Herbal Medicine: Back to the Future: Volume 5, Infectious Diseases E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Serie: Herbal Medicine: Back to the Future
- Sprache: Englisch
Herbal Medicine: Back to the Future compiles expert reviews on the application of herbal medicines (including Ayurveda, Chinese traditional medicines and alternative therapies) to treat different ailments. The book series demonstrates the use of sophisticated methods to understand traditional medicine, while providing readers a glimpse into the future of herbal medicine.
This volume presents reviews of plant based therapies useful for treating different infectious diseases. The list of topics includes some niche reviews in this area including a review of the neem plant, the historical use of herbs in infectious disease therapy in Russia, and natural remedies from garlic, among other topics., The topics included in this volume are:
- Improving anti-microbial activity of allicin and carvacrol through stabilized analogs and nanotechnology
- Plant phenolics as an alternative source of antimicrobial compounds
- Herbal medicine in Russia’s history: the use of herbal medicine for infectious diseases in Russia’s history
- Azadirachta indica (neem) in various infectious diseases
- Contribution of novel delivery systems in the development of phytotherapeutics
This volume is essential reading for all researchers in the field of natural product chemistry and pharmacology. Medical professionals involved in internal medicine who seek to improve their knowledge about herbal medicine and alternative therapies for tropical and other infectious diseases will also benefit from the contents of the volume.
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Veröffentlichungsjahr: 2006
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PREFACE
Herbal Medicine: Back to the Future presents expert reviews on the applications of herbal medicines (including Ayurveda, Chinese traditional medicines, and alternative therapies). This volume demonstrates the use of sophisticated methods to explore traditional medicine, while providing readers a glimpse into the future of herbal medicine.
The book is a valuable resource for pharmaceutical scientists and postgraduate students seeking updated and critically important information regarding natural product chemistry and pharmacology of natural materials in the treatment of infectious diseases. The chapters are written by authorities in the field. Cundell, in chapter 1, reviews the antimicrobial properties of curcumin and eugenol, with their modes of action. Armutcu and Kucukbayrak, in chapter 2, present various herbal medicines that are effective against respiratory tract infections (RTIs) and their therapeutic mechanisms. Arya et al. in chapter 3, provide a comprehensive overview of epidemiological, pathogenesis, diagnostic aspects, and therapeutic interventions to tackle the current outbreak of COVID-19. Severcan et al. in chapter 4, discuss the applications of mid-infrared spectroscopy in the investigation of the constituents of herbal medicines used in infectious diseases. Tahri et al. in chapter 5, focus on the traditional uses of plants in Algerian pharmacopoeia against infectious diseases. In the last chapter of the book, Wang et al. discuss the use of ancient Chinese herbal medicines for treating infectious diseases.
We hope that the readers involved in the study of infectious diseases will find these reviews valuable and thought provoking so that they may trigger further research on herbal medicines and alternative therapies.
We are grateful for the timely efforts made by the editorial personnel, especially Mr. Mahmood Alam (Editorial Director), Mr. Obaid Sadiq (In-charge Books Department), and Ms. Asma Ahmed (Senior Manager Publications), at Bentham Science Publishers for the publication of this book.
List of Contributors
Improving Curcumin and Eugenol Through Computational Chemistry and Nanotechnology
Diana R. Cundell*,1Abstract
Curcumin and eugenol have been appreciated as broad-spectrum antimicrobial agents since the early 20th century, and their parent plants of turmeric and clove have been used in Ayurvedic and traditional Chinese medicines for thousands of years. Although extensive research has identified several antimicrobial mechanisms of action, it is the only eugenol that has become established for dental uses. Curcumin and eugenol have been hard to purify and stabilize and, in their native states, show poor bioavailability. New antimicrobial agents are now needed due to the growth in resistant strains, and this means natural agents are back in vogue. Nanoparticle and antimicrobial-coated surfaces are popular strategies to maximize the consistent delivery of mainstream pharmaceuticals. Computational chemistry and docking analyses are the primary methods used to identify and design novel variants of natural molecules to improve bioavailability and stability. Both curcumin and eugenol have benefitted from the expansion of these fields, and reports of stabilized forms with superior activity are now rapidly appearing in the literature. This chapter will review the antimicrobial spectrum of curcumin and eugenol, explaining their antimicrobial modes of action. Finally, potential and currently available delivery systems will be explored using the semi-synthetic analogs and bioengineered structures that have been created.
