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Leishmanicidal activity of lipophilic extracts of some Hypericum species.

ABSTRACT

Leishmaniasis has emerged as the third most prevalent parasite-borne disease worldwide after malaria and filariasis, with about 350 million people at risk of infection. Antileishmanial drugs currently available have various limitations, mainly because of the parasite resistance and side effects. The search of new antileishmanial drugs is ventured throughout the world.

Purpose: The purpose of this study was to assess the leishmanicidal activity of lipophilic extracts of eight Hypericum species against promastigote forms of Leishmania (Leishmania) amazonensis.

Material and methods: The dried and powered materials of aerial parts of H. andinum Gleason, H. brevistylum Choisy, H. caprifoliatum Cham. & Schltdl., H. carinatum Griseb., H. linoides A. St.-Hil., H. myrianthum Cham. & Schltdl., H. polyanthemum Klotzsch ex Reichardt and H. silenoides Juss. were extracted by static maceration with n-hexane. Extracts were evaporated to dryness under reduced pressure and stored at -20[degrees]C until biological evaluation and HPLC analysis. The metabolites investigated were dimeric phloroglucinol derivatives, benzophenones and benzopyrans. The yields were expressed as mean of three injections in mg of compound per g of extract (mg/g extract). The effect of Hypericum species on the viability of infective forms of L (L) amazonensis was determined using a hemocytometer. Amphotericin B was used as a standard drug. The 50% inhibitory concentration ([IC.sub.50]) values for each extract were determined by linear regression analysis. The cytotoxic effects of extracts were assessed on peritoneal macrophages of BALB/c mice by MTT assay. The concentration that causes 50% of macrophage cytotoxicity ([CC.sub.50]) was determined by linear regression analysis. The selectivity index (SI) of the extracts was determined considering the following equation: [CC.sub.50] against mammalian cells/[IC.sub.50] against L. amazonensis.

Results: We demonstrated that H. carinatum, H. linoides and H. polyanthemum were able to kill the parasites in a dose dependent manner. These extracts presented low cytotoxicity against murine macrophages. At 48 h of incubation H. polyanthemum presented significant leishmanicidal activity with a 50% inhibitory concentration ([IC.sub.50]) of 36.1 [micro]g/ml. The leishmanicidal activity of H. myrianthum was significantly lower than that presented by H. polyanthemum, H. carinatum and H. linoides extracts. H. brevistylum and H. caprifoliatum showed significant leishmanicidal activity only at high concentrations (500 and 1000 [micro]g/ml), while H. andinum and H. silenoides were ineffective.

Conclusion: The promising results demonstrate the importance of the species of the genus Hypericum as source of compounds potentially useful for the treatment of leishmaniasis.

Keywords:

Hypericum species

Leishmaniasis

Leishmania amazonensis

Leishmanicidal activity

Introduction

Parasites of the genus Leishmania cause a spectrum of diseases ranging from self-healing ulcers to disseminate and often fatal visceral diseases, depending on the parasite species transmitted, intrinsic resistance to drugs and mechanisms immune mediated, such as the production of oxygen and nitrogen reactive species (Convit et al., 1993; Garcia-Hernandez et al., 2012; McMahon-Pratt and Alexander, 2004). Leishmaniasis is endemic in 98 countries on five continents. At the moment, approximately 350 million people are considered at risk of contracting leishmaniasis, and about 0.2-0.4 million cases of visceral leishmaniasis (VL) and 0.7-1.2 million cases of cutaneous leishmaniasis (CL) occur each year (Alvar et al., 2012). Leishmania (Leishmania) amazonensis is able to produce a wide spectrum of diseases in humans, including localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), mucocutaneous leishmaniasis (MCL) and VL (Barral et al., 1991).

Despite of the vast amount of research conducted on Leishmania biology, chemotherapeutic interventions are still far from ideal and adequate vaccines have yet to be developed (Garcia-Hernandez et al., 2012; Mutiso et al., 2013). The pentavalent antimonials remain the first-line drugs for the treatment of leishmaniasis regardless their high toxicity, resistance emergency and treatment failure (Sundar, 2001; WHO, 2010). Other drugs such as pentamidine, amphotericin B and miltefosine have limited use due to their high cost, severe side effects or resistance (Sanchez-Canete et al., 2009; WHO, 2010). Plants are a rich source of new molecules for pharmaceutical purposes, either as a source of compounds or as a model for the designing of new ones (Cragg and Newman, 2013). In the last decades, there has been a renewed interest in natural products as source of new drugs, including anti-infective substances (Butler, 2004; Cragg and Newman, 2013).

