The endangered tropical pitcher plant Nepenthes khasiana has evolved mechanisms to lure, capture, and digest prey animals (USDA 2003; Eilenberg et al. 2010). Native to areas with nutrient-poor soil, consuming insects enables Nepenthes to gain additional nourishment (Mithofer 2011). Located at the ends of leaf tendrils, their pitchers’ bright colors and nectar secretions are a powerful attractant for bugs (Mithofer 2011). Venturing insects slip on the pitcher’s waxy mouth and fall inside, ultimately drowning in the liquid (Rischer et al. 2002). Glands located at the base of the pitcher secrete hydrolyzing enzymes (Eilenberg et al. 2006). This serves to break the arthropod’s body apart so it can be absorbed by the plant as sustenance (Eilenberg et al. 2006). Under special circumstances, Nepenthes pitcher liquid will also contain potent antifungal compounds called naph-thoquinones (Eilenberg et al. 2010). Their purpose, it is believed, is to prevent captured prey from being consumed by fungi, a competing organism (Eilenberg et al. 2010). For hundreds of years, naphthoquinones have been utilized in Asia and South America for medicinal purposes (Babula et al. 2009). In the United States, emerging studies are investigating how these compounds can be produced, as well as their efficacy against opportunistic human fungal pathogens (Eilenberg et al. 2010). The importance of this research is underscored by the limited variety of antifungal drugs that are commercially available, as well as their susceptibility to pathogenic resistance and cross-tolerance (Kayser et al. 2003).
Naphthoquinones are secondary metabolites produced by Nepenthes’ pitcher liquid (Rischer et al. 2002). Secondary metabolites are not essential for a plant’s regular growth, but aid in lesser functions like defense against herbivory (Bio-Medicine 2012). For centuries, many cultures have been utilizing this type of organic compound for medicinal applications (Karuppusamy 2009). Antibiotics, widely used to treat bacterial infections, are secondary metabolites of mold (ePlant-science.com 2009). One major drawback of antibiotic medication is that its use can suppress the immune system by killing microbes which ward off infection (Merck 2008). Women taking antibiotics are at risk for developing candidiasis, which is named after its causal fungal strain Candida albicans (NIH 2010). Also known as vaginal yeast infection, candidiasis is responsible for more than 10 million doctor visits in the United States each year (Merck 2008). People with compromised immune systems, like the elderly and those with HIV/AIDs, are at risk for devel-oping a more serious form of candidiasis (Merck 2008). This potentially fatal infection occurs when C. albican spores infiltrate the bloodstream (Merck 2008). There is a limited variety of commercial antimycotics (antifungal drugs) that are currently available (Eilenberg et al. 2010). Over-usage of these antimycotics, which tend to operate by the same mechanism of action, is cre-ating drug-resistant fungal strains (Kayser et al. 2003). Moreover, concomitant use of antifungal drugs is causing cross-resistance in pathogens (Eilenberg et al. 2006). Developing antimycotics which function differently than current ones is needed to ensure our continuing ability to treat fungal infections.
Eilenberg et al. (2006) demonstrated a method of inducing the production of antifungal second-ary metabolites known as naphthoquinones in Nepenthes khasiana. Published by the Journal of Experimental Botany, the article entitled “Isolation and characterization of chitinase genes from pitchers of the carnivorous plant Nepenthes khasiana” showed that naphthoquinone production can be induced via colloidal chitin injections into closed N. khasiana pitchers. Chitin is the main component of insect exoskeleton, and is also an integral part of fungal cell walls (Collinge et al. 1993). Chitinase, an enzyme which degrades chitin, was found to be an implicit component of N. khasiana pitcher liquid. Chitinase appears to function in both carnivory, by breaking-down insects, and defense, by degrading fungi. The liquid of closed N. khasiana pitchers (referred to as “prey-challenged”) did not contain naphthoquinones, but two types of chitinases were present. When immature Nepenthes pitchers were injected with chitin, napthiquinones and a third type of chitinase were detected. Chitinase was also found in open pitchers containing prey, but naphthoquinones were not. Though the pitcher liquid does not appear to contain the potent antifungal naphthoquinones under normal circumstances, it may still exhibit antifungal activities due to the endogenous presence of chitinase. This might explain why there is “anthropological evidence of [people] using the liquid from unopened traps as a curing means” (Eilenberg et al. 2010).
