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DOI: 10.1055/s-0030-1250656
© Georg Thieme Verlag KG Stuttgart · New York
Antimycobacterial Activity and Alkaloid Prospection of Psychotria Species (Rubiaceae) from the Brazilian Atlantic Rainforest
Prof. Dr. Maura Da Cunha
Laboratório de Biologia Celular e Tecidual
Centro de Biociências e Biotecnologia
Universidade Estadual do Norte Fluminense
Av. Alberto Lamego, 2000
CEP 28013-602 Campos dos Goytacazes
Brazil
Phone: +55 22 27 39 72 63
Fax: +55 22 27 39 71 78
Email: maurauenf@gmail.com
Prof. Dr. Michelle Frazão Muzitano
Faculdade de Farmácia, Campus Macaé
Universidade Federal do Rio de Janeiro
R. Aluísio da Silva Gomes, 50
CEP 27930-560 Macaé, RJ
Brazil
Phone: +55 22 27 96 25 39
Fax: +55 22 27 96 25 39
Email: mfmuzitano@yahoo.com.br
Publication History
received August 23, 2010
revised Dec. 1, 2010
accepted Dec. 3, 2010
Publication Date:
17 January 2011 (online)
Abstract
Ten Psychotria species were collected in two fragments of Atlantic Forest in Rio de Janeiro: Psychotria pubigera (P1A and B), P. ruelliifolia (P2), P. suterela (P3), P. stachyoides (P4), P. capitata (P5), P. glaziovii (P6), P. leiocarpa (P7), P. nuda (P8), P. racemosa (P9) and P. vellosiana (P10). Ethanol extracts of these species were evaluated for their antimycobacterial activity, in an attempt to find new antituberculosis agents. Psychotria pubigera (P1A), P. ruelliifolia (P2) and P. stachyoides (P4) were the most active against Mycobacterium. The anti-inflammatory potential of these extracts was also evaluated in vitro to learn if they inhibit nitric oxide (NO) production in macrophages and if they have free-radical scavenging properties, because inflammation is a severe problem caused by tuberculosis, especially when the infection is from M. bovis or M. tuberculosis. Psychotria suterela (P3), P. stachyoides (P4) and P. capitata (P5) were the most active in inhibiting macrophage NO production but they were not the most antioxidant species. This suggests that NO inhibitory activity is not due to the scavenging of NO generated but due to a specific inhibition of iNOS activity or expression. In addition, cytotoxicity was tested in the macrophages (the host cells of the Mycobacterium) and it was verified that the extracts selectively killed the bacteria and not the host cells. When analyzing antimycobacterial, cytotoxicity and NO inhibitory activities in combination, P. stachyoides (P4) was the most promising anti-TB extract tested. Further, indol alkaloids were detected in P. suterela and P. nuda, and 5,6-dihydro-β-carboline alkaloids in all of the species studied, with the highest amounts found in P. capitata and P. racemosa.
Key words
Psychotria (Rubiaceae) - alkaloids - Mycobacterium bovis - anti‐inflammatory - antioxidant - tuberculosis
Introduction
Psychotria L. is comprised of approximately 2000 species that are found in tropical and subtropical rain forests [1]. The genus, which belongs to the Rubiaceae family, is taxonomically complex. Based on morphology and biogeography, it is divided into three subgenera: Psychotria (pantropical), Tetramerae (some species from Africa and Madagascar), and Heteropsychotria (neotropical species) [2]. However, morphological, phytochemical, and molecular similarities between species of the subgenus Heteropsychotria and of the genera Palicourea and Rudgea suggest that these taxa should be placed in a new genus [3].
Studies about the chemistry of Psychotria have helped establish new limits for the genus, which is known for its synthesis of bioactive alkaloids. The main metabolites found in pantropical species of Psychotria (subgenus Psychotria) are polyindole alkaloids [4]. Monoterpenoid indole alkaloids have been found in the neotropical species, and most of these alkaloids contain a glucoside residue that is unique compared to other monoterpene alkaloids (e.g., [5], [6]).
Several species of Psychotria show biological activity by presenting different types of secondary metabolites, especially alkaloids. Psychotria ipecacuanha is a medicinal plant of the understory of forests that is a powerful emetic and amebicide [7]. Psychotria viridis is used in religious rituals, where it is an ingredient in the hallucinogenic drink called ayahuasca [8]. Some species, such as P. beccarioides, P. forsteriana, and P. olenoides, biosynthesize highly cytotoxic alkaloids [9], [10]. Other pharmacological effects have also been reported for some species, such as the following: anti-inflammatory and antipyretic efficacies, and analgesic activity [11], [12]; inhibition of platelet aggregation [13]; antidepressant, anxiolytic, and antipsychotic activities [14]; antioxidant and antimutagenic activities [15], [16]; as well as activities against bacteria, protozoa, and fungi (e.g, [10]).
Natural products extracted from plants have been important in the discovery of new drugs. Between 1981 and 2006, approximately 52 % of the small molecules that were discovered or in development came from natural products [17]. Several compounds with antibacterial activity have been found in plants, fungi, and marine organisms [18].
Tuberculosis (TB), a major cause of death worldwide, is an infectious disease caused by Mycobacterium tuberculosis and other bacilli (e.g., Mycobacterium bovis, M. africanum, and M. microti). The World Health Organization reported that 8.9 million new TB cases occurred in 2004, and 1.7 million people died of this disease in the same year [19]. The available treatment is long (6–9 months), involves several drugs and is difficult to complete for some patients. Moreover, the available drugs do not ensure the destruction of the lesion because the bacillus can remain latent within the macrophage or become resistant to the drugs [20]. An increase in drug-resistant tuberculosis cases and the emergence of additional resistant strains and coinfections with HIV has stimulated the search for and development of new anti-TB drugs. Despite an increase in the number of natural product studies using plants, data show that only 15–17 % of plants worldwide have been studied for their medicinal potential [21].
Protective immunity against tuberculosis is mediated by interactions between specific T cells and activated macrophages. Phagocytic cells, such as macrophages, play a key role in the initiation and direction of the adaptive immune response against mycobacteria because of their antigen presentation, co-stimulatory activity, cytokine, and chemokine production [22]. Previous results obtained by our group showed that inflammation leads to an increase of severity of TB, and in a murine model of tuberculosis leads to the death of animals, especially for M. bovis and M. tuberculosis infections, which are different from M. avium (data not published). Infections with M. tuberculosis (Mtb) result in different clinical outcomes ranging from asymptomatic to rapidly progressing TB. Data has shown that the expression of inflammation-related genes (IL-1β, IL-6, TNF-α, CXCL2) and iNOS increased in mice that were more susceptible to TB, resulting in a relative abundance of these genes being expressed in the lungs [23].
