Asian Pacific Journal of Tropical Biomedicine

: 2018  |  Volume : 8  |  Issue : 5  |  Page : 245--253

Phytochemical bioprospecting, antioxidant, antimicrobial and cytotoxicity activities of saline extract from Tithonia diversifolia (Hemsl) A. Gray leaves

Bruno Rafael Barboza1, Bárbara Rafaela da Silva Barros1, Bárbara de Azevedo Ramos2, Maiara Celine de Moura2, Thiago Henrique Napoleão2, Maria Tereza dos Santos Correia2, Luana Cassandra Breitenbach Barroso Coelho2, Iranildo José da Cruz Filho1, Ana Maria Souto Maior1, Túlio Diego da Silva3, Leylianne de Cássia Rodrigues Nerys4, Edson Renan Barros de Santana4, Cláudia Sampaio de Andrade Lima4, Virgínia Maria Barros de Lorena5, Cristiane Moutinho Lagos de Melo1,  
1 Department of Antibiotics, Biosciences Centre, Federal University of Pernambuco, Pernambuco, Brazil
2 Department of Biochemistry, Biosciences Centre, Federal University of Pernambuco, Pernambuco, Brazil
3 Center for Strategic Technologies of the Northeast, Pernambuco, Brazil
4 Department of Biophysics, Biosciences Centre, Federal University of Pernambuco, Pernambuco, Brazil
5 Aggeu Magalhães Institute, IAM/FIOCRUZ, Pernambuco, Brazil

Correspondence Address:
Cristiane Moutinho Lagos de Melo
Departamento de Antibióticos, Universidade Federal de Pernambuco, Pernambuco


Objective: To evaluate antimicrobial and antioxidant properties of saline extract from Tithonia diversifolia leaves by phytochemical bioprospecting, and investigate its safety against animal cells. Methods: The saline extract was prepared, with NaCl (0.15 M), by constant stirring of the dried and pulverized leaves, followed by volume reduction by lyophilization. The extract was phytochemical characterized using ultra-performance liquid chromatography, and total phenol and flavonoid analysis also was performed. The antioxidant capacity was determined through DPPH· radical, the antimicrobial property was evaluated against standard bacteria and fungi, and the viability assays were performed against mice splenocytes. Results: Fifteen compounds were identified belonging to two main classes terpenoids and phenolics. The extract showed 22.185 mg GAE/g of total phenolic compounds and 3.220 mg QE/g of flavonoid. Moreover, extract showed higher antioxidant ability similar to butylated hydroxytoluene a standard molecule [(3.042±0.019) mg AAE/g and (4.12±0.10) mg AAE/g to saline extract and butylated hydroxytoluene, respectively]. The antimicrobial assays demonstrated that the extract had a significant antifungal potential against Candida species and could be used with safety against mice splenocytes, in concentrations lower than 50 μg/mL, promoting higher proliferation in these cells. Conclusions: Saline extract from Tithonia diversifolia leaves presents potential antioxidant, antifungal properties and induces immunostimulation in mice splenocytes.

How to cite this article:
Barboza BR, da Silva Barros BR, Ramos Bd, de Moura MC, Napoleão TH, dos Santos Correia MT, Barroso Coelho LC, da Cruz Filho IJ, Souto Maior AM, da Silva TD, Rodrigues Nerys Ld, de Santana ER, de Andrade Lima CS, de Lorena VM, de Melo CM. Phytochemical bioprospecting, antioxidant, antimicrobial and cytotoxicity activities of saline extract from Tithonia diversifolia (Hemsl) A. Gray leaves.Asian Pac J Trop Biomed 2018;8:245-253

How to cite this URL:
Barboza BR, da Silva Barros BR, Ramos Bd, de Moura MC, Napoleão TH, dos Santos Correia MT, Barroso Coelho LC, da Cruz Filho IJ, Souto Maior AM, da Silva TD, Rodrigues Nerys Ld, de Santana ER, de Andrade Lima CS, de Lorena VM, de Melo CM. Phytochemical bioprospecting, antioxidant, antimicrobial and cytotoxicity activities of saline extract from Tithonia diversifolia (Hemsl) A. Gray leaves. Asian Pac J Trop Biomed [serial online] 2018 [cited 2022 Jul 3 ];8:245-253
Available from:

Full Text

 1. Introduction

Many therapeutic agents are used in medicine against diseases and different plants comprise of the sources of these compounds[1],[2]. In fact, according to Pan et al.[2], more than 35 000 species of plants possess curative potentials because of their phytochemical constituents. These chemical constituents are functionally classified as primary and secondary metabolites. Primary metabolites, essential for development, growth and reproduction of plant, are proteins, lipids, nucleic acids and carbohydrates. Secondary metabolites, produced in response to the involvement of plant with the environment or as possible defensive mechanisms against aggressive agents, can be represented by many compounds as alkaloids, flavonoids and terpenoids[3].

