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Chemical composition of essential oils of Piper jacquemontianum and Piper variabile from Guatemala and bioactivity of the dichloromethane and methanol extracts

Abstract

The essential oils from two native species from Guatemala were studied for their chemical composition and the dichloromethane and methanol extracts for their biological activity. A GC-MS analysis of the essential oil from Piper jacquemontianum Kunth, Piperaceae, showed 34 constituents, consisting mainly of linalool (69.4%), while Piper variabile C. DC. essential oil had 36 constituents, camphor (28.4%), camphene (16.6%) and limonene (13.9%) being the major components. Dichloromethane extracts of both species were cytotoxic against MCF-7, H-460 and SF-268 cell lines (<7 µg/mL). Dichloromethane extract of P. jacquemontianum was slightly active against bacteria (0.5 mg/mL), was active against promastigotes of Leishmania (20.4-61.0 µg/mL), and epimastigotes of Trypanosoma cruzi (51.9 µg/mL). The methanol extract of P. variabile showed antimalarial activity against Plasmodium falciparum F32 (4.5 µg/mL), and the dichloromethane extract against Leishmania (55.8-76.3 µg/mL) and T. cruzi (45.8 µg/mL). None of the extracts from the two species was active against Aedes aegypti larvae and Artemia salina nauplii.

antimicrobial activity; cytotoxic activity; essential oil; extracts; Guatemala; Piper


Chemical composition of essential oils of Piper jacquemontianum and Piper variabile from Guatemala and bioactivity of the dichloromethane and methanol extracts

Sully M. CruzI; Armando CáceresII; Luis ÁlvarezIII; Julio MoralesIII; Miriam A. ApelIV; Amelia T. HenriquesIV; Efraín SalamancaV Alberto GiménezV; Yelkaira VásquezVI; Mahabir P. GuptaVI

IEscuela de Química Farmacéutica, Facultad de CCQQ y Farmacia, Universidad de San Carlos, Guatemala

IIEscuela de Química Biológica, Facultad de CCQQ y Farmacia, Guatemala

IIIHerbario USCG, Centro de Estudios Conservacionistas, Universidad de San Carlos, Guatemala

IVFaculdade de Farmácia, Universidad Federal Rio Grande do Sul, Brasil

VInstituto de Investigaciones Fármaco Bioquímicas, Universidad Mayor de San Andrés, Bolivia

VICentro de Estudios Farmacognósticos de la Flora Panameña, Universidad de Panamá, Panamá

Correspondence Correspondence: Armando Cáceres Escuela de Química Biológica, Facultad de CCQQ y Farmacia, Universidad de San Carlos, Edificio T-11 Ciudad Universitaria zona 12, Guatemala caceres-armando@usac.edu.gt Tel.: 502 2418 9410 Fax: 502 2418 9414

ABSTRACT

The essential oils from two native species from Guatemala were studied for their chemical composition and the dichloromethane and methanol extracts for their biological activity. A GC-MS analysis of the essential oil from Piper jacquemontianum Kunth, Piperaceae, showed 34 constituents, consisting mainly of linalool (69.4%), while Piper variabile C. DC. essential oil had 36 constituents, camphor (28.4%), camphene (16.6%) and limonene (13.9%) being the major components. Dichloromethane extracts of both species were cytotoxic against MCF-7, H-460 and SF-268 cell lines (<7 µg/mL). Dichloromethane extract of P. jacquemontianum was slightly active against bacteria (0.5 mg/mL), was active against promastigotes of Leishmania (20.4-61.0 µg/mL), and epimastigotes of Trypanosoma cruzi (51.9 µg/mL). The methanol extract of P. variabile showed antimalarial activity against Plasmodium falciparum F32 (4.5 µg/mL), and the dichloromethane extract against Leishmania (55.8-76.3 µg/mL) and T. cruzi (45.8 µg/mL). None of the extracts from the two species was active against Aedes aegypti larvae and Artemia salina nauplii.

Keywords: antimicrobial activity, cytotoxic activity, essential oil, extracts, Guatemala, Piper

Introduction

Within the context of a multinational Organization of American States sponsored project aimed at studying the potential of Central American biodiversity, species of Piper native to Guatemala were studied for the chemical composition of the essential oil, and the biological activity of dichloromethane and methanol extracts.

