Propolis is a natural pitchy substance collected by Apis mellifera bees from sprouts, flower-buds and exudations of assorted works beginnings ( Ramos et al. , 2007 ) . The coloring material of propolis can be dark green or brown in coloring material with a characteristic spirit of poplar buds, wax, vanilla or honey ( Mahmoud, 2006 ) . Propolis shows adhesive belongingss with the composing of 50 % rosin and vegetable balsam, 30 % wax, 10 % indispensable and aromatic oils, 5 % pollen every bit good as other substances. Bees blend the original propolis with wax after it is partly digested by salivary enzymes and is used as protective substances in their honeycombs, to smooth out the internal walls and surface the carcases of encroachers to forestall their decomposition. Propolis besides acts as a barrier to protect the honeycombs against diseases due to its antiseptic and antimicrobic belongingss ( Sosa et al. , 2007 ; Sforcin, 2007 ) .
The chemical content of propolis is extremely variable and complex where over 300 types of components have been identified and the mixture of its composing is reliant upon the available beginnings in different geographical zones and clime features. The chemical profile of propolis is distinguishable between those collected from temperate, tropical and semitropical zones, besides being affected by the season of aggregation and the types of scrounging bees ( Seidel et al. , 2008 ; Bankova, 2005 ) . Many different components in propolis have been identified, such as phenolic esters, phenolic acids, aromatic aldehydes and intoxicants, flavonoids, sesquiterpenes, naphtalene, ketones, inorganic substances, vitamins et cetera. Amongst all, flavonoid is one of the most widely researched constituents in propolis ( Ramos et al. , 2007 ; Marcucci et al. , 2001 ) .
The usage of propolis as medical specialty can be traced back to antediluvian times where antediluvian Egyptians, Romans and Greeks used it to bring around lesions of the tegument, to mend sores and ulcers every bit good as to embalm the dead. Since the last decennaries, propolis has remained as a prospective campaigner in the research field, motivated by its several biological and pharmacological belongingss, such as immunomodulatory, antitumor, antimicrobic, antiviral, fungicidal, anti-inflammatory and antioxidant effects. At present, eternal list of propolis-containing merchandises has been marketed by the pharmaceutical company as tablets, capsules, sirups, sprays and ethanolic infusions. It is besides often being promoted in health-food shops and a assortment of propolis merchandises are now available worldwide in the signifier of confects, cocoa bars, shampoos, antiseptic picks, skin lotions and toothpastes. Propolis is normally used for the intervention of minor ulcers, thrush, tegument 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 readying because the variableness of its chemical composing has been an obstruction towards achieving its quality confidence.
Up to this clip, the antimicrobic belongingss of propolis have been extensively investigated. One of the researches by Kosalec et Al. on 10 different commercially available propolis samples has proven its antimicrobic effectivity, with entire flavonoids content of above 1 % . The consequences suggested that all 10s propolis samples exert their antimicrobic activities against Gram positive pathogenic strains every bit good as the yeast-like fungus Candida albicans. On the contrary, all merchandises did non demo important disinfectant activity against the Gram negative Escherichia coli. Overall, its activity is more powerful against Gram-positive than Gram-negative bacteriums, and the antimicrobic actions have normally been attributed to the presence of flavonoids in propolis ( Kosalec et al. , 2005 ) . However, the flavonoids content in propolis merchandises have high variableness. An analysis by Watson et Al. ( 2006 ) on several propolis samples shows that merely 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 noteworthy attending given to the antimicrobic belongingss of propolis, a recent research lab research by Sosa et Al. measuring the anti-inflammatory consequence of a few merchandises has demonstrated consequences of high topical anti-inflammatory consequence, particularly for the novel spray preparation ‘Propoli LeniGola Spray Senza Alcool ‘ . These uphold their utility for the intervention of unwritten 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 consequence, galangins act by suppressing the prostaglandin system, where it suppresses the enzyme activity of COX-1 and COX-2, besides suppressing the COX-2 cistron look ( Borrelli et al. , 2002 ) .
Besides that, in recent past the possible application of propolis in unwritten and dental readyings as a cariostatic agent was investigated, and consequences from in vivo surveies have demonstrated a lessening in Streptococcus mutans counts in spit, indissoluble polyose formation and the plaque index, which are the factors doing dental cavities. The research suggested that propolis and its compounds may be utile as an unwritten antiseptic to forestall dental cavities every bit good as other unwritten infective diseases. On top of that, other relevant surveies on propolis besides indicate that it is safer and less toxic to utilize a standardise dosage of propolis as a cariostatic agent as compared to man-made antiseptic medical specialties. However, the fluctuation in the chemical composing of propolis samples from different geographical parts might interfere with the attempt to standardize its chemical profile ( Liberio et al. , 2009 ) .
