In vivo hypotensive effect and in vitro inhibitory activity of some Cyperaceae species

In 1820, French naturalist August Saint Hillaire, during a visit in Espírito Santo (ES), a state in southeastern Brazil, reported a popular use of Cyperaceae species as antidote to snake bites. The plant may even have a hypotensive effect, though it was never properly researched. The in vitro inhibitory of the angiotensin converting enzyme (ACE) activity of eigth ethanolic extracts of Cyperaceae was evaluated by colorimetric assay. Total phenolic and flavonoids were determined using colorimetric assay. The hypotensive effect of the active specie (Rhychonospora exaltata, ERE) and the in vivo ACE assay was measured in vivo using male Wistar Kyoto (ERE, 0.01-100mg/kg), with acetylcholine (ACh) as positive control (5 μg/kg, i.v.). The evaluation of ACE in vivo inhibitory effect was performed comparing the mean arterial pressure before and after ERE (10 mg/kg) in animals which received injection of angiotensin I (ANG I; 0,03, 03 and 300 μg/kg, i.v.). Captopril (30 mg/kg) was used as positive control. Bulbostylis capillaris (86.89 ± 15.20%) and ERE (74.89 ± 11.95%, ERE) were considered active in the in vitro ACE inhibition assay, at 100 μg/mL concentration. ACh lead to a hypotensive effect before and after ERE’s curve (-40±5% and -41±3%). ERE showed a dose-dependent hypotensive effect and a in vivo ACE inhibitory effect. Cyperaceae species showed an inhibitory activity of ACE, in vitro, as well as high content of total phenolic and flavonoids. ERE exhibited an inhibitory effect on both in vitro and in vivo ACE. The selection of species used in popular medicine as antidotes, along with the in vitro assay of ACE inhibition, might be a biomonitoring method for the screening of new medicinal plants with hypotensive properties.


INTRODUCTION
French naturalist August Saint Hillaire, during his stay in ES, noted that locals used some Cyperaceae, Aristilochiaceae and Fabaceae plants to treat wounds caused by snake bites -two of which applied orally and the other one applied locally (Saint-Hillaire, 1820/2002).Regarding the study of antihypertensive plants, the selection can be ethnobotanical, based on species used as diuretics, antidotes and antihypertensives (Ribeiro, et al., 1986).Several species commonly used as antivenins also present anti-inflammatory, antioxidant and hypotensive activity (Balestrin et al., 2008).Species of the Cyperus genus, Cyperaceae family, are reported as anti-inflammatory (Falcão, et al., 2005), antimalarial (Schwikkard, Heerden, 2002) and hepatoprotective extracts (Amani et al., 2012).
Concerning the advanced ecosystem destruction together with the potentialities of the Cyperaceae family, the aim of this study were to identify whether any of the the ethanolic extracts of Cyperaceae species presents an inhibitory effect on in vitro ACE, and whether the most active species in vitro shows an acute hypotensive effect in vivo and ACE inhibitory effect in vivo.

Plant materials and extraction
Ten specimens from Cyperaceae family were harvested (July, 2010) 40°25'10.5"W, VIES 26080).Two of the ten collected specimens were identified as the same (B.capillaris and R. tenuis).

Determination of total phenolic
The total phenolic content was determined using Folin-Ciocalteu method, along with some modifications (Singleton, Rossi, 1965).Solutions at a concentration of 1.5 mg/mL of the ethanolic extracts were prepared.The calibration curve was plotted with a pyrogallol solution (from 10 to 350 µg/mL), and the equation was y = 0.001244x + 0.002889, r 2 = 0.9977.The results were expressed as micrograms of pyrogallol equivalents (PE) per milligram of dry sample.The analyses were performed in triplicates.The limit of quantification was 13.7 µg/mL and the limit of detection was 4.1 µg/mL.