The Atlantic Forest has high plant diversity and could potentially be a good place for natural product research. In addition, it is one of the hot spots of the world, where only about 1 % of the original area remains intact [24], [25]. Due to the richness of plant diversity and the rapid destruction of this biome, it is important to investigate the secondary metabolites found in the plants of this region and their possible biological activity. This study aimed to: (1) evaluate the activity of leaf ethanol extracts of 10 Psychotria species against Mycobacterium; (2) verify the extracts ability to inhibit NO production by the macrophages and their ability to act as an antioxidant, which are anti-inflammatory strategies that are important because tuberculosis causes severe inflammation; (3) evaluate the extracts cytotoxicity in the macrophages, which are the Mycobacterium host cells; and (4) verify the presence of alkaloids in the Psychotria leaves and determine the chemical profile of the ethanol extracts by HPLC/UV.
#Materials and Methods
#Botanical material
Plants were collected in two fragments of the Atlantic Forest in the state of Rio de Janeiro and identified by Dr. S. J. da Silva Neto at the Universidade Estadual do Rio de Janeiro. Psychotria capitata Ruiz & Pav., P. pubigera Schltdl., P. ruelliifolia (Cham. & Schltdl.) Müll. Arg., P. stachyoides Benth., and P. suterela Müll. Arg. were collected in Parque Nacional de Itatiaia, and P. glaziovii Müll. Arg., P. leiocarpa Cham. & Schltdl., P. nuda (Cham. & Schltdl.) Wawra, P. pubigera Schltdl., P. racemosa Rich., and P. vellosiana Benth. in the Reserva Biológica de Tinguá. Voucher specimens (RB-348480, RB-348434, RB-377884, RB-348438, RB-348440, H-5594, H-5595, H-5597, H-5593, H-5596, H-5598, respectively) were deposited at the Herbarium of the Jardim Botânico do Rio de Janeiro and at the Herbarium of the Universidade Estadual do Norte Fluminense Darcy Ribeiro.
#Ethanol extraction
The leaves were dried at room temperature and ground into powder. One gram of macerated samples were placed in 20 mL of EtOH for 24 hours on a shaker (120 rpm). The ethanol extracts were then filtered and ethanol was evaporated using a water bath (50 °C).
#Extracts analysis by thin layer chromatography (TLC)
Extracts were submitted to thin layer chromatography using silica gel F254 60. CH2Cl2/MeOH/NH4OH (85 : 18 : 1) was used as the mobile phase, and the chromatoplates were sprayed with Dragendorff reagent to identify the alkaloids.
#Ethanol extract analysis by high-performance liquid chromatography (HPLC)
Extracts were submitted to high-performance liquid chromatography using a Shimadzu Class – LC10 chromatographer with two LC10AT pumps, a scanning ultraviolet SPD-M10A photodiode array detector, and a Rheodyne 7725i injector. Approximately 1 mg of the ethanol extract was dissolved in 250 µL of MeOH and 250 µL of H2O. 20 µL of the sample was injected into a C18 column (5 µm, 250 × 4.6 mm) and eluted with MeOH and H2O, at a flow of 1 mL/min in a linear gradient. The gradient began with MeOH 0 % to 50 % for 5 minutes, MeOH 50 % to 70 % for 8 minutes, and MeOH 70 % to 100 % for 12 minutes. The components of the samples were detected by UV at 280 nm and 330 nm.
#Antimycobacterium activity of the extracts
All extracts were evaluated for their antimycobacterial activity using a tetrazole salt assay in a 96-well plate [26]. Initially, 300 µL of a suspension of Mycobacterium bovis BCG strain Moreau (3 × 107 CFU/mL) was incubated with 7.2 mL of Middlebrook 7H9 medium supplemented with 0.05 % Tween 80 and ADC. When at logarithmic growth phase, 50 µL were plated in a 96-well plate at 1 × 106 CFU/well, and 50 µL of each sample was added in three concentrations. The sealed plate was incubated at 37 °C and 5 % CO2 for 7 days. After this period, 10 µL of tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazole-5 mg/mL in sterile PBS) was added, and 3 hours after this, 100 µL of lyses buffer (20 % w/v SDS/50 % DMF – dimethylformamide in distilled water – pH 4.7) were added. The plate was incubated overnight, and the reading was made using a spectrophotometer at 570 nm. As a positive control, a culture medium with bacteria and the antibiotic rifampin (Sigma-Aldrich, 95 % purity), at concentrations of 0.0011, 0.0033, 0.01, and 0.03 µg/mL, was used. As a negative control, a culture medium with bacteria not treated with the extracts, was used. The test was performed in triplicate and the average value and standard deviation were calculated.
#Determination of nitric oxide production by macrophage RAW 264.7 and cytotoxicity
The murine peritoneal macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (ATCC) and grown at 37 °C and 5 % CO2 in DMEM F-12 that was supplemented with 10 % FCS and gentamicin (50 µg/mL). RAW 264.7 cells (1 × 105 cells/well) were seeded in flat bottom 96-well tissue culture plates (Corning, Inc.) in the presence or absence of various concentrations of the samples (100, 20, and 4 µg/mL) and/or LPS (Escherichia coli 055:B5; Sigma-Aldrich). After a 24-h incubation period, culture supernatants were collected and nitrite, a stable NO metabolite, was determined by using the Griess test [27]. As a negative control, macrophages not treated and not LPS stimulated were used. As a positive control, macrophages not treated but stimulated with 1 µg/mL LPS were used. L-NMMA (Sigma-Aldrich, 98 % purity) was also used as a positive control at 20 µg/mL inhibiting 54.71 ± 6.21 % NO production. The release of LDH (cytoplasmic enzyme lactate dehydrogenase) was determined using 50 µL of culture supernatant collected at the end of the assay [28]. The LDH content, which represents an indirect indication of cytotoxicity, was determined colorimetrically using a commercial kit (Doles Reagentes e Equipamentos para Laboratorios LTDA). The specific release was calculated as a percentage of the controls (nontreated macrophages as the negative control and 1 % Triton X-100 [Vetec Chem.] detergent treated macrophages as the positive control). Final concentrations of DMSO, used as the solvent of the samples, were tested in parallel as a control. Tests were performed in triplicate, and the mean value and standard deviation were calculated.