INTRODUCTION
Ethnomedicine practices existed at least 60,000 years ago when man sought to alleviate pain and infection using the plant species that grew in his local environment [1-3]. Spices especially provided both flavorings to food and helped to naturally preserve it from spoilage, i.e., they were antibacterial and antifungal [4]. These plants became both food and medicine in countries, like Ancient India
and China, and this concept was preserved even in the Ancient Greek Hippocrates’ medicinal treatises [5, 6]. Two plants emerged as important in these cultures, turmeric (Curcuma longa L.) [7] and the clove tree Syzgium aromaticum [8]. Easy to cultivate in both tropical and semi-tropical climates, turmeric is still used today as a nutraceutical in India and China to treat infections, tumors, gastrointestinal issues, and arthritic conditions [9, 10]. Since the 1st century AD, cloves have been traded from Indonesia around the world to be used as flavoring and medicinal agents [11]. Traditional Chinese Medicine (TCM) practices have established clove essential oils (EO) in the treatment of toothache and as a broad-spectrum antimicrobial, for alleviating gastrointestinal disorders [8]. Extracts of both turmeric and cloves have been successfully and continuously used in an anti-microbial capacity for thousands of years but to evaluate their abilities further, an understanding of their individual phytochemical activities is required.
At the beginning of the early 1900s, chemists began to identify the agents responsible for plant-derived antimicrobials [12] and it soon became apparent that there were numerous groups of secondary metabolites responsible for this activity [13]. It was revealed that plant extracts contain many complex antimicrobial molecules, including flavonoids, alkaloids, phenolics, and terpenoids [13]. Interestingly, the most prominent groups in this respect have been the terpenes and terpenoids, which have demonstrated the most broad-spectrum and potent activities [13]. Terpenoids are branched, lipid-based cyclic molecules found in most plants that are easily able to penetrate and disrupt phospholipid cell membranes [13]. As a result, they have been reported to inhibit the activities of at least 60% of bacterial species investigated and approximately one-third of all fungi [13]. As the individual terpenes from this group were identified, several molecules emerged [13]. Some compounds, such as artemisinin from sweet wormwood, eugenol from cloves, and capsaicin from chili peppers, were subsequently developed into pharmaceutical agents [13]. Others, including curcumin from turmeric, remain primarily nutraceuticals [13], however, it can be seen from this chapter, that situation may be changing.
Antimicrobial Activities of Turmeric and Clove
Sometimes referred to as “the Golden Spice,” turmeric tubers contain a diverse series of compounds, but the most powerful and prevalent antimicrobial is curcumin (5.0-6.6%) [7, 14]. Curcumin constitutes three-quarters of the curcuminoids present in turmeric [15]. Demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) together constitute another 20% of the curcuminoids but are without antimicrobial activity [15]. Vogel and Pelletier named curcumin in 1815 for the yellow discoloration they observed on the surface of turmeric tubers [16]. After several attempts to chemically analyze curcumin, in 1881, Jackson and Menke were able to create the first salts of curcumin, identify its poor solubility in water, and also theorize that it contained a vanillin group [17]. Purified curcumin was finally reported by the Polish scientists Milobedzka and Lampe in 1910 [18], who identified its structure as being a phenolic diaryl heptanoid, diferuloylmethane or (1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione).
Curcumin’s antibacterial and antifungal activity was identified in 1949 by Schraufstätter and Bernt [19]. The gram-positive bacteria Staphylococcus aureus and Mycobacterium tuberculosis, gram-negative bacterium Salmonella paratyphi, and the fungus Trychophyton gypseum were found to be effectively killed by curcumin in vitro [19]. Schraufstätter and Bernt [19] were the first to identify that a dibenzalacetone analog of curcumin (4,4’-dihydroxy-3,3’-dimethoxydi benzalacetone) possessed similar antibacterial and antifungal activity. Since then curcumin has been shown to possess potent antibacterial, antifungal, and antiviral activity [12]. Several reports of anti-protozoal [20, 21] and anti-helminthic [21-24] activity have also appeared in the literature.
