Chemical Constituents , Larvicidal Effects and Radical Scavenging Activity of Tetracera breyniana Schltdl . ( Dilleniaceae )

Article history: Received on: 09/07/2013 Revised on: 29/07/2013 Accepted on: 14/09/2013 Available online: 30/09/2013 Extracts and fractions from the leaves and stems of Tetracera breyniana Schltdl. were evaluated against the fourth instar Aedes aegypti larvae and ability to scavenge free radicals. Fractions that provided the best results were fractionated on silica gel column to afforded three flavonoils (quercetin, 7-O-methylquercetin and 7-Omethylkaempferol) and two terpenoids (β-sitosterol and betulinic acid). These compounds were identified on basis of their physical and NMR spectral data and by comparison with literature data. With exception of quercetin, all other compounds are been described for the first time in the investigated species. In the larvicidal assays, when compared to synthetic insecticide Temephos, only hexane fraction from stem was effective (LD50 72.08 g/mL). In the DPPH assays, EtOAc fractions from the leaves (CI50 74.15 ± 14.73 g/mL) and stem (IC50 39.87 ± 13.46 g/mL), of which quercetin was isolated, showed the best results when compared with the positive standards used while CHCl3 fractions of both plant parts, of which methylated flavonols were isolated, showed only a moderate activity. Possibly these constituents are responsible in part for the radical scavenging activities observed. Subsequently, evaluation of all isolated compounds will be needed to confirm the active component.


INTRODUCTION
Dengue is a major public health problem in the world.According to World Health Organization (WHO) estimates, 0.5 million people with dengue hemorrhagic fever (DHF) require hospitalization each year and approximately 2.5% of these individuals die from DHF (WHO, 2008).Annually, there may be over 50-100 million dengue infections in tropical and subtropical areas (Renjana and Thoppil, 2013), and so these diseases have emerged as a major international health problem and a notable increase in dengue cases (77.7%) has been reported by Brazilian health authorities (Oliveira et al., 2010).
In recent years, there has been a global trend toward the use of natural substances present in vegetables and other sources as antioxidant.Several of these substances may interfere with the oxidation process by reacting with free radicals (Silva et al., 2010).When these species had an excessive formation induces oxidative damage to biomolecules, which eventually causes numerous diseases (Gülçin et al., 2006;Edewor-Kuponiyi, 2013).
Chemical investigations carried out with active extracts of different Tetracera species have reported mainly the isolation of flavonoids (Gurni et al., 1981;Gurni and Kubitzi, 1981;Na et al., 2001;Lee et al., 2009) and terpenoids (Dan and Dan, 1980;Ma et al., 1999;Na et al., 2001;Subramanyam et al., 2009) and studies have showed that some of these compounds possess important biological activities (Ma et al., 1999;Subramanyam et al., 2009;Nguyen and Nguyen, 2013).T. breyniana Schltdl., known as "cipó-de-fogo", is a creeper whose leaf decoction is used in Brazil against lymphatic problems (Souza, 2000).From the leaves of this species, besides proanthocyanidin some common flavonols (azaleatin, kaempferol, 5-methylkaempferol, and quercetin), glycoside (quercetin 3-O-galactopyranoside) and sulphates (kaempferol 3-sulphate and quercetin 3-sulphate) have been isolated (Gurni and Kubitzki, 1981).This work was undertaken in order to determine the phytochemicals, larvicidal effects and free radical scavenging activity of the extracts from the leaves and stem of this species.Among isolated compounds, with exception of quercetin, this work report for the first time the presence of 7-Omethylquercetin and 7-O-methylkaempferol, and terpenoids (betulinic acid and β-sitosterol) as well as evaluation of larvicidal and radical scavenging activities in the investigated species.

Plant Material
Leaves and stem of T. breyniana were collected in the Área de Proteção Ambiental de Santa Rita, Marechal Deodoro, AL, Brazil, in May 2008 and identified by Rosangela P. de Lyra Lemos of the Instituto do Meio Ambiente do Estado de Alagoas (IMA-AL), where a voucher specimen (MAC-23840) was deposited.
The structures of the compounds (1-5) (Fig. 1) were identified by their physical (mp) and by interpretation of their spectral data ( 1 H and 13 C NMR, including DEPT, HSQC and HMBC experiments) and by the comparison with those reported in literature.

Larvicidal assays
Fourth instar of A. aegypti larvae, aged four to six days, were collected from a mosquito colony maintained at insectaria of the Instituto de Química e Biotecnologia da Universidade Federal de Alagoas.The mosquitoes were kept at 27.1 ± 4°C and 69.9 ± 7.8% relative humidity and photoperiod of approximately 12 h.The hatching of larvae occurred in distilled water, and the feeding of adult insects was made with anhydrous glucose solution to 10% in cotton balls changed daily.The blood supply for the females was performed using the species pigeons Columbia livia.Larvicidal assays were performed following the recommendations of the WHO (1981) with some modifications (Oliveira et al., 2010).Initially all samples were screened at 250 μg/mL.Based on the percent mortality values (mortality ≥ 75%), LD50 values in micrograms per milliliter and their 95% fiducial limits of upper and lower limits of the samples were determined.

