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Propolis is a natural resinous substance collected by Apis mellifera bees from sprouts, flower-buds and exudates of various plant sources (Ramos et al., 2007). The colour of propolis can be dark green or brown in colour with a characteristic flavour of poplar buds, wax, vanilla or honey (Mahmoud, 2006). Propolis shows adhesive properties with the composition of 50% resin and vegetable balsam, 30% wax, 10% essential and aromatic oils, 5% pollen as well as other substances. Bees blend the original propolis with wax after it is partially digested by salivary enzymes and is used as protective substances in their honeycombs, to smooth out the internal walls and coat the carcasses of invaders to prevent their decomposition. Propolis also acts as a barrier to protect the honeycombs against diseases due to its antiseptic and antimicrobial properties (Sosa et al., 2007; Sforcin, 2007).
The chemical content of propolis is highly variable and complex where over 300 types of constituents have been identified and the assortment of its composition is reliant upon the available sources in different geographical zones and climate characteristics. The chemical profile of propolis is distinct between those collected from temperate, tropical and subtropical zones, besides being affected by the season of collection and the types of foraging bees (Seidel et al., 2008; Bankova, 2005). Many different constituents in propolis have been identified, such as phenolic esters, phenolic acids, aromatic aldehydes and alcohols, flavonoids, sesquiterpenes, naphtalene, ketones, inorganic substances, vitamins et cetera. Amongst all, flavonoid is one of the most widely researched components in propolis (Ramos et al., 2007; Marcucci et al., 2001).
The use of propolis as medicine can be traced back to ancient times where ancient Egyptians, Romans and Greeks used it to cure lesions of the skin, to heal sores and ulcers as well as to embalm the dead. Since the last decades, propolis has remained as a prospective candidate in the research field, motivated by its several biological and pharmacological properties, such as immunomodulatory, antitumor, antimicrobial, antiviral, antifungal, anti-inflammatory and antioxidant effects. At present, endless list of propolis-containing products has been marketed by the pharmaceutical company as tablets, capsules, syrups, sprays and ethanolic extracts. It is also frequently being promoted in health-food stores and a variety of propolis products are now available worldwide in the form of candies, chocolate bars, shampoos, antiseptic creams, skin lotions and toothpastes. Propolis is commonly used for the treatment of minor ulcers, thrush, skin or respiratory infections. (Sforcin J.M., 2007; Kosalec et al., 2005; Ramos et al., 2007). However, even now propolis is yet to be licensed as an official pharmaceutical preparation because the variability of its chemical composition has been an obstacle towards attaining its quality assurance.
Up to this time, the antimicrobial properties of propolis have been extensively investigated. One of the researches by Kosalec et al. on ten different commercially available propolis samples has proven its antimicrobial effectiveness, with total flavonoids content of above 1%. The results suggested that all ten propolis samples exert their antimicrobial activities against Gram positive pathogenic strains as well as the yeast-like fungus Candida albicans. On the contrary, all products did not show significant bactericidal activity against the Gram negative Escherichia coli. Overall, its activity is more potent against Gram-positive than Gram-negative bacteria, and the antimicrobial actions have usually been attributed to the presence of flavonoids in propolis (Kosalec et al., 2005). Nevertheless, the flavonoids content in propolis products have high variability. An analysis by Watson et al. (2006) on several propolis samples shows that only the European and Asian propolis samples have high flavonoids content, whereas the African samples gave low response for flavones and flavonols (Watson et al., 2006).
Despite the notable attention given to the antimicrobial properties of propolis, a recent laboratory research by Sosa et al. evaluating the anti-inflammatory effect of a few products has demonstrated results of high topical anti-inflammatory effect, especially for the novel spray formulation 'Propoli LeniGola Spray Senza Alcool'. These uphold their usefulness for the treatment of oral and pharyngeal inflammatory diseases (Sosa et al., 2007). The anti-inflammatory activity of propolis can be accredited to the presence of galangins flavonoid. For its anti-inflammatory effect, galangins act by inhibiting the prostaglandin system, where it suppresses the enzyme activity of COX-1 and COX-2, besides inhibiting the COX-2 gene expression (Borrelli et al., 2002).
Besides that, in recent past the potential application of propolis in oral and dental preparations as a cariostatic agent was investigated, and results from in vivo studies have demonstrated a decrease in Streptococcus mutans counts in saliva, insoluble polysaccharide formation and the plaque index, which are the factors causing dental caries. The research suggested that propolis and its compounds may be useful as an oral antiseptic to prevent dental caries as well as other oral infectious diseases. On top of that, other relevant studies on propolis also indicate that it is safer and less toxic to use a standardise dose of propolis as a cariostatic agent as compared to synthetic antiseptic medicines. Nevertheless, the variation in the chemical composition of propolis samples from different geographical regions might interfere with the effort to standardise its chemical profile (Libério et al., 2009).
