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Prof. Akah Peter Achunike Prof. Parveen Bansal Editor-in-chief Department of Biochemistry Department of Pharmacology & Toxicology Postgraduate Institute of Medical Education and University of Nigeria, Nsukka Research Nigeria Chandigarh India.

Associate Editors Dr. Ravichandran Veerasamy AIMST University Dr. Ugur Cakilcioglu Faculty of Pharmacy, AIMST University, Semeling - Elazıg Directorate of National Education 08100, Turkey. Kedah, Malaysia.

Dr. Jianxin Chen Dr. Sayeed Ahmad Information Center, Herbal Medicine Laboratory, Department of Beijing University of Chinese Medicine, Pharmacognosy and Phytochemistry, Beijing, China Faculty of Pharmacy, Jamia Hamdard (Hamdard 100029, University), Hamdard Nagar, New Delhi, 110062, China. India.

Dr. Hassan Sher Dr. Cheng Tan Department of Botany and Microbiology, Department of Dermatology, first Affiliated Hospital College of Science, of Nanjing Univeristy of King Saud University, Riyadh Traditional Chinese Medicine. Kingdom of Saudi Arabia. 155 Hanzhong Road, Nanjing, Jiangsu Province, China. 210029 Dr. Jin Tao Professor and Dong-Wu Scholar, Dr. Naseem Ahmad Department of Neurobiology, Young Scientist (DST, FAST TRACK Scheme) Medical College of Soochow University, Plant Biotechnology Laboratory 199 Ren-Ai Road, Dushu Lake Campus, Department of Botany Suzhou Industrial Park, Aligarh Muslim University Suzhou 215123, Aligarh- 202 002,(UP) P.R.China. India.

Dr. Pongsak Rattanachaikunsopon Dr. Isiaka A. Ogunwande Department of Biological Science, Dept. Of Chemistry, Faculty of Science, Lagos State University, Ojo, Lagos, Ubon Ratchathani University, Nigeria. Ubon Ratchathani 34190, Thailand.

Editorial Board

Prof Hatil Hashim EL-Kamali Dr. Arash Kheradmand Omdurman Islamic University, Botany Department, Lorestan University, Sudan. Iran.

Prof. Dr. Muradiye Nacak Prof Dr Cemşit Karakurt Department of Pharmacology, Faculty of Medicine, Pediatrics and Pediatric Cardiology Gaziantep University, Inonu University Faculty of Medicine, Turkey. Turkey.

Dr. Sadiq Azam Samuel Adelani Babarinde Department of Biotechnology, Department of Crop and Environmental Protection, Abdul Wali Khan University Mardan, Ladoke Akintola University of Technology, Pakistan. Ogbomoso Nigeria. Kongyun Wu Department of Biology and Environment Engineering, Dr.Wafaa Ibrahim Rasheed Guiyang College, Professor of Medical Biochemistry National Research Center China. Cairo Egypt. Prof Swati Sen Mandi Division of plant Biology, Bose Institute India.

Dr. Ujjwal Kumar De Indian Vetreinary Research Institute, Izatnagar, Bareilly, UP-243122 Veterinary Medicine, India.

Journal of Medicinal Plants Research

Table of Contents: Volume 9 Number 31, 17 August, 2015

ARTICLES

Research Articles

An aqueous Citrillus colocynthis peel extract inhibits neutrophil reactive oxygen species production and attenuates lung inflammation in mice 829 Fatma Zioud, Amira Ben Mahmoud, Tarak Boussetta, Jamel El-Benna and Rafik Bachoual

A review on antimicrobial potential of species of the genus Vernonia (Asteraceae) 838 Antonio Carlos Nogueira Sobrinho, Elnatan Bezerra de Souza and Raquel Oliveira dos Santos Fontenelle

Vol. 9(31), pp. 829-837, 17 August, 2015 DOI: 10.5897/JMPR2015.5733 Article Number: 55D6D4554892 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR

Full Length Research Paper

An aqueous Citrillus colocynthis peel extract inhibits neutrophil reactive oxygen species production and attenuates lung inflammation in mice

Fatma Zioud 1, Amira Ben Mahmoud 1, Tarak Boussetta 2,3, Jamel El-Benna 2,3 and Rafik Bachoual 1*

1Faculté des Sciences de Gabès, Université de Gabès, Tunisia. 2Inserm, U1149, CNRS, ERL8252, Centre de Recherche sur l’Inflammation (CRI), Paris, France. 3Université Paris Diderot, Sorbonne Paris Cité, Laboratoire d’Excellence Inflamex Faculté de Médecine, Site Xavier Bichat, Paris, France.

Received 12 January, 2015; Accepted 12 August, 2015

Citrillus colocynthis peel aqueous extract (CCPAE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. The objectives of this work were to study the effect of C. colocynthis peel aqueous extract, on human neutrophil reactive oxygen species (ROS) production in vitro, and to evaluate its protective effect on lipopolysaccharide (LPS)-induced lung inflammation in vivo in mice. Neutrophils were isolated from blood of healthy volunteers. ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H 2O2 was detected by horseradish peroxidase (HRP)-amplified chemiluminescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethylbenzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. CCPAE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl- methionyl-leucyl-phenylalanine (fMLF)- or phorbolmyristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. CCPAE also inhibited superoxide anion generation; and did not scavenge H 2O2 and superoxide anions nor inhibited MPO activity in vitro suggesting that it inhibits nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. In vivo studies showed that CCPAE attenuated LPS-induced lung inflammation in mice. This study shows that CCPAE inhibits neutrophil ROS production and attenuates LPS-induced lung inflammation in mice. Inhibition of NADPH oxidase activation by CCPAE could explain its anti-inflammatory action.

Key words: Citrullus colocynthis , colocynth, inflammation, neutrophils, reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

INTRODUCTION

Citrullus colocynthis (L.) Schrad. (Cucurbitaceae), the Tunisian traditional medicine, this plant has been commonly known as “bitter apple” is a plant that grows used to treat various diseases including, hypertension abundantly in Tunisia (Pottier-Alapetite, 1981), and and rheumatism (Le Flock, 1983; Boukef et al., 1982), widely in other parts of the world (Abbes el al., 2006). In while in other countries it is used to treat constipation,

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oedema bacterial infections, cancer and diabetes, and as Healthcare. phosphate buffered saline (PBS), Hank's balanced salt an abortifacient (Abbes el al., 2006). The ethnobotanical solution (HBSS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic uses of this plant include its use as cathartic, purgative acid (HEPES) and glucose were from Gibco. 2’,7’- dichlorofluoroscein-diacetate (DCFH-DA) was from Acros Fine and vermifuge, and for the treatment of fever, cancer, Chemicals. Stock solutions of fMLF (10 -2 mol/L) and PMA (1 mg/ml) amenorrhea, jaundice, leukemia, rheumatism and tumour were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C. (Abbes el al., 2006). The ethnobotanical use efficiency of The different solutions were diluted in PBS immediately before use. this plant was consolidated by a number of studies which demonstrated that C. colocynthis Schrad has a potent Preparation of CCPAE anti-tumour (Tannin-Spitz et al., 2007), anti-microbial (Marzouk et al., 2009, 2010a) and antioxidant activity In this study, three batch of Tunisian C. colocynthis , collected from (Marzouk et al., 2010b). Many secondary metabolites the island of Jerba in South of Tunisia were used. The colocynth peels were, dried at 37 °C, blended and suspended in sterile 0.9% from C. colocynthis , including cucurbitacins, flavonoids, NaCl, then centrifuged at 2000 rpm for 3 min. From each batch, the caffeic acid derivatives and terpenoids, have been supernatants of different preparations of CCPAE were used for the previously reported (Yankov and Hussein, 1975; Hatam experiments. The results obtained with different preparations from et al., 1989; Maatooq et al., 1997) and could explain the different batches are reproducible and the same dose effect biological activity of this plant. responses were found.