The genus Piper has a large number of species, and has been of worldwide interest due to their wide utilization as aromatic species and their use in the traditional medicine. This genus is of high botanical, chemical and pharmacological complexity (Dyer & Palmer, 2004). The analysis of volatile chemical components of species of Piper has shown the presence of monoterpenes, sesquiterpenes and arylpropanoids with interesting insecticidal, antimicrobial, and antioxidant activities (Parmar et al., 1997; Martins et al., 1998; Moreira et al., 1998). The two species collected from Guatemala, locally known as "cordoncillo" (Standley & Steyermark, 1952), were chosen for their ethnomedical use for treatment of infection, anaemia and body aches (Cleaves, 2001; Michel et al., 2007). The study of these species is relevant, as they have not been studied before, have important medicinal properties, and are a potential source of aroma.

Material and Methods

Plant material

Leaves of Piper jacquemontianum Kunth and P. variabile C. DC., Piperaceae, were collected in Lachuá, Alta Verapaz, botanically identified by one of the authors (JM), and voucher specimens (21304 and 21310, respectively) deposited at the USCG Herbarium at CECON. Plant material (500-900 g) was shade-dried at 35 °C for 5-7 days and ground.

Extraction and analysis of essential oils

Essential oils were obtained by hydrodistillation for 3 h in a modified Clevenger-type apparatus. The yield of the oil was calculated on the basis of dry weight of the plant material. Oil samples were analysed on a Shimadzu Gas Chromatographer (GC-17A) with injector, quantification was achieved by electronic integration by normalization technique. For separation of constituents an apolar Durabond-DB5 column (30 m long and 0.25 mm of I.D.) with polydimethylphenyl siloxane containing 5% of phenyl groups in a 0.25 µm thick film (John Wiley & Sons Scientific, USA); a polar column (LM-120; by L&M, San Carlos, SP, Brazil), filled with propylene glycol; and a flame ionization detector (FID) were used. Conditions of the equipment included: temperature program 60-300 °C, 3 °C/min; analysis time 60 min; injector temperature 220 °C; FID/EM interface: 250 °C; amount of sample injected: 1 µL diluted in diethyl ether. Qualitative analysis was done with the same equipment fitted to a Shimadzu mass spectrophotometer (GC/MS-QP5000), connected with cylindric quadruple and operated by ionization energy of 70 eV, obtained by electronic impact technique. Retention indices were calculated with an aliphatic hydrocarbon series (C8-C22). The chiral isomeric ratios of the main monoterpene components of the essential oil were studied using a Cyclosil B column; temperatures program 60-300 °C, 3 °C/min.

Sequential extraction

Two extracts were obtained by sequential percolation in a stainless steel percolator, first with dichloromethane twice, and then with methanol for three days. The extracts were concentrated under reduced pressure at a temperature <40 °C in a rotary evaporator, and kept in a vacuum dryer.

Antimicrobial screening

Activity against bacteria and yeast was determined by an agar plate dilution method according to Mitscher et al. (1972), microbial strains used were Bacillus subtilis ATCC 6051, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonella typhi ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Mycobacterium smegmatis ATCC 607, Candida albicans ATCC 10231 and Cryptococcus neoformans C13. Minimum inhibitory concentration (MIC) was determined by the same procedure in quadruplicate containing agar-extract dilutions.

Artemia salina cytotoxicity and larvicidal activity

Artemia assay was done according to Solis et al. (1993) using extract dilutions of 1,000, 500 and 250 µg/mL; LC50 was calculated by non parametric regression analysis using a Finney program for Basic. One, two and three instars larvae (Aedes aegypti and Anopheles albimanus) were used against the same dilutions, after 24 h, death larvae were evaluated visually (Araujo et al., 2003).

Antiprotozoal activity

Activity was measured on in vitro cultures of Leishmania parasite in promastigote forms of complex L. amazonensis (strand PH8), complex L. braziliensis (strand M2903) and complex L. donovani (strand PP75), cultivated at 26 °C in Schneider medium supplemented with 5% of inactivated (56 °C x 30 min) calf bovine serum. Parasites in logarithmic phase of growth, at a concentration of 1x106 parasites/mL, were distributed on 96 micro well plates with different concentration of the substances (100, 50 and 25 µg/mL), and incubated for 72 h. Activity was determined by optical counting in an inverted microscope. Pentamidine (100 µg/mL) was used as reference drug during the evaluations that were done by triplicate. Under the same conditions, epimastigote forms (1x104 parasites/mL) of Trypanosoma cruzi (strand Tulahuen), were cultivated in LIT medium supplemented with 5% calf bovine serum. Amphotericin B (10 µg/mL) was used as reference drug (Fornet et al., 1994). The IC50 of the active extract was determined by lineal interpolation of a typical curve obtained by plotting the logarithm of the concentration vs. the % of inhibition, using programs for logit, probit or polynomial regression analysis. Plasmodium falciparum F32 (Indochine, clone W2, chloroquine sensitive strain) was cultivated in glucose-enriched RPMI 1640 medium, supplemented with 10% human serum. Viability was measured by fluorochrome (PicoGreen®) intercalation and read in a micrometric fluorometer according to Corbett et al. (2004).