In short, all propolis products whether they are the conventional or novel ones need to be put through further examinations and tests to establish a more reliable chemical profile and relevant guidelines before propolis can be safely employed into clinical application.
The Disease: Trypanosomes
a) A brief background on the disease
Human African Trypanosomiasis (HAT), which is also known as sleeping sickness, is affecting more than 30 African countries and is threatening the lives of 60 million people. The disease is prevalent in sub-Saharan African countries especially the endemic geographic regions such as Angola, Democratic Republic of Congo, Uganda and southern Sudan. Based on the recent epidemiological figures reported by the World Health Organisation (WHO), a number of 17,500 new cases of HAT has been recorded while the actual number of cases are estimated between 50,000 to 70,000 (Hoet et al., 2004; Rodgers, 2009; WHO, 2006). The amount of cases may be small on a worldwide scale, but it certainly can have a considerable impact on the socioeconomic status of the affected regions in Africa (Brun et al., 2010). In addition, the unsettled issue of the civil war in Africa has also resulted in the negligence of the local health care service to combat the disease, thus increasing the possibility of disease outbreak (Rodgers, 2009).
HAT is caused by the protozoan parasite Trypanosoma brucei, which is transmitted by its vector, the tsetse fly of the genus Glossina. The pathogens that cause the Trypanosoma infection in human occurs in two subspecies: Trypanosoma brucei gambiense, which affects central and west Africa, and Trypanosoma brucei rhodesiense, which brought the infection in east and southern Africa (Brun et al., 2010). The two different forms of parasite show different clinical implications: T. b. gambiense infection is usually chronic and it can progress to bring death within months or years, while the infection by T. b. rhodesiense is acute and can be fatal within weeks if left untreated. African trypanosomes undergo life cycles which alternate between the bloodstream forms in the human host followed by the midgut forms, epimastigote forms, and the infective metacyclic forms in the tsetse fly, and subsequently the developed parasite are injected into the human host in the next blood meal (Hoet et al., 2004; Brun et al., 2010). In the first beginning stage of infection, trypanosomes parasites are restricted to the lymph and blood systems and the primary signs and symptoms are irregular course of fever, headache, joint pains, lymphadenopathy and pruritus. Subsequently the parasites will invade the liver, spleen and heart. After a few weeks or months of infection, trypanosomes are able to cross the blood brain barrier and attack the central nervous system, leading to the encephalitic stage of the sickness. During this stage, the patient will experience neurological symptoms such as headaches, irritability, inconsistent personality and behaviour changes; poor muscle coordination and possibly paralysis; anaesthesia and intense itching, as well as disturbance of the sleep cycle such as daytime somnolence which often go together with night time insomnia (which is the leading symptom that gives the disease its name). All of these clinical presentations give rise to progressive body wasting where the patient will progress to unconsciousness and death in the final stages, if left untreated. (Hoet et al., 2004; Rodgers, 2009)
b) Current drug treatment and its limitations
While HAT disease still remains as a major threat to public health in those affected regions in Africa, the available chemotherapeutic treatment options are still very limited and far from ideal. The available pharmacological therapy was first introduced five decades ago however currently there are only four approved drugs for treatment of HAT (Figure 1), they are: pentamidine, suramin, melarsoprol and eflornithine (Hoet et al., 2004).
Figure 1 Chemical structures of drugs used to treat first-stage and second-stage Human African Trypanosomiasis.
Pentamidine, which is an aromatic diamidine, is the preferred choice of drug for the treatment of first- stage HAT caused by T. b. gambiense. It is given by intramuscular injections for a week, or by intravenous infusion in saline for 2 hours. Generally, pentamidine has poor oral bioavailability because it is highly protonated at physiological pH, whilst severe blood pressure lowering can occur following intravenous administration. Although it is generally well tolerated, pentamidine treatment can result in adverse effects such as gastrointestinal problems, leucopenia, thrombocytopenia, hyperkalaemia, hypoglycaemia and QT-prolongation. T. b. rhodesiense infections can be treated by Suramin, a colourless, polyanionic sulfonated naphthylamine. The trypanocidal effect of Suramin is associated with a reduction in respiration rate of the parasite, and subsequently results in the inhibition of various glycolytic enzymes. With the administration of suramin, adverse drug reactions are common but they are usually mild and reversible, including nausea, vomiting, nephrotoxicity, peripheral neuropathy, agranulocytosis, bone marrow toxicity and thrombocytopenia, all of which are life-threatening. Both of the drugs are highly ionic in nature, neither pentamidine nor suramin can readily penetrate into the CNS, thus they are not used to treat the CNS stage of the disease (Brun et al., 2010; Fairlamb, 2003).