Clove oil was first studied by Carl Jacob Ettling who, in 1834, identified its major constituent as eugenol (4-Allyl-2-methoxyphenol) [8, 25]. Eugenol occurs naturally in plant species, including turmeric, cinnamon, and nutmeg (~ 3 mg/kg) but the major commercial source remains clove flowers and buds (180 mg/kg) [26]. Clove EO consists primarily of eugenol (40-50%) and α-selinene (40%), with lesser amounts of monoterpenes [11]. Selinenes are known to be microbial defense molecules of plants [27] and possess anti-tumor activity [28]. Although less thoroughly investigated than eugenol, it is likely that these entities might be additive to eugenol’s activities.
Since the 1800s, eugenol has been shown to possess potent broad-spectrum antimicrobial activity [29, 30]. Interestingly, the early use of cloves for dentistry led to the incorporation of eugenol into a root canal zinc oxide paste (ZOE) by Chisholm in 1876 [30, 31]. By 1930, Charles Sweet established the use of ZOE for primary teeth, with this same compound still in use today [32]. Studies have shown that eugenol’s phenol group assists with the setting of ZOE and that eugenol gradually dissociates without weakening the complex [30, 31]. Eugenol’s anti-inflammatory and antimicrobial activities persist in the root canal area for at least a month and assist in the healing process [31]. Additionally, eugenol acts on the γ-aminobutyric acid type A (GABAA) receptor, providing pain-relieving and anesthetic properties [33].
Old Barriers for Curcumin and Eugenol and New Directions
Unfortunately for most herbal entities, the discovery of fungal-derived antibiotics in the 1920s began the use of pharmaceuticals that has continued to be focused on natural or synthetic variants of these molecules [34]. Another barrier to the development of plant-derived antimicrobials was their instability, poor bioavailability, and extraction methods resulted in variable levels of product composition [35]. This is certainly the case for curcumin which is rapidly absorbed into the gastrointestinal tract with low or negligible levels found in serum after 2-4 hours of ingestion [36]. Most ingested native pure curcumin appears to be excreted in the feces as glucuronide and sulfate metabolites, with maximal absorption seen in gastrointestinal and liver tissues and poor entry into brain and muscle [37-41]. Most commercial grades of curcumin are not 100% pure and instead mimic the complex of curcumin and curcuminoids found in the natural tuber i.e. they contain curcumin (curcumin I), DMC (curcumin II), and BDMC (curcumin III) [42]. Curcumin is generally considered safe with the EFSA (European Food Safety Authority), JECFA (The Joint United Nations and World Health Organization Expert Committee on Food Additives) supporting a daily intake of up to 3 mg/ kg body weight [43]. Most individuals report no side effects, although trials of individuals taking 500-12,000 mg per day have reported rash and gastrointestinal symptoms, including stool discoloration [43].
Pharmacokinetic studies on eugenol absorption in human and animal studies have demonstrated that it is rapidly metabolized within twenty-hour hours of ingestion [44-47]. Studies by Fischer et al. [49] reported that in humans more than half of the urinary metabolites of eugenol were glucuronide and sulfate conjugates. Rodent studies reported similar data [44, 46, 47] following oral dosing (40 mg/kg) with a plasma half-life of fourteen hours for eugenol [46, 47]. Another consideration in developing eugenol is that some dermatological (contact) allergic reactions to eugenol have been reported as liver toxicity following ingestion of clove EO [8]. Finally, data on herbal medicine efficacy often came from studies that are poorly blinded, contain multiple mixes of plant products, and are not standardized in terms of individual components [49].
With the rapid evolution of resistance to antibacterial and antifungal agents, scientists are now searching for new treatment strategies [50, 51]. Many resistant bacterial or fungal species form biofilms, which are virtually impossible to eradicate even with very high levels of antimicrobial agents [52]. Developing new antifungal and anti-protozoal agents without unwanted side effects has been traditionally more difficult, as these eukaryotic microbes share significant structural homology with human cells [51, 53]. The range of existing antiviral [54] medications is also limited.
As modifications to existing medications using computational chemistry techniques begin to produce fewer leads [50, 51, 55], some researchers are returning to plant-derived molecules [56]. During the past decade, nanotech-nology has become the new, effective method to encapsulate and either retain or improve the bioactivity for antibiotics [57] as well as natural antimicrobial herbal compounds [58]. Various formulations have been made with each providing different advantages of delivery, potency, and bioavailability [57, 58]. Using these same technologies for herbal-derived antimicrobials would increase their tissue bioavailability by decreasing their direct gastrointestinal tract and liver absorption [58].
