, UMAE Hospital de Especialidades, 2° Piso CORCE, Centro Médico Nacional Siglo XXI, México D. F., México 2 Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, México D. F., México 3 Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, UMAE Hospital de Pediatría, 2° Piso, Centro Médico Nacional Siglo XXI, México City, México 4 Unidad de Investigación Médica en Genética Humana, UMAE Hospital de Pediatría, Centro Médico Nacional Siglo XXI, IMSS, I, México City, México
Terpenoids from Salvia species have been identified to possess biological properties as antiprotozoal agents. Here, we evaluated the antiamoebic and antigiardial activities of 14 known clerodane and modified clerodane-type diterpenes isolated from five Mexican Salvia species against Entamoeba histolytica and Giardia lamblia, and analyzed the effects of the functionalities in decalin ring or in the whole clerodane framework to visualize the structural requirements necessary to produce an antiprotozoal activity. Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 μM for E. histolytica and of 28.2 μM in the case of G. lamblia. In this context it may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: antiprotozoal activity; Entamoeba histolytica; Giardia lamblia; clerodane diterpenes; Salvia species.
INTRODUCTION Amoebiasis and giardiasis are parasitic infections caused by pathogenic unicellular protozoan, acquired through the ingestion of contaminated food or water and constitute one of the most widespread human health problems. These infections are prevalent particularly in developing countries and are associated with poor hygienic conditions (Singh et al., 2009; Mineno and Avery, 2003). In addition, both protozoa cause diarrhea, which is considered to be one of the five most common causes of death worldwide (1.6 millions of deaths each year) (Baldursson and Karanis, 2011). Amoebiasis is an endemic disease and a public health problem throughout Mexico, with incidence rates that vary among the geographic regions of the country. It the last six years, it has been a serious health problem, and it was the 10th cause of morbidity among all age groups. In the case of giardiasis, it currently accounts for an estimated nine millions of sicks each year, and it is the leading cause of gastrointestinal parasitosis of medical importance in children (DGE, 2013; Hernandez et al., 2015; GPC, 2012). Drugs, such as metronidazol, used to treat amoebic dysentery and giardiasis, have the disadvantage of causing many undesired effects. This situation highlights the * Correspondence to: Dr. Fernando Calzada Bermejo, Unidad de Investigación Médica en Farmacología. UMAE Hospital de Especialidades, 2° Piso CORCE, Centro Médico Nacional Siglo XXI, México D. F., México. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
need for discovery and development of new effective and safer antiprotozoal agents (Calzada et al., 2010) The name Salvia is derived from Latin ‘Salvare’ meaning ‘to heal or to be safe’ which was associated with the use of these species to treat multiple human ailments, for this reason they were considered ‘magical’ in ancient time (Yi-Bing et al., 2012). Salvia species belong to one of the largest genera of Lamiaceae family, which consists of approximately 1000 species. Plants of this genus have been organized in four sections: Salvia, Leonia, Clarea and Calosphace (Bentham, 1876). Subgenus Calosphace grouped 500 species which are endemic to North and South America; many of them constitute four largest complexes of medicinal plants, they are: Cantueso complex, Mirto complex, Manga– Paqui complex and Ñucchu complex. The first two complexes are endemic to Mexico, and it represents the major diversification center