Searching for effective natural products against Human African Trypanosomiasis (HAT) with special reference to African natural resources

For centuries, African natives have been facing various infectious tropical illnesses, among which African trypanosomiases are some of the most frequent relevant parasitic diseases. African trypanosomiases, commonly called sleeping sickness in humans (HAT; Human African Trypanosomiasis) and Nagana in domestic livestock, affect a huge number of people living in poverty in 36 sub-Saharan countries, resulting in a key socioeconomic impact. After a century of outbreaks, due to political instability and lack of funding, around 70 million people and 50 million cattle are still at risk of exposure in Africa. Trypanosomiasis is transmitted by the bite of insects from the Glossina spp (Glossinidae) and is fatal in humans, if untreated. While taking a blood meal, infected Glossina flies can spread extracellular protozoans from the species Trypanosoma brucei. There are three morphologically indistinguishable subspecies of T. brucei. The subspecies T. b. gambiense is responsible for a chronic form of the human disease, while T. b. rhodesiense causes an acute form, which more rapidly leads to death. Both subspecies are infective to humans, whereas T. b. brucei is only infective to animals. During the early stage of the disease or hemolymphatic phase, the parasite is restricted to the blood and lymph and after months or years it invades the central nervous system resulting in various neurological symptoms including sleeping disturbance. As for other neglected tropical diseases, the chemotherapeutical arsenal against HAT is based on limited, expensive and often toxic medicines that are administered parentally in a context of poverty and lack of qualified personnell in healthcare centers. The few drugs that are available are pentamidine and suramin for the early stage disease and eflornithine (also in combination with nifurtimox) and melarsoprol for the late stage when the parasite infects the brain. Overall, the situation described above highlights the critical nature of this phenomena and the urgent need to explore new sources of potentially effective and safe compounds for therapy. In this scenario the naturally-occurring products may play a crucial role as source of bioactive drug candidates. With this vision in mind, in Chapter 2 I performed a complete phytochemical analysis on both polar and volatile compounds of T. diversifolia collected from a geographically isolated population living in Dschang, Cameroon and I assessed their biological activities (antitrypanosomal and amtimicrobial activities). The main secondary metabolites occurring in the T. diversifolia methanolic extract were isolated by column chromatography and structurally elucidated by MS and NMR techniques. Tagitinins C emerged as the most active compound against T. brucei (TC221) with an IC50 value  of  0.0042  μg/mL.  This  activity  was  4.5  times  better  than  that of the reference drug suramin. Then I analysed the chemical composition and the antimicrobial effects of the essential oil (EO) hydrodistilled from inflorescences of T. diversifolia. Results showed that T. diversifolia EO was mostly active against Staphylococcus aureus and selectively inhibited in vitro the NAD biosynthetic enzyme NadD from S. aureus (IC50 of ∼60 g/mL). Besides its extensive utilizations in the traditional medicine, the plant is believed to have a great potential in agriculture. For this reason, I decided to evaluate the T. diversifolia polar extracts against the two-spotted spider mite Tetranychus urticae (Tetranychidae), which is one of the most economically important arthropod pests worldwide. The ethyl acetate extract resulted as the most active oviposition inhibitor, with an ED50 value of 44.3 µg.cm-3 and an ED90 of 121.5 µg.cm-3. In Chapter 3, I investigated a lipophilic extract of Onosma visianii roots containing 12% of shikonin derivatives. The phytochemical investigation of the lipophilic extract resulted in the isolation of 12 naphtoquinone derivatives which were evaluated against Trypanosoma brucei. Isobutylshikonin and isovalerylshikonin emerged as the most active naphtoquinone derivatives, showing an IC50 of 3.3 and 2.7 g/mL, respectively. Furthermore, isovalerylshikonin provided an inhibition of Glossina palpalis acetylcholinesterase (gpAChE) (IC50 =  7.1  μg/mL),  stronger  than   isobutyrylshikonin (IC50 =  91.3  μg/mL),  with  a  significant  tse-tse fly versus human selectivity (SI = 7.2). In Chapter 4, I oriented my attention to the Apiaceae family, which is a class of aromatic plants rich of EOs. Four out of nine Apiaceae EOs resulted active against T. brucei showing an IC50 in the range 2.7-10.7 g/mL. Terpinolene, one the major isolated component of these oils, was particularly active with an IC50 value of 0.035 g/mL (0.26 µM) and a selectivity index (SI) of 180. As part of the extended family of naturally-occurring products, sesquiterpenes hold promising inhibitory effects against the bloodstream forms of T. brucei. For this reason, in Chapter 5, I decided to explore the potential of Smyrnium olusatrum EOs obtained and its main oxygenated sesquiterpenes,  namely  germacrone,  isofuranodiene,  and  β-acetoxyfuranoeudesm-4(15)-ene, as potential inhibitors of T. brucei. The EOs obtained efficiently inhibited the growth of parasite with IC50 ranging from 1.9 to 4.0 g/mL. Among the isolated main EOs components, isofuranodiene exhibited a significant and selective inhibitory activity against T. brucei (IC50 = 0.6 g/mL, SI = 30). In Chapter 6, I finally selected six medicinal and aromatic plants traditionally used in Cameroon to treat several disorders, including infections and parasitic diseases. Then I evaluated the activity of their EOs against T. brucei TC221 and their selectivity against Balb/3T3 cells, used as counter-screen for cytotoxicity. The most relevant outcomes showed that the EOs from A. indica, A. daniellii and E. giganteus were the most active ones, with IC50 values of 15.21, 7.65 and 10.50 g/mL, respectively. Overall, the results of my PhD thesis provided new insights into the potential of naturallyoccurring compounds as valuable sources for the development of innovative trypanocidal drugs or botanical insecticides.