Antimicrobial drug discovery from plants is now a group process, involving ethnobotanists, computational and physical chemists, who are able to model with some certainty the likelihood of any compound’s success [49, 59]. Stable analogs of curcumin (curcuminoids) [60] and nanoparticle/ micelle encapsulations [61-64] have already have been created. Investigators have developed several interesting new potential commercial eugenol delivery systems for dentistry [48, 65], wastewater treatment [66-68] and to prevent food spoilage [11]. Stable derivatives of eugenol and nanoparticles have both improved their efficacy and identified their antimicrobial mechanisms [11]. This chapter will discuss the mechanisms of action of curcumin and eugenol as well as evaluate their current status in this development process. Their chemical structures are depicted in Fig. (1).
Fig. (1)) Chemical structures of curcumin and eugenol.CURCUMIN (from Turmeric Curcuma Longa L.)
Antimicrobial Activities of Curcumin
Reviewed by Gupta et al [69], curcumin’s structure allows it to undergo keto- to enol- tautomerism that allows it to attach to and disrupt a number of cellular proteins and directly bind to DNA in both prokaryotic and eukaryotic cells. The hydrophobic, branched nature of the molecule allows it to insert itself into cell membranes disrupting the functionality of both bacteria [70-72] and fungal cell walls [73, 74].
Curcumin also binds and inactivates the surface protein anchoring transpeptidase (sortase A) of S. aureus [71] and S. mutans [72] with IC50 of 13.8 μg/ml and 10.2 μM, respectively. Studies in Candida albicans have reported curcumin down-regulates cell wall integrity pathway genes, increasing membrane permeability [73]. Neelofar et al [74] reported that curcumin at MIC doses (250 μg/ml) decreased the percentage of ergosterol in C. albicans and Candida glab-rata cell walls by 70 and 54%, respectively. Once inside cells curcumin impairs bacterial cell division [75-77]. Several gene targets have been suggested, includ-ing the bacterial analog of tubulin, FTsZ [75, 76], and the DNA repair and cell survival gene umuC [77]. Hu et al [78] reported that curcumin targeted the endoplasmic reticulum of Cryptococcus neoformans and significantly impaired (p<0.05) the yeast’s growth, in vitro. The authors suggested this was due to curcumin’s iron-chelating activity [78]. Literature review has identified several modes of action for curcumin that affect bacterial [70-72, 75-77, 79-86], fungal [73, 74, 78, 87], protozoal [88-91] or viral [92-103] viability and repli-cation (Fig. 2).
Biofilm Disrupting Effects of Curcumin
Curcumin has been reported to reduce in vitro biofilm formation by at least ten bacterial species [72, 79-86] and the yeast C. albicans [87]. Several quorum-sensing gene products have been suggested to be implicated in the effects on bacterial biofilms including extracellular polysaccharide production [79, 84, 86] and acyl-homoserine lactone [90]. Raorane et al. [86] demonstrated that curcumin (50μg/ml) significantly decreased (p<0.05) in vitro outer polysaccharide (pellicle) expression and motility of Acinetobacter baumanii. These authors observed that curcumin was bound directly to the biofilm response regulator (BfMr), downregulating all abilities to initiating or perpetuating these resistant structures [86]. Shahzad et al. [87] reported that curcumin (100 μg/ml) decreased the early phase of C. albicans biofilm formation by significantly (p<0.01) decreasing the expression of adhesin and filamentation genes.
Fig. (2)) Possible antimicrobial mechanisms for curcumin.Legend: Five potential targets in bacteria, fungi, protozoa, and viruses have been identified by researchers for curcumin that are responsible for its antimicrobial effects.
Curcumin can Impair Protozoal Attachment and Intracellular Functions
Curcumin inhibits the enzyme glyoxalase in the intracellular glycolysis pathway of Toxoplasma gondii, resulting in in vitro demise of the parasite [88]. Chakrabarti et al. [89] reported that curcumin (< 5 μM) prevented Plasmodium falciparum division by directly inhibiting alpha and beta-tubulin assembly. Similar data have also been reported for Giardia lamblia trophozoites [91]. A second anti-protozoal target for curcumin has been suggested to the parasite orthologue of mammalian sarco (endo) plasmic reticulum Ca2+ ATPase (SERCA) PfATP6 [90]. Curcumin attaches to PfATP6 by its phenol group [90] and, interestingly, is also the target for the gold standard antimalarial artemisinin [104].