Inflammatory disorders are due to excessive production of pro-inflammatory mediators, such as tumor necrosis Isolation of human neutrophils factor (TNF)α, granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin (IL)-1, IL-6, IL-8, Venous blood was collected from healthy adult volunteers and neutrophils were isolated by Dextran sedimentation and density leukotriene B4 and platelet-activating factor (PAF), to the gradient centrifugation as previously described (El Benna and activity of inflammatory cells such as neutrophils, Dang, 2007). Erythrocytes were removed by hypotonic lysis. monocytes and macrophages, and to the excessive Following isolation, the cells were resuspended in appropriate production of reactive oxygen species (ROS) (Ley, 2002; medium, such as HBSS. The cells were counted and their viability Nathan, 2006). Polymorphonuclear neutrophils play a key was determined with the Trypan Blue exclusion method. role in host defenses against invading microorganisms (Hampton et al., 1998), but excessive neutrophil Measurement of ROS production by chemiluminescence activation participates in tissue damage associated with inflammatory disorders (Babior, 2000). In response to a Isolated neutrophils were resuspended in HBSS at a concentration of 1 million per ml. Cell suspensions (5 × 10 5) in 0.5 ml of HBSS variety of agents neutrophils migrate to inflammatory containing 10 µM luminal in the presence or absence of CCPAE sites, where they release proteases, bactericidal were preheated to 37 °C in the thermostatted chamber of a peptides, and large quantities of ROS, in a process luminometer (Berthold-Biolumat LB937) and allowed to stabilize. known as the respiratory burst (Babior, 1984). Oxygen After a baseline reading, cells were stimulated with 10 -6 M fMLF or reduction by neutrophil NADPH oxidase, a 100 ng/ml PMA. Changes in chemiluminescence were measured multicomponent enzyme system, yields superoxide anion over a 30-min period. –. (O 2 ) (El-Benna et al., 2005), while myeloperoxidase (MPO) produces hypochloric acid from hydrogen Measurement of superoxide production peroxide (Klebanoff, 2005). This study was undertaken to analyze the effect of C. Isolated cells were resuspended in HBSS at a concentration of 1 colocynthis million/ml. Cell suspensions in 1 ml of HBSS containing 1 mg/ml peel aqueous extract (CCPAE) on ROS cytochrome c in the presence or absence of CCPAE were production by human neutrophils; and to evaluate the preheated to 37°C in the thermostated chamber of a effect of this product on intratracheal lipopolysaccharide spectrophotometer (Uvikon) and allowed to stabilize. After a (LPS)-induced lung inflammation in mice. baseline reading, cells were stimulated with 10 -6 M fMLF or 100 ng/ml PMA. Changes in absorbance were measured at 550 nm over a 15-min period. MATERIALS AND METHODS

Chemicals and reagents Detection of H 2O2

Luminol, cytochrome c, fMLF, PMA, zymosan, superoxide In order to investigate whether CCPAE reacts directly with H2O2 dismutase (SOD), catalase and HRPO Escherichia coli (O55:B5) CCPAE was incubated in PBS with H 2O2 (80 µM) for 15 min in the lipopolysaccharide (LPS) were from Sigma-Aldrich (Saint-Quentin presence of luminol (10 µM) and the reaction was initiated by Fallavier, France). Ficoll and Dextran T500 were from GE adding HRPO (5U). Changes in chemiluminescence were

*Corresponding author. E-mail: [email protected]. Fax: (00) (216) 75 392 421. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

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measured over a 15 min period. production, human neutrophils were incubated with different CCPAE concentrations and ROS were detected

Preparation of azurophilic granules and measurement of MPO by luminol-amplified chemiluminescence. Results show activity that CCPAE inhibited luminol-amplified chemiluminescence in resting neutrophils (Figure 1A) and Neutrophils were lysed by nitrogen cavitation and the granule in neutrophils stimulated with fMLF (Figure 1B) or PMA fraction was purified by Percol gradient centrifugation (Udby and (Figure 1C). The effect of CCPAE shows an inhibitory Borregaard, 1989). The granules were sonicated in 0.2 cetyltrimethylammonium bromide (CTAB) and MPO activity was effect starting at a concentration between 20 and 40 assessed using the H 2O2-dependent tetramethylbenzedine (TMB) µg/ml. As fMLF and PMA activate neutrophils through oxidation assay at 650 nm. different transduction pathways, these results suggested that CCPAE decreases neutrophil ROS production by

LPS-induced lung inflammation in mice either inhibiting a final common target, such as the NADPH oxidase or MPO, or by scavenging ROS. These experiments were approved by our Institutional Committee on Animal Care and Use and the experimental protocol complied with Tunisian legal requirements for animal studies. Male Balb C CCPAE inhibits fMLF- and PMA-stimulated mice aged 7 weeks and weighing 22 to 26 g were purchased from superoxide anions production by human neutrophils SIFAT (Société des industries pharmaceutique de Tunisie) and housed in standard wire-topped cages and the temperature- controlled units. Food and water were supplied ad libitum . The mice Luminol-amplified chemiluminescence technique used received an intraperitoneal injection of 200 mg/kg CCPAE on day 0 above allows the detection of several ROS molecules (D0), followed by 200 mg/kg on day 1 (D1). Three hours after the (such as superoxide anions, H 2O2) and several neutrophil second injection, the mice received a cocktail of anesthetics [75 functions (such as NADPH oxidase activation, mg/kg ketamin (Virbac Santé Animale) plus 1 mg/kg medetomidine degranulation and MPO activity). To investigate precisely (Pfizer)], before intratracheal LPS instillation (5 g/mouse). The mice were aroused by an intraperitoneal injection of 1 mg/kg the effect of CCPAE on neutrophil NADPH oxidase atipamezol (Pfizer), a medetomidine antagonist, and were killed 24 activation, superoxide anion production was measured h later. using cytochrome c reduction assay. Since superoxide production is not detectable in resting neutrophils, the

Bronchoalveolar lavage (BAL) and lung sampling effect of CCPAE was tested only on PMA- and fMLF- stimulated cells. Results show that CCPAE inhibited The mice were anesthetized by an intraperitoneal injection of 50 mg superoxide anion production by neutrophils stimulated of urethane (sigma) and killed by exsanguination. The lungs were with PMA or fMLF (Figure 2). The effect of CCPAE shows lavaged twice with 1 ml of physiological saline, removed from the an inhibitory effect starting at a concentration as low as chest cavity, and immediately placed at -80°C until use. The lavage fluid (1.8 ml) was immediately placed on ice. Free alveolar cells 20 µg/ml. As fMLF and PMA activate the NADPH oxidase were recovered from the lavage fluid by centrifugation at 400 g for through different transduction pathways, these results 15 min at 4°C. The total protein concentration in the supernatant suggested that CCPAE directly inhibits a final common was measured with the Quick-Start Bradford assay (Bio-Rad, target, such as the NADPH oxidase, or scavenges Marnes-la-Coquette, France). The cell pellet was suspended in 150 superoxide anions. l of physiological saline and an aliquot was used to determine the total white cell count with a hemocytometer. For differential counts, the cell suspension was cytospun (Cytospin-2, Shandon Products Ltd.), fixed in methanol, and stained with Diff Quick solution CCPAE does scavenge superoxide anions nor H 2O2 (Medion Diagnostics, Plaisir, France). One hundred cells were nor inhibit MPO activity counted with an oil immersion lens (1000×). Firstly, to investigate whether CCPAE scavenges

Statistical analysis superoxide anions, xanthine/xanthine oxidase was used to produce superoxide anions, which were then detected Data were reported as mean ± standard error. The Newman-Keuls by the cytochrome c reduction assay. Results show multiple comparisons test was used, and P values <0.05 were (Figure 3A) that CCPAE had no effect on superoxide considered to denote significant differences. anions production by this system. Secondly, to investigate whether CCPAE scavenges H O , 2 2 RESULTS commercial H 2O2 which was detected by the luminol- amplified chemiluminescence assay was used. Results CCPAE inhibits luminol-amplified chemi- show (Figure 3B) that CCPAE had no effect on H 2O2. luminescence in human neutrophils, independently Thirdly, to investigate the effect of CCPAE on MPO of the stimulus activity, azurophilic granules extracts were incubated with different concentrations of CCPAE, and MPO activity was To investigate the effect of CCPAE on neutrophil ROS measured using H2O2-TMB oxidation assay. Results

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Figure 1. Effect of CCPAE on ROS production by human neutrophils. Human neutrophils (5 × 10 5) were incubated in the presence or absence of different CCPAE concentrations in resting conditions (A), and stimulated with fMLF (10 -6 M) (B), or PMA (100 ng/ml) (C). Luminol-amplified chemiluminescence was measured for 30 min (mean ± SEM of 5 experiments, *p<0.05).