Dtermination of total flavonoids
The total flavonoids content was determined by colorimetric aluminum chloride method (Marinova et al., 2005).The calibration curve was prepared with rutin solutions, at the 31.25-1000µg/mL range.The calibration regression equation was y = 0.0033x + 0.0121, r 2 =0.9991.The results were expressed as micrograms of rutin equivalents (RE) per milligram of sample.The analyses were done in triplicates.The limit of quantification was 5.3 µg/mL and the limit of detection was 1.6 µg/mL.

High performance liquid chromatography (HPLC) analysis
The analysis of the eight Cyperaceae extracts, especially of ERE by HPLC was performed in a Waters 1515 separation device, along with a binary pump, and an UV/VIS detector (model 2489).The data were processed using the Breeze software.A C18 XBridge reverse-phase column was used (150 x 4.6 mm i.d., 3.5 µm, Waters), along with an also C-18 XBridge precolumn (20 x 4.6 mm i.d., 3.5 µm, Waters), at room temperature.Two isocratic elution systems were used.The first one was composed by a MeOH:H 2 O (9:1) solution, wavelength detection at 254 and 365 nm, and isocratic flow at 0.8 mL/min; the second was employed a MeOH:H 2 O:ACN: AcOOH: CHOOH (20:10:20:1:1) eluent, same wavelengths, and 1.2 mL/min flow.The solvents were HPLC quality (Merck, Darmstadt, Germany), the water was ultrapure (ELGA, 18.2Ω) and was degassed by sonication and vacuum pump prior to use.Both the reference rutin and apigenin solutions (Sigma, St. Louis, USA) and the ERE sample were dissolved in MeOH, HPLC degree (Merck, Darmstadt, Germany).The sample concentration was 1.0 mg/mL.The final concentration of the reference solutions used on the plotting of the standard curve ranged from 0.03 to 0.6 µg/mL.Samples, as well as reference solutions, were centrifuged at 8400 g for 5 min.Each sample and standard solution (20 µL) was done manually.All analyses were done in triplicates.

ACE in vitro inhibition assay
Effects of the Cyperaceae crude ethanolic extracts on ACE were determined by measuring the accumulation of Gly-Gly (glycine-glycine), the ACE cleavage product of Hip-Gly-Gly.This assay was performed as previously described (Endringer et al., 2014).In brief, the assays were initiated by an addition of 10 µL of the sample (final concentration of 100 μg/mL), 10 µL of MeOH/HEPES (1: 4), pH 8.0 (negative control), or 10 µL of a captopril solution (final concentration of 64 nM; positive control) into a 96-well plate.A rabbit lung solution (10 µL for the final concentration of 9.1 mg/mL) was used as the ACE source.The plate was then homogenized and preincubated at 37 °C for 10 min.The enzymatic reaction was initiated by an addition of 60 µL of the HEPES buffer (final concentration of 27.3 mM), pH 8.15 and 30 µL of a Hip-Gly-Gly solution (final concentration of 27.3 mM).
After the homogenization, the mixture was incubated for 40 min at 37 °C.The reaction was stopped by an addition of 100 µL of 0.3 M sodium tungstate and 100 µL of 0.3 M sulfuric acid.In the sequence, an aliquot (64 µL) was diluted with 100 µL of water and kept in rest for 5 min.Subsequently, the system was mixed with the color reagent 2,4,6trinitrobenzenesulfonic acid solution, TNBS (6.92 mM) (Sigma, USA).The plate was kept in the dark at room temperature for 20 min and its absorbance was measured using a microplate reader (BioRad, Model 550) at 415 nm.The readings were compared against a blank solution, which was prepared in a similar manner with the exception of sodium tungstate and sulfuric acid solutions being added before the rabbit lung solution.Based on the measured ACE inhibitory activity, the compounds were classified as inactive (<30 %), weak (>30 % to 50 %), moderate (>50 % to 80%), and strong (>80 %) inhibitors.