Species of the genus Hypericum (Hypericaceae) have been traditionally used in different parts of the world as antiseptic, diuretic, stomachic, wound healing and antimicrobial agents (von Poser et al., 2006), and several reports have confirmed their therapeutical potential. Recently, our group demonstrated that the lipophilic extract and isolated compounds (benzopyrans HP1, HP2, HP3 and dimeric phloroglucinol uliginosin B) from H. polyanthemum present antiprotozoal activity against Trichomonas vaginalis (Cargnin et al., 2013). Moreover, extracts of H. lanceolatum and phloroglucinol derivatives of H. erectum presented pronounced antiplasmodial activity (Moon, 2010; Zofou et al., 2011), while samples of H. perforatum macerated in olive oil exerted mild inhibitory activity against Trypanosoma rhodesiense (Orhan et al., 2013).

Considering the current problems related to antileishmanial drugs and the increasing need for alternatives that may lead to development of new medicines, this study investigated the leishmanicidal activity of lipophilic extract of eight Hypericum species against L. amazonensis and their cytotoxicity on macrophages. To determine the major compounds present in the extracts (Fig. 1), analyses by high performance liquid chromatography (HPLC) were performed.

Materials and methods

Plant material

Aerial parts of eight Hypericum species in flowering stage were collected (Table 1). Plants were identified by Dr. Sergio Bordignon (UNILASALLE, RS, Brazil) and voucher specimens were deposited in the herbarium of Universidade Federal do Rio Grande do Sul (ICN). Plant collection was authorized by Conselho de Gestao do Patrimonio Genetico (CGEN), Instituto Brasileiro do Meio Ambiente (IBAMA-003/2008 P 02000.001717/2008-60) and Direccion General Forestal y de Fauna Silvestre of the Republic of Peru (0147-2010-AG-DGFFS-DGEFFS).

Extraction procedures

Dried and powered plant materials (50 g) were thoroughly extracted (5 x 24 h at 25[degrees]C) by static maceration with n-hexane (5 x 500 ml) (F. Maia, Cotia, Sao Paulo, Brazil). Extracts were evaporated to dryness under reduced pressure (Rotavapor 8020 Fisatom[R]) yielding 1.9% (H. andinum), 3.0% (H. brevistylum), 2.0% (H. caprifoliatum), 4.0% (H. carinatum), 3.8% (H. linoides), 3.6% (H. myrianthum), 2.5% (H. polyanthemum) and 2.8% (H. silenoides). The extracts were stored at -20[degrees]C until biological evaluation and analysis (Barros et al., 2013a).

HPLC analysis

A Shimadzu liquid chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with a DGU-20A5 degasser, LC-6AD pumps, SIL-10AD auto sampler, CTO-20AC column oven, SPD-20AV UV/VIS detector and CBM-20A communications module was employed. LC Solution 1.24 SP2 software was used to record and process the data.

Acetonitrile (C[H.sub.3]CN) and methanol (MeOH) were HPLC grade (Merck, Darmstadt, Germany); trifluroacetic acid (TFA) was reagent grade (Vetec, Duque de Caxias, RJ, Brazil) and distilled water ([H.sub.2]O) was purified by a Milli-Q system (Millipore, Bedford, MA, USA). The separations were performed with Waters Nova Pack C18 column (4 [micro]m, 3.9 mm x 150 mm) and Waters Nova-Pack C18 60 [Angstrom] guard column (3.9 mm x 20 mm). Benzophenones (1 = cariphenone A; 2 = cariphenone B) and benzopyrans (3 = 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1); 4 = 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2); 5 = 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3)) were eluted with an isocratic mobile phase system consisting of C[H.sub.3]CN:[H.sub.2]O (60:40, v/v) and detection at 270 nm. Dimeric phloroglucinols (6 = hyperbrasilol B; 7 = japonicin A; 8 = uliginosin B) were also eluted with an isocratic mobile phase system but consisting of C[H.sub.3]CN:[H.sub.2]O (95:5, v/v) containing 0.01% of TFA and detection at 220 nm.

