A PRELIMINARY EVALUATION OF ANTIBACTERIAL AND TOXIC POTENTIALS OF P. GUINEENSE SWARTZ. FRUIT PULP FROM DIFFERENT STAGES OF RIPENESS

A PRELIMINARY EVALUATION OF ANTIBACTERIAL AND TOXIC POTENTIALS OF P. GUINEENSE SWARTZ. FRUIT PULP FROM DIFFERENT STAGES OF RIPENESS

Samara Alves Brito , Fabiola Fernandes Galvao Rodrigues , Adriana Rolim Campos , Jose Galberto Martins da Costa*

Molecular Bioprospection Post-graduation Program, Laboratory of Natural Products Research, Regional University of Cariri ¹, Crato, CE, Brazil

Vice-Rectory of Research and Post-Graduation, University of Fortaleza ², Fortaleza-CE, Brazil

ABSTRACT:Psidium guineense Swartz is a shrub native to Brazil. It is consumed in natura or in the form of pulp, which presents a high content of C vitamin and proteins. The chemical prospection of the aqueous extracts from unripe and mature pulp revealed the presence of tannins and flavonoids. The extracts demonstrated moderate antibacterial activity against the most of strains tested, and the best result was obtained with the unripe pulp extract against Staphylococcus aureus (MR358; MIC 256 μg/mL). The extracts were further tested for in vivo brine shrimp lethality test. The mature pulp extract exhibited significant toxicity (LC₅₀=125 μg/mL).

INTRODUCTION: Psidium is a genus of about 100 species of tropical shrubs and small trees ¹, of the genus Psidium, the most important species is classified as Psidium guajava L. (goiabeira). The genus comprises many species that produce comestible fruits, and woody and ornamental plants with potential commercial value. Among these species, the “aracazeiros” deserve more attention, especially because some characteristics of the fruits, like exotic flavor, high content of C vitamin and good consumer acceptance ².

Psidium guineense Swartz, a shrub known popularly as “araca verdadeiro”, belongs to the Myrtaceae family ³. This species is native to Brazil and is cultivated mainly for fruits production ^{2, 4} that are described to possess high content of C vitamin in comparison to citrus fruits and to present more proteins than guava ³. The study of antimicrobial activity, mode of action and potential use of medicinal plants has been increasing in importance,    mainly the evaluation of antimicrobial properties comprising a great variety of microorganisms ⁵. There is any report in literature regarding the antimicrobial and toxicity activities of P. guineense. The absence of data stimulates the emerging of studies in these areas.

Given the need of chemical analysis of natural products ⁶ and the fact that fruits are a source of chemical diversity and the widely domestic and industrial consumption in nature or in the pulp form⁷, this work aimed to realize the phytochemical prospection of aqueous extracts from P. guineense unriped and mature fruits pulps in order to identify the secondary metabolites and to evaluate the antibacterial and toxic potentials.

MATERIAL AND METHODS:

Plant material and extract collection: Fruits from P. guineense Swartz from different stages of ripeness were collected from Grangeiro Municipality, Ceara State (Brazil) and transported to the Natural Products Research Laboratory of Regional University of Cariri. The selected fruits were cleaned and processed in order to obtain the pulp. The pulp extracts were obtained by cold extraction with distillated water ⁸ followed by lyophylization until complete dehydration.

Phytochemical prospection:            The phytochemical prospection of aqueous extracts from unriped (AEUP) and mature (AEMP) fruits to detect the presence of secondary metabolites was performed following the method described by Matos ⁸.