Acute hypotensive assay
The in vivo experiments were performed according to the ethical principles determined by Brazilian School of Animal Experimentation (COBEA, 1991), and were approved by the Ethical, Bioethical and Animal Welfare Committee (CEUA -UVV, 120/2010).Male Wistar Kyoto (WKY; n=6) and Spontaneously Hypertensive Rats (SHR; n=6) were used.The animals were about three months old, with a body weight from 200 to 250 g.The rats were kept at temperatures of 22 ± 3 ºC, in a 12 h light/12 h dark cycle, and had free access to standard pellet diet (Probiotério stockfeed, Moinho Primor SA) and water.
Under sodium pentobarbital anesthetic (50 mg/kg, intraperitonial Hypnol, Crystal, Brasil) (Andrade et al., 2008), both femoral arteries and veins were catheterized (polyethylene catheter, PE 50, Clay Adans, USA).The venous catheter was used for the injection of the raw ethanolic extract from R. exaltata (ERE -267 mg/mL in saline solution) and/or saline, the arterial catheter was connected to pressure pipes (Spectramed, Statham, P23XL, USA) by a flexible catheter, and the register values of the mean arterial pressure (MAP) were collected by a computer (Pentium MMX 233 MHz) and by a biological data acquisition software (Biopac, Biopac Systems, USA, mod.100A/94111065 MP series).
The acute hypotensive effect was evaluated by the plotting of a dose-effect curve of the ERE solution.The WKY and SHR animals received saline 1 mL/kg (Soncini et al., 2011) and, afterwards, increasing doses, in bolus, of ERE (0.01 -100 mg/kg).The subsequent dose was administrated only after MAP returned to the levels observed prior to the first application.ACh was utilized as positive control (5 µg/kg, i.v.;) both before and after the ERE curve (Lahlou et al., 2003).

ACE in vivo inhibition assay
ACE in vivo inhibitory evaluation was performed according to Mangiapane et al. (1994), with slight modifications.Briefly, the effect of angiotensin I (ANGI; 0.03, 03 and 300 µg/kg i.v.) was analyzed before and after the injection of a single dose of ERE ethanolic extract (10 mg/kg, i.v.).As positive control, ANG I was administered before and after captopril (30 mg/kg).

Statistical analysis
The values of the biological evaluation were expressed as the mean ± standard error of the mean (S.E.M.).Basic values of MAP and FC, and MAP after ERE's injection were submitted to analysis of variance (ANOVA), followed by a post-hoc Fisher test, p<0.05.Correlation coefficient of the standard curves was obtained by linear regression by the least squares method and the data were then analyzed by ANOVA, on a 95% LC and degree of freedom of n-1.The limits of detection (LOD) and quantification (LOQ) were computed by the division of the standard deviation of the slope of the curves on the calibration assay.