#Antioxidant activity of the ethanol extracts
The evaluation of antioxidant activity was carried out using the photocolorimetric stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl) [29]. Stock solutions of the extracts were prepared at 2 mg/mL in ethanol and diluted to different concentrations (0.2 and 0.02 mg/mL). 500 µL samples were added to a 500 µL DPPH solution 0.1 mM and were allowed to react at room temperature. After 1 h, the absorbance values were measured at 515 nm against the blank, which only contained the extract and not the reagents. The radical scavenging activity (% inhibition) was expressed as a percentage DPPH radical elimination and was calculated according to the following equation: % inhibition = [(Acontrol – Asample)/Acontrol] × 100, where Acontrol is the absorbance of negative control and Asample is the absorbance of the reaction mixture. All tests were performed in triplicate, and the average value and standard deviation were calculated. As a positive control, BHT was used [2,6-di-(tert-butil)-4-metilfenol] (Sigma-Aldrich, 99 % purity) which is a synthetic antioxidant industrially used.
#Results and Discussion
Ten Psychotria species were collected in two fragments of the Atlantic Forest of Rio de Janeiro: Psychotria pubigera (P1A), P. ruelliifolia (P2), P. suterela (P3), P. stachyoides (P4), and P. capitata (P5) in Parque Nacional de Itatiaia, and P. glaziovii (P6), P. leiocarpa (P7), P. nuda (P8), P. pubigera (P1B), P. racemosa (P9), and P. vellosiana (P10) in Reserva Biológica de Tinguá. Ethanol extracts of these species were evaluated for their antimycobacterial activity in an attempt to find new antituberculosis agents. Previous anti-bacterial activity reported for Psychotria species [10] contributed to our interest about this topic. Anti-inflammatory potential was evaluated, in vitro, to understand two important aspects: inhibition of macrophage NO production and the ability of the extracts to scavenge free radicals. Finding ways to reduce inflammation is important because it is a severe effect of tuberculosis, especially when the infection is from M. bovis or M. tuberculosis. In addition, cytotoxicity was assessed in the macrophages (the host cells of the Mycobacterium) to verify if the extract was selective against the Mycobacterium.
Antimycobacterial activity of the leaf ethanol extracts of the Psychotria species was verified using Mycobacterium bovis BCG culture. It was shown that the Psychotria extracts inhibited the M. bovis BCG growth at 100, 20, and 4 µg/mL ([Fig. 1 A] and [B]). The extracts had dose-dependent activity. All extracts were active in high concentrations, but only P1A, P2, and P4 showed more than 50 % inhibition in growth at the lower concentration (4 µg/mL). IC50 values, the sample concentration necessary to produce 50 % of the maximum response, was shown in [Table 1]. The most active species, P. pubigera (P1A) and P. stachyoides (P4), at a concentration of 4 µg/mL, inhibited the growth of M. bovis BCG by 79.33 ± 7.37 and 92.84 ± 2.57 %, respectively. The extract of P. glaziovii (P6) was the least active, inhibiting growth by only about 7 % at 4 µg/mL (as shown in [Fig. 1]). Rifampin was used as a positive control, and concentrations of 0.0011 and 0.03 µg/mL inhibited, respectively, 60.87 ± 1.31 % and 95.42 ± 1.17 % of the growth of the mycobacteria. Analyses with different concentrations of DMSO were performed to exclude a possible interference in the growth of the mycobacteria. DMSO had no significant effect on the growth of cultures at the concentrations used (≤ 2 %).
Fig. 1 Effect of ethanol extracts at concentrations of 4 µg/mL, 20 µg/mL, and 100 µg/mL, from the Psychotria leaves, on the growth of Mycobacterium bovis BCG. A – (P1A) P. pubigera – Itatiaia; (P1B) P. pubigera – Tinguá; (P2) P. ruelliifolia; (P3) P. suterela; (P4) P. stachyoides; (P5) P. capitata. B – (P6) P. glaziovii; (P7) P. leiocarpa; (P8) P. nuda; (P9) P. racemosa; (P10) P. vellosiana. Positive control – culture medium without bacteria (optical density, O. D. 0.086) and culture medium with bacteria and the antibiotic rifampin at concentrations of 0.0011 µg/mL, 0.0033 µg/mL, 0.01 µg/mL, and 0.03 µg/mL (60.87 % ± 1.31, 83.90 % ± 1.92, 94.94 % ± 3.48, 95.42 ± 1.17, respectively). Negative control – culture medium with bacteria (O. D. 0.785). Arithmetic mean ± SD (n = 3).
Fig. 2 The inhibitory effect of ethanol extracts at the concentrations of 4 µg/mL, 20 µg/mL, and 100 µg/mL, from the Psychotria species, on the production of nitric oxide by LPS-stimulated macrophage RAW 264.7. NO indirectly quantified in culture supernatant as NO2 − by the Griess Method. A – (P1A) P. pubigera – Itatiaia; (P1B) P. pubigera – Tinguá; (P2) P. ruelliifolia; (P3) P. suterela; (P4) P. stachyoides; (P5) P. capitata. B – (P6) P. glaziovii; (P7) P. leiocarpa; (P8) P. nuda; (P9) P. racemosa; (P10) P. vellosiana. Negative control – macrophages in culture medium (8.846 µM NO2 −). Positive control – macrophages in culture medium with stimulus – LPS 1 µg/mL (44.615 µM NO2 −). L‐NMMA was also used as a positive control at 20 µg/mL inhibiting 54.71 ± 6.21 % NO production.
