Antiplasmodial evaluation of Anacardium occidentale and alkyl-phenols

Valéria M.M. Gimenez Tavane A. Alvarenga Milton Groppo Márcio L.A. e Silva Wilson R. Cunha Ana H. Januário Martin J. Smilkstein Michael K. Riscoe Patricia M. Pauletti About the authors

Abstract

The ethanol crude extract from cashew (Anacardium occidentale L. Anacardiaceae) displayed significant antiplasmodial activity (IC50 0.577 µg/ml). Liquid chromatography-high resolution Mass spectrometry analysis was performed to identify the main compounds existing in the ethanol extract. The occurrence of anacardic acids, cardols, and 2-methylcardols derivatives was confirmed in the extract. The IC50 obtained, when the main isolated compounds were evaluated in Plasmodium falciparum D6 strain, ranged from 5.39 µM to >100 µM. Tested here for the first time, the data showed that cardol triene 1 (IC50 = 5.69 µM) and 2-methylcardol triene 4 (IC50 = 5.39 µM) demonstrated good antimalarial activity. In conclusion, Anacardium occidentale nuts presented relevant biological potential, and further studies should be considered.

Keywords
Anacardic acid; Antiplasmodial; Cardol; Cashew; Methylcardol

Introduction

Anacardiaceae is a plant family of mostly tropical trees, comprising around seventy genera and 600 species found predominantly in tropical areas of Africa, Asia, and America (Mitchell and Mori, 1987Mitchell, J.D., Mori, S.A., 1987. The Anacardium occidentale and its relatives (Anacardium: Anacardiaceae). Mem. N. Y. Bot. Gard. 42, 1-76.; Wannan, 2006Wannan, B.S., 2006. In Anacardiaceae. Blumea 51, 165-195.). Phytochemically, the family Anacardiaceae is known by biosynthesis of flavonoids, triterpenes, steroids, xanthones, and alkyl-phenols (Correia et al., 2006Correia, S.J., David, J.P., David, J.M., 2006. Secundary metabolites from species of Anacardiaceae. Quim. Nova. 29, 1287-1300.).

Anacardium occidentale L. (the "cashew") is recognized as an indispensable source of alkyl-phenols, which were isolated from its fruit (the "cashew nuts"). These compounds have several biological activities described among them: antioxidant, larvicidal, cytotoxicity to cancer cell lines, antibacterial, molluscicidal, and schistosomicidal (Kubo et al., 1986Kubo, I., Komatsu, S., Ochi, M., 1986. Molluscicides from the cashew Anacardium occidentale and their large-scale isolation. J. Agric. Food Chem. 34, 970-973.; Himejima and Kubo, 1991Himejima, M., Kubo, I., 1991. Antibacterial agents from the cashew Anacardium occidentale (Anacardiaceae) nut shell oil. J. Agric. Food Chem. 39, 418-421.; Kubo et al., 2011Kubo, I., Nitoda, T., Tocoli, F.E., Green, I.R., 2011. Multifunctional cytotoxic agents from Anacardium occidentale. Phytother. Res. 25, 38-45.; Oliveira et al., 2011Oliveira, M.S., Morais, S.M., Magalhães, D.V., Batista, W.P., Vieira, I.G., Craveiro, A.A., de Manezes, J.E., Carvalho, A.F., de Lima, G.P., 2011. Antioxidant, larvicidal and antiacetylcholinesterase activities of cashew nut shell liquid constituents. Acta Trop. 117, 165-170.; Alvarenga et al., 2016Alvarenga, T.A., de Oliveira, P.F., de Souza, J.M., Tavares, D.C., Andrade, E., Silva, M.L., Cunha, W.R., Groppo, M., Januário, A.H., Magalhães, L.G., Pauletti, P.M., 2016. Schistosomicidal activity of alkyl-phenols from the Cashew Anacardium occidentale against Schistosoma mansoni adult worms. J. Agric. Food Chem. 64, 8821-8827.). They also showed inhibitory activities in enzymatic assays, when evaluated against tyrosinase, acetylcholinesterase, α-glucosidase, aldose reductase, invertase, 15-lipoxygenase, and xanthine oxidase (Toyomizu et al., 1993Toyomizu, M., Sugiyama, S., Jin, R.L., Nakatsu, T., 1993. α-Glucosidase and aldose reductase inhibitors: constituents of cashew, Anacardium occidentale, nut shell liquids. Phytother. Res. 7, 252-254.; Kubo et al., 1994Kubo, I., Kinst-Hori, I., Yokokawa, Y., 1994. Tyrosinase inhibitors from Anacardium occidentale fruits. J. Nat. Prod. 57, 545-551.; Shobha et al., 1994Shobha, S.V., Ramadoss, C.S., Ravindranath, B., 1994. Inhibition of soybean lipoxygenase-1 by anacardic acids, cardols, and cardanols. J. Nat. Prod. 57, 1755-1757.; Oliveira et al., 2011Oliveira, M.S., Morais, S.M., Magalhães, D.V., Batista, W.P., Vieira, I.G., Craveiro, A.A., de Manezes, J.E., Carvalho, A.F., de Lima, G.P., 2011. Antioxidant, larvicidal and antiacetylcholinesterase activities of cashew nut shell liquid constituents. Acta Trop. 117, 165-170.; Masuoka et al., 2015Masuoka, N., Nihei, K., Maeta, A., Yamagiwa, Y., Kubo, I., 2015. Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase. Food Chem. 166, 270-274.).

