Acetone Extract from Streptoverticillium Sp., a Bacterium Isolated from Brazilian Cerrado Soil, Induces Anti-infl Ammatory Activity in Mice

The Streptoverticillium sp. Z1 is an actinomycete isolated from the soil under Cerrado vegetation, the extract of this strain was investigated in nociceptive and infl ammatory models. The Streptoverticillium extract (ExS) 50 and 100 mg/kg (s.c.) produced a signifi cant inhibition of acetic acid-induced abdominal writhings thereby demonstrating an anti-nociceptive effect. In the tail fl ick test the ExS (s.c.) was inactive. This result implited that ExS does not contain opioid-like compounds with central analgesic properties. In the infl ammatory models, ExS 100 and 200 mg/kg (s.c.) were able to inhibit the croton oil-induced ear edema and, ExS 200 and 500 mg/kg (s.c.) inhibited the leukocyte migration on the carrageenan-induced peritonitis. The phospholipase A 2 enzymatic assay showed that the anti-infl ammatory activity of ExS was not due to direct effect on phospholipase A 2 activity. These data suggest that Streptoverticillium sp. produces metabolites with anti-infl ammatory effect and that these metabolites are unable to directly inhibit phospholipase A 2 enzyme.


INTRODUCTION
Infl ammation is a natural host-defensive process in the innate immunity response and is usually associated with pain as a secondary process resulting from the release of algesic mediators (Hunskaar andHole 1987, Osadebe andOkoyé 2003).Generally, the infl ammatory process involves a series of events that can be elicited by numerous stimuli such as infectious agents, ischaemia, antigen-antibody interaction and thermal or physical injury (Osadebe andOkoyé 2003, Insel 1990).Acute infl ammation is a rapid and securely self-terminating process that can, however, be harmful to the host if subclinical infl ammation survives and it is followed by the development of local chronic infl ammation.Such infl ammation provides a cellular microenvironment that favors malignant progressions such as tumor promotion (Balkwill et al. 2005).
Biotechnology research is a viable and promising means of obtaining new substances, for example, from the study of microorganisms.The biochemical heterogeneity of actinomycetes, their ecological diversity and its exceptional ability to produce secondary metabolites make them a suitable target for the discovery of new substances that have biological activity of biotechnological interest (Peckyñska-czoch and Mordarski 1988).The Streptoverticillium sp.Z1 is an actinomycete isolated from soil under Cerrado vegetation, belonging to the microbial culture collection of the Laboratory of Biochemistry and Genetic Engineer, Biologic Sciences Institute -Universidade Federal de Goiás.This microorganism produces substances with antimicrobial activity (K. Hoffmann et al., unpublished data), chitinase and N-acetylglucosaminidase (I.S. Sobrinho, unpublished data).K. Hoffmann et al. (unpublished data) demonstrated that Streptoverticillium sp.Z1 provides a qualitative and quantitative variation in the production of antimicrobial substances when grown in different culture medium.Hence, the aim of this study is to assay the anti-infl ammatory activity of cell biomass extracts of Streptoverticillium culture in different methodologies.

STREPTOVERTICILLIUM SP. Z1 STRAIN
The Streptoverticillium sp.Z1, belonging to the microbial culture collection of Laboratory of Biochemistry and Genetic Engineer, Biologic Sciences Institute, Universidade Federal de Goiás, was isolated from soil of the Brazilian cerrado.

MAINTENANCE AND CULTIVATION OF THE ISOLATED STRAIN
The Streptoverticillium sp.Z1 was kept isolated in Petri dishes containing ISP-2 medium (glucose 4.0 g/L; Yeast extract 4.0 g/L; Malt extract 10.0 g/L; Agar 20.0 g/L, pH 7.2) at 30°C.This microorganism was grown in 2,000 mL Erlemayer fl ask containing 500 mL of medium MPE (soy fl our 20.0 g/L; glucose 20.0 g/L; CaCO3 2.0 g/L; NaCl 5.0 g/L, pH 7.0), and incubated at 30°C under constant agitation at 150 rpm for 10 days.