Control (%) (%) Control (%) Control

Figure 2. Effect of CCPAE on superoxide anion production by neutrophils. Human neutrophils (1 × 10 6) were incubated in the presence or absence of CCPAE, and stimulated with fMLF (10 -6 M) or PMA (100 ng/ml). Cytochrome c reduction was measured at 550 nm in a spectrophotometer for 10 min (mean +/- SEM of 5 experiments, *p<0.05).

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Control (%) (%) Control

Figure 3. Effect of CCPAE on superoxide anions and H 2O2 in vitro . (A) Xanthine oxidase was incubated in the presence or absence of CCPAE, xanthine was added and superoxide anions was detected by the cytochrome c reduction assay. (B) H 2O2 was incubated in the presence or absence of CCPAE and detected using HRP-amplified chemiluminescence (mean +/- SEM of 3 experiments, *p<0.05).

show (Figure 4) that CCPAE had no effect on MPO ROS production, as measured by luminol-amplified activity. chemiluminescence in both resting and stimulated neutrophils. However, CCPAE did not scavenge superoxide anions or H O , but markedly inhibited Effect of CCPAE on BAL fluid protein content and 2 2 superoxide anions production by neutrophils. In addition, cellularity in LPS-treated mice CCPAE attenuated LPS-induced lung inflammation in

mice. Intratracheal administration of 5 g of LPS to mice CCPAE inhibited luminol-amplified chemiluminescence induced a significant increase in BAL protein content after in human neutrophils stimulated with the chemotactic 24 h, compared with animals treated with either the peptide fMLF or the protein kinase C activator PMA. As vehicle or CCPAE alone (Figure 5). Interestingly, the fMLF and PMA induce NADPH oxidase activation BALF protein content after intratracheal LPS challenge through different transduction pathways, these results was significantly lower when animals were pretreated suggest that CCPAE does not affect a specific with 200 mg/kg CCPAE (Figure 5). transduction pathway, but directly inhibits a final common Moreover, intratracheal LPS administration induced a biochemical target, such as the NADPH oxidase or MPO, significant increase in both the BALF total cell count (p < or that it scavenges reactive oxygen species. Besides, it 0.05 vs. vehicle or CCPAE alone, Figure 6A and B) and was found out that CCPAE had an inhibitory effect on the BALF neutrophil count after 24 h (p < 0.05 vs. vehicle cytochrome c reduction, a specific technique for or CCPAE alone, Figure 6A and C). Neither the vehicle superoxide anion detection, suggesting that CCPAE nor CCPAE at 200 mg/kg modified the BALF cell count. could react either by inhibiting NADPH oxidase activity or However, intraperitoneal CCPAE injection at 200 mg/kg by scavenging superoxide anions. significantly reduced both the BALF total cell count after Luminol-amplified chemiluminescence can be used to intratracheal administration of LPS (p < 0.05 vs. LPS assay both intracellular and extracellular ROS production alone, Figure 6A and B), and the neutrophil recruitment by neutrophils, as luminol is a membrane-permeable when compared with LPS alone (p < 0.05 vs. LPS, Figure molecule. Luminol-amplified chemiluminescence is 6A and C). dependent on superoxide anions, H2O2, and on peroxidases, such as cytosolic peroxidases and MPO DISCUSSION (Dahlgren and Karlsson, 1999). To determine whether CCPAE reacted with H 2O2, a more specific technique In this study, it was shown that CCPAE inhibited neutrophil was used to detect H2O2 in vitro . CCPAE did not affect

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Figure 4. Effect of CCPAE on MPO activity. MPO was incubated with or without CCPAE and its activity was measured in terms of tetra-methylbenzidine oxidation at 655 nm (mean +/- SEM of 5 experiments, *p<0.05).

Figure 5. Effect of CCPAE on the protein concentration of mouse bronchoalveolar lavage fluid (BALF). Total content of protein was measured in BALF 24 h after intratracheal instillation of lipopolysaccharide (LPS 5 µg/mouse) or in controls. LPS induced a massive increase in the protein content, which was significantly attenuated by CCPAE (200 mg/kg) (n=8, mean ± SEM, *p<0.05).

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Figure 6. Effect of CCPAE on BALF cell content. BALB/c mice were treated with vehicle, CCPAE (200 mg/kg), LPS (5 µg/mouse) or CCPAE (200 mg/kg) plus LPS (5 µg/mouse), and cells were counted in BALF. A, Total cell counts; B and C, Neutrophil counts (n=8, mean ± SEM *p<0.05).

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the amount of H 2O2, suggesting that CCPAE does not anti-inflammatory action. The intraperitoneal react with H 2O2. However, CCPAE inhibited NADPH administration of aqueous extracts of seeds and fruits of oxidase activity in vitro and exerted a potent anti- C. colocynthis , significantly reduced the paw edema inflammatory effect in the lungs of mice exposed to LPS, induced by the noxious agent (Marzouk et al., 2013). reducing the BALF protein content, total cell number and ROS are important contributors to tissue injury, neutrophil count. The inhibitory effect of CCPAE on inflammation, cancer and many other diseases. The NADPH oxidase activity could thus explain its anti- antioxidant properties of flavonoids especially the inflammatory action in vivo. flavones glucoside (isosaponarin, isovitexin and Persistent asthma, chronic obstructive pulmonary isoorientin 3’-O-methyl ether) and the cucurbitacin disease (COPD) and emphysema are chronic glucosides (2-O-β-D-glucopyranosylcucurbitacin and 2-O- inflammatory lung diseases (Lemanske and Busse, 1997; β-D-glucopyranosyl cucurbitacin L) , probably contribute, Ward, 1997). COPD and asthma involve several types of at least to some extent, to the pharmacological and inflammatory cells and soluble mediators (Barnes et al., traditional medicinal uses of the C. colosynthis (Abbas el 2003). COPD is associated with destruction of the al., 2009). These compounds scavenge free radicals and alveolar epithelium and flooding of the alveolar spaces inhibit lipid oxidation in vitro (Gil et al., 2000; Noda et al., with proteinaceous exudates containing abundant 2002). Further studies are needed to identify the precise neutrophils (Di Stefano et al., 1998). In our study, BALF phenolic compounds responsible for the NADPH oxidase from mice exposed to LPS contained a large amount of inhibition observed in this study. protein, reflecting high-permeability pulmonary oedema. The BALF protein concentration was significantly reduced by CCPAE treatment, suggesting that CCPAE reduces Conclusion lung vascular permeability and oedema, and might therefore protect the integrity of the alveolocapillary As concluded from this study, CCPAE inhibited neutrophil membrane. The reduction in neutrophil infiltration could luminol-amplified chemiluminescence in vitro , by explain these beneficial effects, as neutrophils are inhibiting NADPH oxidase. CCPAE also attenuated considered a primary cellular effector of alveolocapillary inflammation induced by intratracheal endotoxin damage in COPD and asthma (Pesci et al., 1998; Fabbri instillation in mice, leading to a decrease in the BALF et al., 2003). protein concentration, total cellularity and neutrophil It was found out that CCPAE inhibited NADPH oxidase content. activity in vitro , an effect possibly explaining the anti- inflammatory action observed in vivo . NADPH oxidase stimulation triggers murine macrophages to produce ROS Conflict of interest (Lincoln et al., 1995; Gelderman et al., 1998). Secreted ROS enhances the secretion of TNF α, IL-8 and other The authors declare that they have no conflict of interest. proinflammatory cytokines (Nelson et al., 1998). In particular, alveolar macrophage-derived TNF α and IL-8 recruit neutrophils to sites of inflammation (Gibson et al., Abbreviations 2001). NADPH oxidase inhibition by CCPAE could attenuate these inflammatory reactions. fMLF, N-formyl-methionyl-Leucyl-phenylalanine; MPO, Fruit and vegetables are important sources of myeloperoxidase; PMA, phorbolmyristate acetate; ROS, antioxidants, including ascorbic acid, carotenoids, reactive oxygen species. flavonoids and hydrolysable tannins. Epidemiological studies indicate that populations that consume foods rich REFERENCES in specific polyphenols have a lower incidence of inflammatory disorders, such as cardiovascular and Babior BM (2000) Phagocytes and oxidative stress. Am. J. Med. cerebrovascular disease, as well as certain cancers 109:33-44. Barnes PJ, Shapiro SD, Pauwels RA (2003). Chronic obstructive (Huxley and Neil, 2003; Temple and Gladwin, 2003). pulmonary disease: molecular and cellular mechanisms. Eur. Respir. Several studies have demonstrated the high antioxidant J. 22:672-688. activity of colocynth. 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Vol. 9(31), pp. 838-850, 17 August, 2015 DOI: 10.5897/JMPR2015.5868 Article Number: 3AC6F7C54895 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR

Review

A review on antimicrobial potential of species of the genus Vernonia (Asteraceae)

Antonio Carlos Nogueira Sobrinho 1*, Elnatan Bezerra de Souza 2 and Raquel Oliveira dos Santos Fontenelle 2

1Academic Master in Natural Resources, Center for Science and Technology, State University of Ceará, Campus do Itaperi, 60740-903 Fortaleza-CE, Brazil. 2Course of Biological Sciences, Center for Agricultural Sciences and Biological Sciences, State University Vale do Acaraú, Campus da Betânia, 62040-370 Sobral-CE, Brazil.

Received 13 June, 2015; Accepted 4 August, 2015

Natural products are sources of various biologically active chemicals. Therefore, ethnopharmacological and ethnobotanical studies are essential to discover new substances for the treatment of diseases. In this context, many studies have been conducted of the Asteraceae family demonstrating medicinal properties of its representatives, such as species of the genus Vernonia , which are rich in bioactive substances like sesquiterpene lactones, flavonoids, tannins and steroids. This review presents an overview of Vernonia species with antimicrobial potential, their main phytochemical characteristics and ethnomedicinal uses.

Key words: Compositae, Vernonieae, phytochemistry, biological activity, antimicrobial, antibacterial, antifungal.

INTRODUCTION

Humans have always used plants for therapeutic potential, such as assays of essential oils with purposes to control microbial infections and other antimicrobial properties against a variety of medical conditions (Rangel et al., 2001). The increase of microorganisms (Silva et al., 2012). bacterial and fungal infections and the development of The Asteraceae family (Compositae) has about 24,000 microbial resistance to synthetic drugs have led to described species, belonging to 1,600 to 1700 genera renewed interest in recent years to investigate plants as distributed in 17 tribes and three sub-families (Funk et natural sources of substances for therapy against al., 2009; Petacci et al., 2012). They have cosmopolitan microorganisms of medical and veterinary importance distribution and are widely found in the tropics, sub- (Denning, 2002; Rocha et al., 2011; Nascimento et al., tropics and temperate regions (Teles and Bautista, 2006; 2000; Bautista-Baños et al., 2003). Numerous plants Hattori and Nakajima, 2008). Representing to the largest have been used for prophylactic purposes and to cure family of the eudicotyledons, Asteraceae is relevant for infections. In this context, many studies have been its cosmetic, aromatic and therapeutic properties conducted to find plant species with antimicrobial (Nakajima and Semir, 2001; Hattori and Nakajima,

*Corresponding author. E-mail: [email protected]. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Sobrinho et al. 839

2008). Together with Calyceraceae , Campanulaceae , In Brazil; country with the highest genetic diversity of Menyanthaceae and Goodeniaceae, it forms a clade;the the world (Lewinsohn and Prado, 2005), there are over Asterales order (Pozner et al., 2012). The main genera 200 species of Vernonia , in the form of herbs, shrubs or of Asteraceae are Aster L., Inula L., Xanthium L., trees with foliage in various habitats. As for the morpho- Eupatorium L., Carpesium L., Saussurea DC., Vernonia anatomical features, they present solitary capitulums that Schreb. and Taraxacum Zinn. (Wu et al., 2006). are racemose, paniculated or scorpoid with flowers of Of the tribe Vernonieae, the genus Vernonia is one of various colors. The fruits are cipselas and can be angled the largest, predominantly in Africa and South America or costadas , glabrous or sericeous. They nearly always (Costa et al., 2008). Its species have wide have a couble pappus formed by an outer row of short ethnopharmacological use for treatment of several bristles and an inner row of feathery, barbeladas or diseases as snakebite antidote, and as food in some scabrous bristles, persistent or not (Galastri et al., 2010). African regions (Toyang and Verpoorte, 2013). In traditional medicine, many Vernonia species are Numerous phytochemical studies of the genus Vernonia employed to treat various diseases. From the have revealed the presence of diterpenes (Kos et al., pharmacological point of view, species have been 2006), triterpenes (Liang and Min, 2003), steroids investigated revealing many properties, such as (Tchinda et al., 2003), and the most frequently isolated antiplasmodial (Stangeland et al., 2011), analgesic chemical constituents are flavonoids (Carvalho et al., (Frutuoso et al., 1994), anti-inflammatory (Malafronte et 1999) and sesquiterpene lactones (Buskuhl et al., 2010). al., 2009), antimicrobial (Ogundare et al., 2006), anti- This article presents a review of the antimicrobial diabetes (Akinola et al., 2010), antioxidant (Owolabi et potential, focusing on the antibacterial and antifungal al., 2011) and antitumor (Sangeetha and activities of the species of the genus Vernonia , Venkatarathinakumar, 2011). highlighting the biological, phytochemical and Phytochemical studies have indicated that the main ethnopharmacological properties of the genus. constituents of the genus are sesquiterpene lactones, triterpenes, steroids, carotenoids, flavonoids, lignoids, alkaloids and tannins (Toigo et al., 2004). The most METHODOLOGY common constituents are flavonoids, which often have antioxidant activity (Salawu et al., 2011), and The current review was achieved using an organized sesquiterpene lactones, the latter being considered the search of the scientific data published about chemotaxonomic markers in the genus (Albuquerque et antimicrobial activity, focusing on the antibacterial and al., 2007a). Several sesquiterpene lactones have been antifungal activities of the species of the genus Vernonia . isolated from species of the genus with different types of The search was conducted between November 1 and bioactivity, such as molluscicidal, antifungal, antitumor, December 28, 2014, using the keyword search term cytotoxic and insecticidal against herbivores insects "Vernonia antimicrobial activity". The searches were (Wedge et al., 2000; Freire et al., 1996c; Lopes et al., carried out using various databases, including PubMed 1991). In V. amygdalina the sesquiterpene lactones (http://www.ncbi.nlm.nih.gov/pubmed), Science Direct vernolide and vernodalol (Figure 1) were isolated, with (http://www.sciencedirect.com/), Scopus potential antibacterial and antifungal activity (Erasto et (http://www.scopus.com/), Scielo (http://www.scielo.org/) al., 2006). In an investigation of the chemical and Google Scholar (http://www.scholar.google.com/). composition of hexane and ethanol extracts from aerial parts of V. chalybaea Mart by spectroscopic methods, terpenoids, flavonoids, alcohols and aliphatic ethers were Antimicrobial potential of the genus Vernonia isolated for the first time in the genus (Costa et al., 2008). Vernonia is one of the largest genus of plants belonging There have been numerous studies involving extracts, to the tribe Vernonieae (Cichorioideae), Asteraceae fixed oils and essential oils of Vernonia species (Table 1) family, with about 1,500 described species, being the with potential antimicrobial activity against strains of largest genus of the tribe (Silva et al., 2013). Vernonia bacteria, protozoa and fungi that are pathogens to species are found in temperate, tropical and sub-tropical animals and plants, using extracts, fixed oils, and areas, especially in South America, Asia, Africa (Costa et essential oils. In particular, phytochemical screening is al., 2008; Dematteis and Pire, 2006) and North America promising to identify bioactive compounds with (Redonda-Martínez et al., 2012). More than 500 of these antimicrobial activity (Magadula and Erasto, 2009; Hamill Vernonia plants are distributed in Africa and Asia and et al., 2000). approximately 300 species distributed in tropical areas, In Ethiopia, a study of nine plants with the from Mexico to Argentina (Yeap et al., 2010). This genus ethnomedicinal use indicated that Vernonia species are is named in honour of the English botanist William used for the treatment of eye infections, for wound Vernon, who first identified this genus in the region that is healing and the treatment of bone related problems such now Maryland in the USA in the late 1600s (Toyang and as fractures. They are used by decoction of fresh leaves Verpoorte, 2013). and subsequent application on the lesions or intake 840 J. Med. Plants Res.