In 2010, the Journal of Experimental Botany published a compelling article entitled “Induced production of antifungal naphthoquinones in the pitchers of the carnivorous plant Nepenthes kha-siana”. In this study, Eilenberg et al. (2010) examined the antifungal effects of naphthoquinones on various strains of human pathogenic fungi. Similar to previous experiments, the team induced naph-thoquinone production by injecting closed Nepenthes khasiana pitchers with colloidal chitin. The N. khasiana pitcher liquid of chitin-induced, prey-challenged, and open pitchers containing prey was then screened for the presence of naphthoquinones. Only the chitin-induced pitcher liquid con-tained these secondary metabolites. The absence of naphthoquinones from open pitchers containing prey implies that chitin alone is not responsible for its production. However, there is evidence that the components of chitin are needed to form the naphthoquinone compound (Rischer et al. 2002). Eilenberg et al. postulate that injecting the pitchers with a syringe simulates a predatory presence. As previously discussed, the major natural role of secondary metabolites is to aid in defense (Bio-medicine 2012). It is possible that naphthoquinones are synthesized “to avoid the fast consumption of organic compounds by competitors before being absorbed by the pitcher cells” (Eilenberg et al. 2010). That is, Nepenthes may produce naphthoquinones as a way of preventing insects from decomposing due to fungal activity before the plant is able to digest them.
The Eilenberg et al. study in 2010 demonstrated that chitin-induced pitcher liquid exerts an-tifungal and fungicidal effects on the yeast strain Candida albicans. The article cited an in vitro experiment in which naphthoquinone derivatives inhibited C. albicans as effectively as Diflucan, the leading clinical yeast infection treatment (Tandon et al. 2009). Plumbagin is a naphthoquinone derivative that has been isolated and studied extensively due to its proven ability to inhibit and kill human pathogenic fungi (Eilenberg et al. 2010). The major problem with plumbagin is its high cytotoxicy and low therapeutic selectivity (Eilenberg et al. 2010). Droserone, an oxygenated de-rivative of plumbagin, exerted an antifungal effect on opportunistic fungal pathogens with a much lower level of toxicity. In addition, droserone demonstrated a different mechanism of action from that of mainstream antimycotics, like Diflucan. The majority of mainstream antimycotics prevent fungal growth by inhibiting fungal enzymes (Kayser et al. 2003). Droserone instead catabolizes, or breaks-down, fungal spores. This is advantageous considering the growing prevalence of antimy-cotic resistance (Kayser et al. 2003). In addition to inhibiting C. albicans, naphthoquinones dem-onstrated lethality to Aspergillus, a common household fungus which causes the life-threatening disease aspergillosis (Merck 2008).
Extracting secondary metabolites from plant cell cultures can often be a difficult and inefficient process (Eilenberg et al. 2010). When injected with chitin, Nepenthes pitchers produce moderate amounts of medicinal compounds which require no additional purification (Eilenberg et al. 2010). In addition to serving as an antifungal agent, naphthoquinones derived from Nepenthes pitchers have the potential for a wide array of applications. These compounds are so versatile, they demon-strate antifungal, anti-cancer (Eilenberg et al. 2010; Sandur et al. 2006), anti-inflammatory (Lien et al. 1996), antimalarial (Biot et al. 2004), antiviral (Sacau et al. 2003), antiallergic (Lien et al. 1996), and antibacterial (Tandon et al. 2009) activities. Napthoquinones may also serve as a totally “green” insect control because of their ability to inhibit insect ecdysis, or moulting, which is essential for insect survival (Eilenberg et al. 2010). Considering all of the potential applications in which Nepen-thes’ chitin-induced pitcher liquid can be used, it is lucky that this tropical plant is able to produce exceptional yields of pure compounds, relatively simply.
Top image credit: http://www.quickstopmedia.com/bizzare-plants/
This article was featured in the June 2013 Volume 42 Carnivorous Plant Newsletter. For a list of Resources follow the link: https://docs.google.com/file/d/1cH6LLWjHwVneatXZmyhXnzqlkXB6STz8cBFyV8cv_hH0fZYucoTdjVquqRvz/edit
Jess Cuest 