Eflornithine is the most newly registered drug for the treatment of late-stage HAT. This drug is used as the first-line treatment for second-stage T. b. gambiense disease. Eflornithine nevertheless is still far from being an ideal drug due to its complex administration process which demands a fourteen-day course of intravenous infusions of 100mg/kg body weight every 6 hours. Its adverse effects include gastrointestinal upset, convulsions, pancytopenia and hemiparesis. However all of these side effects are reversible upon withdrawal of the drug (Brun et al., 2010; Rodgers, 2009). Another drug, melarsoprol which is first used in 1949 is effective against both T. b. gambiense and T. b. rhodesiense during the late-stage HAT. Melarsoprol is not soluble in water and the usual regimen given comprised of three to four series of intravenous drug solution dissolved in propylene glycol. Melarsoprol causes a few serious adverse drug reactions, including vomiting, fever, abdominal upset, peripheral neuropathy, thrombophlebitis, and even subconjunctival haemorrhages. Above the list, the most critical side effect of melarsoprol treatment is the post-treatment reactive encephalopathy (PTRE) which will normally developed in 5-10% of cases, and can result in death in half of the affected patients. However the mechanism of this destructive incidence remains a mystery (Fairlamb, 2003; Rodgers, 2009).
Anti-trypanosomal active natural products obtained from African savannah region
The current chemotherapy available for HAT is far from being satisfactory. Most of the anti-trypanosomal drug treatment is beset with the problems of parasite resistance and toxicity. Currently very few research groups have been working on the chemotherapy of this disease due to the lack of funding and support of the pharmaceutical industry in this area. Besides that, most of the drugs are not readily accessible to rural African patients who especially suffer the most afflictions from the disease. The conventional drugs available also lack its practicality as the duration of treatment are often very lengthy thus requires special medical care and specialised staff which are usually not available in rural African areas (Ene et al., 2009; Ogbadoyi et al., 2007). Hence, immediate attention is needed to search for effective, non-toxic, easy to administer and inexpensive plant-derived sources for the treatment of HAT. Natural plant products are the best alternative to orthodox drugs because of the diversity of plant species with a great variety of chemical structures and therapeutic activities (Ene et al., 2009).
In Africa, the savannah region is blessed with a great variety of plant species that shows antitrypanosomal activity. Many of the plants are widely used by the native people as traditional remedies to treat HAT and other parasitic diseases (Ogbadoyi et al., 2007). This therefore has motivated the scientists to evaluate the natural products in vitro for their antitrypanosomal activity (Bulus et al., 2008; Atawodi & Ogunbusola, 2009; Abu et al., 2009), and in vivo for their therapeutic efficacies in mice and rats (Atawodi & Alafiatayo, 2007; Ogbadoyi et al., 2007; Ene et al., 2009; Antia et al., 2009). The potential of natural plant products with antitrypanosomal activities has been reported by a number of researches and there are several claims of plant-derived compounds with possible pharmacological activities.