Table 1. Species of the genus Vernonia with antimicrobial activity.

Biological Scientific name Part used Bioactive substances Biological activity Reference preparation Antibacterial activity: Gram-positive and gram-negative bacteria Vernonia adoensis L PE Terpenoids Kisangau et al. (2007) Antifungal activity: Candida albicans yeast

Alkaloids, flavonoids, saponins, tannins, AP Vernonia ambigua CE/EE glycosides, cardiac glycosides, steroids and Antibacterial activity: Gram-positive and gram-negative bacteria Aliyu et al. (2011)

triterpenes

L EAE Sesquiterpene lactones Antibacterial activity: Gram-positive and gram-negative bacteria Jisaka et al. (1993) Antibacterial activity: Gram-positive and gram-negative bacteria L ME Saponins, tannins and flavonoids Akinpelu (1999) Antifungal activity: Candida albicans yeast

ME/DEE/AqE L Sesquiterpene lactones and steroid glucosides Antibacterial activity: Gram-positive and gram-negative bacteria Otshudi et al. (1999)

Vernonia amygdalina AP ME/AE - Antibacterial activity: Gram-positive and gram-negative bacteria Kambizi and Afolayan (2001) L EE Sesquiterpene lactones Antibacterial activity: Gram-positive and gram-negative bacteria Erasto et al. (2006) L AqE - Antifungal activity: Fusarium sp filamentous fungi Suleiman et al. (2008) L ME Sesquiterpene lactones and steroid glucosides Antibacterial activity: Gram-positive and gram-negative bacteria Cheruiyot et al. (2009)

L AE - Antibacterial activity: Gram-positive Mycoplasma mycoides subsp. mycoides Muraina et al. (2010)

L EE/AqE Phenolic compounds Antibacterial activity: Gram-positive and gram-negative bacteria Salawu et al. (2011) L EE/AqE Sesquiterpene lactones Antibacterial activity: Gram-positive and gram-negative bacteria Adetutu et al. (2011)

Alkaloids, phenols, tannins, phytosterols, L ME Antibacterial activity: Gram-negative bacteria Noumedem et al. (2013) flavonoids and saponins

Antibacterial activity: Gram-positive and gram-negative bacteria Vernonia anthelmintica S ME - Jahan et al. (2010) Antifungal activity: yeast and filamentous fungi

HE/DE/EAE/ME Vernonia auriculifera L/St/R Triterpenes and sesquiterpenes Antibacterial activity: Gram-positive and gram-negative bacteria Kiplimo et al. (2011)

alkaloids, flavonoids, saponins, tannins, AP CE/EE glycosides, cardiac glycosides, steroids and Antibacterial activity: Gram-positive and gram-negative bacteria Aliyu et al. (2011) triterpenes Vernonia blumeoides

Antitrypanosomal activity: L CE Lacones Ibrahim et al. (2013) Trypanosoma brucei brucei

Vernonia brasiliana L EO Sesquiterpenes Antibacterial activity: gram-positive and gram-negative bacteria Maia et al. (2010) AP ME - Antibacterial activity: Gram-negative Pseudomonas aeruginosa Latha et al. (2010) Vernonia cinerea L ME - Antifungal activity: Candida albicans and Cryptococcus neoformans yeast Latha et al. (2011)

L/St/R ME/EAE - Antibacterial activity: Gram-positive and gram-negative bacteria Kelmanson et al. (2000) Vernonia colorata L HE/EAE Sesquiterpene lactones Antibacterial activity: Gram-positive and gram-negative bacteria Rabe et al. (2002) Vernonia condensate AP ME - Antibacterial activity: Gram-positive and gram-negative bacteria Brasileiro et al. (2006) Sobrinho et al. 841

Table 1 . cont’d

Antibacterial activity: Gram-positive and gram-negative bacteria Vernonia galamensis S FO Amino compounds Mbugua et al. (2007) Antifungal activity: Yeast and filamentous fungi

Antibacterial activity: Gram-positive and gram-negative bacteria Vernonia glabra AP/St/R DE/ME - Kitonde et al. (2013) Antifungal activity: Yeast and filamentous fungi

Antibacterial activity: Gram-positive and gram-negative bacteria Vernonia guineenses R DE/ME/AqE - Toyang et al. (2012) Antifungal activity: Yeast and filamentous fungi

Vernonia hymenolepis L ME Alkaloids, phenols and flavonoids Antibacterial activity: Gram-negative bacteria Noumedem et al. (2013) Vernonia lasiopus L/St AqE - Antibacterial activity: Gram-positive and gram-negative bacteria Kareru et al. (2008)

alkaloids, flavonoids, saponins, tannins, Vernonia oocephala AP CE/EE glycosides, cardiac glycosides, steroids and Antibacterial activity: Gram-positive and gram-negative bacteria Aliyu et al. (2011) triterpenes

Alkaloids, triterpenoids, coumarins and Antifungal activity: Candida albicans and Cryptococcus neoformans yeast Vernonia polyanthes L ME Braga et al. (2007) flavonoids Antileishmanial activity: Leishmania amazonensis and Leishmania chagasi

Vernonia remotiflora L EO Sesquiterpenes Antibacterial activity: Gram-positive and gram-negative bacteria Maia et al. (2010) St HE/CE Sesquiterpene lactones Antifungal activity: Penicilium citrinum Freire et al. (1996a) L HE/CE Sesquiterpene lactones Antifungal activity: Penicilium citrinum and Aspergillus alutaceos Freire et al. (1996b)

Vernonia scorpioides Antibacterial activity: Gram-positive and gram-negative bacteria AP EO Terpenoids Toigo et al. (2004) Antifungal activity: Candida albicans yeast

L EAE - Antibacterial activity: Gram-positive bacteria Kreuger et al. (2012)

Antibacterial activity: Gram-positive and gram-negative bacteria Vernonia smithiana AP EO Terpenoids Vagionas et al. (2007) Antifungal activity: Yeast

Vernonia species L ME - Antibacterial activity: Gram-positive and gram-negative bacteria Kalayou et al. (2012)

Alkaloids, tannins (including phlobaphenic Antibacterial activity: Gram-positive and gram-negative bacteria Vernonia tenoreana L/St ME/EAE/AE/HE Ogundare et al. (2006) tannins), cardiac glycosides and anthraquinones Antifungal activity: Yeast and filamentous fungi

AP - Aereal part; L - Leaves; R - Root; S - Seed; St – Stems; AE - Acetone extract;; AqE - Aqueous extract; CE - Chloroform extract; DE - Dichloromethane extract; DEE - Diethyl ether extract; EAE - Ethyl acetate extract; EE - ethanol extract; EO - Essential oil; FO - Fixed oil; HE - Hexane extract; ME - Methanol extract; PE - Petroleum ether extract.

(Kalayou et al., 2012). This article presents an Vernonia adoensis Sch. Bip. ex Walp. et al., 2010), tuberculosis and gonorrhea overview of ethnopharmacological uses, local (Kisangau et al., 2007) and malaria (Ragunathan prevalence and antimicrobial potential of species In Africa, this species is used in folk medicine for and Solomon, 2009). In Tanzania the petroleum of the genus Vernonia , with corresponding thetreatment of various diseases, especially ether extract of the leaves against the bacteria information in Table 1. infectious diseases such as HIV/AIDS (Lamorde Staphylococcus aureus , Bacillus subtilis , 842 J. Med. Plants Res.

Figure 1. Chemical structure of vernolide (I) and vernodalol (II), sesquiterpene lactone found in V. amygdalina .