The extracts, completely dissolved in MeOH, were filtered (0.22 [micro]m pore size, Merck) and analyzed at room temperature (25[degrees]C), at a flow rate of 1 ml/min. The injection volume was of 20 [micro]l. Peaks were identified by comparison of their retention times (Rt) (1 = 8.0 min; 2 = 6.2 min; 3 = 8.2 min; 4 = 11.5 min; 5 = 19.4 min; 6 = 8.8 min; 7 = 5.7 min; 8 = 7.3 min) and co-injection with authentic standards isolated from Hypericum species (benzophenones 1 and 2 from H. carinatum; benzopyrans 3-5 from H. polyanthemum: dimeric phloroglucinol 6 from H. caprifoliatum, 7 and 8 from H. myrianthum) (Bernardi et al., 2005; Dall'agnol et al., 2005; Ferraz et al., 2001; Nor et al., 2004). The identity and purity of compounds were confirmed by [sup.1]H-NMR spectroscopy (Eft-60[R], Anasazi Instruments). Calibration curves described by Barros et al. (2013a,b) were used to quantify the metabolites. The benzophenone 2 and dimeric phloroglucinols 6 and 7 were determined with 1 and 8 calibration curves, respectively. The yields were expressed as mean of three injections in mg of compound per g of extract (mg/g extract).

L. (L). amazonensis culture

The MHOM/BR/73/M2269 strain of L. (L). amazonensis were routinely isolated from draining popliteal lymph node of footpad lesions of infected BALB/c mice and maintained as promastigotes in M199 medium containing 40 mM of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES), 0.1 mM adenine, 7.7 mM hemin, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 50 U/ml of penicillin and 50 [micro]g/ml of streptomycin. Cultures were incubated at 26[degrees]C, and cells were kept at densities ranging between 5 x [10.sup.5] and 3 x [10.sup.7] parasites/ml (Romao et al., 2006). All experimental procedures were performed in accordance with the guidelines of the National Institute of Health and Brazilian Society for Science on Animals of Laboratory with the approval of local Ethics Committee from Federal University of Health Sciences of Porto Alegre (process number 11-061).

Determination of leishmanicidal activity in vitro

Promastigote forms of L amazonensis (3 x [10.sup.6] on stationary phase) were plated in 96 well microtiter plates in four replicates and incubated with M199 medium supplemented with 10% FBS in the presence or absence Hypericum extracts (0-1000 [micro]g/ml) for 48 h. Amphotericin B (Sigma, USA) at 4 [micro]g/ml was used as standard antileishmanial drug (100% of mortality). Control cells were incubated with M199 medium containing less than 0.05% of polysorbate 80 (Tween 80). Parasites viability was evaluated from motility after 48 h and cell density was determined using a hemocytometer. The survival rate was calculated according to the formula: percentage survival = (average number of viable parasites in treated group/average number of viable untreated parasites) x 100. The 50% inhibitory concentration ([IC.sub.50]) values for each extract were determined by linear regression analysis using GraphPad Software, version 5.0.

[FIGURE 1 OMITTED]

Macrophages viability

The macrophages viability was measured by a colorimetric assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Peritoneal macrophages were harvested from BALB/c mice which 3 days previously had been injected i.p. with 2 ml of sterile thioglycollate solution (3% w/v in PBS). The cells were plated at 2 x [10.sup.5] cells/0.2 ml in RPM1 1640 plus 10% FBS, 100 U/ml penicillin and streptomycin (100 [micro]g/ml) in 96 well microtiter plates and were incubated overnight at 37[degrees]C in an atmosphere of 5% C[O.sub.2]. Then, plates were washed prior to being cultured for 44 h at 37[degrees]C in RPMI or medium plus different concentrations of Hypericum extracts (0-1000 [micro]g/ml). Subsequently, MTT (5 mg/ml in PBS) was added (20 [micro]l/well) and incubations were continued 4 h afterwards. The purple formazan product which is formed by the action of mitochondrial enzymes in living cells was solubilized by addition of acidic isopropanol, and the absorbance at 570 nm measured using a Spectramax M2 (Pal et al., 2011; Romao et al., 1999). The concentration of extract that causes 50% of macrophage cytotoxicity ([CC.sub.50]) was determined by linear regression analysis using GraphPad Software, version 5.0. The selectivity index (SI) of the extracts was determined considering the following equation: [CC.sub.50] against mammalian cells/[IC.sub.50] against L. amazonensis.