In short, all propolis merchandises whether they are the conventional or fresh 1s need to be put through farther scrutinies and trials to set up a more dependable 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 besides known as kiping illness, is impacting more than 30 African states and is endangering the lives of 60 million people. The disease is prevailing in sub-Saharan African states particularly the endemic geographic parts 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 figure of 17,500 new instances of HAT has been recorded while the existent figure of instances are estimated between 50,000 to 70,000 ( Hoet et al. , 2004 ; Rodgers, 2009 ; WHO, 2006 ) . The sum of instances may be little on a world-wide graduated table, but it surely can hold a considerable impact on the socioeconomic position of the affected parts in Africa ( Brun et al. , 2010 ) . In add-on, the unsettled issue of the civil war in Africa has besides resulted in the carelessness of the local wellness attention service to battle the disease, therefore increasing the possibility of disease eruption ( Rodgers, 2009 ) .
HAT is caused by the protozoon parasite Trypanosoma brucei, which is transmitted by its vector, the tzetze fly fly of the genus Glossina. The pathogens that cause the Trypanosoma infection in human occurs in two races: Trypanosoma brucei gambiense, which affects cardinal and west Africa, and Trypanosoma brucei rhodesiense, which brought the infection in east and southern Africa ( Brun et al. , 2010 ) . The two different signifiers of parasite show different clinical deductions: T. b. gambiense infection is normally chronic and it can come on to convey decease within months or old ages, while the infection by T. b. rhodesiense is acute and can be fatal within hebdomads if left untreated. African trypanosomes undergo life rhythms which alternate between the blood stream signifiers in the human host followed by the midgut signifiers, epimastigote signifiers, and the morbific metacyclic signifiers in the tzetze fly fly, and later the developed parasite are injected into the human host in the following blood repast ( Hoet et al. , 2004 ; Brun et al. , 2010 ) . In the first beginning phase of infection, trypanosomes parasites are restricted to the lymph and blood systems and the primary marks and symptoms are irregular class of febrility, concern, joint strivings, lymphadenopathy and pruritus. Subsequently the parasites will occupy the liver, spleen and bosom. After a few hebdomads or months of infection, trypanosomes are able to traverse the blood encephalon barrier and onslaught the cardinal nervous system, taking to the encephalitic phase of the illness. During this phase, the patient will see neurological symptoms such as concerns, crossness, inconsistent personality and behavior alterations ; hapless musculus coordination and perchance palsy ; anesthesia and intense itchiness, every bit good as perturbation of the sleep rhythm such as daylight sleepiness which frequently go together with dark clip insomnia ( which is the taking symptom that gives the disease its name ) . All of these clinical presentations give rise to progressive organic structure blowing where the patient will come on to unconsciousness and decease in the concluding phases, if left untreated. ( Hoet et al. , 2004 ; Rodgers, 2009 )
B ) Current drug intervention and its restrictions
While HAT disease still remains as a major menace to public wellness in those affected parts in Africa, the available chemotherapeutic intervention options are still really limited and far from ideal. The available pharmacological therapy was foremost introduced five decennaries ago nevertheless presently there are merely four approved drugs for intervention of HAT ( Figure 1 ) , they are: pentamidine, suramin, melarsoprol and eflornithine ( Hoet et al. , 2004 ) .
Figure 1 Chemical constructions of drugs used to handle first-stage and second-stage Human African Trypanosomiasis.
Pentamidine, which is an aromatic diamidine, is the preferable pick of drug for the intervention of first- phase HAT caused by T. b. gambiense. It is given by intramuscular injections for a hebdomad, or by endovenous extract in saline for 2 hours. By and large, pentamidine has hapless unwritten bioavailability because it is extremely protonated at physiological pH, whilst terrible blood force per unit area take downing can happen following endovenous disposal. Although pentamidine is by and large good tolerated by patients, its intervention can ensue in inauspicious effects such as GI jobs, leukopenia, thrombopenia, hyperkalaemia, hypoglycemia and QT-prolongation. T. b. rhodesiense infections can be treated by Suramin, a colourless, polyanionic sulfonated naphthylamine. The trypanocidal consequence of Suramin is associated with a decrease in respiration rate of the parasite, and later consequences in the suppression of assorted glycolytic enzymes. With the disposal of suramin, inauspicious drug reactions are common but they are normally mild and reversible, including sickness, purging, nephrotoxicity, peripheral neuropathy, agranulosis, bone marrow toxicity and thrombopenia, all of which are dangerous. Both of the drugs are extremely ionic in nature, neither pentamidine nor suramin can readily perforate into the CNS, therefore they are non used to handle the CNS phase of the disease ( Brun et al. , 2010 ; Fairlamb, 2003 ) .