of Salvia species with approximately 275. In this region, some Mexican Salvia species are used in the traditional medicine to treat disorders of the central nervous system, obstetrical and gynecological, as well as gastrointestinal ailments (Jenks and Kim, 2013). Some salvias are used to treat particularly diarrhea, they are: Salvia adenophora, Salvia coccinea, Salvia divinorum, Salvia mexicana, Salvia microphylla, Salvia pinguifolia, Salvia polystachya and Salvia reptans (Aguilar et al., 1996). Of all these species, S. divinorum, known as ska María Pastora, ska Pastora, ska María or hierba de la Pastora, is also used as pyschotomimetic agent in magical rites by the Mazatec healers in Oaxaca (Valdes et al., 1983). Received 13 March 2015 Revised 01 July 2015 Accepted 02 July 2015
ANTIPROTOZOAL ACTIVITY OF CLERODANE DITERPENE FROM SALVIA SPECIES
Additional uses of this plant to stop diarrhea and relieve headache and rheumatism have been described (Prisinzano, 2009). Salvinorin A, a clerodane diterpene isolated previously from leaves of this plant, is the substance responsible of its hallucinogenic effect (Ortega et al., 1982; Roth et al., 2002). Recently, a study of salvinorin A described the potent antiinflammatory effect of this compound (Aviello et al., 2011). Importantly, the intestinal antisecretory and anti-motility effects of Salvinorin A and Salvia extract have been also documented in rodents (Capasso et al., 2008a; Capasso et al., 2008b; Fichna et al., 2009). However, the antiprotozoal properties, which can strengthen the traditional antidiarrheal use of Salvia divinorum, have been not reported to date. Among the Salvia species used in Mexico to relieve gastrointestinal disorders, only the antiprotozoal activity of the acetone soluble extract of the aerial parts of S. polystachya has been described. This study led to the isolation of linearolactone (14) (Fig. 1) as the active ingredient (Calzada et al., 2010). In addition, Salvia species are a rich source of terpenoid compounds, mainly diterpenes and triterpenes. This kind of secondary metabolites possesses a widespread variety of biological activities; one of them, recently investigated, is the antiprotozoal activity; this type of compounds has shown to be active against Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum and Trypanosoma brucei (Calzada et al., 2010; Ebrahimi et al., 2013; Moridi et al., 2011). This background prompted us to evaluate the antiprotozoal activity of fourteen clerodane and rearranged clerodane type diterpenoids isolated from Mexican Salvia species as a part of our search of new bioactive natural products.
Figure 1. Chemical structures of clerodane and rearranged clerodanetype diterpenes assayed. Copyright © 2015 John Wiley & Sons, Ltd.
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MATERIALS AND METHODS Chemicals. Metronidazole and emetine were used as positive controls and purchased from Sigma. 1(10)Dehydrosalviarin (1; 0.0105% dry weight), 1β,10βepoxysalviarin (2; 0.0006% dry weight), tehuanines D-F (3–5; 0.2288%, 0.0203% and 0.0145% dry weight respectively) and tehuanine H (6; 0.0012% dry weight) were isolated from S. herbacea (Bautista et al., 2012); salvinorin A (7; 0.0900% dry weight) was obtained from S. divinorum following the method previously described (Ortega et al., 1982); microphyllandiolide (8; 0.0510% dry weight), salvimicrophyllin B (9; 0.0160% dry weight) and salvimicrophyllin D (10; 0.0064% dry weight) were isolated from S. microphylla (Bautista et al., 2013; 2014); polystachyne E (13; 0.0064% dry weight) and linearolactone (14; 0.2221% dry weight) were obtained from S. polystachya using the method described (Maldonado and Ortega, 2000; Calzada et al., 2010).