Most of the commercially available drugs are derived from plants or generated from natural products. Some compounds of plant and/or derived from plant were discovered throughout history. Among these compounds, we can find capsaicin (Capsicum spp.), aspirin (Salix alba L.), atropin (Atropa belladonna), morphine (Papaver somniferum L.), quinine (Cinchona sp.), pilocarpine (Pilocarpus jaborandi and Pilocarpus microphyllus), colchinine (Colchicum autumnale), etoposide (Podophyllum peltatum L.), masoprocol (Larrea divaricata Cav.), metformin (Galega officinalis), levodopa (Mucuna pruriens), dicumarol (Melilotus officinalis), vinblastine, vincristine, vinrosidine, vinorelbine and vinflunine (Catharanthus roseus), solamargine (Solanumdu lcamara), taxol (Taxus brevifolia), artemisinin (Artemisia annua), maytansine (Maytenus ovatus), irinotecan (Camptotheca acuminata), and arglabin (Artemisia glabella Kar.)[4-30]

Currently, about 80% of the immunosuppressive, cardiovascular, antimicrobial and antitumor drugs are originated from plants. The identification and isolation of compounds and/or phytochemical groups are crucial for the discovery of new therapies[1],[2],[31],[32],[33].

Tithonia diversifolia (T. diversifolia) (Hemsl) A. Gray, is a perennial and shrub species of the family Asteraceae, tribe Heliantheae, which is native in Central America and Mexico and popularly known as Mexican sunflower[34],[35],[36]. Many representatives of this species are found throughout the world, especially in Asia, Africa and America[35].

In the literature, T. diversifolia is described as an important source of biologically active molecules[35], which possesses many compounds in leaves and inflorescences belonging to sesquiterpene lactones and flavonoids classes, and derivatives of trans-cinnamic acid[37],[38]. Extracts and secondary metabolites isolated from inflorescences and leaves showed significant biological properties, such as antitumor[39], anti-inflammatory[37], antihyperglycemic[40] and antimicrobial[41].

In this study we explore the phytochemical characterization as well as investigate antioxidant profile, antimicrobial properties against pathogenic species of bacteria and Candida sp. and cytotoxicity against mice splenocytes promoted by saline extract of T. diversifolia leaves.

 2. Materials and methods

2.1. Processing of plant leaves and extract preparation

The plant was collected in city of Paudalho, interior of the State of Pernambuco, Brazil. The botanical identification was carried by Herbarium Geraldo Mariz of the Center of Biosciences of the Federal University of Pernambuco. Plant was deposited, under identification number 82.703. For two days, the humidity was removed for obtainment of dried leaves. These leaves were crushed in reverse black oster and the pulverized material (20 g) was diluted in 200 mL of NaCl (0.15 M/10% (w/v). Therefore, using an orbital and alternative incubator shaker (Lab Companion™ IS-971) the material was kept under agitation (400 rpm) in constant temperature (28 °C) for 16 hours. After the stirring time, the material was filtered using paper filter and centrifuged (15 min, 10 000 × g). The obtained supernatant was collected and called saline extract from T. diversifolia leaves. After this, the extract was lyophilized, and 9.2 g of crude material was obtained.

2.2. Phytochemical bioprospecting

2.2.1. Determination of total phenolic compounds

The contents of total phenols were determined through method described by Li et al.[42] with few modifications. One mg/mL of diluted extract was dissolved in distilled water. A total of 100 μL of Folin solution (1:10 v/v) was added to 200 μL of this diluted extract. After incubation, the extracts were protected from light for 3 min, and 0.08 mL of Na2CO3 (7.5%) was added. Extracts were then incubated again and protected from light, at 21 °C , for 120 min. After incubation time, the extracts were read in the spectrophotometer (765 nm). Distilled water was the blank on the calibration curve. A calibration curve of the graphical representation of the absorbance as a function of the concentration of gallic acid (0-500 μg/mL) was prepared and the linear equation (Y = 0.004 8x + 0.001 6, R2 = 0.999 9). Phenols were indicated in gallic acid equivalent (mg GAE/g of extract).

2.2.2. Investigation of total flavonoids

The measurement of total flavonoid amount was performed similarly to the method used by Woisky and Salatino[43], with some modifications. Using test tubes, 1 mL of the extract, previously diluted (1 mg/mL) was added. After this, 1 mL of the 3% solution of aluminum chloride (AlCl3) prepared with methanol was added in tubes. After incubation (30 min), in dark (21 °C), the absorbance was obtained (425 nm). A standard curve with quercetin (0-500 μg/mL) was performed to obtain the equation (Y = 0.023x + 0.150 9, R2 = 0.995 6). The assays were obtained in quintuplicate. Flavonoids were indicated in quercetin equivalent (mg QE/g of extract).

2.2.3. Ultra-High Performance Liquid Chromatography coupled to Mass Spectrometry

Ultra-High Performance Liquid Chromatography was performed with an Acquity H-Class (Waters) employing a 2.1 mm × 100 mm BEH column with a particle size of 1.7 μm. The column was maintained under a constant temperature of 40 °C and the auto injector at 10 °C. We used an aqueous solution (eluent A) containing 2% methanol (MeOH), 5 mM ammonium formate and 0.1% formic acid and a methanol solution (eluent B) containing 0.1% formic acid, which were pumped at a flow rate of 0.3 mL/min. Ten microliters of the T. diversifolia extract was injected. Elution was performed in gradient mode and the initial condition (98% A / 2% B) was maintained for 0.25 min. The B ratio increased linearly to 99% in 8.5 min, remaining at 99% B for 1 min, followed by an immediate decrease to 2% B, where it was maintained for up to 11 min. The ultra-performance liquid chromatography system was coupled to a single quadrupole mass spectrometer SQ Detector 2 (Waters). The data were obtained in fullscan mode, analyzing masses between 100 and 1 000 Da, in negative ionization. The acquisition of the chromatograms and mass spectra was obtained through MassLynx™ software (Waters).