As reported by the World Health Organization, malaria affected 216 million people in 2016. The number of estimated deaths, in 2016, was 445 thousand, and the records point to 407 thousand deaths in Africa alone (WHO, 2017WHO, 2017. World Malaria Report 2017. World Health Organization, Geneva.). Additionally, drug resistance, particularly to artemisinin-based combination therapies is also an increasing concern (Haldar et al., 2018Haldar, K., Bhattacharjee, S., Safeukui, I., 2018. Drug resistance in Plasmodium. Nat. Rev. Microbiol. 16, 156-170.). These facts make the continued search for new antimalarials important.

Because of the promising activity presented by the alkyl-phenols when evaluated against the adult worms of Schistosoma mansoni, but also because cashew seed oil is used in folk medicine to treat malaria (Duke, 2000Duke, J.A., 2000. Handbook of Nuts: Herbal Reference Library. CRC Press, New York.), we sought to assess the extract and alkyl-phenols from A. occidentale against Plasmodium falciparum, the causative agent of malaria.

Materials and methods

We purchased cashew nut and apple (Anacardium occidentale L., Anacardiaceae) from "Varejão Irmãos Patrocínio" market, Franca, São Paulo State, in October 2013. Prof. Dr. Milton Groppo authenticated the plant sample, and a voucher specimen (SPFR 16040) has been stored in the Herbarium of the Department of Biology, Faculdade de Ciências e Letras de Ribeirão Preto, University of São Paulo, Brazil (Herbarium SPFR).

The air-dried and powdered cashews (34.9 g) were extracted with ethanol using ultrasound, yielding 12 g of crude extract. Previously, the ethanol extract (5 g) was purified, as described by Alvarenga et al. (2016)Alvarenga, T.A., de Oliveira, P.F., de Souza, J.M., Tavares, D.C., Andrade, E., Silva, M.L., Cunha, W.R., Groppo, M., Januário, A.H., Magalhães, L.G., Pauletti, P.M., 2016. Schistosomicidal activity of alkyl-phenols from the Cashew Anacardium occidentale against Schistosoma mansoni adult worms. J. Agric. Food Chem. 64, 8821-8827., resulting in seven compounds: 1 (63.9 mg, cardol triene), 2 (45.8 mg, cardol diene), 3 (8.5 mg, cardol monoene), 4 (29.9 mg, 2-methylcardol triene), 5 (11.0 mg, 2-methylcardol diene), 6 (8.9 mg, anacardic acid triene), and 7 (4.1 mg, anacardic acid diene).

