Ethanolic extract of Casearia sylvestris Sw exhibits in vitro antioxidant and antimicrobial activities and in vivo hypolipidemic effect in rats

The Casearia sylvestris Sw (Flacourtiaceae) is a shrub that occurs in forests of Southern Brazil; its leaves are widely used in folk medicine as a depurative, analgesic, anti-inflammatory and antiulcerogenic agent. The objective of this study was to perform the phytochemical description and to evaluate the pharmacological activities (antimicrobial, antifungal, antioxidant and toxicity) of the ethanolic extract (EE) of C. sylvestris Sw. In addition, we also evaluated the effect of the EE of C. sylvestris Sw on the glucose levels and lipid profile in blood serum of rats submitted to a model of streptozotocin-induced diabetes. Material and Methods: In vitro assay: the detection of chemical groups was done through chemical reactions with the development of color or precipitate and by chromatographic profile; the antioxidant activity was measured by the method of reduction of DPPH free radical (2,2-diphenyl-1picrylhydrazyl); the Minimum Inhibitory Concentration was evaluated by the broth microdilution method, and the Minimum Bactericide Concentration and the Minimum Fungicide Concentration were performed in Petri dishes; the cytotoxic activity was measured by the Artemia salina test. In vivo assay: diabetic and non-diabetic rats were treated with EE of C. sylvestris Sw (300 mg/ kg) for 45 days, and the glycaemia and lipid profile were analyzed. Results: The EE showed a Lethal Dose50 of 724.76 μg.mL-1 and important antioxidant, fungicide and fungistatic activities. The EE showed better antimicrobial activity regarding the microorganisms Staphylococcus aureus, Escherichia coli and Salmonella setubal. Conclusion: The EE of C. sylvestris Sw produces a significant decrease in triglycerides, total cholesterol and VLDL levels without any significant alteration in the glycaemia. The EE of C. sylvestris Sw presents antioxidant and antimicrobial activities and it exhibits a potent hypolipidemic effect.


INTRODUCTION
Herbal medicine has been used throughout centuries similarly to the modern pharmaceuticals are used nowadays.The Brazilian territory covers a wide range of climates and soil types, providing a large biodiversity.Herbal medicines provide rational means for the treatment of many diseases (Vermani & Garg, 2002); between them, we call attention to diabetes.This disease is associated with impaired glucose tolerance and lipid metabolism.It is also linked to other common health problems, such as obesity, hypertension and atherosclerosis (Saltiel & Kahn, 2001;Modak et al., 2007).Although there are many approaches to reduce the ill effects of diabetes and its secondary complications, a lot of people prefer herbal formulations due to lesser side effects and low cost.Thus, screen for natural compounds or their derivatives with biological activity against diseases could provide a thorough knowledge of ethno botanical based on therapies and a rational exploration of new metabolites of therapeutic value.
Previous study showed that flavonoids are able to improve the lipid profile in normal rats (Ito et al., 2008); and the C. sylvestris extract is a flavonoid-rich plant, as demonstrated in phytochemical screening in this study.The antihyperlipidemic activity of C. sylvestris was reported in olive oil-loaded mice (Schoenfelder et al., 2008).Nevertheless, there are no studies of C. sylvestris action on lipid profile and glycaemia in animal models of diabetes.Therefore, our objective was described phytochemical screening and evaluated the pharmacological activities as antimicrobial, antifungal, antioxidant and toxicity of ethanolic extract of C. sylvestris Sw.In addition, we also evaluated the effect of the ethanolic extract of C. sylvestris Sw on glucose levels and lipid profile in blood serum of male rats submitted to a model of streptozotocin-induced diabetes.

Plant material
The material was collected for analysis in Lajeado, Rio Grande do Sul, in February 2008.And, it was identified by Professor André Jasper PhD (PPGAD -UNIVATES) and voucher material, under number 2267, was filed in Herbarium of the Museum of Natural Sciences -Centro Universitário UNIVATES.