Antibacterial activity evaluation: The antibacterial activities of the extracts were investigated by employing a microdilution method, recommended by NCCCLS M7-A6 ⁹. Brain Hear Infusion Broth (BHI 3.8%) was used for bacterial growth (24 h, 35±2^oC). The inoculum was an overnight culture of each bacterial species in BHI broth diluted in the same media to a final concentration of approximately 1 x 10 8 UFC/mL (0.5 nephelometric turbidity units - McFarland scale). After this, the suspension was diluted to 1 x 10 6 UFC/mL in 10% BHI. 100 µL of each dilution were distributed in 96-well plates plus extracts in different concentrations, achieving 5x10 5 UFC/mL as the final concentration of the inoculum. Six standard strains were used: two gram-positive (multiresistant Staphylococcus aureus MR 358 and Staphylococcus aureus MR 6538) and four gram-negative (Bacilus cereus ATCC 33018, Pseudomonas aeruginosa ATCC 5442, Escherichia coli MR 27 e Escherichia coli ATCC 2992.

The extracts were dissolved in distilled water and dimethyl sulfoxide (DMSO) to a concentration of 1024μg/mL. Further serial dilutions were performed by addition of BHI broth to reach a final concentration in the range of 512 a 8µg/mL. All experiments were performed in triplicate, and the microdilution trays were incubated at 35±2^oC for 24h. Antibacterial activity was detected using a colorimetric method by adding 25µL of resauzurin staining (0.01%) aqueous solution in each well at the end of the incubation period. The minimal inhibitory concentration (MIC) was defined as the lowest the extracts able to inhibit the bacteria growth, as indicated by resauzurin staining (bacteria died cells are not able to change the staining color by visual observation-blue to red).

Both extracts were tested for brine shrimp lethality. The assay was performed basically according to the simplified Meyer's et al ¹⁰. Briefly, brine shrimp, Artemia salina L. encysted eggs were incubated in artificial seawater at 28 ^OC. Samples were dissolved in DMSO plus artificial seawater. Serial dilutions (triplicate) were prepared in the same solution. Metanauplii (10 units) was added to each set of tubes containing samples and the cultures further incubated for 24h. Controls containing DMSO were included on each set of experiments. Potassium dichromate was used as reference standard. LD₅₀ (after 24h) were calculated by Probit analysis. When found value of LC₅₀<1000 μg/mL the assayed product was regarded as toxic bioactive compound ¹¹.

RESULTS AND DISCUSSION: Phytochemical prospection of P. guineense fruits aqueous extracts indicated the presence of the same secondary metabolites classes as tannins and flavonoids. These chemical constituents present a wide range of biological activities as antimicrobial ^{12, 13, 14}, antitumoral ¹⁵ and antiofidic ¹⁶. Especially the antioxidant potential of phenolic compounds, described to be significantly found in fruits from Psidium genus ¹⁷.

The extracts here studied presented moderate activity against gram-positive and gram-negative strains and the best result was obtained with the unripe pulp extract against Staphylococcus aureus (MR358), MIC 256 μg/mL (Table 1). According to the literature, extracts from roots and leaves from Psidium species showed antimicrobial properties ^{18, 19, 20, 21, 22} and only the pulps fruits of Psidium guajava ²³ and Psidium sartorianum ²⁴ are antifungal.

TABLE 1: ANTIMICROBIAL ACTIVITY OF THE AQUEOUS EXTRACTS FROM P. GUINEENSE PULPS FRUITS

 The larval mortality index obtained on toxicity bioassay ranged from 27 to 100%. The LC₅₀ found was 125 μg/mL to AEMP, indicating significant cytotoxic activity against Artemia salina and 1000 μg/mL to AEUP.

CONCLUSION: The chemical prospection of P. guineense extracts detected the presence of tannins and flavonoids. The antibacterial activity might be related to the presence of tannins as these metabolites have proved antibacterial properties. Concerning the antibacterial evaluation, the extracts presented moderate activity against the tested bacteria and the best result was obtained with the unripe pulp extract against Staphylococcus aureus (MR358), MIC 256 μg/mL. The extract from mature fruits was classified as toxic to Artemia salina as the observed LC₅₀ was minor than standard limit (1000 μg/mL). According to the preliminary bioassays, the extracts presented significant differences in relation to the toxicity and a moderate biological potential against pathogenic bacteria. The results can propel new researches with P. guineense isolated substances in the search of therapeutic alternatives from Brazilian natural products.

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