|
Species |
IC50 (µg/mL) |
||
|
Antimycobacterial activity |
NO production inhibitory activity |
LDH specific release – cytotoxic activity |
|
|
P. pubigera (P1A) |
< 4 |
27.33 ± 1.45 |
42.39 ± 1.51 |
|
P. pubigera (P1B) |
6.26 ± 1.90 |
36.37 ± 1.49 |
> 100 |
|
P. ruelliifolia (P2) |
< 4 |
29.71 ± 1.45 |
41.70 ± 1.44 |
|
P. suterela (P3) |
13.59 ± 1.26 |
4.09 ± 1.28 |
32.82 ± 1.31 |
|
P. stachyoides (P4) |
< 4 |
18.74 ± 1.25 |
49.18 ± 1.46 |
|
P. capitata (P5) |
21.00 ± 1.27 |
10.51 ± 1.35 |
12.45 ± 1.35 |
|
P. glaziovii (P6) |
26.75 ± 1.10 |
38.74 ± 1.82 |
48.15 ± 1.64 |
|
P. leiocarpa (P7) |
14.24 ± 1.08 |
43.09 ± 1.75 |
74.54 ± 1.64 |
|
P. nuda (P8) |
8.32 ± 2.39 |
38.57 ± 1.66 |
> 100 |
|
P. racemosa (P9) |
9.91 ± 1.21 |
30.96 ± 1.36 |
> 100 |
|
P. vellosiana (P10) |
8.79 ± 1.17 |
30.10 ± 1.42 |
54.01 ± 1.83 |
This was the first time that the antimycobacterial activity of Psychotria species has been reported, except for P. vellosiana. In a previous study using plants from the Brazilian Atlantic Forest, the antimycobacterial activity of thirty-six plant extracts was evaluated, and five of these extracts were active against M. tuberculosis (including an extract from P. vellosiana), with a minimum inhibitory concentration (MIC) that was ≤ 0.2 µg/mL [26]. The presence of indole alkaloids, commonly found in the Rubiaceae [30], and β-carboline alkaloids in Psychotria species (e.g, [5]), could explain the activity of the extracts evaluated against Mycobacterium. Alkaloids are a very promising secondary metabolite class that should be investigated during the search for new anti-TB drugs. Between 2003 and 2006, 37 alkaloids were reported as growth inhibitors of Mycobacterium [31].
Psychotria species could inhibit Mycobacterium growth as seen in [Fig. 1]. In addition, the activity of ethanol extracts of the Psychotria leaves in the RAW 264.7 macrophages stimulated by LPS was evaluated to verify if, despite the antimycobacterial activity, they exhibit anti-inflammatory activity. These two activities together could be useful in the treatment of TB. [Fig. 2 A] and [B] show that all of the ethanol extracts at 100 µg/mL inhibited nitric oxide production by the RAW 264.7 macrophages. The extracts of P. suterela (P3), P. stachyoides (P4), and P. capitata (P5) were the most active in inhibiting NO production and even showed activity at lower concentrations, especially for P. suterela (P3). IC50 values were showed in [Table 1] and P. suterela presented the lower one, 4.09 ± 1.28 µg/mL. These results were supported by a lower release of LDH by the macrophages, at low concentration, indicating a low cytotoxicity against the macrophages ([Tables 1] and [2]). As seen in [Table 2], all extracts were toxic at the highest concentration (100 µg/mL). This interfered with the NO results and was not true for the other concentrations tested (4 and 20 µg/mL). When analyzing antimycobacterial and NO inhibitory activities together, P. stachyoides (P4) was the most promising extract tested.
|
Species |
LDH specific release (% control) |
||
|
4 µg/mL |
20 µg/mL |
100 µg/mL |
|
|
P. pubigera (P1A) |
1.27 ± 1.94 |
14.45 ± 2.90 |
91.38 ± 3.16 |
|
P. pubigera (P1B) |
−4.36 ± 0.72 |
17.30 ± 7.10 |
14.51 ± 5.74 |
|
P. ruelliifolia (P2) |
23.12 ± 5.31 |
23.02 ± 11.62 |
74.95 ± 22.37 |
|
P. suterela (P3) |
11.76 ± 2.44 |
25.86 ± 1.48 |
90.01 ± 0.93 |
|
P. stachyoides (P4) |
2.74 ± 11.33 |
12.02 ± 0.07 |
84.28 ± 7.60 |
|
P. capitata (P5) |
19.93 ± 11.55 |
62.93 ± 25.74 |
94.02 ± 1.86 |
|
P. glaziovii (P6) |
−6.08 ± 1.58 |
9.13 ± 9.61 |
90.92 ± 2.51 |
|
P. leiocarpa (P7) |
−9.84 ± 0.14 |
−1.77 ± 3.37 |
75.91 ± 0.22 |
|
P. nuda (P8) |
−8.72 ± 0.43 |
−4.06 ± 3.15 |
29.41 ± 1.00 |
|
P. racemosa (P9) |
−1.37 ± 8.53 |
3.19 ± 6.96 |
56.44 ± 10.11 |
|
P. vellosiana (P10) |
−7.86 ± 0.64 |
−0.10 ± 0.14 |
92.80 ± 0.43 |
In a previous study, the evaluation of nitric oxide activity was tested using methanol extracts of seven Malaysian rain forest plants, including P. rostrata [32]. These authors showed that the extracts from P. rostrata inhibited nitric oxide production because of their cytotoxic effects on RAW 264.7 cells. This data is in agreement with our study but only when comparing it to the highest concentration we used.
Scavenger activity is beneficial to immune response during an inflammatory process because it decreases oxidative stress, principally when it is associated with the inhibition of inflammatory mediator production. Antioxidant activity of the ethanol extracts of the Psychotria leaves was determined by using a DPPH antioxidant assay. This assay is based on the ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical, to decolorize in the presence of antioxidants. The extracts that had higher antioxidant activity were P. pubigera (P1A and P1B) and P. racemosa (P10) ([Table 3]). Psychotria suterela (P3) and P. capitata (P5) were very active in inhibiting macrophage NO production, but they did not have the highest antioxidant activity. This suggests that the NO inhibitory activity is not due to the scavenging of NO generated but due to a specific inhibition of iNOS activity or expression. The amount of ethanol extract of P. stachyoides was very small, just enough for antimycobacterial activity, macrophages cytotoxicity and NO assay, and for TLC evaluation.
|
Species |
100 µg/mL (%) |
10 µg/mL (%) |
|
P. pubigera (P1A) |
82.63 ± 1.88 |
77.29 ± 8.73 |
|
P. pubigera (P1B) |
79.29 ± 1.36 |
64.21 ± 10.21 |
|
P. ruelliifolia (P2) |
80.41 ± 0.42 |
31.94 ± 17.43 |
|
P. suterela (P3) |
52.19 ± 4.53 |
20.92 ± 0.75 |
|
P. capitata (P5) |
82.30 ± 1.92 |
35.39 ± 1.45 |
|
P. glaziovii (P6) |
50.02 ± 5.20 |
18.69 ± 1.36 |
|
P. leiocarpa (P7) |
70.89 ± 5.17 |
23.42 ± 2.11 |
|
P. nuda (P8) |
42.68 ± 5.15 |
17.08 ± 0.34 |
|
P. racemosa (P9) |
83.08 ± 1.51 |
69.83 ± 4.69 |
|
P. vellosiana (P10) |
79.85 ± 3.52 |
27.26 ± 2.70 |
|
BHT |
52.1 ± 2.2 |
43.6 ± 1.5 |
In addition, chemical aspects such as alkaloid content were investigated to verify if there was a relationship between them and the different biological activities observed.