2.3. Antioxidant activities in vitro

2.3.1. Free radical sequestration by 2,2-diphenyl-l- picrylhydrazyl (DPPH•)

The antioxidant ability of saline extract was investigated through the stable radical DPPH•, as described by Blois[44]. We performed a serial dilution with 400 μL of the saline extract (3.9; 7.8; 15.6; 31.3; 62.5; 125; 250 and 500 μg/mL). In each concentration was added 0.25 mL of the DPPH solution (1 mM and OD517 = 0.650 ± 0.50). After incubation time (25 min/21 °C/in the dark), the absorbance was obtained in 517 nm. The DPPH solution added to water was used as control. The sequestration of DPPH radicals was measured by using the equation:

DPPH· (%) = [(As - Ac)/Ac] × 100

Where As is the sample absorbance and Ac is the control absorbance.

2.3.2. Total antioxidant activity

The total antioxidant activity of saline extract was measured as a function of ascorbic acid, according to Pietro et al.[45]. Saline extract (100 μL in 500 μg/mL) was mixed with ascorbic acid (1 mg/mL) and with 1 mL of phosphomolybdenum solution (600 mM sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). This mixture was incubated in water bath (95 °C / 90 min). After cooling, the absorbance was obtained at 695 nm against a blank (1 mL solution with 0.1 mL of water). A standard curve with ascorbic acid (0-500 μg/mL) was performed to obtain the equation (Y = 0.019x + 0.072 3, R2 = 0.993 7). The total antioxidant activity measured by the equation:

ATT (%) = [(As - Ac)/(Aaa - Ac)] × 100.

Where Ac is the control absorbance, As is the sample absorbance and Aaa is the Ascorbic acid absorbance.

2.3.3. FRAP assay

The stock solution of the FRAP assay was prepared using acetate buffer (300 mM / 16 mL of CH3COOH and 3.1 g of CH3COONa) at 3.6 pH, 10 mM TPTZ (2,4,6-tripyridyl-triazine) solubilized in FeCl3 solution (20 mM) and HCl (40 mM) as described by Benzie et al.[46]. The assay solution was prepared by combination of the acetate buffer, TPTZ and FeCl3 in a ratio of 10:1:1 (v/v/v) and incubated for 5 min at 37 °C. Saline extract (25 μL in 1 mg/mL) was added in 0.180 mL of FRAP solution. After stirring, the extract was rested for 30 min at 37 °C, protected from light. Subsequently the absorbance was obtained at 593 nm. A standard curve with FeSO4 (0–1 000 μg/mL) was performed to obtain the equation (Y = 0.002 4x + 0.001 9, R2 = 0.995 3). The results were obtained as mg EFeSO4 (II)/g of extract.

2.4. Antimicrobial activity test

2.4.1. Bacterial and fungal strains, culture conditions and preparation of samples

Fungal strains were obtained from the culture collections at Mycology Department from Federal University of Pernambuco and bacteria isolates were provided by the culture collection of the Departamento de Antibióticos da Universidade Federal de Pernambuco. Stock cultures were kept under refrigeration (-20 °C) in sterilized skim milk containing 10% (v/v) glycerol. Microorganisms pathogens used in this investigation were Candida albicans (C. albicans), Candida krusei, Candida tropicalis, Candida parapsilosis (C. parapsilosis), Candida glabrata, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Staphylococcus saprophyticus. For antimicrobial activity assay, bacterial and yeast species were cultured in Mueller Hinton and Sabourand Dextrose agar medium, respectively, overnight at 36 °C, and subsequently the colonies were resuspended in sterile saline solution (0.15M NaCl) and turbidimetrically adjusted at a wavelength of 600 nm (OD600) to obtain the suspension equivalent to 106 CFU/mL. For the assay, the samples were filtered on the sterile polyvinylidene difluoride syringe filter (pore size: 0.22 μm; diameter: 13 mm).

2.4.2. Determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC)

The MIC of the leaf extract of T. diversifolia was determined by the microtiter test based on CLSI[47] criteria. In 96-well microtiter plates, the extract (12 mg/mL) was added (80 μL) into the fourth well from which a serial dilution in sterile Milli-Q water was performed to the twelfth well of the same row. Subsequently, 40 μL of Mueller Hinton (bacteria) or Sabourand Dextrose (yeast) broths were added to all wells, but to the first one was filled with 200 μL of the culture medium, corresponding thus to the sterility medium control. Antibiotics ampicillin and tetracycline (8 μg/mL) and antifungal fluconazole (64 μg/mL), obtained from Sigma-Aldrich (USA), were used as positive controls in the second well. Finally, the bacterial or yeast suspensions (80 μL 106 CFU/mL) were added in the second well to the last well in the row. The third well (containing microorganisms in the absence of the sample) corresponded to the 100% growth control or negative control. The plates were incubated at 36 °C and the optical density was measured at time zero and after 24 h of incubation. The MTC90 and MTC50 corresponded to the lowest concentration of the sample capable of promoting a reduction of ≥90% or ≥50%, respectively, in optical density, as compared to the 100% growth control. For determination of the MBC and the MFC, aliquots of the wells containing concentrations of ≥MTC50 samples were inoculated into petri dishes containing Mueller Hinton or Sabourand Dextrose agar medium, which were subsequently incubated at 36 °C for 24 h. The MBC and MFC corresponded to the lower concentration of the sample which was able to reduce the number of CFU in 99.9% in relation to the initial inoculum. For this study, three independent experiments, performed in triplicate, were used.