Statistical analysis
The average larval mortality data were subjected to probit analysis for calculating LD 50 , and other statistics at 95% fiducial limits of upper and lower confidence limits were calculated by using Probit analysis of Finney (1971).

Free radical scavenging activity (DPPH)
These assays for all samples were conducted in triplicate, according to the methods described by Silva et al. (2010).Samples and standards were measured at 515 nm, using at least five different concentrations (12.5 to 100 μg/mL), against a blank (MeOH HPLC grade).In a cuvette, 0.1 mL of the samples was mixed with 0.9 mL DPPH• dissolved in MeOH (100 μmol/L) and the absorbance was monitored for 1 h.The percentage inhibition was calculated using the equation, (%) = [(Abs control -Abs samples )/ Abs control ] x 100, and IC 50 values were estimated by a nonlinear regression algorithm using Origin TM version 7.0 (Micronal, Northampton, MA, USA).Data were analyzed by ANOVA followed by Tukey test (p < 0.05).Ascorbic acid and BHT were used as controls.

RESULTS AND DISCUSSION
In the preliminary larvicidal assays, among the extracts tested only one of them showed 100% mortality against fourth instar A. aegypti larvae.All other no caused mortality (≤ 25%) after 48 h exposure.As shown in Table 1, when compared to synthetic insecticide Temephos (LD 50 8.7 μg/mL), only hexane fraction from stem (LD 50 72.08g/mL; 95% CL 60.92-82.47)was effective.All others were inactive (LD 50 > 280 g/mL; results not shown in Table 1) (Oliveira et al., 2010).The isolation and purification of this extract and evaluation of these compounds will be needed to identify the active component.
Despite significant advances in the techniques used for mosquitoes control during recent decades, it continues to pose serious public health problems and many infectious diseases for humans and animals such as malaria, dengue fever, yellow fever, filariasis, and others can be transmitted by vectors. A. aegypti acts as a vector for the arboviruses responsible for yellow fever and also for dengue and dengue fevers (Garcez et al., 2009).Since there is no vaccine currently available for dengue and DHF prevention, the eradication of the mosquito adults and larvae, which is primarily relied on applications of synthetic insecticides or larvicides, is the most commonly adopted strategy for reducing morbidity (WHO, 2008).According to the literature, the organophosphate temephos is one of the most employed commercial insecticides for the control of this vector (Macoris et al., 2007;Garcez et al., 2009;Oliveira et al., 2010).However, these chemicals can affect non target population and may increase mosquito resistance to insecticides.So, plants may be an alternative source of mosquito control agents because they constitute a rich source of bioactive chemicals.
In this study, free radical scavenging activity of extract and fractions from T. breyniana, as well as of the standards used (ascorbic acid and BHT) were evaluated by DPPH.As shown in Table 1, among the extracts tested, with exception of hexane fractions from the leaves and stems, all other samples tested showed significant antioxidant activity (IC 50 < 350.78 ± 2.71 μg/mL) and the best results were obtained with the EtOAc fractions from the leaves (74.15 ± 14.73 μg/mL) and stems (39.87 ± 13.46 μg/mL), with IC 50 values comparable to standards used, of which quercetin (3) was isolated.Comparatively, CHCl 3 fractions of both parts of the plant showed only a moderate activity (Table 1).These fractions, less hydroxylated flavonoids (7-Omethylquercetin and 7-O-methylkaempferol) were isolated.Subsequently, evaluation of all isolated compounds will be needed to confirm the active component.37.37 ± 3.18 97.86 ± 2.52 CL = confidence limits (lower and upper confidence limits).a Mean value of three replicates; LD50 lethal dose that kills 50% of the exposed larvae; NT = not tested.
Many studies have shown that antioxidant properties, especially radical scavenging activities, are very important due to the deleterious role of free radicals in foods and in biological systems (Gülçin et al., 2006) and antioxidant capacity is widely used as a parameter to characterize food or medicinal plants and their bioactive components (Silva et al., 2010).The hydroxyl radical which is a very reactive species that is formed in biological systems has been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells.Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity (Edewor-Kuponiyi, 2013).According Silva et al. (2010), anti-radical activities of plant extracts are often associated with the presence of polyphenol compounds, like flavonoids, which have an important role in stabilizing lipid oxidation.

CONCLUSION
There are no reports on larvicidal and radical scavenging activities to species studied.The results of this study showed that hexane fraction from stem of T. breyniana possess potential for control of mosquito A. aegypti and open the possibility of further investigations on the efficacy of the larvicidal properties of plant product extracts.This study also showed that some extracts were effectively able to inhibit the formation of free radicals.Among the extracts tested, the best radical scavenging activities was found mainly by the most polar extracts and in some case, this activity was comparable with that of BHT or ascorbic acid.Although there is the possibility that the tannins and others pigments takes a role in scavenging activity, previous studies showed that the effects of some extracts seem to be correlated with the presence of phenolic compounds such as flavonoids.Then, among compounds isolated, quercetin, 7-O-methylkaempferol, 7-O-methylquercetin may mediate the radical scavenging activity observed in the extracts from leaves and stem of T. breyniana.

Table . 1
: Effects of extracts and fractions against forth instar A. aegypti larvae and DPPH.