The evaluation of the extracts chromatographic profiles using HPLC/DAD allowed the verification of peaks with typical chromophores of indole alkaloids (223 and 280 nm) and 5,6-dihydro-β-carboline alkaloids (236 and 320 nm) as shown in [Table 4]. The chromatographic profile of P. suterela (P3) revealed two peaks that showed the characteristic UV spectra of the indole alkaloid and one 5,6-dihydro-β-carboline. Psychotria capitata (P5) and P. racemosa (P9) showed higher total percentages of area of the 5,6-dihydro-β-carboline. In both extracts of P. pubigera (P1A and P1B) the presence of 5,6-dihydro-β-carboline alkaloids was detected; however, a greater percentage was detected in P1A ([Table 4]). This result may be due to the influence of the environment, because the species were collected in different locations. The chromatographic profile of Psychotria extracts, using thin layer chromatography (TLC) and Dragendorff reagent, revealed the presence of one or more alkaloids that seemed to be common in all species, corroborating the HPLC data (data not shown).
|
Species |
Indole alkaloids (number of peaks) |
Indole alkaloids (total %area) |
5,6-dihydro-β-carboline alkaloids (number of peaks) |
5,6-dihydro-β-carboline alkaloids (total % area) |
|
P. pubigera (P1A) |
– |
– |
2 |
11.191 |
|
P. pubigera (P1B) |
2 |
4.653 |
||
|
P. ruelliifolia (P2) |
– |
– |
1 |
11.288 |
|
P. suterela (P3) |
1 |
8.588 |
1 |
4.392 |
|
P. capitata (P5) |
– |
– |
5 |
44.348 |
|
P. glaziovii (P6) |
– |
– |
1 |
6.024 |
|
P. leiocarpa (P7) |
– |
– |
1 |
4.299 |
|
P. nuda (P8) |
1 |
5.176 |
– |
– |
|
P. racemosa (P9) |
– |
– |
4 |
28.660 |
|
P. vellosiana (P10) |
– |
– |
1 |
7.794 |
A previous study analyzed the total alkaloid fraction of the leaves of 14 Brazilian Psychotria species using HPLC/DAD, which showed peaks of the UV spectra that are characteristic of the indole chromophore [33]. Compounds with the UV spectra for β-carboline and 5,6-dihydro-β-carboline were also found in P. suterela and P. umbellata [9], [10].
Studies have demonstrated that β-carboline alkaloids can act as scavengers of reactive oxygen species. The aromatic and dihydro-β-carbolines show a protective effect against oxidative agents in yeast (Sccharomyces cerevisiae), and their hydroxyl radical-scavenging property appears to contribute to their antimutagenic and antigenotoxic effects in yeast and mammalian cells (i.e., Chinese hamster lung fibroblasts – V79 cells) [34]. There are only a few reports published about the antioxidant activity of crude extracts or compounds isolated from the Psychotria species [15], [16]. Studies done with crude foliar extracts and psychollatine and brachycerine alkaloids from P. umbelata and P. brachyceras, respectively, showed antioxidant and antimutagenic effects in Saccharomyces cerevisiae. These activities appear to be related to their scavenging capacity toward the hidroxyl radical [15], [16].
Psychotria is considered a “hot genus” with regard to its cytotoxic potential of extracts and fractions used in studies conducted by the National Cancer Institute-USA (NCI) [35]. In the future, studies of cytotoxicity in tumor cells will be performed to evaluate the antitumor potential of these plant extracts.
In conclusion, our results showed that the extracts from the Psychotria species studied were active against Mycobacterium bovis, inhibiting NO production and lowering the cytotoxicity of the RAW 264.7 macrophage, especially at 20 and 4 µg/mL. Extracts from P. pubigera (P1A) and P. stachyoides (P4) were the most active against Mycobacterium. Extracts from P. suterela (P3), P. stachyoides (P4), and P. capitata (P5) were the most active in inhibiting macrophage NO production, but they were not the most antioxidant extracts in the DPPH assay. This suggests that NO inhibitory activity is not due to the scavenging of NO generated but due to a specific inhibition of iNOS activity or expression. Further, indole alkaloids were detected in P. suterela (P3) and P. nuda (P8), and β-carboline alkaloids in all of the species studied, with a higher amount found in P. capitata (P5) and P. racemosa (P9). When analyzing antimycobacterial, cytotoxic, and NO inhibitory activities together, P. stachyoides (P4) was the most promising anti-TB extract tested. Further purification studies on the extracts of these species are being carried out in order to identify the active compounds.
#Acknowledgements
We thank the Coordenação the Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ) for financial support; Msc. Sebastião José da Silva Neto for the species identification. This study was part of the PhD thesis of the first author, presented to the Programa de Pós-Graduação em Biociências e Biotecnologia/UENF.