Statistical analysis

The statistical analysis was determined by one-way ANOVA followed by Bonferroni's test. The analyses were performed with GraphPad Software and values of p < 0.05 were considered significant. All experiments were performed at least three times and in quadruplicate.

Results

The addition of H. linoides, H. carinatum and H. polyanthemum extracts directly to promastigotes of L. amazonensis resulted in dose-dependent parasite killing (Fig. 2A). After 48 h incubation, H. polyanthemum at concentrations 31.2 and 62.5 [micro]g/ml showed moderate to high leishmanicidal activity reducing the parasite viability in an order of 27.5% and 98.8%, respectively (Fig. 2A). The [IC.sub.50] value of H. polyanthemum was calculated to be 36.1 [micro]g/ml (Table 2). As illustrated in Fig. 2C and D, H. polyanthemum at 125 [micro]g/ml led to total lysis of the parasites. In addition, it was observed that H. carinatum and H. linoides at 62.5 [micro]g/ml killed the parasite in 42.2% and 31.6%, respectively when compared to the control group (Fig. 2A). As expected, amphotericin B, the reference antileishmanial drug, was the most active against L amazonensis ([IC.sub.50] 48 h = 0.12 [micro]g/ml) (data not shown).

The effect on the viability of macrophages of the most active extracts (H. carinatum, H. linoides and H. polyanthemum) against infective forms of L. amazonensis was also investigated. In general, these extracts showed low cytotoxicity (Fig. 2B). It was verified that the 50% cytotoxic concentration ([CC.sub.50]), and selectivity index values ([CC.sub.50]/[IC.sub.50]) ranged from 118.6 to 144.7 [micro]g/ml and 1.2 to 4, respectively (Table 2).

[FIGURE 2 OMITTED]

The leishmanicidai activity of H. myrianthum was significantly lower than those found for H. polyanthemum, H. carinatum and H. linoides independently of the tested concentration (data not shown). Hypericum brevistylum and H. caprifoliatum showed significant leishmanicidal activity only at high concentrations. These extracts at 500 [micro]g/ml were able to reduce the viability in an order of 90% and 55%, respectively. In addition, the concentration of 1000 [micro]g/ml induced 100% and 82.5% of mortality, respectively. In contrast, H. andinum and H. silenoides did not show significant leishmanicidai effect even at the highest concentration (data not shown).

The results of HPLC analysis are shown in Table 3. HPLC profile of the most active extract (H. polyanthemum) can be visualized in Fig. 3. All the plants presented the dimeric phloroglucinol 8. The extracts of H. linoides and H. myrianthum presented the highest concentration of 6 and 7, respectively. Confirming previous reports (Barros et al., 2013b; Bernardi et al., 2005; Cargnin et al., 2013; Ferraz et al., 2001), benzophenones 1 and 2 were found exclusively in H. carinatum, while benzopyrans 3, 4 and 5 were detected only in H. polyanthemum.

Discussion

This is the first study reporting the leishmanicidai activity for Hypericum species on L amazonensis. Here, it was demonstrated that depending on the dose, the lipophilic extracts of H. polyanthemum, H. carinatum, H. linoides, H. myrianthum, H. caprifoliatum and H. brevistylum were able to kill infective forms of L. amazonensis, one of the main agent of cutaneous leishmaniasis in the world (Carvalho et al., 1994; Franca-Costa et al., 2012). The current treatment of leishmaniasis presents problems such as drug resistance, variable or low efficacy, high cost, side effects, besides others (Sanchez-Canete et al., 2009; Sundar, 2001; WHO, 2010). Considering this, the search for new active compounds may lead to the discovery of more efficient medicines for the treatment of leishmaniasis. It is interesting to note that different extracts of Hypericum spp. present activity against protozoa as Plasmodium sp., Trypanosoma sp. and Trichomonas sp. (Cargnin et al., 2013; Moon, 2010; Orhan et al., 2013; Verotta et al., 2007; Zofou et al., 2011), confirming this potential.