Eflornithine is the most freshly registered drug for the intervention of late-stage HAT. This drug is used as the first-line intervention for second-stage T. b. gambiense disease. Eflornithine nevertheless is still far from being an ideal drug due to its complex disposal procedure which demands a fourteen-day class of endovenous extracts of 100mg/kg organic structure weight every 6 hours. Its inauspicious effects include GI disturbance, paroxysms, pancytopenia and hemiparesis. However all of these side effects are reversible upon backdown of the drug ( Brun et al. , 2010 ; Rodgers, 2009 ) . Another drug, melarsoprol which is foremost used in 1949 is effectual against both T. b. gambiense and T. b. rhodesiense during the late-stage HAT. Melarsoprol is non soluble in H2O and the usual regimen given comprised of three to four series of endovenous drug solution dissolved in propene ethanediol. Melarsoprol causes a few serious inauspicious drug reactions, including emesis, febrility, abdominal disturbance, peripheral neuropathy, thrombophlebitis, and even subconjunctival bleedings. Above the list, the most critical side consequence of melarsoprol intervention is the post-treatment reactive brain disorder ( PTRE ) which will usually developed in 5-10 % of instances, and can ensue in decease in half of the affected patients. However the mechanism of this destructive incidence remains a enigma ( Fairlamb, 2003 ; Rodgers, 2009 ) .
Anti-trypanosomal active natural merchandises obtained from African Savannah part
The current chemotherapy available for HAT is far from being satisfactory. Most of the anti-trypanosomal drug intervention is beset with the jobs of parasite opposition and toxicity. Presently really few research groups have been working on the chemotherapy of this disease due to the deficiency of support and support of the pharmaceutical industry in this country. Besides that, most of the drugs are non readily accessible to rural African patients who particularly suffer the most afflictions from the disease. The conventional drugs available besides lack its practicality as the continuance of intervention are frequently really drawn-out therefore requires particular medical attention and specialised staff which are normally non available in rural African countries ( Ene et al. , 2009 ; Ogbadoyi et al. , 2007 ) . Hence, immediate attending is needed to seek for effectual, non-toxic, easy to administrate and cheap plant-derived beginnings for the intervention of HAT. Natural works merchandises are the best alternate to orthodox drugs because of the diverseness of works species with a great assortment of chemical constructions and curative activities ( Ene et al. , 2009 ) .
In Africa, the savannah part is blessed with a great assortment of works species that shows antitrypanosomal activity. Many of the workss are widely used by the native people as traditional redresss to handle HAT and other parasitic diseases ( Ogbadoyi et al. , 2007 ) . This therefore has motivated the scientists to measure the natural merchandises in vitro for their antitrypanosomal activity ( Bulus et al. , 2008 ; Atawodi & A ; Ogunbusola, 2009 ; Abu et al. , 2009 ) , and in vivo for their curative efficaciousnesss in mice and rats ( Atawodi & A ; Alafiatayo, 2007 ; Ogbadoyi et al. , 2007 ; Ene et al. , 2009 ; Antia et al. , 2009 ) . The potency of natural works merchandises with antitrypanosomal activities has been reported by a figure of researches and there are several claims of plant-derived compounds with possible pharmacological activities.