Casearia sylvestris
The plant was collect in the city of Lajeado, Rio Grande do Sul, Brazil, during the summer of 2008 and was identified by Professor Eduardo Ethur PhD (Department of Chemistry of Centro Universitário Univates).The dried leaves were minced and extracted with 90% ethanol/water solution (ethanol:water 9:1), at room temperature, for 7 days.The solution was then totality evaporated and the extract was stored at -8 °C.The extracts were dissolved in a 3% Tween 80 solution to the desired concentration, just before use.The ethanolic extract of C. sylvestris was administered at concentration of 300 mg/kg (de Mattos et al., 2007).

Phytochemical Screening
It consisted of a set of qualitative analysis  Pharmacopoeia (1988).

Pharmacological activities Antibacterial and antifungal activities of the ethanolic extract (EE) of Casearia sylvestris
The analysis of the antimicrobial activity of ethanolic extract of C. sylvestris was performed using the microdilution method, using ATCC strains (American Type Culture Collection) of the following organisms -  (CBS 7987).The minimum inhibitory concentration (MIC) was determined by the microdilution method, realized in triplicate.The microbial suspension was prepared on the day of analysis, obtained by adding the inoculum in 5.0 mL of sterile saline 0.8% to a standardized amount of microorganisms in solution.This standardization was carried out according to the McFarland nephelometric scale at 0.5, equivalent to approximately 1.5 x 10 8 UFC.mL -1 (colony forming units per milliliter).For greater accuracy the amount of inoculum present, followed the recommendations of the NCCLS, whose reading of turbidity was performed in UV-VIS spectrophotometer at 625 nm wavelengths.We adapted the technique described by NCCLS (2003)[20], using a 96-microwell sterile.From solutions of the ethanolic extract of C. sylvestris at a concentration of 20.0 mg.mL 1 in dimethyl sulfoxide (DMSO) were applied in microliter aliquots of 100.0 microwells in triplicate.Then there was a series of dilutions of these samples in casein soy broth for bacteria and Sabouraud broth for fungi, previously inoculated, resulting in the following test concentrations: 10, 5, 2.5, 1.25, 0.625 and 0.3125 mg.mL -1 .The standard antibiotic was also applied in serial dilutions from a solution of chloramphenicol 0.2 mg.mL -1 , while the standard antifungal ketoconazole was used in a solution of 1 mg.mL -1 .During the test was also carried out control of the medium, inoculum and DMSO.The plates were incubated for 24 hours at 35 °C for bacteria and 48 hours at 25 °C for fungi.After this, there was microbial (bacteria and fungi) growth through the presence of turbidity in the wells.The well contains the lowest concentration where no more microbial growth was observed corresponded to the value of the minimum inhibitory concentration (MIC).For the visualization of microbial growth, were applied 10 mL of a solution of triphenyltetrazolium chloride (TTC) 5 mg.mL -1 , which results in red coloration in the wells where there was growth.The culture showed no growth plate was transferred to solid medium (nutrient agar and Sabouraud agar), to determine the minimum bactericidal concentration (MBC).

Antioxidant activity of the ethanolic extract (EE) of Casearia sylvestris
T h e r e d u c t i o n p o t e n t i a l o f D P P H (2,2-diphenyl-1-picrylhydrazyl) from ethanolic extract of C. sylvestris was assessed spectrophotometrically as described by Elmastas et al. (2006).It was prepared a methanol solution of DPPH 0.051 mg.mL -1 , in 1.0 mL of this solution was added to 3.0 mL of methanol solution of ethanolic extract in concentrations of 0.10, 0.06, 0.04, 0.02, 0.01 and 0.005 mg.mL 1 .The mixtures were shaken and kept in the dark at room temperature for 30 minutes.The absorbance of the samples and controls were read in UV-VIS spectrophotometer (Perkin Elmer λ 25) at a wavelength of 517 nm (VIS), which corresponds to the maximum absorption of the free radical under study.The experiment was performed in triplicate, with the control the butylhydroxytoluene (BHT), synthetic antioxidant and quercetin (QUE), a natural antioxidant.The ability of the ethanolic extract of C. sylvestris to reduce the free radical DPPH was calculated according to the following equation: % Inhibition of DPPH = [(A0 -A1) / A0 × 100].Where A0 is equal to the absorbance of the control and A1 is the absorbance of the samples.For the analysis of the results were used spreadsheet graphics to express the percentage of antioxidant activity.