Escherichia coli and Pseudomonas aeruginosa was Vernonia amygdalina Delile investigated, as well as against the yeast Candida albicans . V. amygdalina is a species with wide medicinal use in The authors used the disk diffusion and agar diffusion African countries, to treat malaria, helminth infections, methods. The extracts only showed bioactivity against gastrointestinal disorders and fever (Hamill et al., 2000; the strain of E. coli (Kisangau et al., 2007). Magadula and Erasto, 2009), to promote wound healing by decoction (Adetutu et al., 2011) and to treat microbial infections (Noumedem et al., 2013). Three sesquiterpene Vernonia ambigua Kotschy and Peyr (Asteraceae) lactones were isolated from the ethyl acetate extract of the leaves: vernodalin, vernolide and hydroxyvernolide. In Africa, particularly in Nigeria, Cameroon and In turn, vernodalol was isolated from the methanol Tanzania, this species is used in folk medicine to treat extract. All were tested against strains of B. subtilis , urinary tract infections, cough and colds (Burkill, 1985) Micrococcus luteus , E. coli and Agrobacterium as well as malaria (Builders et al., 2011) tumefaciens , showing significant results, indicated by In Northern Nigeria, it is widely used in traditional growth inhibition zone (Jisaka et al., 1993). Akinpelu medicine for the treatment of various infectious diseases (1999), isolated saponins, tannins and flavonoids of the (Aliyu et al., 2011). V. ambigua is an annual shrub, in methanol extract of the leaves. Sensitivity tests were “Yoruba” is known as “Orungo” and in “Hausa” as "Tab- performed by the agar diffusion and broth dilution taba or Tattaba" (Builders et al., 2011). A phytochemical methods for the bacteria Klebsiella pneumoniae , B. screening revealed the presence of secondary subtilis , E. coli , P. aeruginosa , Proteus vulgaris , Serratia metabolites alkaloids, flavonoids, saponins, tannins, marcescens , Shigella dysenteriae and Staphylococcus glycosides, cardiac glycosides, steroids and triterpenes aureus , along with antifungal testing against the yeast (Aliyu et al., 2011). Candida albicans . Antibacterial activity of ethanolic extract of leaves was The results showed antimicrobial activity against all reported against ten clinical bacterial strains, Klebsiella tested microorganisms except S. marcescens , E. coli and pneumoniae (16 mm), Streptococcus pyogenes (16 mm), C. albicans . Several studies have investigated the S. aureus (18 mm), Corynbacterium ulcerans (16 mm), antimicrobial potential of solvent extracts of the aerial methicillin resistant S. aureus (14 mm), Salmonella typhi parts against gram-positive and gram-negative bacteria, (20 mm), P. aeruginosa (18 mm), Shigella dysentriae (0 using methanol, ethanol and water (Otshudi et al., 1999); mm), Proteus mirabilis (0 mm) and Pseudomonas methanol and acetone (Kambizi and Afolayan, 2001); fluorescence (0 mm), by the disk diffusion and broth ethanol (Erasto et al., 2006); methanol (Cheruiyot et al., microdilution methods. The MIC values ranged from 1.25 2009); ethanol and water (Salawu et al., 2011); ethanol to 2.5 mg/mL for all the organisms tested (Aliyu et al., and water (Adetutu et al., 2011); methanol (Noumedem 2011). et al., 2013); and acetone (Muraina et al., 2010). There Sobrinho et al. 843

are also reports of the antifungal activity of V. aerial parts against gram-positive bacteria B. subtilis , S. amygdalina against C. albicans (Akinpelu, 1999), as well pyogenes , Staphylococcus saprophyticus , as fungi of the genus Fusarium , which cause superficial Staphylococcus sciuri , Staphylococcus xylosus , S. and systemic human infections, in addition to food aureus and S. epidermidis , by the disk diffusion method. contamination by the mycotoxins produced (Suleiman et The sesquiterpene lactone blumeoidolide - A al., 2008). demonstrated the better antibacterial activity (Aliyu et al., 2015). In a previous study, a phytochemical screening revealed the presence of secondary metabolites Vernonia anthelmintica (L.) Willd. alkaloids, flavonoids, saponins, tannins, glycosides, cardiac glycosides, steroids and triterpenes (Aliyu et al., V. anthelmintica [syn. Centhraterum anthelminticum (L.) 2011). Kuntze], native to Africa and Asia, has been studied to In other studies in Nigeria, the ethanolic extracts of treat diabetes (Rao et al., 2010; Fatima et al., 2010) and leaves was reported against the clinical bacterial strains, helminth infections (Mali and Mehta, 2008). The K. pneumoniae (22 mm), S. pyogenes (16 mm), S. methanol extract of the seeds was tested against strains aureus (22 mm), C. ulcerans (24 mm), methicillin of the bacteria P. aeruginosa , Yersinia aldovae , resistant S. aureus (17 mm), S. typhi (18 mm) and S. Citrobacter , Shigella flexneri , E. coli and S. aureus . The dysentriae (14 mm), by the disk diffusion and broth antifungal activity was also tested against microdilution methods. The MIC values ranged from 1.25 Saccharomyces cerevisiae , C. albicans , Aspergillus to 2.5 mg/ml for all the organisms tested (Aliyu et al., parasiticus , Macrophomina , Fusarium solani , 2011). Trichophyton rubrum and Trichophyton occidentalis . The extracts showed antibacterial activity against all strains tested except Y. aldovae . However the antifungal assay Vernonia brasiliana (L.) Druce. showed only inhibitory action against the dermatophyte T. rubrum (Jahan et al., 2010). V. brasiliana is a species found in north eastern Brazil whose antiplasmodial activity was investigated against Plasmodium berghei and P. falciparum (Carvalho et al., Vernonia auriculifera Hiern. 1991). The essential oil of leaves, extracted by hydrodistillation, was tested against P. aeruginosa , V. auriculifera is a shrubby species whose height varies Enterobacter aerogenes , Salmonella choleraeasuis , K. between 1 and 7.5 m. It is native to Africa and is used in pneumoniae , S. aureus and B. subtilis , by the disk folk medicine to relieve headaches (Kusamba, 2001), to diffusion method, finding broad-spectrum antibacterial treat conjunctivitis, fever, viral and bacterial infections activity against all tested microorganisms (Maia et al., (Muthaura et al., 2007), and for various purposes by 2010). decoction in Cameroon (Focho et al., 2009). Extracts using the organic solvents hexane, dichloromethane, ethyl acetate and methanol of roots, stems and leaves of Vernonia cinerea (L.) Less. V. auriculifera were conducted. Eight triterpenes and one aminated sesquiterpene were V. cinerea is an herbaceous species with isolated. All compounds were tested by the broth ethnopharmacological use in South America, Africa and microdilution method, against E. coli , P. aeruginosa , K. Asia, to treat various diseases or ailments such as pneumoniae , Stenotrophomonas maltophilia , S. aureus , malaria (Jain and Puri, 1984; Moshi et al., 2009; Padal et B. subtilis , Enterococcus faecium , Staphylococcus al., 2010), helminths infections (Johri et al., 1995; epidermidis and Staphylococcus saprophyticus . The Alagesaboopathi, 2009) and skin infections (Maregesi et results showed moderate antibacterial activity (Kiplimo et al., 2007; Moshi et al., 2009). In Asia it has been al., 2011). documented and recommended in Thai and Indian traditional medicine, as in other countries, for smoking cessation, and relief of urinary calculi, cough, fever, Vernonia blumeoides Hook. F. asthma, malaria, and arthritis (Leelarungrayub et al., 2010; Kitikannakorn et al., 2013). In India the V. blumenoides is a perennial herb native to Africa in undergrowth includes shrub species such as V. cinera “Hausa” known as ‘‘Bagashi’’, used in traditional medicine (Behera and Misra, 2006). Studies on the chemical for the treatment of various protozoans (malaria), composition of extracts of the aerial parts from V. cinera diarrhea and other unspecified infectious diseases in revealed the presence of triterpenes like α-amyrin, β- Nigeria (Ibrahim et al., 2013; Aliyu et al., 2015). A study amyrin and lupeol (Gaikwad et al., 2012; Sreedevi et al., have investigated the antibacterial activity of two 2011). sesquiterpene lactones isolated and of crude extracts The methanol extract of the aerial parts was tested using hexane, dichloromethane and ethyl acetate of against the bacterium P. aeruginosa , a prominent 844 J. Med. Plants Res.