Neoplastic cell lines cytotoxicity

Human cancer cell lines from breast (MCF-7), lung (H-460) and central nervous system (SF-268) were kindly donated by National Cancer Institute, USA, cultivated in vitro and the bioassay performed according to the standard procedure described by Monks et al. (1991; 1997), using adriamycin as reference drug.

Results

The yields of the essential oils obtained from leaves of P. jacquemontianum and P. variabile by hydrodistillation were 0.8 and 0.7% respectively. The GC/MS analysis of P. jacquemontianum showed at least 34 constituents, linalool (69.4%), (E)-nerolidol (8.0%), and α-pinene (3.2%) being the major constituents while P. variabile showed at least 36 constituents, mainly camphor (28.4%), camphene (16.6%), limonene (13.9%), and o-cymene (6.3%) (Table 1). In the chiral characterization, linalool, camphene and limonene had strong chiral isomeric preference, with more than 84% for (+)-linalool and 94% of (+)-limonene form, while camphene showed the preference for the (-)-camphene form. For camphor, only a racemic mixture could be observed.

Cytotoxic activity against MCF-7, H-460 and SF-268 cell lines was very good in both of the dichloromethane extracts (<7 µg/mL). The methanol extract of P. jacquemontianum showed activity against promastigotes of L. amazonensis (61 µg/mL), L. braziliensis (20.4 µg/mL) and L. donovani (20.8 µg/mL), and epimastigotes of T. cruzi (51.9 µg/mL), while the dichloromethane extract showed activity against L. amazonensis and L. braziliensis at a higher concentration (102.8 µg/mL). The methanol extract of P. variabile showed good activity against P. falciparum F32 (4.5 µg/mL), and the dichloromethane extract was active against L. donovani (55.8 µg/mL), L. braziliensis (66.3 µg/mL), L. amazonensis (76.3 µg/mL) and T. cruzi (45.8 µg/mL).

The dichloromethane extract showed little activity against B. subtilis and M. smegmatis; no activity was shown against A. aegypti, A. albimanus larvae and against A. salina nauplii (Table 2).

Discussion

The literature on the genus Piper shows a great variability in chemical composition among different species, for Latin-American species the main constituents reported are monoterpenes and sesquiterpenes, while African and Asian species are rich in phenylpropanoids (Dyer & Parmar, 2004). The main constituents described in several organs from Piper species are: camphor, apiole, myristicin, safrole, sarisan, dilapiole, linalool, α-pinene, α-humulene, β-caryophyllene, as well as alkaloids, lignans, terpenes, steroids, propenyphenols, kavapyones, chalcones, flavonoids and piperolides (Von Poser et al., 1994; Martins et al., 1998; Parmar et al., 1997; Moreira et al., 1998).

The linalool content in P. jacquemontianum (69.4%) is the highest reported for any Piper species and could be a valuable resource for perfumery industry. Mundina et al. (1997) showed the presence of linalool in three species from Panama (Piper fimbriulatum C. DC., Piper arboreum Aublet and Piper obliquum Ruiz & Pavón), and Guerrini et al. (2009) from Piper aduncum L. from Ecuador, although their contents were low. All four species from Saint Tomé and Príncipe had low amount, particularly Piper capense L. and Piper guineense Schumach. & Thonn. (Martins et al., 1998). From three species from Cameroon, Piper umbellatum L. contained linalool (14.4%) as major constituent (Françoise et al. 2009). In Brazil, from seven species, only Piper goesii Yunck showed traces of linalool (Dias dos Santos et al., 2001); Constantin et al. (2001) showed that myrcene (52.6%) and linalool (15.8%) were the major constituents of the essential oil of leaves of Piper regnelli (Miq.) C. DC.; de Almeida et al. (2009) showed that linalool was the major constituent of the essential oil of leaves from Piper divaricatum G. Mey. (23.4-29.7%); and, Autran et al. (2009) showed low content in the leaf of Piper marginatum Jacq.