Cytotoxic activity of the ethanolic extract (EE) of Casearia sylvestris
For analysis of cytoxicity activity (Figure 4) was used the microcrustacean Artemia saline, a methodology was adapted from Meyer et al. (1992).The samples were diluted to obtain concentrations of 10, 100 and 1000 µg.mL -1 , was applied along the positive control test tubes, in triplicate, leaving rest for 24 hours, drying the solvent.After this, it was applied 10 specimens of A. saline in each tube and 0.05 mL of yeast.The tube was sealed and left to stand for 24 hours to evaluate the Lethal Dose 50 (LD50).

Experimental model Animals
The experiments were carried out on 60 day-old male Wistar (180-190g) rats that was randomized according to the weight and housed in groups of five in home cages made of Plexiglass material (65 x 25 x 15 cm).The animals were maintained at 22 ± 2 °C with water and food ad libitum, under a 12:12h light/dark cycle.Experiments were conducted between 08h00 and 17h00.

Experimental design
Before the beginning of experiment, the rats were habituated to the maintenance room by one week.The animals were firstly divided into two groups: non-diabetic (received citrate buffer, 1mL/ Kg, i.p.) and diabetic groups [received an unique dose of streptozotocin (STZ), 65 mg/Kg, i.p.].After, each group was divided into three groups, in total six groups: (CC): non-diabetic receiving drinking water by gavage (1 mL/kg); (CT): non-diabetic receiving 3% Tween 80 solution by gavage (extract of C. sylvestris Sw vehicle -1 mL/kg); (CE): non-diabetic rats treated with ethanolic extract of C. sylvestris Sw by gavage (300 mg/kg); (DS): diabetic receiving drinking water by gavage (1 mL/kg) and saline s.c.; (DI): diabetic rats receiving drinking water by gavage (1 mL/kg) and injections insulin s.c.(2 UI/ kg of regular insulin at 08:00 a.m., and 2-4 UI/kg of NPH insulin at 05:00 p.m.) dose of insulin was adjusted based on the severity of hyperglycemia, in order to maintain their blood glucose below 150 mg/ dL (Kowluru, 2003); (DE): diabetic rats treated with ethanolic extract of C. sylvestris by gavage (300 mg/ kg) and saline s.c.

Experimental induction of diabetes in rats
Rats were injected intraperitoneally with a single dose of 65 mg/kg STZ (Sigma Chemical Co., St Louis, MO, USA), freshly dissolved in citrate buffer (0.1 mol/L, pH 4.5) as previous described (Vats et al., 2004).Diabetes induction in rats was identified by polydipsia, polyuria and by measuring non-fasting serum glucose concentrations 48 h after injection of STZ.Rats with a serum glucose level above 300 mg/ dL were selected for experimentation.

Pharmacological Treatment
The pharmacologic treatment started after the diabetes induction, i.e., 48 hours after STZ administration, and it was applied for 45 days consecutives: once a day, the animals received water (CC and DI), 3% Tween 80 solution (CT) and ethanolic extract of C. sylvestris Sw (CE and DE) by gavage; and twice a day the animals received saline (s.c., DS) and insulin (s.c., 08:00 a.m. and 05:00 p.m., DI).