opportunistic pathogen in hospital infections. The disk with minimal inhibitory concentration greater than the diffusion and broth dilution methods were used. The positive control used, chloramphenicol (Brasileiro et al., extract showed dose-dependent antimicrobial activity 2006). against the tested bacterium (Latha et al., 2010). The antifungal activity was tested using the methanol extract of the aerial parts against strains of the yeasts C. Vernonia galamensis Less. albicans and Cryptococcus neoformans . Assays were performed using the disk diffusion method, showing V. galamensis is found in Africa and has added activity against both yeasts, but better activity against C. commercial value because the oil extracted from its albicans (Latha et al., 2011). seeds has important medicinal use for treating diabetes and other organic dysfunctions (Autamashih et al., 2011). These oils have high vernolic acid content, an Vernonia colorata (Willd) Drake. unsaturated fat composed of triglycerides such as the trivernolin, present as the major constituent (Figure 2). V. colorata is a shrub native to Africa especially found in The derivatives of fixed oils of V. galamensis such as Cameroon, Ivory Coast, Senegal, Togo, South Africa and aminated fats are important intermediate chemicals, also Benin (Chukwujekwu et al., 2009), known as “Ibozane” in acting as additives for polyethylene films, water-based South Africa (Kelmanson et al., 2000). Senegalese and insect repellent and fungicidal compounds, among others Togolese traditional medicine practitioners employ the (Watanabe et al., 1993). decoction of V. colorata leaves to cure diabetes mellitus A study of the fixed oil of the seeds isolated (Sy et al., 2004). It is used for the treatment of cough, unsaturated fats, subsequently converted into aminated fever, hepatitis, gastritis, stomach pain, gastrointestinal fats, vernolamide, tested the antimicrobial activity disorders, venereal diseases and skin eruptions against the bacteria E. coli , B. subtilis and S. aureus , and (Hutchings et al., 1996; Cioffi et al., 2004). Its leaves are the fungi S. cerevisiae , Microsporum gypseum and used in culinary art in Benin, Cameroon and Togo (Sy et Trichophyton mentagrophytes . The authors have used al., 2005). Phytochemical studies reported the presence the disk diffusion method and the results showed activity of sesquiterpene lactones vernolide, 11 β, 13- against strains of E. coli and B. subtilis , but not against dihydrovernolide and vernodalin (Rabe et al., 2002). Also identified were tannins and saponosides (Sy et al., 2005). the yeast and dermatophyte fungi tested (Mbugua et al., In a study with 14 plants used in traditional Zulu 2007). medicine for treatment of ailments of an infectious nature, the methanolic and ethyl acetate extracts of V. colorata revealed antibacterial activity against gram- Vernonia glabra (Steetz) Oliv. & Hiern positive bacterium S. epidermidis , S. aureus , M. luteus and B. subtilis and gram-negative E. coli and P. A herbaceous species found in Africa, V. glabra has aeruginosa , by the disk diffusion and broth microdilution therapeutic use in Kenya for gastrointestinal problems by methods. The ratios of the inhibition zone were drinking tea from decoction of leaves and roots (Johns et expressed between 0.52 to 1.36 mm. The MIC values al., 1995). It is also used in the same country as an ranged from 1.0 to 4.0 mg/mL for S. aureus , M. luteus , antidote for snakebite (Owuor and Kisangau, 2006). B. subtilis and P. aeruginosa (Kelmanson et al., 2000). In Dichloromethane and methanolic extracts of the aerial other study in South Africa, the sesquiterpene lactones parts, stems and roots were tested against strains of the vernolide, 11 β, 13-dihydrovernolide and vernodalin were gram-positive bacterium S. aureus and gram-negative E. identified and tested against the bacterium S. aureus , B. coli and fungi C. albicans and Aspergillus niger by the subtilis , E. coli and K. pneumoniae by the direct bio- disk diffusion method. The results showed moderate autography and broth microdilution methods. The results antimicrobial potential against all tested microorganisms, showed that all sesquiterpene lactones were active at dependent upon the plant part used (Kitonde et al., inhibiting bacterial growth (Rabe et al., 2002). 2013).

Vernonia condensata Baker. Vernonia guineensis Benth.

V. condensata is a species found in Brazil and Africa with This species is widely used in folk medicine in the medicinal use reported in Nigeria against the snakebite Central African Republic, Congo and Cameroon. It has (Pereira et al., 1994). Known in Brazil as “aluma” and ethnopharmacological use of the roots as a stimulant, “alcachofra”, and its leaves are mainly used to relieve aphrodisiac, antimicrobial, snakebite antidote and to treat muscle pain along with gastrointestinal and hepatic helminth infections (Tchinda et al., 2002; Noumi, 2010). problems (Albuquerque et al., 2007b). A study of the Extracts obtained with dichloromethane, methanol and methanol extract of the aerial parts showed antibacterial distilled water of the roots of V. guineensis var. activity against S. aureus by the broth dilution method, cameroonica C. D. Adams were tested against the Sobrinho et al. 845

Figure 2. Trivernolin, a triglyceride of vernolic acid (cis-12,13-epoxy-cis-9-octadecenoic acid).

bacteria Acinetobacter baumannii , E. coli , P. aeruginosa , 2008). Salmonella typhimurium , S. epidermidis and S. aureus as well as a methicillin-resistant strain of S. aureus. In turn, antifungal tests were conducted against Aspergillus Vernonia oocephala Baker. fumigatus , C. albicans , C. neoformans and T. mentagrophytes . The authors used the microdilution Distributed across Northern part of Nigeria, V. oocephala broth method for determination of the minimal inhibitory is an erect perennial shrub used in the folk medicine to concentration. All extracts showed antimicrobial activity treat malaria and a number of unspecified infectious against all tested strains (Toyang et al., 2012). diseases (Aliyu et al., 2014). A study of phytochemical screening with native plants from Nigeria revealed the presence of secondary metabolites alkaloids, flavonoids, Vernonia hymenolepis Vatke. saponins, tannins, glycosides, cardiac glycosides, steroids and triterpenes (Aliyu et al., 2011). Found in western Africa, the species V. Hymenolepis has The antibacterial activity of the ethanolic extracts of medicinal use in Angola for treatment of respiratory leaves of V. oocephala was reported against the clinical system disorders and high blood pressure (Mengome et bacterial strains, K. pneumoniae (20 mm), S. pyogenes al., 2010). The methanol extract of the fresh leaves (16 mm), S. aureus (18 mm), C. ulcerans (20 mm), allowed isolation of alkaloids, phenols and flavonoids. methicillin resistant S. aureus (20 mm) and P. mirabilis The extracts were tested against the gram-negative (22 mm) by the disk diffusion and broth microdilution bacteria Providencia stuartii , P. aeruginosa , K. methods. The MIC values ranged from 1.25 to 2.5 pneumoniae , E. coli , Enterobacter cloacae and E. mg/mL for all the organisms tested (Aliyu et al., 2011). aerogenes . The assays were performed by the microdilution broth method, with promising results for all the tested strains of microorganisms (Noumedem et al., Vernonia polyanthes Less. 2013). V. polyanthes is native to Brazil. Extracts of its leaves and roots are used in folk for the treatment of Vernonia lasiopus O. Hoffm. rheumatism, bronchitis and cough (Lorenzi and Matos, 2002). The methanol extract of the leaves was found to V. lasiopus is a species native to Africa, especially found contain alkaloids, triterpenoids, coumarins and in Central African countries such as in Kenya, Tanzania, flavonoids. The extracts were tested for antifungal and Uganda and Rwanda. It shows biological potential, being antileishmanial activity. In the antifungal assay, strains of used for the treatment of malaria and helminth infections, the yeast C. albicans and C. neoformans were tested by by decoction of leaves and bark (Kareru et al., 2007). the broth dilution and agar diffusion methods to The aqueous extract of the leaves and bark was tested determine the minimum inhibitory concentration. For the against strains of E. coli , S. aureus and B. subtilis by disk antileishmanial tests, the protozoa Leishmania diffusion method, with positive results for E. coli and B. amazonensis and L. chagasi were used. In all cases, the subtilis , indicated by growth inhibition zone (Kareru et al., results were indicated by inhibition of parasite growth. 846 J. Med. Plants Res.

