A significant antihypertensive effect of Holarrhena floribunda supported by an exploratory phytochemical study

Introduction Cardiovascular diseases (CVD) are pathologies that affect the heart and blood vessels. They are the main cause of deaths in the world (1). More than three-quarters of deaths in lowand middle-income countries are due to CVD (2). In Togo, the situation of CVD is of great concern while their incidence is not fully known (3). Risk factors for CVD include poor nutrition and physical inactivity, smoking, oxidative stress, heavy drinking, diabetes, hyperlipidemia, and hypertension. Indeed, hypertension is a chronic pathology that constitutes a real public health problem with a global prevalence in the world for adults estimated at 26.4% in 2000 (972 million people including 639 million in resource-limited countries) and could reach 29% by 2025 (1.56 billion of peoples) (4). The conventional treatments of hypertension require synergy of several http://www.herbmedpharmacol.com doi: 10.15171/jhp.2018.27


Introduction
Cardiovascular diseases (CVD) are pathologies that affect the heart and blood vessels.They are the main cause of deaths in the world (1).More than three-quarters of deaths in low-and middle-income countries are due to CVD (2).In Togo, the situation of CVD is of great concern while their incidence is not fully known (3).Risk factors for CVD include poor nutrition and physical inactivity, smoking, oxidative stress, heavy drinking, diabetes, hyperlipidemia, and hypertension.Indeed, hypertension is a chronic pathology that constitutes a real public health problem with a global prevalence in the world for adults estimated at 26.4% in 2000 (972 million people including 639 million in resource-limited countries) and could reach 29% by 2025 (1.56 billion of peoples) (4).The conventional treatments of hypertension require synergy of several classes of antihypertensive agents; even so none of those treatments definitely cured this pathology.Moreover, the high cost when facing the treatment of hypertension with conventional drugs makes it hardly affordable for common peoples.For that, common people prefer using traditional medicines.Indeed, some studies have shown the positive effects of antioxidants from plants as good agents for the prevention of hypertension diseases (5).It is known that several plants are used in traditional medicine to treat high blood pressure (6).Holarrhena floribunda is one of those species which natural ecological zone covers many African countries namely Togo, Senegal, Guinea-Bisseau, Guinea, Mali; Burkina Faso, Sierra Leonne, Liberia, Ivory Coast, Ghana, Benin, Nigeria, Chad, Cameroon, Gabon, Congo, Central Africa.The many uses of H. floribunda in traditional medicine in those African countries include the treatment of dysentery, diarrhea, fever, snake bite, infertility, venereal disease, diabetes, hypertension, and malaria (7,8).So, the present study aimed mainly the assessment of the antihypertensive effects of the ethanolic extract of the trunk bark of H. floribunda.For that, the phytochemical groups of the leaves and the trunk bark were firstly investigated.Then, the anti-radical activity and the effect on the blood pressure of normal strained guinea pigs of the trunk bark hydro-ethanolic extract were evaluated.

General information
Optical densities (absorption spectra) were determined on a Shimadzu UV-1601PC spectrophotometer.Samples were weighted using a Sartorius lab instruments balance with maximum weight of 1000 ± 0.01 g.Evaporation was carried out on an R-100 BÜCHI Rotavapor.An electric shredder and a transducer connected to Biopac System (Model MP100, Harvard Apparatus) allowed the assessment of extract effects on sound guinea pigs blood pressure.Analytical grade solvents and all others materials were used as received.

Phyto-constituents characterization
Phytochemical screening refers to all methods and techniques for the detection of active chemical groups in the plant.In this study, the whole phytochemical screening was performed according to standard methods described by Badiaga et al (9).So, the presence of alkaloids has been revealed by Mayer or Dragendorff 's reagent.STIANSY reagent or 1% ferric chloride was used to characterize tannin derivatives.Flavonoids derivatives and buffy anthocyanins were evidenced by the cyanidine reaction (SHINODA reagent).Quinone compounds were evidenced by the Bornträger reaction.The detection of sterols and triterpenes were possible with Liebermann-Burchard reaction.Cardiotonic glycosides were revealed when green or purple color came out after adding the acetic and sulfuric acid in chloroformic extract.The appearance of a flaky precipitate revealed the presence of mucilages.Saponosides were characterized by their foaming power.Reducing constituents were characterized using A and B Fehling liquors.The anthracites derivatives were revealed using a 50% ammonia solution.