Significant differences in the leishmanicidai activity of extracts obtained from different Hypericum species were observed. Hypericum polyanthemum presented the highest activity (H. polyanthemum > H. carinatum > H. linoides > H. myrianthum > H. brevistylum > H. caprifoliatum). It is noteworthy to notice that this is the only species that accumulate benzopyrans 3-5 (Table 3, Fig. 3) (Barros et al., 2013a,b; Cargnin et al., 2013; Ferraz et al., 2001).

In the present study, the [IC.sub.50] values of H. polyanthemum, H. carinatum and H. linoides n-hexane extracts against promastigotes of L. amazonensis ranged from 36 to 112 [micro]g/ml. Recently, Cargnin et al. (2013) demonstrated that the supercritical fluid extract of H. polyanthemum at 206.98 [micro]g/ml reduced the viability of Trichomonas vaginalis in 50%, while its main isolated compounds (3, 4, 5 and 8) at 62.5 [micro]g/ml decreased the parasite viability in 50%, 20%, 10% and 50%, respectively. In the present study, at 62.5 [micro]g/ml the n-hexane extract of H. polyanthemum caused an inhibition of 90% in the Leishmania viability, showing an [IC.sub.50] value of 5.7 times lower. It is important to consider that the difference in the susceptibility of these pathogens might be due to a higher selectivity of H. polyanthemum extract against L. amazonensis and/or a synergistic effect of its compounds. In this context, Grivicich et al. (2008) demonstrated that in association, 3, 4 and 5 were more potent in inhibiting the growth of U-373 MG cells, a glioblastoma cell line, when compared to the independent effect of each isolated one. Thus, such compounds seem to be very interesting candidates to future studies against protozoa pathogens.

[FIGURE 3 OMITTED]

According to the literature, extracts and more lipophilic compounds tend to favor the anti-protozoa activity (Cargnin et al., 2013; Maciel-Rezende et al., 2013). Alkyl-substituted benzophenones, for example, demonstrated to be more active than their hydroxylated precursors suggesting that an increase in Iipophilicity of these compounds could facilitate the protozoa membrane permeation (Maciel-Rezende et al., 2013). Similar hypothesis was confirmed by Cargnin et al. (2013) that demonstrated the action of 3, 4, 5 and 8 on membranes of T. vaginalis. Alterations in respiratory chain of L. donovani were also observed to benzophenone-derived bisphosphonium salts with intermediate hydrophobicity (Luque-Ortega et al., 2010).

Considering the wide occurrence of phloroglucinol derivatives in Hypericum, other species of the genus should be investigated. The well-studied species H. perforatum, for example, contains very lipophilic phloroglucinol derivatives such as hyperforin and adhyperforin that were not still investigated for leishmanicidal activity.

Conclusion

The results of the present research exhibit for the first time that H. polyanthemum, H. carinatum and H. linoides present significant activity against L. amazonensis. Under this point of view H. polyanthemum extract might be investigated as a promising candidate for antileishmanial treatment. Further studies are being conducted to elucidate the mechanism of action.

Conflicts of interest

The authors declare that they have no conflict of interest.

ARTICLE INFO

Article history:

Received 2 March 2014

Revised 29 August 2014

Accepted 17 October 2014

Acknowledgments

The authors are grateful to the Brazilian agencies Coordenacao de Aperfeiqoamento de Pessoal de Nivel Superior (CAPES, CAPES-PNPD-23038007200/11-08), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-307277/2013-5) and Fundacao de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS-1017479) for financial support and by fellowships from CAPES (APD, FMCB, JSP) and CNPq (PRTR, GLvP) and the PEC-PG program of CAPES/CNPq (GVCC).

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Ana Paula Dagnino (a), Francisco Maikon Correa de Barros (b), Gari Vidal Ccana-Ccapatinta (b), Josiane Somariva Prophiro (a), Gilsane Lino von Poser (b), Pedro R.T. Romao (a), *

(a) Laboratdrio de Imunologia, Programa de Pos-Graduafao em Ciencias da Saude, Departamento de Ciencias Basicas da Saude, Universidade Federal de Ciencias da Saude de Porto Alegre (UFCSPA), Rua Sarmento Leite, 245, sala 206, CEP 90050-170, Porto Alegre, RS, Brazil

(b) Programa de Pos-Graduacao em Ciencias Farmaceuticas, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil

* Corresponding author. Tel.: +55 51 3303 8746; fax: +55 51 3303 8810.