Determination of blood glucose levels
Blood glucose levels were monitored using blood glucose test strips and a blood glucose meter (Accu-Check, Roche, Brazil).Non-fasting blood glucose was measured on day 1 (basal), 15, 30 and 45 from blood obtained from the tail vein in all groups.

Determination of serum lipids
Triglycerides (TG), total cholesterol (TC), LDL cholesterol (LDL-C) and HDL cholesterol (HDL-C) were determined on day 45 using commercial enzymatic kits (Diagnostic Labtest, MG, Brazil).VLDL cholesterol (VLDL-C) was calculated by the Friedewald equation (Friedewald et al., 1972) after measurement of total serum cholesterol, triglycerides and HDL cholesterol as previously reported (Wood et al., 2006).After fasting for 12h, animals were submitted to anesthesia with xylazine (0.5mg/kg) and ketamine (10mg/kg) and blood samples collected by cardiac puncture, centrifuged at 3000 rpm for 10 min and serum samples were obtained.

Statistical analysis
Data were expressed as mean + Standard Error of the Mean (S.E.M.) or Standard Deviation (S.D.) as indicated.For the antioxidant activity, it was used one-way ANOVA test, followed by Student-Newman-Keuls test when necessary.The cytotoxic activity was calculated from linear regression obtained by Origin Graph version 5.0 program, which allows the calculation of LD50.For confirm the diabetes condition was applied Student t test for independent samples.Repeated measures two-way ANOVA with independent factors diabetes and treatment was performed followed by multiple comparisons test (Bonferroni test) when indicated for glucose measures, and two-way ANOVA followed by multiple comparisons (Student-Newman-Keuls test -SNK) for lipid profile.Differences were considered to be statistically significant if P<0.05.

Income
After removing the solvent in a rotary evaporator, we obtained dark brown extract with a yield of approximately 3.7%.

Phytochemical Screening
Through the phytochemical screening tests detected the following constituent groups: phenolic compounds, flavonoids, alkaloids, saponins, coumarins and tannins.

Pharmacological activities Antibacterial and antifungal activities of ethanolic extract (EE) of C. sylvestris
The EE of C. sylvestris showed activity against the Staphylococcus aureus, Escherichia coli and Salmonella setubal (Table 1).In addition, the EE of C. sylvestris presented also a fungicide activity and be lethal to Saccharomyces cerevisiae and Cryptococcus neoformans, and fungistatic activity characterized by inhibition the growth of Candida albicans, Candida glabrata and Candida dubliniensis (Table 2).For the standard MBC, was found 3,12x10 -3 mg.mL -1 .For the standard MFC was found 1.25x10 -1 mg.mL -1 .

Antioxidant activity of ethanolic extract (EE) of C. sylvestris
We observed an important antioxidant activity of this extract, where the EE at the lowest dose tested showed a significant antioxidant activity.Importantly, BHT concentrations 0.005 and 0.01 mg.mL -1 , like QUE 0.005 mg.mL 1 had lower potential to reduce the DPPH radical than in all of the EE concentrations (ANOVA, SNK P<0.05).

Cytotoxicity activity of ethanolic extract (EE) of C. sylvestris
This test it was possible to assess the cytotoxic activity and calculate the LD50 value provided by Figure 4.The behavior of the extract was different in each test performed.According to Meyer et al. (1982), the larger the LD50 value, the lower the degree of toxicity to extract evaluated, as such, the lower the LD50 value found, the greater the toxicity.Then, test the value found for the LD50 was 724.76 μg.mL -1 , and values of LD50 less than 1000 μg.mL -1 are consider toxic samples, however our EE presented moderate toxicity.