A recent study by Emmanuel et al. has found that the aqueous leaf extract of a savannah plant Annona senegalensis, which is claimed to be used traditionally to treat sleeping sickness in Northern Nigeria, had very promising antitrypanosomal effect. The plant extracts were also shown to be effective in the treatment of late stage HAT as the parasites has been shown to be cleared completely from both the blood and cerebrospinal fluid (CSF) of the infected animals (Ogbadoyi et al., 2007). Another plant, Terminalia avicennioides which grows abundantly in the Savannah region of West Africa, was tested in vitro by a group of researches on its antitrypanosomal efficacy. The study reported that the stem and root extract of Terminalia avicennioides can effectively immobilised the test organisms and the crude extracts were found to possess a few major phytochemicals which are anthraquinones, saponins, flavonoids and tannins. A further screening on the columns has revealed that the active ethyl acetate/methanol fractions were rich in saponins and this gives the hypothesis that saponins may be the bioactive compounds which accounts for the antitrypanosomal effect. However, further fractionation and in vitro studies on the antitrypanosomal effect of saponins are required to confirm this hypothesis (Bulus et al., 2008). Atawodi and Ogunbusola, in a similar study evaluated the antitrypanosomal properties of another African Savannah plant Prosopis africana. The solvent extracts of Prosopis africana displayed strong antitrypanosomal activity in both in vitro and in vivo condition, at 4mg/ml concentration and 200mg/kg dose respectively. The extracts that displayed antitrypanosomal activity also contained anthraquinones, saponins, flavonoids and tannins. This shows an agreement with the results observed for the active phytochemicals found in the extract of Terminalia avicennioides (Atawodi & Ogunbusola, 2009), as well as of the plant Artemesia maciverae. (Ene et al., 2009)
Yet, a further investigation by Ene et al. showed that the chemical fractions which possess high in vitro and in vivo antitrypanosomal activity was found to contain triterpenes and alkaloids, suggesting that the bioactive compounds is either one or both of these two compounds, while the mechanism of antitrypanosomal activity by the phytochemicals is still unknown by the researchers (Ene et al., 2009). However one of the recent journals by Hoet et al. reported a more in depth investigation of the active phytochemicals. Hoet et al. carried out a bioassay-guided fractionation of an antitrypanosomal lipophilic leaf extract of Strychnos spinosa. The experiment has successfully isolated four triterpenoids (Figure 2) found in the leaf extracts of Strychnos spinosa. Of all the active fractions investigated, the triterpene with the lupane-type skeleton was found to be the most potent on Trypanososma brucei brucei. The antitrypanosomal activity of isolated triterpenoids and sterols were reported to be attributable to the presence of either an oxygenated moiety at C-17 side chain or an oxygenated function at C-28 (Hoet et al., 2007).
Figure 2 Triterpenoids identified in lipophilic leaf extract of Strychnos spinosa - each with different types of skeleton, i.e. the triterpene with ursane-type skeleton (1 & 4), the oleanane-type skeleton (2) and the ursane-type skeleton (3). They all exhibit selective activities on trypanosomes.
These studies has shown that different phytochemicals may exhibit variable antitrypanosomal effect and it is important to identify the mechanisms behind the chemical activity. A more in depth knowledge of the secondary metabolites that have antitrypanosomal activity will be useful in the management of trypanosomiasis in the future.
Ecological implication: Infection with the trypanosome Crithidia bombi in bees
Most social bees and insects frequently faced the menace of parasitism, and the infection by parasites often can have severe fitness implications on the host (Schluns et al., 2010). In recent years, there were dramatic losses of managed honeybees' colonies due to colony collapse disorder, which is the consequence of parasitism (Gillespie, 2010). One of the host-parasite systems that is especially well-characterised and has significant implications on the ecology system is that of the bumblebee host, Bombus spp., and its trypanosome parasite, Crithidia bombi (Schluns et al., 2010). Crithidia bombi is a protozoan also from the family Trypanosomatidae and comprises of unicellular eukaryotic kinetoplastid flagellates and exhibits a single-host life cycle: the parasite cells infect the gut wall of bumble bees, they multiplied after ingestion and are transmitted through the host faeces (Schluns et al., 2010; Ruiz-Gonzalez & Brown, 2006). C. bombi has the ability to infect numerous bumble bee species, such as Bombus impatient and Bombus terrestris (Ruiz- Gonzalez & Brown, 2006).
During early summer, the prevalence of C. bombi among the field population of bumblebees such as Bombus terrestris can be as high as 80%. Bombus terrestris are annual social bees and their colonies normally get started in spring by a single queen (Yourth et al., 2008). Spring queens and their colonies will become infected when the queens or workers forage on contaminated flowers in the field, where the parasite cells Crithidia bombi can be picked up on the bee's mouthparts, legs, hair or abdomen and then transported to or among flowers (Ruiz- Gonzalez & Brown, 2006). When the queen or forage workers return to the colonies with infected cells, the parasite can be transmitted through faeces within the nest, or between different colonies where the contaminated faeces can fall randomly on soil or flowers in the field when the bees are flying (Yourth et al., 2008; Ruiz- Gonzalez & Brown, 2006). A study by Logan et al. observed that the faeces of infected bumblebees were highly infected by hundreds to thousands of parasite cell, which is a great risk of infection to the bumblebees (Logan et al., 2005). Infected colonies were found to grow slower than normal, as the parasite C. bombi will delay the development of worker ovaries and oviposition, which consequently affect the consistency of intra-colony co-operation. The infected queens are also found to produce lesser workers, gynes and males, resulting in lower amount of resource acquisition, with an overall 40% decrease in fitness when compared with uninfected queens. (Ruiz- Gonzalez & Brown, 2006; Yourth et al., 2008).