#References
- 1 Davis A P, Bridson D, Jarvis C, Govaerts R L. The typification and characterization of the genus Psychotria L. (Rubiaceae). Bot J Linn Soc. 2001; 135 35-42
- 2 Petit E. Les espèces africaines du genre Psychotria L. (Rubiaceae) – II. Bull Jard Bot Brux. 1966; 36 65-189
- 3 Nepokroeff M, Bremer B, Sytsma K J. Reorganization of the genus Psychotria and tribe Psychotrieae (Rubiaceae) inferred from ITS and rbcL sequence data. Syst Bot. 1999; 24 5-27
- 4 Kerber V A, Rech S B, Elisabetsky E, Henriques A T. Análise de alcalóides de Psychotria de ocorrência no sul do Brasil. Caderno de Farmácia. 1997; 13 165-166
- 5 De Santos L V, Fett-Neto A G, Kerber V A, Elisabetsky E, Quirion J C, Henriques A T. Indole monoterpene alkaloids from the leaves of Psychotria suterella Müll Arg. (Rubiaceae). Biochem Syst Ecol. 2001; 29 1185-1187
- 6 Henriques A T, Lopes S O, Paranhos J T, Gregianini T S, Von Poser G L, Fett-Neto A G, Schripsema J. N,β-D-Glucopyranosyl vincosamide, a light regulated indole alkaloid from the shoots of Psychotria leiocarpa. Phytochemistry. 2004; 65 449-454
- 7 Assis M C, Giulietti A M. Diferenciação morfológica e anatômica em populações de “ipecacuanha” – Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae). Rev Bras Bot. 1999; 22 205-216
- 8 Quinteiro M M C, Teixeira D C, Moraes M G, Silva J G. Anatomia foliar de Psychotria viridis Ruiz & Pav. (Rubiaceae). Rev Univ Rural Sér Ci da Vida. 2006; 26 30-41
- 9 Roth A, Kuballa B, Bonthanh C, Caballion P, Sévenet T, Beck J P, Anton R. Cytotoxic activity of polyindoline alkaloids of Psychotria forsteriana (Rubiaceae). Planta Med. 1986; 5 450-453
- 10 Benevides P J C, Young M C M, Bolzani V D. Biological activities of constituents from Psychotria spectabilis. Pharm Biol. 2004; 42 565-569
- 11 Verotta L, Orsini F, Sbacchi M, Scheildler M A, Amador T A, Elisabetsky E. Synthesis and antinociceptive activity of chimonanthines and pyrrolidinoindoline-type alkaloids. Bioorg Med Chem. 2002; 10 2133-2142
- 12 Both F L, Farias F M, Nicoláo L L, Misturini J, Henriques A T, Elisabetsky E. Avaliação da atividade analgésica de extratos alcaloídicos de espécies de Psychotria. Rev Bras Pl Med. 2002; 5 41-45
- 13 Beretz A, Roth-Georger A, Corre G, Kuballa B, Anton R, Cazenale J P. Polyindolinic alkaloids from Psychotria fosteriana. Potent inhibitors of the aggregation of human platelets. Planta Med. 1985; 4 300-303
- 14 Both F L, Meneghini L, Kerber V A, Henriques A T, Elizabetsky E. Psychopharmacological profile of the alkaloid psychollatine as a 5-HT2A/C serotonin modulator. J Nat Prod. 2005; 68 374-380
- 15 Nascimento N C, Fragoso V, Moura D J, Silva A C R, Fett-Neto A G, Saffi J. Antioxidant and antimutagenic effects of the crude foliar extract and the alkaloid brachycerine of Psychotria brachyceras. Environ Mol Mutat. 2007; 48 728-734
- 16 Fragoso V, Nascimento N C, Moura D J, Silva A C R, Richter M F, Saffi J, Fett-Neto A G. Antioxidant and antimutagenic properties of the monoterpene indole alkaloid psychollatine and the crude foliar extract of Psychotria umbellata Vell. Toxicol In Vitro. 2008; 22 559-566
- 17 Newman D J, Cragg G M. Natural products as sources of new drugs over the last 25 years. J Nat Prod. 2007; 70 461-477
- 18 Pauli G F, Case R J, Inui T, Wang Y, Cho S, Fischer N H, Franzblau S G. New perspectives on natural products in TB drug research. Life Sci. 2005; 78 485-494
- 19 World Health Organization (WHO) .Global tuberculosis control: surveillance, planning, financing: WHO report 2007. Available
at. http://www.who.int Accessed March 15, 2008
- 20 Zhang X, Kuramitsu Y, Fujimoto M, Hayashi E, Yuan X, Nakamura K. Proteomic analysis of macrophages stimulated by lipopolysaccharide: lipopolysaccharide inhibits the cleavage of nucleophosmin. Electrophoresis. 2006; 27 1659-1668
- 21 Guerra M P, Nodari R O.
Biodiversidade: aspectos biológicos, geográficos, legais e éticos. Schenkel E, Simões CMO Farmacognosia: da planta ao medicamento, 5th ed. Porto Alegre/Florianópolis; Universidade Federal do Rio Grande do Sul and Universidade Federal de Santa Catarina Press 2004 - 22 Mueller P, Pieters J. Modulation of macrophage antimicrobial mechanisms by pathogenic mycobacteria. Immunobiology. 2006; 211 549-556
- 23 Lyadova I V, Tsiganov E N, Kapina M A, Shepelkova G S, Sosunov V V, Radaeva T V, Majorov K B, Shmitova N S, van den Ham H J, Ganusov V V, De Boer R J, Racine R, Winslow G M. In mice, tuberculosis progression is associated with intensive inflammatory response and the accumulation of Gr-1 cells in the lungs. PLoS One. 2010; 5 e10469
- 24 Myers N, Mittermeier R A, Mittermeier C G, Fonseca G A B, Kent J. Biodiversity hotspots for conservation priorities. Nature. 2000; 403 853-858
- 25 Ribeiro M C, Metzger J P, Martensen A C, Ponzoni F J, Hirota M M. The Brazilian Atlantic Forest: How much is left, and how is the remaining forest distributed? Implications for conservation. Biol Conserv. 2009; 142 1141-1153
- 26 Gomez-Flores R, Gupta S, Tamez-Guerra R, Mehta R T. Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method. J Clin Microbiol. 1995; 33 1842-1846
- 27 Da Silva S A G, Costa S S, Rossi-Bergmann B. The anti-leishmanial effect of Kalanchoe is mediated by nitric oxide intermediates. Parasitology. 1999; 118 575-582
- 28 Muzitano M F, Cruz E A, Almeida A P, Silva S A G, Kaiser C R, Guette C, Rossi-Bergmann B, Costa S S. Quercitrin: an antileishmanial flavonoid glycoside from Kalanchoe pinnata. Planta Med. 2006; 72 81-83
- 29 Duarte C D, Tributino J L M, Lacerda D I, Martins M V, Alexandre-Moreira M S, Dutra F, Bechara E J H, De-Paula F S, Goulart M O F, Ferreira J, Calixto J B, Nunes M P, Bertho A L, Miranda A L P, Barreiro E J, Fraga C A M. Synthesis, pharmacological evaluation and electrochemical studies of novel 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives: Discovery of LASSBio-881, a new ligand of cannabinoid receptors. Bioorg Med Chem Lett. 2007; 15 2421-2433
- 30 Schripsema J, Dagnino D, Gosmann G.