EXTRACT PRODUCTION
The medium was vacuum fi ltrated (Whatman number 1 fi lter) then the liquid phases were discarded and the resulted biomass was resuspended in acetone and maintained in agitation for two hours to extract the polar compounds and pigments.The acetone extract obtained (acetone with cell biomass) was concentrated under reduced pressure resulting in the Streptoverticillium acetone extract (ExS).This extract was reconstituted in saline (NaCl 0.9%) at the required concentrations for pharmacological tests.

EFFECT ON GROSS BEHAVIOR
The effect on spontaneous mouse behavior was determined using the Hippocratic procedure (Malone 1977).Groups of eight adult albino mice were treated per orus (p.o.), subcutaneously (s.c.) and intraperitoneally (i.p.) with vehicle (0.9 % NaCl, 10 mL/kg) or ExS 1, 10 and 100 mg/kg and kept under observation for seven days.With this method the doses and administration route were defi ned.

Acetic acid-induced abdominal writhing test
The response to an intraperitoneal injection of acetic acid solution (i.e. the contractions of the abdominal muscles and stretching of hind limbs) was studied according to Koster et al. (1959) and Hendershot and Forsarth (1959).Experimental groups of mice (n = 8) were treated subcutaneously with vehicle (10 mL/kg), ExS 50 and 100 mg/kg, or indomethacin (10 mg/kg), as positive control, 30 min before the administration of a 1.2% (v/v) acetic acid solution (10 mL/kg, i.p.).The number of writhes produced in each group was counted during 30 min of observation.The results obtained were expressed as the percentages relative to the control group.A signifi cant reduction in the number of writhing movements in the groups treated with ExS compared with the control was considered to be a positive anti-nociceptive response.

Tail fl ick test
The reaction of mice to thermal stimulation of the tail tip by immersion in water maintained at 55.5 ± 0.5°C was recorded at -30, -15, 0, 15, 30, 45, 60 minutes of treatment.Experimental groups of mice (n = 8) were treated with vehicle (10 mL/kg, s.c.), ExS 50 and 100 mg/kg (s.c.), or morphine 5 mg/kg, (s.c.), as positive control.The anti-nociceptive data were expressed as mean ± SEM, relative to 0 time, according to the technique of Janssen et al. (1963), as modifi ed by Grotto and Sulman (1967).

Croton oil-induced ear edema test
The animals were treated (s.c.) with vehicle (10 mL/ kg), dexamethasone (2 mg/kg), or ExS 100 and 200 mg/kg (n = 8), and 60 min later, cutaneous infl ammation was induced by applying 25 µL of croton oil (2.5% v/v in acetone) solution to the inner surface of the right ears of the mice.The same volume of acetone was applied to the left ears by the method of Tubaro et al. (1985) and Zanini et al. (1992).Four hours after treatment, mice were killed by cervical dislocation, and a plug (6 mm in diameter) was taken from both treated and untreated ears with a punch.The infl ammatory response (edema) was monitored by measuring the differences in weight (mg) between the two plugs (Δ).The results were expressed as the percentages relative to the control group.

Carrageenan-induced peritonitis test
Animals were treated (s.c.) with vehicle 10 mL/kg, dexamethasone 2 mg/kg or ExS 200 and 500 mg/ kg (n = 8) 45 min before an injected of carrageenan (1%; 0.25 mL, i.p.).Four hours after carrageenan administration, mice were killed and 2 mL of modifi ed PBS (with heparin, 10 IU/mL, and without calcium and magnesium) was injected into the peritoneal cavity.Total cell counts in the lavage fl uid were performed in a Neubauer chamber (Ferrándiz and Alcaraz 1991).The results were expressed as the percentages relative to the control group.