Viral Attachment and Replication are Directly Prevented by Curcumin
Curcumin has been reported to inhibit gene expression in human immunodeficiency virus (HIV) [92-94], hepatitis B (HPB) [96], and human papillomavirus (HPV) [97]. Curcumin (80 μM) pre-treatment of a human kidney cell line (HEK293T cells) transfected with transcription regulator trans-activator of transcription (tat) genes reduced their expression [92]. In vitro docking studies showed curcumin attached to acidic residues in the HIV integrase core [93]. Computational docking studies by Vajragupta et al. [94] identified the terminal o-hydroxyl group of curcumin bound to these residues. Tsvetkov et al. [103] found curcumin (40 μM) inhibited the in vitro activity of the ubiquitin degradation pathway regulator NAD(P)H: quinone oxidoreductase 1 enzyme (NQO1). These data indicate curcumin prevents both viral infection and the transformation of infected cells to tumors [103]. Curcumin may also affect the integrity of the hepatitis C viral envelope, preventing its binding to and penetration of host cells [99, 101]. Computer modeling studies have suggested that curcumin can block the attachment of viral hemagglutinin A to the host cell surface receptor [102].
Other Potential Activities for Curcumin
Curcumin has achieved much of its recognition not from its antimicrobial but immunomodulatory activities through direct binding to nuclear factor kappa beta (NFκβ) in eukaryotic cells [105]. This decreases arachidonic acid activation, nitric oxide (NO), and reactive oxygen species (ROS) production [105]. Interestingly, protozoal colonization both results in and requires an uptick of inflammation, without which these parasites cannot survive [106]. Curcumin may also act as an epigenetic modulator [105]. Certainly, curcumin’s in vitro effects on P. falciparum have been attributed to an ability to inactivate both histone acetyltransferases (HAT) [107] and histone deacetylases (HDAC) [108].
Lopresti [109] has also suggested that curcumin’s effects on the gastrointestinal microbiome are a major driver in its effects on chronic inflammatory syndromes such as tumors and cardiovascular disease. Animal studies reported orally ingested curcumin increased gut microbiome floral diversity and improved probiotic bacterial levels [110-113]. Studies by several groups have suggested curcumin decreased intestinal permeability both in vitro [114-116] and animal in vivo models [117-119]. Curcumin can also eliminate several gastrointestinal pathogens, including Escherichia coli [120], C. albicans [121-123], Giardia [124], and T. gondi [124]. Dysbiosis of the gut flora, through poor diet or lacking complex carbohydrates, results in an increase in the secretion of pro-inflammatory metabolites of phosphatidylcholine, trimethylamine N-oxide (TMAO) [125]. TMAO upregulates bacterial binding and damage to endothelial cells lining the gut, thereby facilitating their egress into the circulation [125]. Further support for a “crosstalk between the gut and heart” can be seen by the finding that inhibitors of TMAO reduce the cardiac enlargement and damage that accompanies cardiovascular disease [125].
Spectrum of Curcumin’s Antimicrobial Activity
Antibacterial
Curcumin has antibacterial activity against at least fourteen gram-positive or gram-negative bacteria [69-72, 75-77, 79-86, 117-127]. Antibacterial MIC reported for curcumin in vitro is far higher than those for standard antibiotics for both gram-positive and gram-negative species with MIC of between 163 and 239 μg/ml [126, 127]. Curcumin MIC for Helicobacter pylori has been reported as between 5 and 50 μg/ml [128]. This is significantly lower than the antibiotics used for the management of the infection [129]. Curcumin (25 mg/kg gavage, seven days) eliminated H. pylori from the gastric mucosa of colonized mice and restored its functionality [129]. Curcumin has been reported to block the efflux pumps in bacteria [130, 131]. Teow and Ali [132] also suggested that curcumin decreased antibiotic loss from the bacterial cell.