RESULTS AND DISCUSSION
There are just a few taxonomical studies about the Cyperaceae family, which makes it hard to identify and/ or differentiate the species et al., 1999).From the gathered Cyperaceae specimens, two of them were of the same species (B.capillaris and R. tenuis), and one of them (B.capillaris) is on August de Saint-Hillaire Virtual Herbarium species list (Virtual Herbarium, 2012).B. capillaris as well as R. exaltata were considered active on the ACE inhibition assay (Table I).
The 50% inhibitory in vitro ACE concentration (CI 50 ) of the active species could not be calculated, as the observed inhibitory effect was dose-independent.The presence of total polyphenols, including tannins, on the extracts might be related to this not specific in vitro inhibition.Nyman et al. (1998), observed that only four species, from the thirteen considered active in the ACE assay, remained presenting dose-dependent inhibitory ACE activity after tannins were removed.Polyphenols, distinctly from flavonoids, might be related to the inhibitory ACE activity of B. capillaris species.However, it is not possible to establish a correlation between the in vitro ACE inhibition percentage and the content of total phenolics, as they might be the constituents related to the ACE inhibitory effect noted for R. exaltata (table I).Barbosa-Filho et al. (2006), summarized several studies of the natural products with inhibitory ACE activity and observed that flavonoids and tannins were related to the activity of several species among them: Erythroxylum laurifolium, "Korean" green tea, Allophylus edulis.In the present study, C. rotundus, Cyperaceae, was cited, but it did not show ACE inhibition (Barbosa-Filho et al., 2006).
The two species showing the most effective ACE inhibition were B. cappilaris and R. exaltata.Due to the available amount of active samples, R. exaltata was chosen for the in vivo hypotensive study.The chromatographic profile obtained for ERE was simple, on the analyzed wavelengths, with a dominance of polar substances, suggesting the presence flavonoid-like compounds.Using particular flavonoid eluents, in comparison with rutin and apigenin, peaks related to aglycones, but an overlap of peaks related to glycolized flavonoids (rutin and others).
As expected MAP of SHR (182±8 mmHg; p<0.01) animals was higher compared with WKY (99±5 mmHg).During the acute hypotensive assay, the ethanolic crude extract of R. exaltata determined a reduction of the pressure levels in a dose-dependent way in both normotensive and hypertensive animals (Figure 1), which were statistically different from the reduction caused by the saline injection (WKY: -7.6±3 mmHg; SHR: -8±4 mmHg).Reductions elicited in SHR animals were greater compared with WKY (Figure 1).ACh induced a hypotensive effect before and after ERE's curve (WKY: -43.2±4 mmHg and -44.3±3 mmHg; SHR: -74±5 mmHg and -78 mmHg; p<0.01 compared with WKY), suggesting vascular integrity, as the vascular relaxation induced by ACh is dependent on the integrity of endothelial cells (Batlouni, 2001).The in vivo ACE inhibition of ERE is reported in Table II.ANGI at all tested doses (0.03, 3.0, 300.00 µg/kg) increased the MAP (Table II, before CAP and before ERE).ERE decreased around 50% of the ANGI effect (Table II), indicating that ERE inhibits ACE in vivo.However, the design of the in vivo inhibition assay applied in the present study does not differ between the two ACE isoforms and also not between AngII and its metabolite, Ang 1-7.ACE and ACE2 isoforms are 40% similar (Watermeyer et al., 2006;Sturrock et al., 2004).ACE2 acts as a carboxylpeptidase, whereas ACE acts as a peptidylpeptidase (Brown, Hall, 2005).Several studies indicate that both enzymes, ACE and ACE2, have similar catalytic mechanisms.However, the ACE inhibitory agents, such as captopril, lisinopril, are inactive against ACE2.These data suggest that the proteolytic activity of ACE2 is different from the isoform ACE.Unlike ACE,  ACE2 is the monopeptidilcarboxipeptidase (Guy et al., 2003;Warner et al., 2004;Guy et al., 2005).
The data reported in the present study indicate that some Cyparaceae species have ACE in vitro inhibitory activity, being related with the high content of total flavonoids and phenolics.ERE exhibited an inhibitory effect on both in vitro and in vivo ACE.The selection of species used in popular medicine as antidotes, along with the in vitro assay of ACE inhibition, might be a biomonitoring method for the screening of new medicinal plants with hypotensive properties.

TABLE I -
ACE inhibition percentages, total polyphenols and flavonoids amounts for the analyzed Cyperaceae species Inhibition percentage of ACE.b Results presented as micrograms of pyrogallol equivalents per milligram of sample.c Results presented as micrograms of rutin equivalents per milligram of sample.d NA: not evaluated due to the available mass. a

TABLE II -
Mean arterial pressure (MAP) increase induced by the injection of ANG I (0.03, 3 and 300 µg/kg, i.v.) before and after injection of the ethanolic extract of Rhynchospora exaltata (ERE; 10 mg/kg) and captopril (CAP; 30 mg/kg, triangles) : MAP before ANG I injection; MAP A : MAP after ANG I injection; ΔMAP: difference between MAP A and MAP B .The values are presented as the mean ± S.E.M. ** p<0.01 compared with MAP values before CAP or ERE administration.++ p<0.01 compared with MAP after CAP administration. B