E-mail address: pedror@ufcspa.edu.br, prtorres@usp.br (P.R.T. Romao).

http://dx.doi.org/10.1016/phymed.2014.10.004
Table 1 Collection data of the Hypericum species.

Species (voucher number)             Collection locality (harvest)

H. andinum Gleason                   Amparaes, Cuzco, Peru (May, 2008)
   (Ccana-Ccapatinta et al. 05)
H. brevistylum Choisy                Pumahuanca, Cuzco, Peru (May,
   (Ccana-Ccapatinta et al. 03)        2008)
H. caprifoliatum Cham. & Schltdl.    Porto Alegre, RS, Brazil
   (Bordignon et al. 2287)             (October-December, 2008)
H. carinatum Griseb. (Bordignon      Glorinha, RS, Brazil
   and Ferraz 2309)                    (October-December, 2008)
H. linoides A. St.-Hil.              SaoJose dos Ausentes, RS, Brazil
   (Bordignon et al. 3317)             (October-December, 2008)
H. myrianthum Cham. & Schltdl.       Paralso do Sul, RS, Brazil
   (Bordignon et al. 3059)             (October-December, 2008)
H. polyanthemum Klotzschex           Cappava do Sul, RS, Brazil
   Reichardt (Bordignon et al.,        (October-December, 2008)
   3118)

Table 2
Leishmanicidai activity and macrophages cytotoxicity of extracts of
Hypericum species at 48 h.

Hypericum         [IC.sub.50] (a),       [CC.sub.50] (b),      SI (d)
species           [micro]g/ml (95% CI)   [micro]g/ml
                                         (95% CI (c))

H. polyanthemum   36.12 (30.25-43.13)    144.7 (91.68-228.3)   4
H. carinatum      64.99 (51.90-81.38)    118.6 (87.34-161)     1.8
H. linoides       111.9 (65.04-192.70)   137.1 (108.4-173.4)   1.2

(a) [IC.sub.50]: concentration of extract that causes 50% of
mortality of L. amazonensis.

(b) [CC.sub.50]: concentration of extract that causes 50% of
macrophage cytotoxicity.

(c) 95 CI: 95% confidence interval.

(d) SI: selectivity index, calculated as ratio of [CC.sub.50] for
macrophage/[IC.sub.50] for L. amazonensis.

Table 3
Content of the major compounds present in the lipophilic extract of
the investigated Hypericum species.

Hypericum species    Compounds (mg/g extract)

                     1        2       3        4

H. andinum           --       --      --       --
H. brevistylum       --       --      --       --
H. caprifoliatum     --       --      --       --
H. carinatum         0.83     5.76    --       --
H. linoides          --       --      --       --
H. myrianthum        --       --      --       --
H. polyanthemum      --       --      356.99   426.84
H. silenoides        --       --      --       --

Hypericum species    Compounds (mg/g extract)

                     5        6       7       8

H. andinum           --       --      --      16.60
H. brevistylum       --       1.08    --      68.98
H. caprifoliatum     --       --      2.79     0.60
H. carinatum         --       --      --      16.48
H. linoides          --       13.98   14.41    1.92
H. myrianthum        --       --      70.51   35.97
H. polyanthemum      109.77   --      --       0.54
H. silenoides        --       --      --      42.27

1 = cariphenone A; 2 = cariphenone B; 3 = 6-isobutyryl-5,7-
dimethoxy-2,2-dimethyl-benzopyran (HP1); 4 = 7-hydroxy-6-isobutyryl-
5-methoxy-2,2-dimethyl-benzopyran (HP2); 5 = 5-hydroxy-6-isobutyryl-
7-methoxy-2,2-dimethyl-benzopyran (HP3); 6 = hyperbrasilol B; 7 =
japonicin A; 8 = uliginosin B; not detected.
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Author:Dagnino, Ana Paula; de Barros, Francisco Maikon Correa; Ccana-Ccapatinta, Gari Vidal; Prophiro, Josi
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jan 15, 2015
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