Isolation of compounds 11 and 12 Plant material. The aerial parts of Salvia gesneriflora were collected in Huitzilac, Morelos, México near km 64 of México-Cuernavaca highway in November 2011. A voucher specimen (MEXU-1320390) was deposited at Nacional Herbarium, Instituto de Biología, Universidad Nacional Autónoma de México. Extraction and isolation. The aerial parts dried and powered (2.1 kg) were extracted by percolation with acetone (12 L) to obtain a gummy residue (96.3 g, 5.6% dry weight). The soluble acetone extract was dissolved in 0.5 L of a mixture of MeOH–H2O (4:1) and partitioned with hexanes (0.4 L × 10). The hydroalcoholic fraction was concentrated to one fifth of its volume and partitioned again with EtOAc (0.5 L × 3). The EtOAc fraction (34 g) was submitted to vacuum column chromatography (VCC) eluted with hexane–EtOAc, 4:1 (fraction A), hexane–EtOAc, 3:1 (fraction B), hexane– EtOAc, 7:3 (fraction C) and hexane:EtOAc, 3:2 (fraction D). Fraction A (2.86 g, 0.14%) was submitted to successive VCC eluted with CHCl3–EtOAc 97:3, hexane–EtOAc, 3:1 and hexane–EtOAc 7:3 to obtain by crystallization from EtOAc–hexane, 173 mg (8 × 10 3%) of compound 11. Fraction B (3.42 g, 0.16%) was subjected to successive VCC eluted with hexane–CHCl3–MeOH, 60:40:1 and hexane–EtOAc–MeOH, 80:20:1 to obtain by crystallization from EtOAc–hexane 87.6 mg (4 × 10 4%) of compound 12. Compounds 11 and 12 were identified by comparison of their 1H and 13C NMR spectra with those described for salvifulgenolide (Narukawa et al., 2006) and isosalvixalapadiene (Esquivel et al., 2005), respectively.
Evaluation of antiprotozoal activity. E.histolytica strain HM1-IMSS used in all experiments was grown axenically at 37 °C in TYI-S-33 medium supplemented with 10% heat inactivated bovine serum. In the case of G. lamblia, strain IMSS: 8909:1 was grown in TYIS-33 modified medium supplemented with 10% Phytother. Res. 29: 1600–1604 (2015)
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calf serum and bovine bile. The trophozoites were axenically maintained and for assays were employed in the log phase of growth. In vitro susceptibility tests were performed using a subculture method described previously (Calzada et al., 2010). Briefly, E. histolytica (6 × 103) or G. lamblia (5 × 104) trophozoites were incubated for 48 h at 37 °C in the presence of different concentrations (2.5–200 μg/mL) of pure compounds in dimethyl sulfoxide (DMSO). Each test included emetine and metronidazole as standard amoebicidal and giardicidal drugs, a control (culture medium plus trophozoites and DMSO) and a blank (culture medium). After incubation, the trophozoites were detached by chilling and 50-μL samples of each tube were subcultured in fresh medium for another 48 h, without antiprotozoal samples. The final number of parasites was determined with a hemocytometer and the percentages of trophozoites growth inhibition were calculated by comparison with the control culture. The results were confirmed by a colorimetric method: the trophozoites were washed and incubated for 45 min at 37 °C in phosphate buffer saline with MTT (3-[4, 5-dimethylhiazol-2-il]-2, 5-diphenyl tetrazolium bromide) and phenazine methosulfate. The dye produced (formazan) was extracted, and the absorbance was determined at 570 nm. The experiments were performed in duplicate for each protozoan and repeated at least three times. The in vitro results were classified as follows: if the samples displayed an IC50 less than 30 μM, the antiprotozoal activity was considered good, from 31 to 160 μM the antiprotozoal activity was considered moderate, from 161 to 200 μM the antiprotozoal activity was considered weak and over 200 μg/mL the samples were considered inactive (Calzada et al., 2010).
Statistical analysis. Data were analyzed using probit analysis. The percentage of trophozoites surviving was calculated by comparison with the growth in the control group. The plot of probit against log concentration was made; the best straight line was determined by regression analysis, and the 50% inhibitory concentration (IC50) values were calculated. The regression coefficient, its level of significance (P < 0.05 indicates significant difference between group) and correlation coefficient were calculated and 95% CI values determined (Finney, 1977; Keene et al., 1986; Calzada et al., 2010).