Antifungal
Most antifungal studies of curcumin have been conducted on candida [73, 74, 133-135]. Most investigators agree with a MIC < 200 μg/ml for C. albicans [73, 133, 135], which is the only candida species sensitive to curcumin [133]. Curcumin cream (1%) significantly decreased (p<0.05) C. albicans colonization in an immunosuppressed rodent model of vulvovaginal candidiasis [134]. A human trial reported that curcumin cream (10%) was as effective as clotrimazole [135]. Curcumin decreased the MIC of commonly used anti-Candida agents [136]. Martin et al. [133] reported that Sporothrix schenkii and Paracoccidiodes brasiliensis possessed curcumin MIC that was between 2- to 8-fold lower than fluconazole. Curcumin showed weak activity against C. neoformans [78, 133] with MIC several-fold lower than fluconazole [133]. Aspergillus growth was unaffected by curcumin (MIC > 256 μg/ml) [129], but its ability to secrete aflatoxin was impaired [138]. Studies have reported that curcumin inhibition of animal damage induced by in vivo aflatoxin ingestion [139, 140].
Anti-protozoal and Anti-Helminthic
Anti-protozoal activity has been reported for curcumin against nine protozoal species [88-91, 141, 146]. From these studies, low curcumin IC50 were reported for five protozoa; Neospora caninum (1.1 μM after 24 hours) [145], G. lamblia (15 μM after 24 hours) [91], Cryptosporidium parvum (26.0 μM after 24 h) [141], Leishmania major (37.6 μM at 18 h) [144], and Plasmodium falciparum (50 μM after 96 hours) [89]. Efficacy of curcumin against G. lamblia, C. parvum, L. major and P. falciparum was similar to those of the anti-protozoal agents, metronidazole and chloroquine [53]. Schistosoma mansoni in vitro curcumin-induced DNA damage was reported [21-23] and a reduction in viability and fertility [21] through ROS-associated mechanisms was also observed [23]. Hussein et al. [23] found similar S. mansoni numbers in mice given praziquantel (500 mg/kg) and turmeric (400 mg/kg). Cervantes-Valencia et al. [24] reported an IC50 for curcumin (5.93 μM) against Besnoitia besnoitii.
Antiviral
Curcumin exhibited in vitro antiviral activity against at least nineteen RNA-containing viruses [92-94, 99-102, 147-161] and seven DNA-containing viruses [95-98, 147, 162-166]. Animal models of influenza A (30 or 100 mg/kg curcumin) [150], Rift Valley fever (10 μM curcumin) [159] and human papilloma virus (HPV) (20% curcumin cream) [166] reported significant (p<0.05) reductions in viral load following treatment. Most studies found an IC50 for curcumin of between 5 and 20 μM [100, 101, 147-149, 151-153, 156, 158-162]. In vitro reproduction of five Herpesviridae was reported with curcumin [95, 99, 153, 162-165] with IC50 of between 2 and 10 μM. Curcumin prevented in vitro HSV-2 viruses shedding from infected cells [153] and latent Epstein Barr virus (EBV) reactivation [164]. These studies suggest curcumin may provide a novel broad-spectrum anti-herpes agent. Docking studies reported curcumin has high selectivity for at least three potential targets in the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [167-169]. An important caveat is to recognize that the effects of curcumin on animal viral infections may not correspond to human physiology [170-172]. Human double-blind, randomized placebo-controlled trials (DBRPCT) of curcumin cream (10%) on HPV infection have failed to show any effect [170]. No effect on HIV viral load was reported in human subjects who ingested curcumin [171, 172]. Curcumin potentiated the in
vitro effects of the antiviral agents, boceprevir and cyclosporin [101], geldanamycin [95], and lamivudine [96].
Semi-Synthetic Curcumin Analogs
Literature studies have reported several non-toxic curcuminoids with potent activity [103, 165, 173-188]. Important analogs are shown in Table 1.
They are compatible with currently used chemotherapeutic agents [103, 176, 181, 183] and all are several-fold more active and stable than the parent compound [108, 165, 173-189]. Monocarbonyl diene and enone analogs possessed broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria [178-180], fungi [183], and protozoa [182, 184-186]. Ou et al. [190] have suggested that enones can also act as cross-link viral surface proteins. One compound, 1,7-bis(4-hydroxy-3-methoxyphenyl) hept-4-en-3-one, is found naturally in ginger as gingerenone A [186]. Molecular docking studies showed gingerenone A uniquely attaches to a virulence factor in S. aureus and has been suggested as a novel antibiotic candidate [191]. Alkhaldi et al. [192] reported that a monocarbonyl analog previously described as lethal for T. cruzi [186], killed trypanosomes by inactivating trypanothione and causing redox balance dysbiosis. This mechanism is unique to the analog and a novel target against these protozoa [192]. Synthetic acetylated curcuminoids display low bacterial MIC [174, 175] and leishmanial IC50 [176], with lesser antiviral activity [177]. Curcumin-diazepine coupling significantly (p<0.05) increased its antibiotic effect against S. aureus [188]. The most extensively studied curcuminoid is EP24, which possesses some antiviral activity but is most notable for its anti-inflammatory and anti-tumor effects [187]. All studies performed to date have been in vitro, and the safety profiles of these novel formulations have yet to be confirmed in vivo.