The crude extract was analyzed by high-performance liquid chromatography-high resolution Mass spectrometry (HPLC-HRMS) using a micrOTOF-Q II-ESI-TOF Mass Spectrometer (Bruker Daltonics), Shimadzu HPLC system with an LC 20 AD pump, automatic injection SIL20AHT, DAD (200-600 nm) detector, and using a C18 column (Phenomenex® Luna, 5 µm 250 × 4.6 mm). The following conditions were applied: capillary voltage 3.5 kV, dry temperature 220 ºC, nebulizer gas 60 psi, dry gas 10 l/min, mass range 50-1300 Da Nitrogen was used as drying, nebulizing and collision gas. A linear solvent gradient from 30% H2O/0.1% acetic acid (solvent A) and 70% methanol (solvent B) to 100% solvent B over 10 min followed by 5 min 100% solvent B, with 1.0 ml/min flow rate. All analyses were processed using Bruker Daltonics Data Analysis 4.3 software.

The Plasmodium falciparum D6 strains (chloroquine-sensitive), were maintained, with slight adaptations, according to Trager and Jensen (1976)Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673-675.. They were cultivated in culture flasks at 37 ºC in an atmosphere of 5% O2, 5% CO2, and 90% N2, with group A-positive human erythrocytes at 2% hematocrit in RPMI 1640 medium, supplemented with Albumax II (0.5%), glucose (3 g/l), hypoxanthine (45 µg/l), and gentamicin (50 µg/l). Every 3-4 days, the percent of parasitemia was calculated by light microscopy counting of 500 erythrocytes on a Giemsa-stained blood smear. Then, infected erythrocytes were transferred to fresh medium containing erythrocytes.

Stock solutions of the compounds (1-7), and crude extract were prepared at a concentration of 10 mM and 180 µg/ml, respective, and were dissolved in 1 ml DMSO. The drugs atovaquone (ATV), ELQ-300, and chloroquine (CQ) were used as positive controls and were prepared in a 10 mM stock solution. They were dissolved in 1 ml DMSO, exception chloroquine, which was prepared in 1 ml water. Serial dilutions of test compounds, extract, and positive controls were made with RPMI 1640 medium in 96 well plates and combined with stock parasite culture and erythrocytes to yield a final 2% hematocrit and 0.2% parasitemia. The maximum percentage of DMSO, after the serial dilution, was 0.1%. Negative controls with DMSO-only were included on each assay plate. After a 72 h growth period, the assay was performed as previously described (Smilkstein et al., 2004Smilkstein, M., Sriwilaijaroen, N., Kelly, J.X., Wilairat, P., Riscoe, M., 2004. Simple and inexpensive fluorescence-based technique for highthroughput antimalarial drug screening. Antimicrob. Agents Chemother. 48, 1803-1806.).

Antiplasmodial activity was obtained using IC50 value (50% P. falciparum growth inhibition), which was calculated with GraphPad Prism software by plotting the % of observed growing against the logarithm of the sample concentrations and curve fitting by nonlinear regression.

Results and discussion

The obtained data (Table 1) indicated that the cashew crude ethanol extract showed good antiplasmodial activity, when compared to Lemma et al. (2017) ranked data, which established that good activity is observed when the IC50 falls in the range of 0.1-1 µg/ml.

Table 1
Antiplasmodial activity of crude ethanol extract of cashew and alkyl-phenols (1-7).

The bioactive extract was analyzed by LC-HRMS, aiming better characterization of the crude extract components, since, in most cases, the traditional phytochemical study results only in the isolation of the abundant compounds, thus providing additional information about the metabolite profile. The LC-HRMS data of crude extract were analyzed, and the peaks with high intensity in the HRMS chromatogram were selected for detailed examination. The mass spectra were investigated to identify the ions corresponding to each peak. In all cases, one or two protonated and/or sodiated ions ([M+H]+ and/or [M+Na]+) were observed. The exact mass obtained was used to determine the possible molecular formulae, and these formulae were searched in Natural Products Dictionary online database together with Anacardium and confirmed the presence of the seven alkyl-phenols, which were usually found in cashew nuts (Table 2).

Table 2
LC-HRMS analysis of cashew ethanol crude extract.

The results of the in vitro tests with isolated compounds against P. falciparum D6 strain were also presented in Table 1. The IC50 of compounds ranged from 5.39 µM to >100 µM. According to Batista et al. (2009)Batista, R., Silva, A., de, J., de Oliveira, A.B., 2009. Plant-derived antimalarial agents: new leads and efficient phytomedicines. Part II. Non-alkaloidal natural products. Molecules 14, 3037-3072., the obtained alkyl-phenols showed good activity as for 1 (IC50 = 5.69 µM) and 4 (IC50 = 5.39 µM), and moderate activity as for compounds 7 (IC50 = 21.28 µM), 2 (IC50 = 27.83 µM), 5 (IC50 = 41.82 µM), and 6 (IC50 = 64.89 µM).