RESULTS AND DISCUSSION As a part of our search to obtain new antiprotozoal agents by medicinal plants several diterpenes isolated from Mexican Salvia species were tested for their antiprotozoal activity against E. histolytica and G. lamblia and also to understand the structural requirements needed for this effect. The following diterpenes: 1(10)-dehydrosalviarin (1), 1β,10β-epoxysalviarin (2), tehuanines D-F (3–5), tehuanine H (6), salvinorin A (7), microphyllandiolide (8), salvimicrophyllin B (9), salvimicrophyllin D (10), salvifulgenolide (11), isosalvixalapadiene (12), polystachyne E (13) and linearolactone (14) were tested (Fig. 1). Among these compounds there are conventional neo-clerodanes with vinilyc and conjugated double bonds: acetate, epoxy, hydroxyl and ketone groups in the ring A; and the rearranged clerodanes: 5, 10-seco-neo-clerodanes and 5, 6-seco-salvigenanes. Results of the antiprotozoal activity for these compounds are shown in Table 1.
Table 1. Antiprotozoal activity data of compounds 1–13 against E. histolytica and G. lamblia trophozoites IC50 μM (CI)a Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 Linearolactone (14)b Kaempferolb Emetine Metronidazole
E. histolytica
G. lamblia
210.4 (211.6–210.1)#$ 188.9 (189.8–188.4) #$ 191.8 (194.0–191.7) #$ 184.8 (185.7–183.5) #$ 204.5 (205.3–204.2) #$ 167.6 (168.1–166.2) #$ 49.0 (49.5–48.8) #$ 182.2 (182.8–180.2) #$ 172.9 (173.5–172.1) #$ 187.2 (187.8–186.4) #$ 183.5 (184.7–182.9) #$ 200.5 (201.4–198.7) #$ 76.6 (77.2–75.8) #$ 22.9 (23.5–22.4)$ 27.7 2.18 (2.2–2.14) 0.23 (0.58–0–17)
241.3 (242.7–240.9) * & 228.0 (228.6–227.4) * & 208.6 (209.2–208.4) * & 221.7 (222.5–220.8) * & 219.5 (220.3–219.0) * & 186.4 (187.5–185.6) * & 64.8 (65.3–64.3)* & 201.3(201.6–200.4) * & 161.4(162.5–160.3) * & 215.3 (215.6–213.6) * & 259.7 (260.9–259.1) * & 241.9 (242.2–240.1) * & 83.6 (84.2–83.1)* & 28.2 (28.5–27.9) 30.5 0.83 (0.87–0.82) 1.22 (1.57–0.81)
&
a
Results are expressed as mean (n = 6), CI = 95% confidence intervals. Calzada et al., 2010. # P < 0.05 compared to linearolactone. $ P < 0.05 compared to emetine and metronidazole. *P < 0.05 compared to linearolactone. & P < 0.05 compared to emetine and metronidazole. **Correlation coefficient >0.874. b
Copyright © 2015 John Wiley & Sons, Ltd.
Phytother. Res. 29: 1600–1604 (2015)
ANTIPROTOZOAL ACTIVITY OF CLERODANE DITERPENE FROM SALVIA SPECIES
Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 μM for E. histolytica and from 28.2 μg/ml in the case for G. lamblia. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. Compounds 7 (E. histolytica IC50 = 49.0 μM and G. lamblia IC50 = 64.8 μM) and 13(E. histolytica IC50 = 76.6 μM and G. lamblia IC50 = 83.6 μM) showed moderate activity against both protozoa (Table 1). Compound 7 possesses a trans-fused decalin ring system with a ketone carbonyl and an acetate group at C-1 and C-2, respectively. Only this compound possesses different oxygenated functionalities other than epoxy and hydroxyl groups, which could explain its antiprotozoal activity. Compound 13 is closely related to linearolactone (14), a neo-clerodane active against E. histolytica and G. lamblia (Calzada et al., 2010). Both compounds were isolated from S. polystachya as a mixture being 13 the minor component (Maldonado and Ortega, 2000). Comparison between the data of antiprotozoal activity and structural features of the neo-clerodanes tested suggested that the following functionalities in ring A of the framework are preferred: a 1,3-diene over a 2,10-diene, a C-3 conjugated double bond over a C-2 vinyl double bond, as well as an 1,2-epoxy group over an 1,10-epoxy group. The hydroxyl group at C-10 in compound 10 decreases the activity in comparison to 14 with hydrogen atom. The 5,10-seco-neo-clerodanes and 5,6-seco-salvigenanes tested showed weak antiprotozoal activity. In conclusion, all the diterpenes assayed were less active than linearolactone (14) and antiprotozoal drugs
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used as controls (emetine and metronidazole). Salvinorin A (7) showed moderate activity against both parasites and it could be the responsible to stop diarrhea caused by protozoa. So the above also could explain the use of S. divinorum in the Mexican traditional medicine to treat this kind of ailment. However, additional studies are necessary. Polystachyne E (13) a minor component from S. polystachya showed weak activity. This result suggested that the antiprotozoal effect of the acetone soluble extract is mainly because of 14 (IC50 values of 22.9 μM for E. histolytica and 28.2 μM for G. lamblia). Also, data obtained in this investigation, combined with our previous results (Calzada et al., 2010, Barbosa et al., 2007), confirm that linearolactone may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea.