Alcalóides indólicos. Schenkel E, Simões CMO Farmacognosia: da planta ao medicamento, 5th edition. Porto Alegre/Florianópolis; Universidade Federal do Rio Grande do Sul and Universidade Federal de Santa Catarina Press 2004: 918-958 - 31 Copp B R, Pearce A N. Natural product growth inhibitors of Mycobacterium tuberculosis. Nat Prod Rep. 2007; 24 278-297
- 32 Saha K, Lajis N H, Israf D A, Hamzah A S, Khozirah S, Khamis S, Syahida A. Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malasyan medicinal plants. J Ethnopharmacol. 2004; 92 263-267
- 33 Lopes S, Von Poser G L, Kerber V A, Farias F M, Konrath E L, Moreno P, Sobral M E, Zuanazzi J A S, Henriques A T. Taxonomic significance of alkaloids and iridoid glucosides in the tribe Psychotrieae (Rubiaceae). Biochem Syst Ecol. 2004; 32 1187-1195
- 34 Moura D J, Richter M F, Boeira J M, Henriques J A P, Saffi J. Antioxidant properties of β-carboline alkaloids are related to their antimutagenic and antigenotoxic activities. Mutagenesis. 2007; 22 293-302
- 35 Cragg G M, Newman D J, Yang S S. Natural product extracts of plant and marine origin having antileukemia potential. The NCI experience. J Nat Prod. 2006; 69 488-498
Prof. Dr. Maura Da Cunha
Laboratório de Biologia Celular e Tecidual
Centro de Biociências e Biotecnologia
Universidade Estadual do Norte Fluminense
Av. Alberto Lamego, 2000
CEP 28013-602 Campos dos Goytacazes
Brazil
Phone: +55 22 27 39 72 63
Fax: +55 22 27 39 71 78
Email: maurauenf@gmail.com
Prof. Dr. Michelle Frazão Muzitano
Faculdade de Farmácia, Campus Macaé
Universidade Federal do Rio de Janeiro
R. Aluísio da Silva Gomes, 50
CEP 27930-560 Macaé, RJ
Brazil
Phone: +55 22 27 96 25 39
Fax: +55 22 27 96 25 39
Email: mfmuzitano@yahoo.com.br
References
- 1 Davis A P, Bridson D, Jarvis C, Govaerts R L. The typification and characterization of the genus Psychotria L. (Rubiaceae). Bot J Linn Soc. 2001; 135 35-42
- 2 Petit E. Les espèces africaines du genre Psychotria L. (Rubiaceae) – II. Bull Jard Bot Brux. 1966; 36 65-189
- 3 Nepokroeff M, Bremer B, Sytsma K J. Reorganization of the genus Psychotria and tribe Psychotrieae (Rubiaceae) inferred from ITS and rbcL sequence data. Syst Bot. 1999; 24 5-27
- 4 Kerber V A, Rech S B, Elisabetsky E, Henriques A T. Análise de alcalóides de Psychotria de ocorrência no sul do Brasil. Caderno de Farmácia. 1997; 13 165-166
- 5 De Santos L V, Fett-Neto A G, Kerber V A, Elisabetsky E, Quirion J C, Henriques A T. Indole monoterpene alkaloids from the leaves of Psychotria suterella Müll Arg. (Rubiaceae). Biochem Syst Ecol. 2001; 29 1185-1187
- 6 Henriques A T, Lopes S O, Paranhos J T, Gregianini T S, Von Poser G L, Fett-Neto A G, Schripsema J. N,β-D-Glucopyranosyl vincosamide, a light regulated indole alkaloid from the shoots of Psychotria leiocarpa. Phytochemistry. 2004; 65 449-454
- 7 Assis M C, Giulietti A M. Diferenciação morfológica e anatômica em populações de “ipecacuanha” – Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae). Rev Bras Bot. 1999; 22 205-216
- 8 Quinteiro M M C, Teixeira D C, Moraes M G, Silva J G. Anatomia foliar de Psychotria viridis Ruiz & Pav. (Rubiaceae). Rev Univ Rural Sér Ci da Vida. 2006; 26 30-41
- 9 Roth A, Kuballa B, Bonthanh C, Caballion P, Sévenet T, Beck J P, Anton R. Cytotoxic activity of polyindoline alkaloids of Psychotria forsteriana (Rubiaceae). Planta Med. 1986; 5 450-453
- 10 Benevides P J C, Young M C M, Bolzani V D. Biological activities of constituents from Psychotria spectabilis. Pharm Biol. 2004; 42 565-569
- 11 Verotta L, Orsini F, Sbacchi M, Scheildler M A, Amador T A, Elisabetsky E. Synthesis and antinociceptive activity of chimonanthines and pyrrolidinoindoline-type alkaloids. Bioorg Med Chem. 2002; 10 2133-2142
- 12 Both F L, Farias F M, Nicoláo L L, Misturini J, Henriques A T, Elisabetsky E. Avaliação da atividade analgésica de extratos alcaloídicos de espécies de Psychotria. Rev Bras Pl Med. 2002; 5 41-45
- 13 Beretz A, Roth-Georger A, Corre G, Kuballa B, Anton R, Cazenale J P. Polyindolinic alkaloids from Psychotria fosteriana. Potent inhibitors of the aggregation of human platelets. Planta Med. 1985; 4 300-303
- 14 Both F L, Meneghini L, Kerber V A, Henriques A T, Elizabetsky E. Psychopharmacological profile of the alkaloid psychollatine as a 5-HT2A/C serotonin modulator. J Nat Prod. 2005; 68 374-380
- 15 Nascimento N C, Fragoso V, Moura D J, Silva A C R, Fett-Neto A G, Saffi J. Antioxidant and antimutagenic effects of the crude foliar extract and the alkaloid brachycerine of Psychotria brachyceras. Environ Mol Mutat. 2007; 48 728-734
- 16 Fragoso V, Nascimento N C, Moura D J, Silva A C R, Richter M F, Saffi J, Fett-Neto A G. Antioxidant and antimutagenic properties of the monoterpene indole alkaloid psychollatine and the crude foliar extract of Psychotria umbellata Vell. Toxicol In Vitro. 2008; 22 559-566
- 17 Newman D J, Cragg G M. Natural products as sources of new drugs over the last 25 years. J Nat Prod. 2007; 70 461-477
- 18 Pauli G F, Case R J, Inui T, Wang Y, Cho S, Fischer N H, Franzblau S G. New perspectives on natural products in TB drug research. Life Sci. 2005; 78 485-494
- 19 World Health Organization (WHO) .Global tuberculosis control: surveillance, planning, financing: WHO report 2007. Available
at. http://www.who.int Accessed March 15, 2008
- 20 Zhang X, Kuramitsu Y, Fujimoto M, Hayashi E, Yuan X, Nakamura K. Proteomic analysis of macrophages stimulated by lipopolysaccharide: lipopolysaccharide inhibits the cleavage of nucleophosmin. Electrophoresis. 2006; 27 1659-1668
- 21 Guerra M P, Nodari R O.