Biological materials
The bark of the trunk and the leaves of H. floribunda were harvested in March 2015 at Danyi Mountain in the plateaus region (Zone IV) of Togo.The plant is locally called "Afèkèti" or "Kpomiti" in "Ewé".It was (the plant species) identified by the Herbarium of "Université de Lomé" (TOGO; TOGO02074) and previously registered under the identification number 1987/03 by K. Akpagana (K.Akpagana, #1522).The plant samples were left at room temperature for two weeks.After this drying process, the samples were reduced in powder using a lab scale grinder.Hydro-ethanolic extract of the trunk was prepared to assess pharmacological effects of H. floribunda on guinea pigs under normal blood pressure (systolic blood pressure ≤120 mm Hg) weighting about 400-550 g.They were bought in Gbonsimé market in Lomé city (Togo) and placed at animal house of the Laboratory of physiology and pharmacology of natural products of the Faculty of Sciences, "Université de Lomé" (Togo), for a week before the experiments.These guinea-pigs had free access to water and were fed with fresh grass.

Cardiovascular effects assessment via bloody method
The so-called direct bloody method is an invasive technique for measuring blood pressure using a catheter introduced into the carotid artery of an anesthetized animal (10).The measurement of invasive pressure presents some advantages, notably the beat-by-beat tracking of blood pressure and the achievement of a very precise pressure even at very low extent.The invasive pressure was measured using a transducer (connected to the catheter) with an ultra-sensitive membrane capable of converting the slightest mechanical variations of the animal's blood pressure into electrical signals.The recording of the cardiovascular parameters was done according to this method exhaustively described previously (11).The guinea pig, anesthetized intraperitoneally with 20% of urethane (ethyl carbamate) at a dose of 1 g/kg (that is 1 mL/200 g body weight), was placed in supine position (12).The jugular vein and the carotid artery were exposed, dissected, and then intubated towards the heart using fine polyethylene catheters.The catheter of the jugular vein connected to one syringe and that of the carotid artery linked to a pressure sensor connected to a transducer were used to visualize the hemodynamic parameters such as the systolic blood pressure (SBP, mm Hg) and the diastolic blood pressure (DBP, mm Hg).Those two types of blood pressure correspond to the maximum and the minimum pressures, respectively.Mean arterial pressure (MAP, mmHg) does not an arithmetic average of SBP and DBP, because blood pressure values in the arteries are more closed to DBP value for a long time.For that, the average pressure was obtained from the following stated Pelletier' relation (13): After stabilization of the cardiovascular parameters for about 45 minutes, the extracts of H. floribunda at cumulative doses of 10; 20; 40; 80 mg/kg (taking into account the toxicity studies of H. floribunda by Bogne et al (14) and Antwi ( 15)) were administered intravenously through the syringe connected to catheter of the jugular vein of a guinea pig under normal blood pressure.The NaCl solution (9‰) containing 10% of heparin was injected at a rate of 1 mL/1000 g via this jugular vein to prevent blood from clotting.The decrease rate in MAP was calculated using the following relation:

Determination of total phenols and polyphenolic antioxidants
The determination of total phenols content is an in vitro colorimetric assay based on the ability to reduce the Folin-Ciocalteu reagent (FCR) ( 16) which is a yellow acid solution consisting of a mixture of phosphotungstic and phosphomolybdic polyheterocyclic acids.In a basic medium, the FCR oxidizes the hydroxyl groups of polyphenolic compounds giving a blue coloration.This determination was carried out according to the method described by Al-Farsi et al (17).A mixture consisting of 100 μL of extract in methanol at 1 mg/mL and 750 μL of the FCR 10 times diluted was prepared.After stirring for 5 minutes, 750 μL of a saturated sodium carbonate (Na 2 CO 3 ) solution at 60 g/L was added.The obtained solution was incubated for 90 minutes at room temperature in the darkness.The absorption spectra (optical densities) were recorded with respect to a blank at 725 nm using a UVvisible spectrophotometer.Referring to the calibration regression equation established with gallic acid in a concentration range of 0-500 μg/mL (Figure 1), the content of polyphenols in the extract was determined.The test was performed in triplicate.The following formula was used to calculate the total phenol contents expressed in mg Equivalent of Gallic Acid (EGA) over 1 g of dry extract: Free DPPH • radical trapping activity The antiradical activity was determined using the method described by Brand-Williams et al (18), modified by Agbonon et al (19).This method relies on the trapping of the stable free radical DPPH in the presence of antioxidant molecules.
The DPPH solution of about 10 -4 mol/L was prepared (about 1 to 2 hours in advance) by making soluble the pure DPPH in methanol so that the solution exhibited an optical density between 0.787 and 0.788 at 517 nm.A range of concentrations (1 to 50 μg/mL) of H. floribunda extract or quercetin solutions was prepared in methanol.A volume of 250 μL of each of those prepared solutions was added to 1.5 mL of the DPPH solution (C = 10 -4 mol/L), the mixture was homogenized and incubated at room temperature for 30 minutes.The optical density was then recorded at 517 nm using a UV-visible spectrophotometer.The control was recorded under the same conditions.The inhibition percent (%I) of the DPPH was calculated according to the following formula: Antioxidant power by the FRAP The ferric ion reducing antioxidant power (FRAP) method consists of reducing ferric ions from the ferric tripyridyltriazine complex (Fe 3+ -TPTZ) to ferrous ions under a low pH and temperature of about 37°C.The change in absorbance at 593 nm is directly associated with The statistical significance between results for different samples and between results for samples and controls were set at P < 0.05.

Phytochemical screening of Holarrhena floribunda
In order to investigate the phytochemical composition of H. floribunda, infusion, maceration, and decoction were used and many extracts obtained.For that, the leaves and bark of trunk powder samples were firstly subjected to infusion and decoction.The similar powder samples were then macerated in many organic solvents (ethanol, chloroform, hydrochloric acid, ethyl acetate, and petroleum ether).The various phytochemical groups detected in infused and decocted samples are recorded on the Table 1, and those from the maceration are drawn up in Table 2.
From infusion method (Table 1), one can see that anthocyanins were not detected.In the same time, tannins (catechics and gallic), saponosides, and flavonoids have been detected.From the decocted phases, the leucoanthocyanins, saponosides, mucilages, flavonoids, and reducing compounds were revealed in the two parts.Moreover, foam index performed on the decocted phase was 500 in the leaves against 333.33 in the trunk bark.
In macerated samples and when using some specific tests, various phytochemical compounds were detected (Table 2).So, only alkaloids and saponosides were detected in ethanol while cardiotonic heterosides, c-heteroside, and o-heteroside anthracenic were found in chloroformic macerated phases.Alkaloids were found in hydrochloric acid; sterols and terpenes in ethyl acetate, and free quinones in petroleum.
Anti-radical activity by DPPH, antioxidant power by FRAP and total phenol content Based on the calibration regression equation established in Figure 1, the total phenol content (in equivalent of gallic acid per gram of dry extract) of the trunk bark hydro-ethanolic extract was 139±0.053mg EGA/g dry extract.The antioxidant activity of the same extract was evaluated by the DPPH and the FRAP methods.The DPPH test allows getting the anti-radical activity.Indeed, it is known that an extract is more antioxidant if its IC 50 is lower.From Table 3, the IC 50 of this extract is 29.80±0.001μg/mL while that of quercetin is 11.19±0.23 μg/mL.The reducing power assessed by the FRAP method gave the value of 1009.9 ± 0.6 μmol Equivalent Fe 2+ /g (Table 3).Furthermore, the kinetics of the reduction of DPPH under different concentrations of the extract was monitored.As shown in Figure 4, the decrease in absorbance is more pronounced for large concentrated sample.The kinetic profiles are dependent on the concentration of the extract.
Close observation shows that the extract reacted quickly with the DPPH reaching a steady state in about 5 minutes.
Assessment of the extract effect on guinea-pigs blood pressure Intravenous injection of the hydro-ethanolic extract (by cumulative dose in the order of 10; 20; 40; 80 mg/kg)    resulted in immediate decreases in mean arterial pressure (MAP) of normal strained guinea pigs (Figures 5 and 6).Indeed, a few seconds after injection of 10 mg/kg of the prepared dose, MAP decreased from 82.734 mm Hg to 65.067 mm Hg, which is an immediate and significant decrease of about 21.35%.Then, the pressure rose to hit the initial value in about 10 minutes later.Administering the dose of 20 mg/kg resulted in a sharp and significant decrease in MAP of about 27.86%; 15 minutes later, the MAP rose and stabilized at about 70.6 mm Hg.The 40 mg/kg dose resulted in a sharp and significant decrease in MAP of about 36.41%;20 minutes later the MAP rose and steadied at about 61.33 mm Hg.When 80 mg/kg was injected, a drastic and significant drop in MAP of about 57.68% was observed, 20 minutes later, the MAP rose slowly and steadied at about 47.67 mm Hg.It should be noted that the physiological solution of NaCl 9% with 10% of heparin had no significant effect on MAP (a variation of 0.025% has been obtained).