The presence of camphor as a major constituent in P. variabile (28.4%) is noteworthy, since previous studies showed from four species from Saint Tomé and Príncipe, and Ecuador, the essential oils from P. guineense (0.8%) (Martins et al., 1998), P. obliquum (0.25%) and P. aduncum (0.05%) had very little amount (Guerrini et al., 2009).

The main bioactivity demonstrated was the cytotoxicity against cancer cell lines. In a previous study, Calderon et al. (2006) reported cytotoxicity in the whole ethanol extract of P. jacquemontianum, however, the cytotoxicity in dichloromethane fraction of P. variabile is shown for the first time.

Cytotoxic activity has been demonstrated in other Piper species. Fractions and compounds from Piper hostmannianum var. berbicense (Miq.) C. DC. showed no cytotoxic activity against MCF-7 cell lines (Portet et al., 2007); ethanol extract of Piper sarmentosum Roxb. inhibited HepG2 cells and non-malignant Chang's liver cell lines (Ariffin et al., 2009); dichloromethane and methanol extracts from fruits of Piper nigrum L. and Piper chaba Hunter were inactive against C32 and HeLa cell lines (Atjanasuppat et al., 2009); although, E-piplartine isolated from roots of P. chaba showed cytotoxicity against several cell lines (BC-8, PCC4, P388S1, IMR32), significantly increased by curcumin (Jyothi et al., 2009). Essential oil from leaves of Piper gaudichaudianum Kunth was cytotoxic to Chinese hamster lung fibroblast (V79) cells (Péres et al., 2009).

With respect to antiprotozoal activity, in a previous study we demonstrated that the whole ethanol extract of P. jacquemontianum was active against P. falciparum (IC50 12 µg/mL), T. cruzi (IC50 12 µg/mL), and L. mexicana (IC50 22 µg/mL) (Calderon et al., 2010), in this study we were not able to confirm the activity against P. falciparum, but the other activities were confirmed in dichloromethane and methanol extracts. In the case of P. variabile it is the first time that the activity against Leishmania and T. cruzi is reported.

The antiprotozoal activity of both species is interesting, although other species have shown similar activity. From twelve extracts from three species of Piper from Comores, only the dichloromethane extract of P. capense showed activity against P. falciparum W2 (7 µg/mL) (Kaou et al., 2008). By bioassay-guided purification from hexane extract of P. hostmannianum var. berbicense in vitro activity to P. falciparum (IC50 5.64 µM) and in vivo activity against Plasmodium vinckei petteri was demonstrated by (-)-methyllinderatin (Portet et al., 2007). From 94 species detected by ethnobotany in Peru, eight species of Piper were detected, P. aduncum showed activity against P. falciparum (9.6±1.7 µg/mL), and Piper dennisii Trel. against L. amazonensis (IC50 10±0.15 µg/mL) (Valadeau et al., 2009).

Activity against bacteria and yeast is considered small, since only activity against B. subtilis and M. smegmatis were demonstrated at a high MIC. No activity was demonstrated against insect larvae as well as against A. salina nauplii. Other species have shown larvicidal activity against A. aegypti, such as P. fimbriulatum (Solis et al., 2005) and P. marginatum (Autran et al., 2009).

It is evident that the two species or Piper native to sub-tropical Guatemala are potential resources for development of new crops that might help in the search for alternative products. Further studies are being conducted for bioguided evaluation of these extracts in order to determine the compounds responsible for such activities, as well as agrotechnological development for sustainable utilization of this native resource.

Acknowledgements

The authors thank the financial support of General Directorate of Research (DIGI) at USAC, Guatemala, the Organization of American States (OAS, grant SEDI/AICD/106/01), and the cooperation within RIBIOFAR (Red 0284) Network of the Ibero-American Program of Science and Technology for Development (CYTED). The support of Ricardo Véliz MSc, School of Chemistry, USAC, and the technical assistance of Ana Gómez and Vinicio García are kindly appreciated.

Received 5 Nov 2010

Accepted 21 Jan 2011

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  • Correspondence:
    Armando Cáceres
    Escuela de Química Biológica, Facultad de CCQQ y Farmacia, Universidad de San Carlos, Edificio T-11
    Ciudad Universitaria zona 12, Guatemala
    caceres-armando@usac.edu.gt Tel.: 502 2418 9410
    Fax: 502 2418 9414
  • Publication Dates

    • Publication in this collection
      17 June 2011
    • Date of issue
      Aug 2011

    History

    • Accepted
      21 Jan 2011
    • Received
      05 Nov 2010
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