Biodiversidade: aspectos biológicos, geográficos, legais e éticos. Schenkel E, Simões CMO Farmacognosia: da planta ao medicamento, 5th ed. Porto Alegre/Florianópolis; Universidade Federal do Rio Grande do Sul and Universidade Federal de Santa Catarina Press 2004 - 22 Mueller P, Pieters J. Modulation of macrophage antimicrobial mechanisms by pathogenic mycobacteria. Immunobiology. 2006; 211 549-556
- 23 Lyadova I V, Tsiganov E N, Kapina M A, Shepelkova G S, Sosunov V V, Radaeva T V, Majorov K B, Shmitova N S, van den Ham H J, Ganusov V V, De Boer R J, Racine R, Winslow G M. In mice, tuberculosis progression is associated with intensive inflammatory response and the accumulation of Gr-1 cells in the lungs. PLoS One. 2010; 5 e10469
- 24 Myers N, Mittermeier R A, Mittermeier C G, Fonseca G A B, Kent J. Biodiversity hotspots for conservation priorities. Nature. 2000; 403 853-858
- 25 Ribeiro M C, Metzger J P, Martensen A C, Ponzoni F J, Hirota M M. The Brazilian Atlantic Forest: How much is left, and how is the remaining forest distributed? Implications for conservation. Biol Conserv. 2009; 142 1141-1153
- 26 Gomez-Flores R, Gupta S, Tamez-Guerra R, Mehta R T. Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method. J Clin Microbiol. 1995; 33 1842-1846
- 27 Da Silva S A G, Costa S S, Rossi-Bergmann B. The anti-leishmanial effect of Kalanchoe is mediated by nitric oxide intermediates. Parasitology. 1999; 118 575-582
- 28 Muzitano M F, Cruz E A, Almeida A P, Silva S A G, Kaiser C R, Guette C, Rossi-Bergmann B, Costa S S. Quercitrin: an antileishmanial flavonoid glycoside from Kalanchoe pinnata. Planta Med. 2006; 72 81-83
- 29 Duarte C D, Tributino J L M, Lacerda D I, Martins M V, Alexandre-Moreira M S, Dutra F, Bechara E J H, De-Paula F S, Goulart M O F, Ferreira J, Calixto J B, Nunes M P, Bertho A L, Miranda A L P, Barreiro E J, Fraga C A M. Synthesis, pharmacological evaluation and electrochemical studies of novel 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives: Discovery of LASSBio-881, a new ligand of cannabinoid receptors. Bioorg Med Chem Lett. 2007; 15 2421-2433
- 30 Schripsema J, Dagnino D, Gosmann G.
Alcalóides indólicos. Schenkel E, Simões CMO Farmacognosia: da planta ao medicamento, 5th edition. Porto Alegre/Florianópolis; Universidade Federal do Rio Grande do Sul and Universidade Federal de Santa Catarina Press 2004: 918-958 - 31 Copp B R, Pearce A N. Natural product growth inhibitors of Mycobacterium tuberculosis. Nat Prod Rep. 2007; 24 278-297
- 32 Saha K, Lajis N H, Israf D A, Hamzah A S, Khozirah S, Khamis S, Syahida A. Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malasyan medicinal plants. J Ethnopharmacol. 2004; 92 263-267
- 33 Lopes S, Von Poser G L, Kerber V A, Farias F M, Konrath E L, Moreno P, Sobral M E, Zuanazzi J A S, Henriques A T. Taxonomic significance of alkaloids and iridoid glucosides in the tribe Psychotrieae (Rubiaceae). Biochem Syst Ecol. 2004; 32 1187-1195
- 34 Moura D J, Richter M F, Boeira J M, Henriques J A P, Saffi J. Antioxidant properties of β-carboline alkaloids are related to their antimutagenic and antigenotoxic activities. Mutagenesis. 2007; 22 293-302
- 35 Cragg G M, Newman D J, Yang S S. Natural product extracts of plant and marine origin having antileukemia potential. The NCI experience. J Nat Prod. 2006; 69 488-498
Prof. Dr. Maura Da Cunha
Laboratório de Biologia Celular e Tecidual
Centro de Biociências e Biotecnologia
Universidade Estadual do Norte Fluminense
Av. Alberto Lamego, 2000
CEP 28013-602 Campos dos Goytacazes
Brazil
Phone: +55 22 27 39 72 63
Fax: +55 22 27 39 71 78
Email: maurauenf@gmail.com
Prof. Dr. Michelle Frazão Muzitano
Faculdade de Farmácia, Campus Macaé
Universidade Federal do Rio de Janeiro
R. Aluísio da Silva Gomes, 50
CEP 27930-560 Macaé, RJ
Brazil
Phone: +55 22 27 96 25 39
Fax: +55 22 27 96 25 39
Email: mfmuzitano@yahoo.com.br
Fig. 1 Effect of ethanol extracts at concentrations of 4 µg/mL, 20 µg/mL, and 100 µg/mL, from the Psychotria leaves, on the growth of Mycobacterium bovis BCG. A – (P1A) P. pubigera – Itatiaia; (P1B) P. pubigera – Tinguá; (P2) P. ruelliifolia; (P3) P. suterela; (P4) P. stachyoides; (P5) P. capitata. B – (P6) P. glaziovii; (P7) P. leiocarpa; (P8) P. nuda; (P9) P. racemosa; (P10) P. vellosiana. Positive control – culture medium without bacteria (optical density, O. D. 0.086) and culture medium with bacteria and the antibiotic rifampin at concentrations of 0.0011 µg/mL, 0.0033 µg/mL, 0.01 µg/mL, and 0.03 µg/mL (60.87 % ± 1.31, 83.90 % ± 1.92, 94.94 % ± 3.48, 95.42 ± 1.17, respectively). Negative control – culture medium with bacteria (O. D. 0.785). Arithmetic mean ± SD (n = 3).
Fig. 2 The inhibitory effect of ethanol extracts at the concentrations of 4 µg/mL, 20 µg/mL, and 100 µg/mL, from the Psychotria species, on the production of nitric oxide by LPS-stimulated macrophage RAW 264.7. NO indirectly quantified in culture supernatant as NO2 − by the Griess Method. A – (P1A) P. pubigera – Itatiaia; (P1B) P. pubigera – Tinguá; (P2) P. ruelliifolia; (P3) P. suterela; (P4) P. stachyoides; (P5) P. capitata. B – (P6) P. glaziovii; (P7) P. leiocarpa; (P8) P. nuda; (P9) P. racemosa; (P10) P. vellosiana. Negative control – macrophages in culture medium (8.846 µM NO2 −). Positive control – macrophages in culture medium with stimulus – LPS 1 µg/mL (44.615 µM NO2 −). L‐NMMA was also used as a positive control at 20 µg/mL inhibiting 54.71 ± 6.21 % NO production.