Phospholipase A 2 Enzymatic Assay
To assay the inhibition activity of ExS on PLA 2 activity we used a modifi ed method based on Harbermann and Hardt (1972).Briefl y, the PLA 2 activity was evaluated by the hemolysis of erythrocytes suspension incorporated into agarose gels.Agar plates (0.8%) were prepared with 30 mL of Tris-HCl 0.05M buffer, 0.25 mL of CaCl 2 0.01M, RODRIGO B. DA CRUZ et al. 0.3 mL of egg yolk in saline (1:4) and 0.3 mL human erythrocytes washed.7.5 µL of Crotallus durissus collilineatus venom solution (0.03 mg/mL) plus an equal volume of ExS (50 mg/mL), dexamethasone (0.2 mg/mL) or indomethacin solutions (1 mg/mL) were incubated at 37°C for one hour.Afterwards, the incubated was placed in 2 mm of diameter equidistance well and the plates were incubated at 37°C for 20 hours.After incubation the halos produced by hemolysis (PLA 2 activity) were measured.The results were expressed as mean ± SEM of halo area.

STATISTICAL ANALYSIS
All the results were expressed as mean ± S.E.M. and treated groups were compared with the control and differences were estimated by means of ANOVA followed by Tukey as the post hoc test.All analyses were performed using the software GraphPad Prism 3.0 for Windows.Effects were considered signifi cant at P < 0.05.

EFFECT ON GROSS BEHAVIOR
In the general test of pharmacological activity, the animals treated with ExS (s.c. or i.p.) shown antinociception (assessed by pain reaction caused by the compression of distal portion of the tail), increase diuresis and decrease in motor activity (spontaneous ambulation) within 30 min of treatments, in a dose response manner.Other parameters suggested by Malone (1977), indicative of pharmacological activity, and did not show signifi cant difference.The extract was ineffective by the oral route.

TAIL FLICK TEST
The ExS (s.c.) was inactive in the tail fl ick test of nociception in both doses tested (50 and 100 mg/ kg).Morphine, used as a reference drug, produced a signifi cant anti-nociceptive effect at all observation times compared to control group.These results suggest that the ExS does not contain opioid-like compounds with central analgesic properties (Fig. 2).

CROTON OIL-INDUCED EAR EDEMA TEST
In the Croton oil-induced ear edema test, the ExS 100 and 200 mg/kg (s.c.) were able to inhibit the edema to 44.77 ± 2.3% and 36.14 ± 3.1%, respectively; from control value of 13.5 ± 0.84 mg of edema.Indomethacin (10 mg/kg) produced a signifi cant inhibition to 15.03 ± 3.80% of the edema (Figure 3).ANTI-INFLAMMATORY ACTIVITY OF Streptoverticillium sp.

CARRAGEENAN-INDUCED PERITONITIS
The treatment with ExS signifi cantly reduced the total leukocyte migration to the peritoneum induced by carrageenan compared with control group.ExS administered subcutaneously at doses of 200 and 500 mg/kg caused inhibition of total leukocyte migration to 45.8 ± 7.8% and 29.7 ± 3.4%, respectively, when compared with control value 1.47 ± 0.17 × 10 7 leukocytes/mL, and dexamethasone 2 mg/kg inhibited the total leukocyte migrated to 22.25 ± 3.9% (Figure 4).

PHOSPHOLIPASE A 2 ENZYMATIC ASSAY
In this methodology, ExS and dexamethasone were not able to inhibit the PLA 2 activity.Only indomethacin inhibit the halo area to 0.667 ± 0.1778 cm 2 from control value of 0.929 ± 0.040 cm 2 (Table I).