2.5. Obtainment of murine splenocytes for cytotoxicity assay

2.5.1. Animals

Female BALB/c mice (6–8 weeks old) were obtained from the animal facilities of the Keizo Asami Immunopathology Laboratory– LIKA located in Federal University of Pernambuco, Brazil. Mice were kept under standard laboratory conditions (20–22 °C/ 12 hours of day and night cycle / standard diet and water). All experimental procedures were performed in accordance with Ethics Committee of Animal Use of Federal University of Pernambuco, Brazil (protocol number: 0048 approved in December of 2016).

2.5.2. Preparation of splenocytes

This procedure was performed in accordance to Melo et al.[48]. Cells of the spleens of mice were processed in polystyrene conic tubes (15 mL) with Ficoll-PaqueTM PLUS solution with the density adjusted to 1.076 g/mL. After three centrifugations, cells were counted in a Neubauer chamber, using the trypan blue solution. Cells were only used when viability was >95%.

2.5.3. Investigation of cell death

Mice splenocytes (106 cells) were treated with T. diversifolia saline extract in 50, 25, 12.5, 6 and 3 μg/mL and were maintained in 48-well plates for 24 h to evaluate the cytotoxicity power of the extract. Untreated cells, only in RPMI 1640 medium, were used as control. After this, lymphocytes were centrifuged twice at 26 °C, 450 × g for 10 min. The pellet was resuspended in a cytometer tube containing 300 μL of binding buffer of cell viability kit (Becton Dickinson Biosciences). Annexin V-FTTC (1:500) and propidium iodide-PE (20 μg/mL) were added to tube. Samples was obtained in FACSCalibur flow cytometer (BD®) and analyzed using Cell Quest Pro software (BD®).

2.5.4. Cell proliferation analysis

Same protocol to obtained splenocytes was used for proliferation assay. After splenocytes obtainment, cell solution was centrifuged at 300 × g (21 °C/5 min) with sterile PBS 1X added with SFB 5% (pH 7.2). After this, the cell solution was adjusted to 1 106 cells/mL and was incubated (for 10 min) with 5mM of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester. Cells were centrifuged at 300 × g/5 min with sterile PBS 1 ×. Cells stained were cultured for 24 and 48 h with 12.5 μg/mL of saline extract from T. diversifolia leaves and only culture complete RPMI 1640 medium (negative control). After culture time cells were centrifuged (300 × g/5 min), they were carried on FACSCalibur flow cytometer (BD®) and results were analyzed using Cell Quest Pro software (BD®).

2.6. Statistical analysis

Shapiro–Wilke test was applied to evaluate normal hypothesis. The statistical difference between two groups was analyzed by Wilcoxon test and among more than three groups by ANOVA. Significance level considered was 5%. GraphPad Prim 5.01 software was used in our statistical tests.

 3. Results

3.1. Phytochemical analysis

The chromatogram and mass spectrum of this extract ([Figure 1]A and [Figure 1]B, respectively) indicated the presence of important classes of biologically active compounds. Identified fifteen compounds were between terpenes and phenolics, based on retention time, peak area and mass characteristics [Table 1]. Total phenol compounds and total flavonoids of saline extract from T. diversifolia leaves showed (22.185 ± 0.201) mg GAE/g and (3.220 ± 1.085) mg QE/g, respectively.{Figure 1}{Table 1}

3.2. Antioxidant activities in vitro

Results of antioxidant capacity promoted by saline extract of T. diversifolia leaves showed that the extract had an antioxidant capacity (3.042±0.019 mg AAE/g) similar to butylated hydroxytoluene (BHT) standard [(4.12±0.10) mg AAE/g]. The extract also promoted lower free radical sequestration [(10.11±1.15)% mg/mL; (93.13±0.58)% mg/mL; (90.04±0.16)% mg/mL, to saline extract, BHT and ascorbic acid, respectively] and was able to induce a reduction of ferric ion [(236.53±26.97) mg EFeSO4 ( Π )/g; (679.17±25.98) mg EFeSO4 ( Π )/g; (1 215.00±48.55) mg EFeSO4 ( Π )/g to saline extract, BHT and ascorbic acid, respectively].

3.3. Antimicrobial assay

The saline extract of T. diversifolia did not show antibacterial activity against bacterial isolates tested (data not shown). However, this compound demonstrated potential fungistatic and fungicidal properties in vitro against pathogenic species of Candida. Results showed that saline extract induced MIC50 in C. albicans (0.293 μg/mL), Candida krusei (0.002 4 μg/mL) and C. parapsilosis (0.293 μg/mL). It also induced MIC90 in the above same fungal strains (0.293 μg/mL; 0.004 8 μg/mL and 0.293 μg/mL, respectively) and MFC in C. albicans (150 μg/mL) and C. parapsilosis (0.375 μg/mL). Fluconazole, used as positive control, showed potential effects to all strains tested (except Candida tropicalis) and MIC50 values obtained were 0.25 μg/mL, 32 μg/mL, 4 μg/mL and 64 μg/mL to C. albicans, Candida krusei, C. parapsilosis and Candida glabrata, respectively.