Biochemical analysis Effect of ethanolic extract (EE) of C. sylvestris on blood glucose
Initially, 48h after the induction of diabetes with STZ i.p., and before the first dose of ethanolic extract or its vehicle, we observed difference between the level of glucose in relation to diabetics (DS, DI and DE) and non-diabetics groups (CC, CT and CE; one-way ANOVA/SNK, P<0.001, data not shown) in the first time (day 1).
We found a significant difference in the mean of glycaemia level between diabetic and nondiabetic rats, when diabetic rats presented higher level of glycaemia (P<0.001).We found a significant difference in the mean of glycaemia level among treatment, the animals that received vehicle (tween 80) and insulin presented lower level of glycaemia in comparison to other groups (two-way ANOVA, P<0.001).The C. sylvestris treatment for 45 days had no effect on glycaemia in normal or streptozotocininduced diabetic rats (repeated measures two-way

Effect of ethanolic extract (EE) of C. sylvestris on lipid profile
We found significant difference on lipid profile between diabetic and non-diabetic rats, where diabetic rats presented higher levels of triglycerides and VLDL, associated to lower levels of total cholesterol, LDL and HDL (two-way ANOVA, P<0.01, Table 3).
We observed effect of the treatment by two-way ANOVA (P<0.05).It was observed which animals that received ethanolic extract of C. sylvestris presented lower triglycerides and VLDL levels in comparison to other groups (two-way ANOVA/SNK, P<0.05).The total cholesterol level of the animals that received ethanolic extract of C. sylvestris presented lower levels in comparison to animals that received water and vehicle (tween 80) by gavage (two-way ANOVA/SNK, P<0.05).While, the animals that received insulin presented even total cholesterol lower level in relation to other groups (two-way ANOVA/SNK, P<0.05), and animals that received vehicle (tween 80) by gavage presented higher levels than other groups.In the HDL level, animals that received ethanolic extract of C. sylvestris and vehicle (tween 80) presented higher level in comparison to animals that received water or insulin (two-way ANOVA/SNK, P<0.05).In relation to the LDL level, animals that received ethanolic extract of C. Sylvestris and insulin presented lower level in relation to animals that received water, and animals that received vehicle (tween 80) by gavage presented higher levels than other groups (two-way ANOVA/SNK, P<0.05, Table 3).
In addition, we observed interaction between diabetes and treatment in relation to level of triglycerides, total cholesterol, LDL and VLDL (two-way ANOVA, P<0.05).