DISCUSSION
The attention given to the actinomycetes in biotechnological applications is a natural result of the great metabolic diversity of these organisms and their long association with the environment  and human needs.However, it is becoming increasingly diffi cult to discover commercially signifi cant secondary metabolites from well known actinomycetes as this practice leads to the wasteful rediscovery of already known bioactive compounds, thereby emphasizing the need to isolate, characterize and screen representatives of undiscovered actinomycete taxa.It is also becoming increasingly clear that un-and underexplored habitats, such as desert biomes and marine ecosystems, are rich sources of novel actinomycetes which have the capacity to produce interesting new bioactive compounds, including antibiotics (Bredholt 2008).
The analgesic activity observed in general test of pharmacological activity should be the result of a motor activity reduction, also observed in this same test, thus it became necessary to perform different specifi c models to assay the analgesic and antiinfl ammatory effect in order to demonstrate the effects produced with Streptoverticillium biomass extract treatment.Considering that the extract showed activity when administered either by i.p. and s.c.routes, the following tests were conducted with treatments performed by the subcutaneous route because this route is safer to avoid false positive results in some models such as writhing and peritonitis tests.
The antinociceptive effect of Streptoverticillium sp.acetone extract (ExS) was tested in two different models of analgesia, i.e., the acetic acid-induced writhing test and tail fl ick test in mice.The acetic acid-induced abdominal writhing is commonly used as a screening method for compounds with potential anti-nociceptive and/or anti-infl ammator.In this method the injected acetic acid produces nociception directly by stimulation of terminal nervous and indirectly by leading to the release of endogenous mediators involved in pain modulation, for example: bradykinin, serotonin, histamine, prostaglandin (Berkenkopf  and Weichman 1988, Chau 1989).This is widely used because of the high sensitivity to drugs with anti-nociceptive action in different drug classes such as aspirin, antagonists of kinin receptors, central and peripheral-acting opioid analgesics (Hendershot andForsaith 1959, Vacher et al. 1964).
In this model of nociception, we demonstrated that the previous treatments with ExS was effective both at a dose of 100 and 50 mg/kg in reducing the abdominal writhes, thereby demonstrating anti-nociceptive and/or anti-infl ammatory activity (Figure 1).In order to evaluate the involvement of a central antinociceptive activity, the same doses of ExS were used in the tail fl ick test.
In the tail fl ick test, morphine is used to induce analgesia.Within 1-30 min, when morphine induced maximum analgesia, since its response involves essentially spinal receptors (μ 2 , κ 1 , δ 2 ) (Reisine and Pasternack 1996).ExS did not produce any analgesia suggesting that its extract has no central analgesic properties (Figure 2).Therefore, it is not probable that ExS exerted its effect through central opioid receptors or promoted release of endogenous opiopeptides.
It is known that the acute infl ammatory response consists of three main vascular effects: (I) vasodilatation and increasing vascular fl ow; (II) increased vascular permeability; and (III) leukocyte migration to the injured tissues.Therefore, the antiinfl ammatory activity of ExS was evaluated on the croton oil-induced ear edema and carrageenaninduced peritonitis tests.In these infl ammation models, higher doses (100 and 200 mg/kg) were used taking into account the lower sensitivity of these methodologies used.
The croton oil-induced ear edema was carried out by topical application of a phlogistic agent to the inner surface of the ear.A reduction in this edema as a result of ExS treatment was assessed as a parameter of anti-infl ammatory activity.
The inhibition of croton oil-induced ear edema caused by treatment with ExS (Figure 3) may be associated with interference in factors that infl uence edema formation such as tissue vascular fl ow and systemic blood pressure (Rates and Barros 1994) and prostaglandins synthesis, important to the genesis of edema and pain (Morrow and Roberts 2001).
Considered that an inhibitory activity only on cyclooxygenase activity has less potential to reduce cell migration and can reduce ear edema, we decided to raise the dose in the peritonitis model.
In the carrageenan-induced peritonitis, the carrageenan administration act as a stimulus to produce an acute infl ammatory response after 4 hours in the peritoneal cavity of mice, with a large number of leukocytes in the exudate.The treatment with ExS inhibited cell migration to the peritoneal cavity in a signifi cant manner when compared with the control group (Figure 4).The results in these two methods suggest that ExS may contain active compounds with anti-infl ammatory effects.
The anti-infl ammatory effect of a drug may occur by different mechanisms of action among which there are the inhibition cyclooxygenase (COX) or phospholipase A 2 (PLA 2 ).The inhibition of PLA 2 in vivo is an important mechanism in the anti-infl ammatory effect of glucocorticoids, this enzyme can also be inhibited directly as occur with indomethacin (Lobo andHoult 1994, Singh et al. 2009) or indirectly by induction of protein synthesis as occurs with dexamethasone (Flower and Blackwell 1979).
Meanwhile, phospholipase A 2 enzymatic assay was performed in order to evaluate possible direct inhibitory effect of ExS on PLA 2 activity, and try to propose a mechanism of action.In the in vitro model, the drugs solutions were used at the same concentrations as they were used in the in vivo test, and the extract solution was used at the same concentration prepared to in vivo highest dose.
As expected, dexamethasone in this in vitro test cannot inhibit the PLA 2 activity, because this inhibition involves cellular mechanisms such as RODRIGO B. DA CRUZ et al. protein synthesis of anexin-1 and gene regulation, that cannot occur in the absence of cellular activity (Schimmer and Parker 1996) and indomethacin inhibited the PLA 2 activity because this inhibition is due to its direct binding to the enzyme (Lobo andHoult 1994, Singh et al. 2009).ExS present no inhibition on PLA 2 activity (Table I).
The ExS anti-edematogenic effect shown should be due to a mechanism of action similar to indomethacin by reducing the activity of the cyclooxygenase enzyme as well as a decrease of its expression.On the other hand, an inhibitory effect dependent of cellular mechanisms on PLA2 activity in a similar way as dexamethasone or reduction in expression or inhibition of lipoxygenase could explain the reduction in cell migration observed with ExS treatment on peritonitis model.
The ExS concentration used in the in vitro model is certainly higher than the concentration achieved by the active principles of the extract with anti-infl ammatory activity in infl ammatory processes local of the in vivo models used, in this way, the anti-infl ammatory activity observed may not be associated with direct inhibition of PLA 2 activity.In spite of the exclusion of direct effect on PLA 2 activity, we cannot discard the possibility of an indirect inhibition of this enzyme as occur with steroidal anti-infl ammatory drug as in the case of dexamethasone, that not inhibits directly the PLA 2 activity (Flower and Blackwell 1979).