3.4. Cell viability promoted by saline extract from T. diversifolia leaves

After the antimicrobial properties were evaluated, we performed a cytotoxicity test to investigate if saline extract from T. diversifolia leaves was able to kill mice splenocytes. For this test, cell cultures were treated with different concentrations of saline extract and results showed that this extract might be used with safety in concentrations equal to and lower than 25 μg/mL [Figure 2]. Moreover, we evaluated if saline extract also was able to promote cell activation and proliferation in these cells. Our results showed that saline extract promoted splenocytes proliferation in higher values especially at 24 hours of assay [Figure 3].{Figure 2}{Figure 3}

 4. Discussion

T. diversifolia is commonly used in folk medicine in several continents[9] due to its phytomedicine and ethnopharmacological applicability in the treatment of various organic disturbances[49]. The extracts of T. diversifolia are used orally because of their analgesic capacity[50], antimalarial[51], antipyretic[52] and antidiabetic[53], and are topical applied by their anti-inflammatory potential[52] and in the treatment of microbial infections in the genitals organs[49].

Studies affirm that the species belonging to the Asteraceae family presents high concentrations of sesquiterpenes lactones[35],[54] and also shows that compounds such as sesquiterpenes lactones, caffeine- derived acids, diterpenes and flavonoids are commonly found in the aerial parts of T. diversifolia[38]. Our results demonstrated that, similar to other extracts from leaves of T. diversifolia (using different types of solvents), saline extract used in this study is rich in terpenoids and phenolics compounds[36],[37],[50],[55],[56]. In fact, Gama et al.[57] and Pantoja et al.[58] demonstrated higher antioxidant capacity promoted by ethanol extract of the inflorescences of T. diversifolia which can be attributed to the phenols of this species. The phenolic compounds are agents that induce effectively to the antioxidant action due mainly to their reducing properties, which give them the ability to act as reducing agents, hydrogen and/or oxygen donors[59],[60],[61]. Our results also showed a similar profile of these studies, especially when compared to the commercial antioxidant BHT, when no significant difference between extract and standard was observed.

T. diversifolia species, as well as the entire Asteraceae family present high amounts of terpenoids, a natural compound with antimicrobial effects[37],[38],[54],[62]. Several tests performed with extracts and compounds isolated from various tissues of T. diversifolia proved the potential antimicrobial capacity against many pathogenic species of bacteria and fungi[38],[41].

However, we did not find results of antibacterial activity to our saline extract, but a significant antifungal action was observed. These results were reinforced by Ait-Ouazzou and collaborators[63] and Zengin and Baysal[64] that demonstrated that terpenoids compounds present in their preparations did not show in vitro inhibitory capacity against growth of the bacterial species. Moreover, Duraipandiyan et al.[62] demonstrated that two sesquisterpenic lactones (ostunolide and eremanthin) isolated from Costus speciosus (Asteraceae) were not be able to inhibit bacterial growth, but both compounds showed fungistatic properties. Assays with T. diversifolia, performed by Obafemi et al.[65] and Olayinka et al.[66], also demonstrated that ethanolic extracts, aqueous extracts, fractions and sesquiterpenic compound isolated from leaves, roots and stem, respectively, presented a potential antifungal activity against C. albicans.

Fluconazole is a potent antifungal agent in microbiology field for the treatment of candidiasis[67],[68]. When we compare the fungistatic results observed from saline extract with those of fluconazole, it is indicated that the extract from T. diversifolia leaves also possesses antifungal potentials against Candida species.

Different plant extracts are indicated by traditional medicine for the treatment of virall[69],[70],[71], bacterial and fungal infections[72],[73],[74],[75]. However, the misconception that natural products, besides not being toxic, also not possess side effects, induces the population to indiscriminate and inappropriate use, which in turn results in serious health problems, such as poisoning[76]. Many treatments, promoted by natural compounds, may be immunosuppressive or cause serious side effects[77],[78]. Here we also investigated if saline extract, prepared using an usual physiological concentration (NaCl 0.15M), is dangerous to normal cells because many communities used T. diversifolia tea for some diseases[49],[53]. Our results showed that in appropriate dose, saline extract from T. diversifolia leaves is able to activate mice splenocytes and induce higher cell proliferation in these cells at 24 hours of assay, indicating a possible immunostimulant effect. Similar to our findings, other studies show that extracts from T. diversifolia leaves were able to promote the differentiation of mesenchymal stem cells[79] and did not promote cytotoxicity against human peripheral blood mononuclear cells and mouse macrophages[80].

In conclusion, saline extract from T. diversifolia leaves presented higher amounts of terpenoids and phenolics compounds, being suggestive of the presence of sesquisterpenes lactones. This extract showed antioxidant capacity, reduction of the ferric ion and antifungal properties against Candida species. Moreover, the extract promoted cell proliferation in mice splenocytes. These results indicate that, in future, saline extract from Tithonia diversifolia leaves can be investigated as a prototype candidate for antifungal agent and/or adjuvant of the antimicrobial conventional therapies.

Conflict of interest statement

The authors declare no conflict of interest.


The authors thank Nucleus of Technological Platforms (NPT-1) located at Aggeu Magalhães Institute (IAM).


1Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, Linder T, Wawrosch C, Uhrin P, et al. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Ad 2015; 33(8): 1582-1614.
2Pan SY, Zhou SF, Gao SH, Kyu ZL, Zhang SF, Tang MK, et al. New perspectives on how to discover drugs from herbal medicines: CAM's outstanding contribution to modern therapeutics. Evid Based Complement Alternat Med 2013; 2013: 627375.
3Dias DA, Urban S, Roessner U. A historical overview of natural products in drug discovery. Metabolites 2012; 2(2): 303-336.
4Bode AM, Dong Z. The two faces of Capsaicin. Cancer Res 2011; 71(8): 2809-2814.
5Mahdi JG, Mahdi AJ, Mahdi AJ, Bowen ID. The historical analysis of aspirin discovery, its relation to the willow tree and antiproliferative and anticancer potential. Cell Prolif 2006; 39(2): 147-155.
6Martinez ST, Almeida MR, Pinto AC. Natural hallucinogens: A flight from medieval Europe to Brazil. Quim Nova 2009; 32(9): 2501-2507.
7Rosenblum A, Marsch LA, Joseph HPortenoy, RK. Opioids and the treatment of chronic pain: Controversies, current status, and future directions. Exp Clin Psychopharmacol 2008; 16(5): 405-416.
8Renslo AR. Antimalarial drug discovery: From quinine to the dream of eradication. ACS Med Chem Lett 2013; 4(12): 1126-1128.
9Haas LF. Pierre Joseph Pelletier (1788-1842) and Jean Bienaime Caventou (1795-1887). J Neurol Neurosurg Psychiatry 1994; 57(11): 1333.
10Hardy ME. On the jaborandi (Pilocarpus pinnatus). Bulletin Societe Chimi Paris 1875; 24: 497-500.
11Gerrard AW. Alkaloid and active principle of jaborandi. Pharmaceutical J 1875; 5: 865.
12Santos AP, Moreno RH. Pilocarpus spp.: A survey of its chemical constituents and biological activities. Braz J Pharm Sci 2004; 40(2): 116-137.
13Larsson S, Ronsted N. Reviewing Colchicaceae alkaloids – perspectives of evolution on medicinal chemistry. Curr Top Med Chem 2014; 14(2): 274-289.
14Gordaliza M, Castro MA, Coral JM, Feliciano AS. Antitumor properties of podophyllotoxin and related compounds. Curr Pharm Des 2000; 6(18): 1811-1839.
15Montecucco A, Zanetta F, Biamonti G. Molecular mechanisms of Etoposide. Excli J 2015; 4: 95-108.
16Son JK, Lee SH, Nagarapu L, Jahng Y. A simple synthesis of nordihydroguaiaretic acid and its analogues. Bull Korean Chem Soc 2005; 26: 1117-1120.
17Bailey JC. Metformin: Historical overview. Diabetologia 2017; 60(9): 1566-1576.
18Min K, Park K, Park DH, Yoo YJ. Overview on the biotechnological production of L-DOPA. Appl Microbiol Biotechnol 2015; 99(2): 575-584.
19Apostolakis S, Lip GYH, Lane DA, Shantsila E. The quest for new anticoagulants: From clinical development to clinical practice. Cardiovascular Ther 2011; 29(6): e12-22.
20Noble RL, Beer CT, Cutts JH. Further biological activities of vinca leukoblastine - An alkaloid isolated from Vinca rosea (L.). Biochem Pharm 1958; 1: 347-348.
21Svoboda GH. Alkaloids of Vinca rosea Linn. IX. Extraction and characterisation of leurosidine and leucocristine. Lloyda 1961; 24: 173-178.
22Tyagi AK, Prasad S. Drug discovery inspired by mother nature for cancer therapy. Biochem Physiol 2015; 4(1): e128.
23Elias TS, Korzhenevsky VV. The presence of taxol and related compounds in Taxus Baccata native to the Ukraine (Crimea), Georgia, and Southern Russia. Aliso: J Syst Evol Bot 1992; 13(3): 463-470.
24Wani MC, Taylor HL, Wall ME, Coggon P, Mcphail AT. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agente from Taxus brevifolia. J Am Chem Soc 1971; 93(9): 2325-2327.
25Su XZ, Miller LH. The discovery of artemisinn and Nobel Prize in Physiology or Medicine. Sci China Life Sci 2015; 58(11): 1175-1179.
26Haynes RK. Artemisin and derivatives: The future for malaria treatment. Curr Opin Infect Dis 2001; 14(6): 719-726.
27Kupchan SM, Komoda Y, Court WA, Thomas GJ, Smith RM, Karim A, et al. Maytansine, A novel antileukemic ansa macrolide from Maytenus ovatus. J Am Chem Soc 1972; 94(4): 1354-1356.
28Potmesil M. Camptothecins: From bench researtch to hospital wards. Cancer Res 1994; 54(6): 1431-1439.
29Bahadori F, Ropçu G, Eroglu MS, Onyuksel H. A new lipid-based nano formulation of vinorelbine. AAPS Pharm Sci Tech 2014; 15(5): 1138-1148.
30Fahy J, Hellier P, Breillout F, Bailly C. Vinflunine: Discovery and synthesis of a novel microtubule inhihbitor. Semin Oncol 2008; 35(3): S3-5.
31Lahlou M. The success of natural products in drug discovery. J Pharm Pharmacol 2013; 4(1): 17-31.
32Ahmed MN, Kabidul AMN. Traditional knowledge and formulations of medicinal plants used by the traditional medical practitioners of Bangladesh to treat schizophrenia like psychosis. Schizophr Res Treatment 2014; 2014: 679810.
