Search of Trypanosomicidal Active Principles by Metabolomic-guided Fractionation in Baccharis trimera

The American Trypanosomiasis, also known as Chagas disease is caused by Trypanosoma cruzi, a protozoan of the Trypanosomatidae Family. It is a zoonotic endemic that affects approximately 6-8 million people, it is considered a neglected diseases making it not attractive for pharmaceutical industries. Currently available treatments use the drugs a nitrofurfurylidene-amino (Nifurtimox) and a nitroimidazole acetamide (Benznidazole). Both are not completely effective against the disease. To overcome these problems we are using natural products combined with nuclear magnetic resonance based metabolomic analysis. We could identify the responsible compound of the trypanosomicidal activity in Baccharis trimera, this being a diterpene of the labdane type containing an aldehyde, agreeing with results obtained by the group previously where metabolites of the same nature had been described with trypanosomicidal activity in Aristeguieta glutinosa. In the present work the Baccharis trimera fractionation oriented to verified the above compound is performed using gradient of polar disolvents extractions, and the biological activity of the fractions obtained in the process is monitored by in vitro assays in the epimastigote form of T. cruzi, Tulahuen 2 strain and the 1H NMR metabolomic characterization. The results obtained confirm that the ethyl acetate Baccharis trimera fraction has an important antiT. cruzi activity, and besides that the aldehyde-diterpene is not the only metabolite with biological activity present in the plant, so we can also infer that we are facing a synergistic effect.


Abstract:
The American Trypanosomiasis, also known as Chagas disease is caused by Trypanosoma cruzi, a protozoan of the Trypanosomatidae Family.It is a zoonotic endemic that affects approximately 6-8 million people, it is considered a neglected diseases making it not attractive for pharmaceutical industries.Currently available treatments use the drugs a nitrofurfurylidene-amino (Nifurtimox) and a nitroimidazole acetamide (Benznidazole).Both are not completely effective against the disease.To overcome these problems we are using natural products combined with nuclear magnetic resonance based metabolomic analysis.We could identify the responsible compound of the trypanosomicidal activity in Baccharis trimera, this being a diterpene of the labdane type containing an aldehyde, agreeing with results obtained by the group previously where metabolites of the same nature had been described with trypanosomicidal activity in Aristeguieta glutinosa.In the present work the Baccharis trimera fractionation oriented to verified the above compound is performed using gradient of polar disolvents extractions, and the biological activity of the fractions obtained in the process is monitored by in vitro assays in the epimastigote form of T. cruzi, Tulahuen 2 strain and the 1 H NMR metabolomic characterization.The results obtained confirm that the ethyl acetate Baccharis trimera fraction has an important anti-T.cruzi activity, and besides that the aldehyde-diterpene is not the only metabolite with biological activity present in the plant, so we can also infer that we are facing a synergistic effect.• Flowering period: Late summer to early autumn.
• Popular medicine recommends it to combat digestive and liver problems.

Antiproliferative activity assay:
• Use cultures of T. cruzi, (epimastigote state) Tulahuen 2 strain, at 28 °C (in exponential phase of growth) • 0.6 mL / well is inoculated into a 24-well plate of a suspension of parasites at a concentration of 4 million cells / mL • The parasites are incubated with the extracts at 28 °C for 5 days.• Measure of absorbance at 610 nm, (proportional to the number of cells) on days 0 and 5. • The percent growth inhibition of the parasite is calculated according to:

Aromatic H
Aldehyde olefinic H

H-C-O aliphatic H
The presense of the aldehyde compund was confirmed by 1 H-NMR espectroscopy in the 1:1:1 fraction obtained by column cromatography of BT AcOEt

Antiproliferative activity assay of purified fraction
Fraction IC 50 (ug/ml) 1:1:1 20,2 ± 7,0 • In the initial fractionation carried out with the gradient of increasing polarity, it is observed that the ethyl acetate fraction presents greater biological activity, this coincides with what was expected, since this fraction is the one with the highest concentration of the active principle proposed by the metabolomic analysis.The hexanic fraction also presents an important activity, which may be due to the high presence of sesquiterpenic compounds and diterpenes.Even the methanolic fraction has considerable activity, this also shows that there are several with harmful compounds for the parasite in the plant, which present different polarity • From the 1 H RMN spectra of the Baccharis trimera fractions obtained with first fractionation is seen the precense of two pics in the región of aldehydes, whereby the diterpene may contain two aldehyde groups.
• Since the AcOEt fraction is the one with the highest concentration of the active ingredient and also the one with the highest biological activity, it was decided to purify it from this fraction, the mobile phase used to eluate the metabolite is 1:1:1 (Hexane, Ethyl acetate, Chloroform) The thin layer cromatography shows that with Vanillin reagent the fraction 111 has spots at rf = 0.52 and rf = 0.66, the first one reveals violet in Vanilla and orange in Brady, corresponding to a terpene with an aldehyde in its structure, the second reveals yellow in Vanillin which implies the presence of flavonoids in the area and yellow in Brady, due to ketone groups characteristic of these compounds.
• The spectrum obtainded with de 1 H-RMN analysis alow to confirm the presense of the aldehyde in the fraction but not to elucidate in detail the structure.Also we can see protons corresponding to aromatic rings due to flavonoids putting on evidence the precense of other compunds in the fraction thus more purification steps will be necesary.
• Bacharis trimera, is an important source of anti-trypanosoma cruzi agents.
• Polarity gradient fractionation demonstrates that there are several compounds with trypanosomicidal activity.
• The fraction enriched in the aldehyde presents a relevant activity so we can infer that plays an important role in the observed growth inhibition of the entire extract.
• Further purification steps are necessary to confirm the structure and activity of diterpene it self versus T. cruzi.
after infection • most cases symptoms are absent • May appear skin lesion or a purplish swelling of the lids of one eye (Romania sign), fever, headache, enlarged lymph glands, pallor, muscle pain, difficulty in breathing, swelling, and abdominal or chest pain.• Chronic: • Parasites are hidden mainly in the heart and digestive muscles • Cardiac, digestive and neurological disorders that can lead to sudden death Introduction .nitrofurfurylidene-amino (Nfx), Benznidazole, nitroimidazole acetamide (Bnz) • Mechanism of action: • Formation of nitro-anion radical metabolite that reacts with nucleic acids of the parasite causing significant break down of DNA.• Both are not completely effective against the disease.Carqueja • Scientific name: Bacharis trimera • Popular name: Carqueja • Morphology: Bush up to 70 cm high, stems trialled, lacking leaves or with reduced leaves • Geographic distribution: southern Brazil, Argentina, Paraguay and Uruguay.