The presence of a carboxyl group in phenol ring was associated with a decrease in the antiplasmodial activity in the case of anacardic acid triene (6), when compared to cardol triene (1) and 2-methylcardol triene (4). Additionally, the existence of a double bond between C-14 and C-15, and C-11 and C-12 are essential for the in vitro activity of compounds 1 and 4. In general, a decrease in the number of double bonds in the side chain decreased the activity as in compounds 1, 2 and 3, besides 4 and 5, although this was not observed for compounds 6 and 7, which showed the opposite pattern. Compounds 6 and 7 have a carboxyl in the aromatic ring, which may explain in part these finds.

The antimalarial activity of the cardol and 2-methylcardol derivatives has not previously been reported, and the inhibitory potencies of compounds 1 and 4 are notable.

Anacardic acid derivatives isolated from Viola websteri, 6-(8′Z-pentadecenyl)-salicylic acid and 6-(8′Z, 11′Z, 14′Z-heptadecatrienyl)-salicylic acid, for which antimalarial activity has been previously described with IC50 values of 10.1 and 13.3 µM (Lee et al., 2009Lee, S.J., Park, W.H., Moon, H.I., 2009. Bioassay-guided isolation of antiplasmodial anacardic acids derivatives from the whole plants of Viola websteri Hemsl. Parasitol. Res. 104, 463-466.). Compound 6-(8′Z-pentadecenyl)-salicylic acid was also evaluated in vivo on Plasmodium berghei infections, and the study indicated moderate antimalarial activity (Chung et al., 2009Chung, I.M., Seo, S.H., Kang, E.Y., Park, W.H., Moon, H.I., 2009. Anti-malarial activity of 6-(8'Z-pentadecenyl)-salicylic acid from Viola websteri in mice. Malaria J. 8, 151-154.).

Cui and coworkers (2008)Cui, L., Miao, J., Furuya, T., Fan, Q., Li, X., Rathod, P.K., Su, X.Z., Cui, L., 2008. Histone acetyltransferase inhibitor anacardic acid causes changes in global gene expression during in vitro Plasmodium falciparum development. Eukaryot. Cell 7, 1200-1210. proposed that anacardic acid affect P. falciparum due to partial inhibition of recombinant PfGCN5 histone acetyltransferase (HAT), causing problems to the parasite transcription. Also, the anacardic acid exhibited an IC50 of around 30 µM in parasite strains.

In conclusion, our data add further evidence that cashew is a rich source of bioactive compounds. The crude ethanol extract showed good antiplasmodial activity, thus presenting relevant potential. The activity profile of the cardol and 2-methylcardol derivatives tested here against P. falciparum for the first time, suggested potencial and provides early structure activity relationship information. Further studies based on chemical modifications in the alkyl-phenols could be examined to further improve antiplasmodial activity.

  • Ethical disclosures
    Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.
    Confidentiality of data. The authors declare that no patient data appear in this article.
    Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Acknowledgments

The authors thank FAPESP (São Paulo Research Foundation) for financial support (Grant 2013/09280-6, 2016/10313-4, and 2016/06260-2). CNPq (The National Council for Scientific and Technological Development) is acknowledged for Research Productivity Fellowships granted to M.L.A.S., W.R.C., A.H.J., M.G. and P.M.P. T.A.A. and V.M.M.G. were supported by FAPESP (2013/20280-8) and CAPES scholarships.