Acknowledgements E. Bautista also thanks Dirección General del Personal Académico (DGAPA) for the postdoctoral scholarship and M. Sc. María del Rosario García Peña for identifying the plant material.
Conflict of Interest The authors declare that there are no conflicts of interest.
REFERENCES Aguilar A, Camacho JR, Chino S, Jacquez PE, López ME. 1996. Plantas Medicinales del Herbario IMSS. Instituto Mexicano del Seguro Social: Mexico D. F. 107–110. Aviello G, Borelli F, Guida F, et al. 2011. Ultrapotent effects of salvinorin A, a hallucinogenic compound from Salvia divinorum, on LPS-stimulated murine macrophages and its anti-inflammatory action in vivo, J Mol Med 89: 891–902. Baldursson S, Karanis P. 2011. Waterborne transmission of protozoan parasites: review of worldwide outbreaks—an update 2004–2010. Water Res 45: 6603–6614. Barbosa E, Calzada F, Campos R. 2007. In vivo antigiardial activity of three flavonoids isolated of some medicinal plants used in Mexican traditional medicine for the treatment of diarrhea. J Ethnopharmacol 109: 552–554. Bautista E, Maldonado E, Ortega A. 2012. Neo-clerodane diterpenes from Salvia herbacea. J Nat Prod 75: 951–958 Bautista E, Toscano RA, Ortega A. 2013. Microphyllandiolide, a new diterpene with an unprecedented skeleton from Salvia microphylla. Org Lett 15: 3210–3213 Bautista E, Toscano RA, Ortega A. 2014. 5,10-seco-neo-clerodanes and neo-clerodanes from Salvia microphylla. J Nat Prod 77: 1088–1092. Bentham G 1876. Labiatae. In Genera Plantarum. vol. 2, Bentham G and Hoocker JD (eds). Revve& Co: London pp. 1160–1222. Calzada F, Yépez-Mulia L,Tapia-Contreras A, Bautista E, Maldonado E, Ortega A. 2010. Evaluation of the antiprotozoal activity of neo-clerodane type diterpenes from Salvia polystachya against Entamoeba histolytica and Giardia lamblia. Phytother Res 24: 662–665. Capasso R, Borrelli F, Zjawiony J, et al. 2008a. The hallucinogenic herb Salvia divinorum and its active ingredient salvinorin A reduce inflammation-induced hypermotility in mice. Neurogastroenterol Motil 20(2):142–148. Capasso R, Borrelli F, Cascio MG, et al. 2008b. Inhibitory effect of salvinorin A, from Salvia divinorum, on ileitis-induced hypermotility: cross-talk between kappa-opioid and cannabinoid CB(1) receptors. Br J Pharmacol 155(5):681–689. Copyright © 2015 John Wiley & Sons, Ltd.