Nanoparticle and Immobilized Curcumin Studies
Seventeen formulations of curcumin have been reported to date (Table 2). All formulations significantly improved bioavailability and potency against bacteria [193-197, 201], fungi [196, 197], protozoa [198, 203-208], and viruses [199, 200, 202, 209]. One study, using 5 mg/ml curcumin-nisin nanoparticles, has also reported a 40% decrease in in vitro hatching of Fasciola sp. eggs [210]. Silver-coupled NP were successfully used as antibacterial agents [193-197] and PLGA NP as optimal anti-protozoals [206-208].
Several animal studies of protozoal infection [198, 204, 206-208] demonstrated significant efficacy of PLGA-NP or Ag-NP against Plasmodium sp [206-208], L. donovani [204], and Giardia [198]. Animal studies reported that the curcumin NP used not only prolonged their survival following the protozoal infections (i.e. were directly non-toxic) but that this was associated with a direct improvement in liver function as assessed by hepatic enzymes [198, 204, 206-208]. Interestingly, although animals received curcumin by gavage, no reports of vomiting or altered stool color were reported by these authors [198, 204, 206-208]. These five studies are small, involving only a total of just over 40 animals [198, 204, 206-208], but the combination of efficacy and an absence of any adverse effects suggests this is a delivery method warranting further study.
EUGENOL (Primarily from Cloves Syzgium Aromaticum L.)
Antimicrobial Mechanisms of Action of Eugenol
Unlike the other phytomedicines described in this chapter, fewer mechanistic studies have been performed on eugenol with many researchers focusing on what it does rather than its underlying chemistry [8, 9]. Eugenol’s antibacterial and antifungal effects involve the binding of its OH group to cell membranes [211] with the subsequent creation of pores and leakage of intracellular components [211-213]. In fungi and yeasts, eugenol inhibits the branched-chain amino acid building blocks of membrane permeases [214].
Inside the microbial cell, eugenol downregulates motility and adhesin genes [215-218] by directly binding to DNA [219-221]. Eugenol has been shown to increase the expression of bacterial oxidative stress proteins [222], deplete bacterial intracellular ATP [223], and disrupt in vitro protozoal [224] and fungal [225] mitochondrial function. Its amphipathic nature allows eugenol to easily penetrate and disrupt pre-formed bacterial [215, 216, 226-230] and fungal biofilms [231] and kill protozoal oocysts [232]. Eugenol’s effects on NFκβ are well documented [8, 11] and downregulation of the apoptotic mitogen-activated kinase pathways (MAPK) ERK1/2 and p38MAPK may be responsible for its antiviral effects [233]. Membrane damage in bacteria may result from eugenol’s capacity to activate bacterial cell ROS production [234]. These mechanisms are summarized in Fig. (3).
Biofilm Disrupting Effects of Eugenol
Sublethal concentrations of eugenol (0.1325 mg/ml) inhibited the expression of ten biofilm regulating genes, including relA, in the dental pathogen S. mutans [216]. These data support eugenol’s current usage in dental practices given its efficacy against the most common cariogenic pathogen [30, 31]. Eugenol’s effects on relA are of importance given the centrality of this gene in regulating biofilm formation in E. coli, Bacillus subtilis, L. monocytogenes, and streptococcal species [235]. Eugenol at sublethal concentrations (0.005%) did not affect planktonic forms of E. coli O157:H7 (EHEC), in vitro, but impaired the production of seventeen of twenty-eight quorum sensing genes [218]. In vitro studies by Rasinath et al. [228] suggested that eugenol (400 μM) targeted the quorum-sensing receptor LasR in P. aeruginosa. A significant decrease (p<0.001) in P. aeruginosa biofilm virulence factors expression was reported by these authors [228] and others [229]. Jayalekshmi et al. [236] performed molecular modeling of eugenol binding to the LasR receptor and reported that it was attached to two central amino acid residues. Yadav et al. [226] found that eugenol (0.08%) inhibited pre-formed biofilms of S. aureus with an associated downregulation of icaD and sarA gene expression using 0.02% [227]. Upadahay et al. [215] found that ten-fold MIC eugenol dispersed L. moncytogenes on stainless steel surfaces, in vitro. Eugenol dispersed S. enteritidis biofilms at high MIC (between 64 and 128 μg/ml) [230]. At lower MIC, eugenol dispersed C. neoformans and Cryptococcus laurenti biofilms (128 μg/ml and 32 μg/ml, respectively) [231]. Eugenol (0.13 μM) also decreased the expression of the oxidative stress protein NapA [222].