References

  • Alvarenga, T.A., de Oliveira, P.F., de Souza, J.M., Tavares, D.C., Andrade, E., Silva, M.L., Cunha, W.R., Groppo, M., Januário, A.H., Magalhães, L.G., Pauletti, P.M., 2016. Schistosomicidal activity of alkyl-phenols from the Cashew Anacardium occidentale against Schistosoma mansoni adult worms. J. Agric. Food Chem. 64, 8821-8827.
  • Batista, R., Silva, A., de, J., de Oliveira, A.B., 2009. Plant-derived antimalarial agents: new leads and efficient phytomedicines. Part II. Non-alkaloidal natural products. Molecules 14, 3037-3072.
  • Chung, I.M., Seo, S.H., Kang, E.Y., Park, W.H., Moon, H.I., 2009. Anti-malarial activity of 6-(8'Z-pentadecenyl)-salicylic acid from Viola websteri in mice. Malaria J. 8, 151-154.
  • Correia, S.J., David, J.P., David, J.M., 2006. Secundary metabolites from species of Anacardiaceae. Quim. Nova. 29, 1287-1300.
  • Cui, L., Miao, J., Furuya, T., Fan, Q., Li, X., Rathod, P.K., Su, X.Z., Cui, L., 2008. Histone acetyltransferase inhibitor anacardic acid causes changes in global gene expression during in vitro Plasmodium falciparum development. Eukaryot. Cell 7, 1200-1210.
  • Duke, J.A., 2000. Handbook of Nuts: Herbal Reference Library. CRC Press, New York.
  • Haldar, K., Bhattacharjee, S., Safeukui, I., 2018. Drug resistance in Plasmodium Nat. Rev. Microbiol. 16, 156-170.
  • Himejima, M., Kubo, I., 1991. Antibacterial agents from the cashew Anacardium occidentale (Anacardiaceae) nut shell oil. J. Agric. Food Chem. 39, 418-421.
  • Kubo, I., Kinst-Hori, I., Yokokawa, Y., 1994. Tyrosinase inhibitors from Anacardium occidentale fruits. J. Nat. Prod. 57, 545-551.
  • Kubo, I., Nitoda, T., Tocoli, F.E., Green, I.R., 2011. Multifunctional cytotoxic agents from Anacardium occidentale Phytother. Res. 25, 38-45.
  • Kubo, I., Komatsu, S., Ochi, M., 1986. Molluscicides from the cashew Anacardium occidentale and their large-scale isolation. J. Agric. Food Chem. 34, 970-973.
  • Lee, S.J., Park, W.H., Moon, H.I., 2009. Bioassay-guided isolation of antiplasmodial anacardic acids derivatives from the whole plants of Viola websteri Hemsl. Parasitol. Res. 104, 463-466.
  • Masuoka, N., Nihei, K., Maeta, A., Yamagiwa, Y., Kubo, I., 2015. Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase. Food Chem. 166, 270-274.
  • Mitchell, J.D., Mori, S.A., 1987. The Anacardium occidentale and its relatives (Anacardium: Anacardiaceae). Mem. N. Y. Bot. Gard. 42, 1-76.
  • Oliveira, M.S., Morais, S.M., Magalhães, D.V., Batista, W.P., Vieira, I.G., Craveiro, A.A., de Manezes, J.E., Carvalho, A.F., de Lima, G.P., 2011. Antioxidant, larvicidal and antiacetylcholinesterase activities of cashew nut shell liquid constituents. Acta Trop. 117, 165-170.
  • Shobha, S.V., Ramadoss, C.S., Ravindranath, B., 1994. Inhibition of soybean lipoxygenase-1 by anacardic acids, cardols, and cardanols. J. Nat. Prod. 57, 1755-1757.
  • Smilkstein, M., Sriwilaijaroen, N., Kelly, J.X., Wilairat, P., Riscoe, M., 2004. Simple and inexpensive fluorescence-based technique for highthroughput antimalarial drug screening. Antimicrob. Agents Chemother. 48, 1803-1806.
  • Toyomizu, M., Sugiyama, S., Jin, R.L., Nakatsu, T., 1993. α-Glucosidase and aldose reductase inhibitors: constituents of cashew, Anacardium occidentale, nut shell liquids. Phytother. Res. 7, 252-254.
  • Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673-675.
  • Wannan, B.S., 2006. In Anacardiaceae. Blumea 51, 165-195.
  • WHO, 2017. World Malaria Report 2017. World Health Organization, Geneva.

Publication Dates

  • Publication in this collection
    Jan-Feb 2019

History

  • Received
    3 Sept 2018
  • Accepted
    13 Nov 2018
  • Published
    7 Dec 2018
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