Dirección General de Epidemiología (DGE). 2013. Anuarios de morbilidad 2008–2013. www.epidemiologia.salud.gob.mx/ anuario/html/anuarios.html. Ebrahimi SN, Zimmermann S, Zaugg J, Smiesko M, Reto B, Hamburger M. 2013. Abietane diterpennoids from Salvia sahendica—antiprotozoal activity and determination of their absolute configurations. Planta Med 79:150–156. Esquivel B, Tello R, Sánchez AA. 2005. Unsaturated diterpenoids with a novel carbocyclic skeleton from Salvia xalapensis. J Nat Prod 68: 787–790. Fichna J, Schicho R, Andrews CN, et al. 2009. Salvinorin A inhibits colonic transit and neurogenic ion transport in mice by activating kappa-opioid and cannabinoid receptors. Neurogastroenterol Motil 21(12):1326-e128. doi: 10.1111/j.1365-2982.2009.01369.x. Finney DL. 1977. Probit Analysis. Cambridge University Press: New York. Hernandez EG, Granados J, Partida-Rodriguez O, et al. 2015. Prevalent HLA class II alleles in Mexico City appear to confer resistance to the development of amebic liver abscess. Plos One May 4;10(5): e0126195. doi: 10.1371/journal. pone.0126195.eCollection. Jenks AA, Kim SC. 2013. Medicinal plant complexes of Salvia subgenus Calosphace: an ethnobotanical study of new world sages. J Ethnopharmacol 146: 214–224. Keene AT, Harris A, Phillipson JD, Warhurst DC. 1986. In vitro amoebicidal testing of natural products; Part I. Methodology. Planta Med 4: 278–285. Maldonado E, Ortega A. 2000. Polystachynes A–E, five cis-neo-clerodane diterpenoids from Salvia polystachya. Phytochemistry 53: 103–109. Mineno T, Avery MA. 2003. Giardiasis: recent progress in chemotherapy and drug development. Curr Pharm Des 9: 841–855. Moridi FM, Bahadori MB, Taheri S, Ebrahimi SN, Zimmermann S, Brun R, Amin G, Hamburger M. 2011. Triterpenoids with rare carbon skeletons from Salvia hydrangea: antiprotozoal activity and absolute configurations. J Nat Prod 74: 2200–2205. Narukawa Y, Fukui M, Hatano K, Takeda T. 2006. Four new diterpenoids from Salvia fulgens Cav. J Nat Med 60: 58–63. Phytother. Res. 29: 1600–1604 (2015)
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CALZADA ET AL.
Ortega A, Blount JF, Manchand PS. 1982. Salvinorin, a new transneoclerodane diterpene from Salvia divinorum (Labiatae). J Chem Soc Perkin Trans 1: 2505–2508. Prisinzano TE. 2009. Natural products as tools for neuroscience: discovery and development of novel agents to treat drug abuse. J Nat Prod 72: 581–587. Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P, Rothman RB. 2002. Salvinorin A: a potent naturally occurring non-nitro genousκ opioid selective agonist. Proc Natl Acad Sci U S A 99: 11,934–11,939 Singh S, Bharti N, Mohapatra PP. 2009. Chemistry and biology of synthetic and naturally occurring antiamoebic agents. Chem Rev 109: 1900–1947.
Copyright © 2015 John Wiley & Sons, Ltd.
Guía práctica clínica (GPC). 2012. Prevención, diagnóstico y tratamiento farmacológico de la giardiasis en niños y adolescentes de 1 a 18 años en el primer y segundo nivel de atención. Evidencias y recomendaciones. Publicado por CENETEC. ISSSTE-252-12. www.cenetec.salud.gob.mx/interior/gpc.html. Valdes III JL, Díaz JL, Paul AG. 1983. Ethnopharmacology of ska María Pastora (Salvia divinorum, Epling and Jativa M.). J Ethnopharmacol 7: 287–312. Yi-Bing W, Zhi-Yu N, Qing-When S, et al. 2012. Constituents from Salvia species and their biological activities. Chem Rev 112: 5967–6026.
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