The virulence of pathogenic effects of C. bombi is found to be condition-dependent where the mortality of the bumblebee host increases dramatically by 50% under stressful condition. A study by Brown et al. (2003) shows that the bumblebees are more vulnerable to the negative impact of infection when the colonies experience an energy shortfall due to food starvation especially during adverse weather conditions. This is especially true for smaller colonies at the start of foraging season, where their activities are often interrupted by rain and cold weather (Brown et al., 2000; Brown et al., 2003). Besides the impact of environmental condition, the suppression of the bee's immune system because of malnutrition was found to predispose bumblebees to a more severe parasitism, as the parasites can mature and reproduce themselves within the host with greater success (Logan et al., 2005). When the bumblebees are infected, the pro-phenoloxidase in the haemolymph of the infected bees will be activated upon recognition of an appropriate antigenic signal. This constitutive immune responds provide an effective first-line defence against the parasites. Besides that, another factor which could influence parasitism in bumblebee population is the geographical position. In Europe, the prevalence of Crithidia bombi was found to be between 50-80% in lowland sites, in contrary to only 10% prevalence in alpine sites (Gillespie, 2010). All these factors that are mentioned above, as well as other conditional factors such as humidity and temperature all play a part to explain the variation in the virulence of the parasites (Brown et al., 2003).
In the habitual environment of bumblebees, a wide range of other animals including ants, honey bees, beetles, butterflies and flies may be the potential vectors of parasites. Ruiz Gonzalez and his colleague have studied about the possible transmission of parasites between the bumblebees and honey bees. The study has shown that honey bees could vector the transmission of trypanosomatids to its specific host - bumblebees, through two different routes: through defecation of contaminated faeces or by the deposition of the parasite cells when their bodies contact with flowers. Crithidia bombi can survive within the honey-bee guts for as long as 5 days, consequently honey bees have a high potential to infect bumblebees as they both share similar ecology, foraging behaviour and they are also phylogenetically close (Ruiz- Gonzalez & Brown, 2006).
Overall, trypanosomatids infections are widespread and common among bumblebee species. The parasite Crithidia bombi plays an important role in regulating pollinator populations (Brown et al., 2000). The infected bees and its colonies as a whole will have decreased fitness and consequently affect the balance of the ecological system, especially in areas like the African Savannah region which has abundant botanical sources (Schluns et al., 2010; Ogbadoyi et al., 2007).
Background on Research
Both honey bees and bumblebees are important pollinators and in fact propolis has helped the bees society to withstand trypanosomal infection. The propolis forms a defence at the entrance of the bee hives which helps to protect the beehive against the intruders from the outside (Seidel et al., 2008), including the parasite Crithidia bombi. However, the bee host is found to be vulnerable to the infection by trypanosome parasite when it is foraging in the field (out of the beehive), and both bumblebees and honey bees have been shown to have varying levels of immune competence against the parasite Crithidia bombi, due to different immune gene expression among host genotypes (Schluns et al., 2010). Thus, it is evident that propolis which is present in the honeycombs plays pivotal role in protecting the bees against trypanosomal infection. The bioactive compounds with high antitrypanosomal activity was found in previous studies to be triterpenoids (Hoet et al., 2007; Ene et al., 2009). The current conventional therapies available for trypanosomal disease are still very limited and have life-threateing side effects and toxicities. Therefore much research is needed to explore phytochemicals with active anti-trypanosomal properties.
Aims of the study
This research aims to carry out dereplication studies of antitrypanosomal active triterpenoids from African Propolis samples. Previous studies have revealed the presence of triterpenoids as the compounds which have active anti-trypanosomal properties. Therefore this research was undertaken to:
Perform a phytochemical investigation on African propolis samples by qualitative analysis of the chemical and biological constituents present in the extract of African propolis, using gas and liquid chromatography coupled to high resolution mass spectrometry (GC/LC-HRMS) and proton nuclear magnetic resonance (NMR) spectroscopy.
Perform bioassay-guided chemical analysis on the isolated active compounds to test for the antitrypanosomal potency in African propolis.
Interpret the chemical structure of the compounds with potent antitrypanosomal activity based on 1D and 2D NMR spectral data, thus profiling the samples.
Compare obtained analytical data with online GCMS-NIST database and other literature data, evaluate the pharmacological profile of African propolis, and formulating recommendations for antitrypanosomal therapy.