33Singh M, Pandey N, Agnihotri V, Singh KK, Pandey A. Antioxidant, antimicrobial activity and bioactive compounds of Bergenia ciliata Sternb.: A valuable medicinal herb of Sikkim Himalaya. J Tradit Complement Med 2016; 7(2): 152-157.
34Toledo JS, Ambrósio SR, Borges CHG, Manfrim V, Cerri DG, Cruz AK, et al. In vitro leishmanicidal activities of sesquiterpene lactones from Tithonia diversifolia against Leishmania braziliensis promastigotes and amastigotes. Molecules 2014; 19(5): 6070-6079.
35Chagas-Paula DA, Oliveira RB, da Silva VC, Gobbo-Neto L, Gasparoto TH, Campanelli AP, et al. Chlorogenic acids from Tithonia diversifolia demonstrate better anti-inflammatory effect than indomethacin and its sesquiterpene lactones. J. Ethnopharmacol 2011; 136(2): 355-362.
36Passoni FD, Oliveira RB, Chagas-Paula DA, Gobbo-Neto L, Costa FB. Repeated-dose toxicological studies of Tithonia diversifolia (Hemsl.) A. Gray and identification of the toxic compounds. J Ethnopharmacol 2013; 147(2): 389-394.
37Abe AE, Oliveira CE, Dalboni TM, Chagas-Paula DA, Roca BA, Oliveira RB, et al. Inflammatory sesquiterpene lactones from Tithonia diversifolia tigger different effects on human neutrophils. Rev Bras Farmacogn 2015; 25(2): 111-116.
38Chagas-Paula DA, Oliveira RB, Rocha BA, da Costa FB. Ethnobotany, chemistry, and biological activities of the genus Tithonia (Asteraceae). Chem Biodivers 2012; 9(2): 210-235.
39Gu JQ, Gills JJ, Park EJ, Mata-Greenwood E, Hawtorne ME, Axelrod F, et al. Sesquiterpenoids from Tithonia diversifolia with potential cancer chemopreventive activity. J Nat Prod 2002; 65(4): 532-536.
40Zhao G, Li X, Chen W, Xi Z, Sun L. Three new sesquiterpenes from Tithonia diversifolia and their antihyperglycemic activity. Fitoterapia 2012; 83(3): 1590-1597.
41Linthoingambi W, Mutum SS. Antimicrobial activities of different solvent extracts of Tithonia diversifolia (Hemsley) A. Gray. Asian J Plant Sci Res 2013; 3(5): 50-54.
42Li HB, Wong CC, Cheng KW, Chen F. Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. LWT Food Sci Technol 2008; 41(8): 385-390.
43Woisky RG, Salatino A. Analysis of própolis: Some parameters and procedures for chemical quality control. J Api Res 1998; 37(2): 99-105.
44Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958; 181: 1199-1200.
45Pietro P, Pineda M, Aguilar M. Spectrophotometric quantification of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal Biochem 1999; 269(2): 337-341.
46Benzie IFF, Strain JJ. Ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Anal Biochem 1996; 239(1): 70-76.
47CLSI. CLSI document M100-S22. Perfomance standards for antimicrobial susceptibility testing; twenty-second informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
48Melo CML, Melo H, Correia MT, Coelho LC, da Silva MB, Pereira VR. Mitogenic response and cytokine production induced by cramoll 1,4 lectin in splenocytes of inoculated mice. Scand J Immunol 2011; 73(2):112-121.
49Ajao AA, Moteetee AN. Tithonia diversifolia (Hemsl) A. Gray. (Asteraceae: Heliantheae), an invasive plant of significant ethnopharmacological importance: A review. S Afr J Bot 2017; 113: 396-403.
50Owyelle VB, Waraola CO, Soladoye AO, Olaleye SB. Studies on the anti-inflammatory and analgesic properties of Tithonia diversifolia leaf extract. J Ethnopharm 2004; 90(2-3): 317-321.
51Goffin E, Ziemons E, Mol P, Madureira MC, Martins AP, Cunha AP, et al. In vitro antiplasmodial activity of Tithonia diversifolia and identification of its main acctive constituent: Tagintinin C. Planta Med 2002; 68(6): 543-545.
52Rüngeler P, Ly G, Castro V, Mora G, Pahl HL, Merfort I. Study of three sesquiterpene lactones from Tithonia diversifolia on their anti- inflammatory activity using the transcription factor NF-kB and enzymes of the arachidonic acid pathway as targets. Planta Med 1998; 64(7): 588-593.
53Wanzala W, Osundwa EM, Alwala OJ, Gakuubi MM. Chemical composition of essential oil of Tithonia diversifolia (Hemsl.) A. Gray from the Southern slopes of Mount Elgon in Western Kenya. IJEPP 2016; 2(2): 72-83.
54Salapovic H, Geier J, Reznicek G. Quantification of sesquiterpene lactones in Asteraceae plant extracts: Evaluation of their allergenic potential. Sci Pharm 2013; 81(3): 807-818.
55Tona L, Kambu K, Ngimbi N, Cianga K, Vlietinck AJ. Antiamoebic and phytochemical screening of some Congolese medicinal plants. J Ethnopharm 1998; 61(1): 57-65.
56Kuo YH, Chen CH. Sesquiterpenes from the leaves of Tithonia diversifolia. J Nat Prod 1998; 61(6): 827-828.
57Gama RM, Guimarães M, Abreu LC, Armando-Júnior J. Phytochemical screening and antioxidant activity of ethanol extract of Tithonia diversifolia (Hemsl) A. Gray dry flowers. Asian Pac J Trop Biomed 2014; 4(9): 740-742.