The results were satisfactory for leishmanicidal activity, Aspergillus alutaceos and P. citrinum (Freire et al., though the extract showed no antifungal activity (Braga 1996b). et al., 2007). Another study investigated the antimicrobial activity of the ethyl acetate extract obtained from fresh leaves of V. scorpioides for healing of excisional wounds in tissues of Vernonia remotiflora Rich. rats with inflammatory lesions caused by S. aureus . Rifamycin B diethylamide was used as positive control V. remotiflora [syn. Lepidaploa remotiflora ] is a species and saline as negative control. The results pointed to found in north eastern Brazil. Preliminary phytochemical effective antimicrobial activity for wound healing in studies allowed the identification and isolation of infected mice with increased wound contraction, smaller sesquiterpene lactones (Valdés et al., 1998) and a area of necrotic tissue, good development of granulation flavone (Jacobs et al., 1986). A study of the essential oil, tissue, extensive array of extracellular matrix and extracted by hydrodistillation from the fresh leaves, epithelial regeneration (Kreuger et al., 2012). The investigated the antibacterial activity to P. aeruginosa , E. ethanol extract of V. scorpioides showed antimicrobial aerogenes , Salmonella choleraeasuis , K. pneumoniae , S. activity against a strain of the yeast C. albicans and the aureus and B. subtilis , by the disk diffusion method. The bacterium S. aureus, with inhibition zones greater than essential oil inhibited the growth of all tested 16 mm that were concentration dependent for gram- microorganisms (Maia et al., 2010). positive cocci and greater than 15 mm for the tested yeast. These tests were performed by agar diffusion method (Toigo et al., 2004). Vernonia scorpioides (Lam.) Pers.

It is popularly known in Brazil by many names, V. Vernonia smithiana Less. scorpioides is a lianous, perennial, branched herb, considered to be a weed widely distributed throughout Native to the African continent, V. Smithian, which the Brazil, usually growing in poor soils and deforested common name in Tanzania is “umwanzuranya”, has areas, behaving as ruderal parantropophyte plant (Rauh therapeutic use for dysentery, gastrointestinal problems, et al., 2011, Toigo et al., 2004). A study of its essential eye diseases and urinary problems, mainly using extracts oil by gas chromatography and mass spectrometry from the aerial parts and roots (Adjanohoun et al., 1988). indicated the predominant presence of sesquiterpene A study of the essential oil extracted from the aerial parts hydrocarbons, with the main constituents being β- by hydrodistillation led to isolation of 39 volatile caryophyllene, germacrene-D and bicyclogermacrene compounds. The main constituents were sesquiterpenes (Albuquerque et al., 2007a). Investigations of extracts of and monoterpenes. the aerial parts, using ethanol and hexane, allowed the The essential oil was tested by the agar dilution isolation of triterpenes, steroids, a flavonoid and a method, compared to a panel of microorganisms polyacetylene lactone (Machado et al., 2013). including the bacteria S. aureus , S. epidermidis , E. coli , V. scorpioides is commonly used externally to treat a E. cloacae , K. pneumoniae , P. aeruginosa , variety of skin disorders such as allergies, irritations, Streptococcus mutans and Streptococcus viridans , the parasitosis, skin lesions, chronic wounds including ulcers latter two oral mucosal pathogens. For the antifungal of the lower extremities and itching (Rauh et al., 2011). It assays, the yeasts Candida glabrata , C. albicans and C. is also popularly used as an anti-hemorrhoidal and a tropicalis were used. The results showed excellent potent antidiarrheal, employed in the form of teas for antimicrobial activity, inhibiting all the species of internal use and infusions for external use (Freire et al., microorganisms tested (Vagionas et al., 2007). 1996c). Evaluation of the antifungal activity of the chloroform extract of the stems of V. scorpioides against the fungus Penicillium citrinum showed the formation of Vernonia tenoreana Oliv. growth inhibition zones up to 80 mm in diameter. Assays were performed by the agar diffusion method. The use of V. tenoreana is a wild growing shrub found in African a low-polar solvent for the preparation of the extract savannas, whose common name in the Yoruba language allowed satisfactory obtainment of active extracts, of Nigeria is “Ewuro Igbo”. The bark is used in some supporting the possibility of the presence of parts of Nigeria as emergency food (Sofowora, 1993). sesquiterpene lactones, constituents with potent The antidiabetic biological activity was investigated in a antifungal action (Freire et al., 1996a). A study with prospective study through random biological screening chloroform and hexane extracts of the stems and leaves (Taiwo et al., 2008). A phytochemical study of the extract demonstrated that the development of hyphae was of fresh leaves, using the organic solvents methanol, incipient in the presence of extracts and that the extracts ethyl acetate, acetone and n-hexane allowed isolating from the leaves showed less intensity, since the growth alkaloids, tannins (including phlobaphenic tannins), inhibition halos were smaller for both tested fungi, cardiac glycosides and anthraquinones, which demon- Sobrinho et al. 847

strates the phytochemical richness of this species, (1988). Contribution aux études ethnobotaniques et floristiques en making it a promising target for subsequent biological République Populaire du Congo. Collection Médecine Traditionnelle et Pharmacopée, Agence de Coopération Culturelle et Technique (ACCT), studies (Ogundare et al., 2006). Paris, France p 605. The extracts above were used in antimicrobial Akinola OS, Caxton-Martins EA, Akinola OB (2010). Ethanolic leaf extract susceptibility tests against bacteria and fungi by the agar of Vernonia amygdalina improves islet morphology and upregulates diffusion method. The bacteria tested were S. aureus , pancreatic G6PDH activity instrptozotocin-induced diabetic wistar rats. Pharmacology online 2:932-942. Streptococcus faecalis , B. subtilis , P, aeruginosa , E. coli , Akinpelu DA (1999). Antimicrobial activity of Vernonia amygdalina leaves. K. pnuemoniae , Bacillus cereus , Proteus vulgaris , Fitoterapia 70:432-434. Shigella dysenteriae and Serratia marcesens and the Alagesaboopathi C (2009). Ethnomedicinal plants and their utilization by fungi were C. albicans , Aspergillus flavus , A. niger , villagers in Kumaragiri Hills of Salem District of Tamilnadu, India. Afr. J. Tradit. Complement. Altern. Med. 6:222-227. Rhizopus stolonifer and Fusarium poae . The results Albuquerque MRJR, Lemos TLG, Pessoa ODL, Nunes EP, Nascimento showed growth inhibition for all bacteria tested, but no RF, Silveira ER (2007a). Chemical composition of the essential oil from activity against the fungi R. stolonifer and F. poae Vernonia scorpioides Asteraceae. Flavour Frag. J. 22:249-250. (Ogundare et al., 2006). Albuquerque UP, Medeiros PM, Almeida ALS, Monteiro JM, Neto EMFL, Melo JG, Santos JP (2007b). Medicinal plants of the caatinga (semi- arid) vegetation of NE Brazil: a quantitative approach. J. Ethnopharmacol. 114:325-354. CONCLUSION Aliyu AB, Adeyemi MMH, Abdulkadir I, Dambatta MB, Amupitan JO, Oyewale AO (2015). Antifeedant activity of Vernonia oocephala against This review shows that the genus Vernonia is rich in stored product pest Tribolium casteneum (Herbst). Bangladesh J. Sci. species with extensive ethnomedicinal use, especially in Ind. Res. 49:243-248. Africa, and extracts obtained by various preparation Aliyu AB, Moodley B, Chenia H, Koorbanally NA (2015). Sesquiterpene lactones from the aerial parts of Vernonia blumeoides growing in methods are used, especially from the leaves, bark and Nigeria. Phytochemistry 111:163-168. roots. There are extensive studies of V. amygdalina and Aliyu AB, Musa AM, Abdullahi MS, Ibrahim H, Oyewale AO (2011). 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