A recent survey by Emmanuel et Al. has found that the aqueous foliage infusion of a Savannah works Annona senegalensis, which is claimed to be used traditionally to handle kiping illness in Northern Nigeria, had really promising antitrypanosomal consequence. The works infusions were besides shown to be effectual in the intervention of late phase HAT as the parasites has been shown to be cleared wholly from both the blood and cerebrospinal fluid ( CSF ) of the septic animate beings ( Ogbadoyi et al. , 2007 ) . Another works, Terminalia avicennioides which grows copiously in the Savannah part of West Africa, was tested in vitro by a group of researches on its antitrypanosomal efficaciousness. The survey reported that the root and root infusion of Terminalia avicennioides can efficaciously immobilised the trial being and the petroleum infusions were found to possess a few major phytochemicals which are anthraquinones, saponins, flavonoids and tannic acids. A farther showing 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 histories for the antitrypanosomal consequence. However, farther fractional process and in vitro surveies on the antitrypanosomal consequence of saponins are required to corroborate this hypothesis ( Bulus et al. , 2008 ) . Atawodi and Ogunbusola, in a similar survey evaluated the antitrypanosomal belongingss of another African Savannah works Prosopis africana. The solvent infusions of Prosopis africana displayed strong antitrypanosomal activity in both in vitro and in vivo status, at 4mg/ml concentration and 200mg/kg dosage severally. The infusions that displayed antitrypanosomal activity besides contained anthraquinones, saponins, flavonoids and tannic acids. This shows an understanding with the consequences observed for the active phytochemicals found in the infusion of Terminalia avicennioides ( Atawodi & A ; Ogunbusola, 2009 ) , every bit good as of the works Artemesia maciverae. ( Ene et al. , 2009 )
Yet, a farther probe by Ene et Al. showed that the chemical fractions which possess high in vitro and in vivo antitrypanosomal activity was found to incorporate triterpenes and alkaloids, proposing 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 research workers ( Ene et al. , 2009 ) . However one of the recent diaries by Hoet et Al. reported a more in depth probe of the active phytochemicals. Hoet et Al. carried out a bioassay-guided fractional process of an antitrypanosomal lipotropic foliage infusion of Strychnos spinosa. The experiment has successfully isolated four triterpenoids ( Figure 2 ) found in the leaf infusions of Strychnos spinosa. Of all the active fractions investigated, the triterpene with the lupane-type skeleton was found to be the most powerful on Trypanososma brucei brucei. The antitrypanosomal activity of stray triterpenoids and steroid alcohols were reported to be attributable to the presence of either an oxygenated mediety at C-17 side concatenation or an oxygenated map at C-28 ( Hoet et al. , 2007 ) .
Figure 2 Triterpenoids identified in lipotropic leaf infusion of Strychnos spinosa – each with different types of skeleton, i.e. the triterpene with ursane-type skeleton ( 1 & A ; 4 ) , the oleanane-type skeleton ( 2 ) and the ursane-type skeleton ( 3 ) . They all exhibit selective activities on trypanosomes.
These surveies has shown that different phytochemicals may exhibit variable antitrypanosomal consequence and it is of import to place the mechanisms behind the chemical activity. A more in depth cognition of the secondary metabolites that have antitrypanosomal activity will be utile in the direction of trypanosomiasis in the hereafter.
Ecological deduction: Infection with the trypanosome Crithidia bombi in bees
Most societal bees and insects often faced the threat of parasitism, and the infection by parasites frequently can hold terrible fittingness deductions on the host ( Schluns et al. , 2010 ) . In recent old ages, there were dramatic losingss of managed Apis melliferas ‘ settlements due to colony prostration upset, which is the effect of parasitism ( Gillespie, 2010 ) . One of the host-parasite systems that is particularly well-characterised and has important deductions on the ecology system is that of the humblebee host, Bombus spp. , and its trypanosome parasite, Crithidia bombi ( Schluns et al. , 2010 ) . Crithidia bombi is a protozoon besides from the household Trypanosomatidae and comprises of unicellular eucaryotic kinetoplastid mastigophorans and exhibits a single-host life rhythm: the parasite cells infect the intestine wall of bumble bees, they multiplied after consumption and are transmitted through the host fecal matters ( Schluns et al. , 2010 ; Ruiz-Gonzalez & A ; Brown, 2006 ) . C. bombi has the ability to infect legion bumble bee species, such as Bombus impatient and Bombus terrestris ( Ruiz- Gonzalez & A ; Brown, 2006 ) .
During early summer, the prevalence of C. bombi among the field population of humblebees such as Bombus terrestris can be every bit high as 80 % . Bombus terrestris are one-year societal bees and their settlements usually get started in spring by a individual queen ( Yourth et al. , 2008 ) . Spring Queenss and their settlements will go septic when the Queenss 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 venters and so transported to or among flowers ( Ruiz- Gonzalez & A ; Brown, 2006 ) . When the queen or eatage workers return to the settlements with septic cells, the parasite can be transmitted through fecal matters within the nest, or between different settlements where the contaminated fecal matters can fall indiscriminately on dirt or flowers in the field when the bees are winging ( Yourth et al. , 2008 ; Ruiz- Gonzalez & A ; Brown, 2006 ) . A survey by Logan et Al. observed that the fecal matters of septic humblebees were extremely infected by 100s to 1000s of parasite cell, which is a great hazard of infection to the humblebees ( Logan et al. , 2005 ) . Infected settlements were found to turn slower than normal, as the parasite C. bombi will detain the development of worker ovaries and oviposition, which accordingly affect the consistence of intra-colony co-operation. The septic Queenss are besides found to bring forth lesser workers, gynes and males, ensuing in lower sum of resource acquisition, with an overall 40 % lessening in fittingness when compared with clean Queenss. ( Ruiz- Gonzalez & A ; Brown, 2006 ; Yourth et al. , 2008 ) .