DISCUSSION
In this study, the EE extract showed an important antioxidant activity, checked by the test of DPPH.In addition, it showed an antioxidant activity higher than the standards BHT and quercetin in lower concentrations, corroborating a recent study using hydroalcoholic extract of C. sylvestris (Albano et al., 2013).In the last years, it has been described the role of free radicals and pro-oxidant molecules in aging, cancer, cardiovascular diseases, and brain dysfunction (Molyneux, 2004).Thus, it is need to study new compounds to protect the body against free radical, and based on our results, we suggest that the potential antioxidant activity observed in our study can be related to the chemical composition of C. sylvestris.It is known that coumarins, flavonoids, lignans and several diterpenes, especially the clerodane (de Carvalho et al., 1998;Tininis et al., 2006) are present, in its composition.The results of the phytochemical screening confirmed the presence of phenolic compounds, alkaloids, flavonoids, saponins, tannins and coumarins.And, phenols compounds, tannins, flavonoids and coumarin, are generally described with the antioxidant activity (Martín-Sánchez at al., 2014).Deserving emphasis on phenolic compounds and flavonoids that determine the antioxidant activity of the extract of C. sylvestris (Menezes et al., 2004), acting as free radical scavenger.In addition, the EE showed an antimicrobial activity against the microorganisms Staphylococcus aureus, Escherichia coli and Salmonella Setubal.Likewise, the antimicrobial activities can be linked to the presence of saponins, which presents bactericidal and fungicidal activities (Chung et al, 2006).
Additionally, in opposing to popular medicine, the chronic treatment of EE (45 days) did not promote any benefits in the glycaemia serum level of rats.However, it is important to highlight that our model of diabetes induction (STZ) worked very well, the diabetic rats presented high levels of glycaemia.It is important to bring to light that the diabetes model used is involved to the inhibition of biosynthesis and secretion of insulin, culminating in the β cells death; and we can not discard that the folk used of C. sylvestris may be related to at least in part the production, releasing or action of endogenous insulin.Interestingly, the vehicle (tween 80) presented lower levels of glycaemia in comparison to other treatments (two-way ANOVA); this effect observed showed a limitation of our study, since, we did not have diabetic rats treated with the vehicle (tween 80).In the present study, we did not evaluate a possible synergic effect between insulin and EE of C. sylvestris Sw, that it can be considered a limitation of the present protocol.
In relation to diabetic condition, we observed that diabetic rats presented lower levels of total cholesterol and LDL in relation to non-diabetic rats, and these levels were decreased in high proportion after insulin treatment revealed by two-way ANOVA corroborating previous study (Pinheiro et al., 2011).
These rats also presented higher serum levels of triglycerides and VLDL, and this condition was reverted by insulin treatment.In addition, the same animals showed lower level of HDL that did not suffer any influence of insulin treatment.Interestingly, we found reduced levels of total serum cholesterol in diabetic rats in comparison to non-diabetic rats.This finding can be interpreted as high absorption and low synthesis of cholesterol.Streptozotocininduced diabetes in rats hypertrophies intestinal mucosal function, enhancing fat and cholesterol absorption and reducing cholesterol synthesis (Young et al., 1988).It is also known that fasting down regulates cholesterol synthesis and reduction of dietary cholesterol has been observed to effectively reduce serum cholesterol in type 1 diabetic subjects (Kaufmann et al., 1975).
Important results were found in relation to experimental model, we observed that the EE of C. sylvestris treatment presented a significant improvement in lipidic profile values in rats (decrease total cholesterol, LDL cholesterol, VLDL cholesterol and triglycerides levels), this corroborates previous study that demonstrated the antihyperlipidemic activity of C. sylvestris (Schoenfelder et al., 2008).We highlight the hypolipidemic activity by ethanolic extract of C. sylvestris including in a not specific model to develop lipid profile alterations.Moreover, it was observed that the HDL levels were increased for both vehicle tween 80 and ethanolic extract of C. sylvestris treatments, highlighting the vehicle action.However, we call attention that tween 80 also increased the total cholesterol and LDL levels (Table 1) while the ethanolic extract of C. sylvestris decreased them, highlighting the action of extract of C. sylvestris.This difference observed by treatment brings to light the protective effect of ethanolic extract of C. sylvestris against cardiovascular diseases.
Previous study showed that flavonoids are FIGURE 3. Antioxidant activity.

FIGURE 5 .
FIGURE 5. Effect of ethanolic extract of Casearia sylvestris on serum blood glucose of rats treated for 45 days.
The protocol was approved by the on the Ethics and Research Committee of Federal University of Health Sciences of Porto Alegre (Number: 608/08), with procedures in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985), the UK Animals Scientific Procedures Act 1986 and the European Community's Council Directive of 24 november 1986 (86/609/EEC).

TABLE 3 .
Effect of ethanolic extract of Casearia sylvestris on serum lipid profile of rats treated for 45 days.Values expressed in mg/dL and data were expressed as mean ± S.D. (n = 4-8).*effect of diabetes (two-way ANOVA/SNK, P<0.05).a different from other groups (two-way ANOVA/SNK, P<0.05).b different from water group (two-way ANOVA/SNK, P<0.05).c different from insulin group (two-way ANOVA/SNK, P<0.05).(CC): non-diabetic receiving drinking water by gavage; (CT): non-diabetic receiving 3% Tween 80 solution by gavage; (CE): non-diabetic rats treated with EE of C. sylvestris Sw by gavage; (DS): diabetic receiving drinking water by gavage and saline s.c.; (DI): diabetic rats receiving drinking water by gavage and injections insulin s.c.; (DE): diabetic rats treated with EE of C. sylvestris by gavage and saline s.c.