Fig. (3)) Possible antimicrobial mechanisms for eugenol.Legend: Seven potential targets in bacteria, fungi, protozoa, and viruses have been identified by researchers for eugenol that are responsible for its antimicrobial effects. Abbreviations used: ERK (extracellular cell regulated signal kinase), MAPK (membrane-associated protein kinase)
Flagellar, Adhesin, and Virulence Gene Inhibition by Eugenol
Several literature studies found that eugenol in vitro simultaneously affects multiple biofilm targets [215, 217, 236, 237]. Eugenol reduced expression of the flagellar and fimbrial genes in the gram-negative bacteria E. coli 0157:H7 (fimA, fliC, lpfA) [216] and in S. enteritidis in vitro (flgG, fimD) [236] and in vivo (flhC, motA) [237]. Exposure of L. monocytogenes to eugenol decreased motA, flaA, flgE, and fliPgene expression [215]. Several studies reported a simultaneous decrease in motility, adhesin molecules, and bacterial virulence factors [215, 217, 229236]. Eugenol (0.02%) decreases the expression of S. aureus enterotoxin A [227].
Mitochondrial, Ergosterol, and Phospholipid Disruption by Eugenol
Ueda-Nakamura et al. [224] reported L. amazonensis promastigotes and amastigotes exposed to eugenol in vitro (135 and 100 μg/ml, respectively) displayed abnormal changes in cell division, mitochondrial swelling, and cristae. Similar observations have been reported in studies of tumor cell mitochondria [238], where eugenol disrupts fatty acid synthesis pathways. Eugenol could thereby impair fungal sterol and phospholipid synthesis [214, 225, 239]. Pereira et al., [225]. reported that eugenol (256 μg/ml) distorted T. rubrum cell walls in vitro and suggested that this resulted from the disruption of ergosterol synthesis. Darvishi et al., [214] saw similar effects on Saccharomyces cerevisiae and connected these with phospholipid perturbation through decreased expression of the membrane permeases Tat1p and Gap1p. Lone et al. [239], using eugenol derivatives, confirmed that they bound to the lanosterol 14-α demethylase enzyme (CYP51).
Antiviral Effects of Eugenol on MAPK Pathways
Dai et al [233] reported that eugenol (5 μg/ml) decreased oxidative stress in influenza A-infected canine kidney (MDCK) cells and fertilized chicken eggs These authors observed eugenol prevented dissociation of the Beclin-1-Bcl2 complex by decreasing oxidative stress [233]. MAPK pathways inhibited by eugenol included ERK 1/2 and p38 and this translated into a decrease in cellular autophagy [233]. Similar findings have been reported for tumor cells [240].
Other Effects of Eugenol
Eugenol inhibits sortase A generation from S. aureus [71, 241] and disrupts the bacterial tubulin analog FtsZ [75, 76, 242]. Its potent effects on inflammatory pathways in eukaryotic cells have been extensively studied [8, 11, 26, 240]. Similarities between these systems and protozoa probably account for the ability of eugenol to be effective.
Spectrum of Eugenol’s Antimicrobial Activity
Antibacterial
Recently reviewed by Mak et al. [29], eugenol has been shown to be effective against at least twenty common bacterial pathogens in vitro. Joshi et al. [243] reported that eugenol displayed a MIC of between 0.33 and 1.34 mg/ml against gram-positive bacteria, with a higher MIC for gram-negative species (2.08-3.1 mg/ml). Similar in vitro MIC were reported for nine gram-positive bacterial isolates of pacific flounder (0.125-1.0%) [244