CONCLUSION
Based on the fi nding from the preset studies, we can summarize that the Streptoverticillium sp.Z1 in appropriate growth conditions produces metabolites with analgesic and/or anti-infl ammatory effects.This anti-infl ammatory effect is not due to a direct inhibition of the phospholipase A 2 enzyme.The anti-infl ammatory activity of these metabolites may involve a reduction in the production of various infl ammatory mediators including those involved in chemotaxis.

Figure 1 -
Figure 1 -Effect of Streptoverticillium sp.extract (ExS 50 and 100 mg/kg, s.c.) on the number of acetic acid-induced abdominal writhes in mice.The vertical bars indicate the mean ± SEM, expressed in relative percentage to the control group.Indomethacin (10 mg/ kg, s.c.) was used as a positive control.** p < 0.01, *** p < 0.001, vs control group (vehicle).

Figure 2 -
Figure 2 -Effect of Streptoverticillium sp.extract (ExS 50 and 100 mg/kg, s.c.) on the time to pain reaction in mice.The vertical bars indicate the mean ± SEM, expressed in relative percentage to the control group.Morphine (5 mg/kg, s.c.) was used as a positive control.** p < 0.01, *** p < 0.001, vs control group (vehicle).

Figure 3 -
Figure 3 -Effect of Streptoverticillium sp.extracts, ExS 100 and 200 mg/ kg, s.c., on croton oil-induced ear edema in mice.The vertical bars indicate the mean ± SEM of differences in weight between right and left ear plugs.Indomethacin (10 mg/kg, s.c.) was used as a positive control.*** p < 0.001, vs control group (vehicle).

TABLE I Effect of Streptoverticillium sp. extract (ExS), Indomethacin and Dexamethasone onphospholipase A 2 enzymatic assay in vitro.
* p < 0.05, vs control group.ANTI-INFLAMMATORY ACTIVITY OF Streptoverticillium sp.