The virulency of infective effects of C. bombi is found to be condition-dependent where the mortality of the humblebee host additions dramatically by 50 % under nerve-racking status. A survey by Brown et Al. ( 2003 ) shows that the humblebees are more vulnerable to the negative impact of infection when the settlements experience an energy deficit due to nutrient famishment particularly during inauspicious conditions conditions. This is particularly true for smaller settlements at the start of scrounging season, where their activities are frequently interrupted by rain and cold conditions ( Brown et al. , 2000 ; Brown et al. , 2003 ) . Besides the impact of environmental status, the suppression of the bee ‘s immune system because of malnutrition was found to predispose humblebees to a more terrible parasitism, as the parasites can maturate and reproduce themselves within the host with greater success ( Logan et al. , 2005 ) . When the humblebees are infected, the pro-phenoloxidase in the haemolymph of the septic bees will be activated upon acknowledgment of an appropriate antigenic signal. This constituent immune responds provide an effectual first-line defense mechanism against the parasites. Besides that, another factor which could act upon parasitism in bumblebee population is the geographical place. In Europe, the prevalence of Crithidia bombi was found to be between 50-80 % in lowland sites, in contrary to merely 10 % prevalence in alpine sites ( Gillespie, 2010 ) . All these factors that are mentioned supra, every bit good as other conditional factors such as humidness and temperature all play a portion to explicate the fluctuation in the virulency of the parasites ( Brown et al. , 2003 ) .
In the accustomed environment of humblebees, a broad scope of other animate beings including emmets, honey bees, beetles, butterflies and flies may be the possible vectors of parasites. Ruiz Gonzalez and his co-worker have studied about the possible transmittal of parasites between the humblebees and honey bees. The survey has shown that honey bees could vector the transmittal of trypanosomatids to its specific host – humblebees, through two different paths: through laxation of contaminated fecal matters or by the deposition of the parasite cells when their organic structures contact with flowers. Crithidia bombi can last within the honey-bee backbones for every bit long as 5 yearss, accordingly honey bees have a high potency to infect humblebees as they both portion similar ecology, scrounging behaviors and they are besides phylogenetically close ( Ruiz- Gonzalez & A ; Brown, 2006 ) .
Overall, trypanosomatids infections are widespread and common among bumblebee species. The parasite Crithidia bombi plays an of import function in modulating pollinator populations ( Brown et al. , 2000 ) . The septic bees and its settlements as a whole will hold decreased fittingness and accordingly impact the balance of the ecological system, particularly in countries like the African Savannah part which has abundant botanical beginnings ( Schluns et al. , 2010 ; Ogbadoyi et al. , 2007 ) .
Background on Research
Both honey bees and humblebees are of import pollinators and in fact propolis has helped the bees society to defy trypanosomal infection. The propolis forms a defense mechanism at the entryway of the bee hives which helps to protect the beehive against the interlopers from the exterior ( 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 scrounging in the field ( out of the beehive ) , and both humblebees and honey bees have been shown to hold changing degrees of immune competency against the parasite Crithidia bombi, due to different immune cistron look among host genotypes ( Schluns et al. , 2010 ) . Therefore, it is apparent that propolis which is present in the honeycombs plays polar function in protecting the bees against trypanosomal infection. The bioactive compounds with high antitrypanosomal activity was found in old surveies to be triterpenoids ( Hoet et al. , 2007 ; Ene et al. , 2009 ) . The current conventional therapies available for trypanosomal disease are still really limited and have life-threateing side effects and toxicities. Therefore much research is needed to research phytochemicals with active anti-trypanosomal belongingss.
Purposes of the survey
This research aims to transport out dereplication surveies of antitrypanosomal active triterpenoids from African Propolis samples. Previous surveies have revealed the presence of triterpenoids as the compounds which have active anti-trypanosomal belongingss. Therefore this research was undertaken to:
Perform a phytochemical probe on African propolis samples by qualitative analysis of the chemical and biological components present in the infusion of African propolis, utilizing gas and liquid chromatography coupled to high declaration mass spectroscopy ( GC/LC-HRMS ) and proton atomic magnetic resonance ( NMR ) spectrometry.
Perform bioassay-guided chemical analysis on the stray active compounds to prove for the antitrypanosomal authority in African propolis.
Interpret the chemical construction of the compounds with powerful antitrypanosomal activity based on 1D and 2D NMR spectral informations, therefore profiling the samples.
Compare obtained analytical informations with on-line GCMS-NIST database and other literature informations, measure the pharmacological profile of African propolis, and explicating recommendations for antitrypanosomal therapy.