Banca de DEFESA: ALEXSANDRA FRAZÃO DE ANDRADE

EVALUATION OF BIOACTIVES FROM THE MICROALGAE Chlorella vulgaris FOR HEALING INJURIES RESULTING FROM AMERICAN TEGUMENTARY LEISHMANIASIS

microalgae, Leishmania, lectin

Leishmaniasis is a neglected tropical disease caused by parasites of the genus
Leishmania. This disease shows two main forms: visceral leishmaniasis (VL) and
tegumentary leishmaniasis (TL). The different clinical sub-forms of TL are associated
with the different species of Leishmania, as well as the genetic, nutritional and/or
immunological factors of the host. The immune response plays an important role in
defense, since an exacerbated Th1 response leads to tissue damage and the persistence
of the lesion. The therapeutic strategies used have shown low efficacy, high toxicity,
adverse effects, lack of immunomodulation, parasite resistance and difficulty in
administering the drug. Therapeutic alternatives using natural products can improve the
therapeutic regimen, and microalgae have shown potential results, which can be
explained by the diversity of metabolites such as carbohydrates, terpenes and proteins,
including lectins. The aim of this study was to evaluate the potential of the bioactives of
the microalga C. vulgaris as an adjuvant to the current therapy for American
Tegumentary Leishmaniasis. C. vulgaris (UTEX 1803) was cultivated in Bold Basal
medium supplemented with 1% corn steep liquor, concentrated, resuspended in 0.15 M
Tris-HCl-NaCl, pH 7.5 at 50 mg/mL, sonicated using ten 1 min pulses with a 1 min
interval. The liquid fraction obtained was called cell extract (CE). The proteins present
in the CE were precipitated by adding 0-60% ammonium sulphate and purified by anion
exchange chromatography. The specificity of the lectin (CVU) to carbohydrates was
determined by its ability to reduce hemagglutinating activity (HA) with 10 mM of D-
fructose, D-galactose, D-glucose, D-glucosamine, D-rafinose and L-arabinose. The
effects of temperature variation on the protein were verified at 40 to 70°C for 30, 60 and
90 minutes and in relation to pH in the 7-12 range, while the effect of ions on the
protein was determined with 10 mM of MgSO 4 , Na 2 S 2 O and CaCl 2. EC (39.06 to 1250
µg/mL), CVU (4.68 to 150 µg/mL) and Glucantime® (7.8 to 250 µg/mL) were
evaluated for their inhibitory potential (IC 50 ) in L. braziliensis promastigote cells (strain
MHOM/BR/1975/M2903) at a concentration of 3.10 6 for 48h and the ultrastructural
analysis of the treated parasites was evaluated by scanning electron microscopy (SEM).
The cytotoxicity (CC 50 ) of EC (140 to 2250 µg/mL), CVU (4.68 to 150 µg/mL) and
Glucantime® (25 to 400 µg/mL) was verified in peripheral blood mononuclear cells
(PBMC) at a concentration of 1.10 6 for 24h, as well as in macrophage cells (J774.A1)
with EC (14.06 to 450 µg/mL), CVU (4.5 to 72 µg/mL) and Glucantime® (25 to 400
µg/mL) at a concentration of 1.10 5 for 24h. The selectivity indices (SI) of EC, CVU and
Glucantime® were calculated from the ratio of CC 50 (50% cytotoxic concentration in
PBMC and macrophages) to IC 50 against the promastigote forms of L. braziliensis. CVU
showed affinity for the carbohydrates tested, except glucose and raffinose. From these
results it was possible to understand the specificity of the protein domain and its
interaction with the Leishmania parasite membrane. CVU lost activity at 60°C and pH
above 8, showing that CVU may be more efficient at temperatures below 60°C and
neutral pH. CVU activity was reduced by Mg 2+ , Ca 2+ and Na + ions, which shows that the
protein has a greater interaction with these ions. EC was used on L. braziliensis
promastigote cells, which showed inhibition of 48.52% at 156.25 µg/mL and 68.86% at

312.5 µg/mL, obtaining an IC 50 value of 161.4 µg/mL. CVU showed an IC 50 value of
66.32 µg/mL, which can be attributed to the presence of galactose residues, a
carbohydrate for which CVU showed affinity. The reference drug used (Glucantime®)
inhibited the growth of promastigotes with an IC 50 value of 129.18 µg/mL. SEM
analysis showed that the untreated promastigote cells had an elongated, fusiform cell
body, while the cells treated with EC and CVU had a rounded morphology, reduced
flagella and cell shrinkage. These alterations can cause a reduction in the parasite's
mobility and impact on its survival. The CC 50 in PBMC of EC was 1210 µg/mL, of
CVU 116.9 µg/mL, while that of Glucantime® was >400 µg/mL. EC, CVU and
Glucantime® exhibited IS for PBMC of 7.49, 1.76 and >3, respectively. The CC 50 in
macrophages of EC was 220.98 µg/mL, CVU 35.73 µg/mL, while that of Glucantime®
was >400 µg/mL. EC, CVU and Glucantime® exhibited IS for macrophages of 1.01,
1.15 and >3, respectively. The cell extract showed a higher selectivity index when
compared to the reference drug; the complex mixture of molecules in the cell extract of
the microalgae C. vulgaris may act synergistically to perform anti-Leishmania activity.
The microalgae C. vulgaris has the potential to be explored as a candidate for new anti-
Leishmania drugs, which offers possibilities for research using natural products for the
treatment of neglected diseases.