58Pantoja KDP, Dulcey AJC, Martínez JHI. New caffeic acid derivative from Tithonia diversifolia (Hemsl.) A. Gray butanolic extract and its antioxidant activity. Food Chem Toxicol 2017; 109(Pt 2): 1079-1085.
59Negri G, Teixeira EW, Alves MLTMF, Moreti ACCC, Otsuk IP, Borguini RG, et al. Hydroxycinnamic acid amide derivatives, phenolic compounds and antioxidant activities of extracts of pollen samples from Southeast Brazil. J Agric Food Chem 2011; 59(10): 5516–5522.
60Dwivedi D, Dwivedi M, Malviya S, Singh V. Evaluation of wound healing, anti-microbial and antioxidant potential of Pongamia pinnata in wistar rats. J Tradit Complement Med 2016; 7(1): 79-85.
61Kahl R, Kappus H. Toxicology of the synthetic antioxidants BHA and BHT in comparison with the natural antioxidant vitamin E. Z Lebensm Unters Forsch 1993; 196(4): 329-338.
62Duraipandiyan V, Al-Harbi NA, Ignacimuthu S, Muthukumar C. Antimicrobial activity of sesquiterpene lactones isolated from traditional medicinal plant, Costus speciosus (Koen ex.Retz.) Sm. BMC Complem Altern Med 2012; 12(13): 1-6.
63Ait-Ouazzou A, Cherrat L, Espina L, Lorán S, Rota C, Pagán R. The antimicrobial activity of hydrophobic essential oil constituents acting alone or in combined processes of food preservation. Innovative Food Sci Emerging Technol 2011; 12(3): 320-329.
64Zengin H, Baysal AHC. Antibacterial and antioxidant activity of essential oil terpenes against pathogenic and spoilage-forming bacteria and cell structure-activity relationships evaluated by SEM microscopy. Molecules 2014; 19(11): 17773-17798.
65Obafemi CA, Sulaimon TO, Akinpelu DA, Olugbade TA. Antimicrobial activity of extracts and a germacranolidetype sesquiterpene lactone from Tithonia diversifolia leaf extract. Afr J Biotechnol 2006; 5(12): 1254-1258.
66Olayinka BU, Raiyemol DA, Etejere EO, Udeze AO. In vitro antimicrobial activities of Tithonia diversifolia (Hemsl) A. gray extracts on two bacteria and fungus isolates. J Chem Pharm Res 2014; 6(6): 2765-2768.
67Pappas PG, Kauffman CA, Andes DR, Clancy CJ, Marr KA, Oztrosky- Zeichner L, et al. Clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of America. Clin Infect Dis 2016; 62(4): e1-50.
68Kett DH, Shorr AF, Reboli AC, Reisman AL, Biswas P, Schlamm HT. Anidulafungin compared with fluconazole in severely ill patients with candidemia and other forms of invasive candidiasis: Support for the 2009 IDSA treatment guidelines for candidiasis. Crit Care 2011; 15(5): R253.
69Rajbhandari M, Mentel R, Jha PK, Chaudhary RP, Bhattarai S, Gewali MB, et al. Antiviral activity of some plants used in nepalese traditional medicine. Evid Based Complement Alternat Med 2009; 6(4): 517-522.
70Rajasekaran D, Palombo EA, Chia Yeo T, Lim Siok Ley D, Lee Tu C, Malherbe F, et al. Identification of traditional medicinal plant extracts with novel anti-influenza activity. PLoS One 2013; 8(11): e79293.
71Tzung LL, Hsu WC, Lin CC. Antiviral natural products and herbal medicines. J Tradit Complement Med 2014; 4(1): 24-35.
72Elamathi R, Kavitha R, Kamalakannan P, Deepa T, Sridhar S. Preliminary phytochemical and antimicrobial studies on the leaf of Ecbolium viride. World J Med Pharm Biol Sci 2012; 2(1): 5-10.
73Mafioleti L, Junior IFS, Colodel EM, Flach A, Martins DTO. Evaluation of the toxicity and antimicrobial activity of hydroethanolic extract of Arrabidaea chica (Humb. & Bonpl.) B. Verl. J Ethnopharm 2013; 150(2): 576-582.
74Marasini BP, Baral P, Aryal P, Ghimire KR, Neupane S, Dahal N, et al. Evaluation of antibacterial activity of some traditionally used medicinal plantas against human pathogenic bacteria. Biomed Res Int 2015; 2015: 265425.
75Tenpaisan R, Kawsud P, Pahumunto N, Puripattanavong J. Screening for antibacterial and antibiofilm activity in Thai medicinal plant extracts against oral microorganisms. J Tradit Complement Med 2017; 7(2): 172-177.
76Ekor M. The growing use of herbal medicines: Issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol 2013; 4: 177.
77Demain AL, Sanchez S. Microbial drug discovery: 80 years of progress. J Antibiot 2009; 62(1): 5-16.
78Arshad L, Jantan I, Bujhari SN, Haque MA. Immunosuppressive effects of natural α, β -unsaturated carbonyl-based compounds, and their analogs and derivatives, on immune cells: A review. Front Pharmacol 2017; 30: 8-22.
79Di Giacomo C, Vanella L, Sorrenti V, Santangelo R, Barbagallo I, Calabrese G, et al. Effects of Tithonia diversifolia (Hemsl.) A. Gray extract on adipocyte differentiation of human mesenchymal stem cells. PLoS One 2015; 10(4): e0122320.
80Hiransai P, Tangpong J, Kumbuar C, Hoonheang N, Rodpech O, Sangsuk P, et al. Anti-nitric oxide production, anti-proliferation and antioxidant effects of the aqueous extract from Tithonia diversifolia. Asian Pac J Trop Biomed 2016; 6(11): 950-956.