Discussion
First of all, the results of phytochemical screening of the leaves and the trunk bark confirmed the previous works of Badmus et al (21) and of Gnangoran et al (22).Indeed, most of the metabolites found in the current study possess significant pharmacological properties, including analgesics, anesthetics, antibiotics, and anticancer (23).For instance, plants containing flavonoids are known potent antioxidants, antiradicals, diuretics, purgatives, disinfectants, analgesics, anti-inflammatories, antipyretics, and antispasmodics (24).The revealed antioxidant properties (Table 3) of the hydro-  ethanolic extract of the trunk bark of H. floribunda could have beneficial effects on the preventive treatment of CVD.Indeed, antioxidants have the ability to trap and inhibit the formation of free radicals.Flavonoids as antioxidants have effects on veins and arteries by decreasing permeability and increasing capillary resistance (23).It is known that the regular feeding of fruits and vegetables decreases the risk of developing CVD (25), as they contain antioxidants.According to the total polyphenols content and the screening study, the antioxidant properties might be mainly due to the presence of polyphenolic compounds such as tannins, flavonoids, and leuco-anthocyanins.Polyphenols are known as antioxidants and include five main families, namely phenolic acids, phenolic alcohols, flavonoids, tannins, and lignans (26).This work confirms that of Badmus et al (21) who showed that the leaves of H. floribunda possess antioxidant activity.Moreover, according to N'guessan et al (27), the anti-radical capacity is often correlated with the total polyphenols content of plant extracts.The result of the kinetics of the reduction obtained in the current study (Figure 4) is similar to those of Popovici et al (28).These observations show that the hydro-ethanolic extract of H. floribunda bark exhibited strong anti-radical efficiency.The fall in MAP was based on the administered dose (Figures 5 and 6).Intravenous injection of the hydroethanolic extract of H. floribunda induced a significant decrease in DBP, in SBP, and thus in MAP.It should be noted that the dose of 40 mg/kg was seen as a best one (induce a better effect) because the return back effect under this dose is not only very slow but also weak.These results are somewhat similar to those obtained on the extracts of Bidens pilosa by Dimo et al (29) and Kouakou et al (30), and might be due to the presence of polyphenols, alkaloids, and saponosides in the current extract.The extract of H. floribunda therefore exhibits antihypertensive (hypothensive) effects, as shown in similar studies (12,31).Indeed, similar development has been obtained by Titrikou et al (12) on the extracts of Biophytum petersianum and Etou et al (31) on the extracts of Lippia multiflora.Furthermore, the possible mechanisms involved in this positive effect (hypothensive) could be an inhibition of orthosympathetic activity or a decrease in oxidative stress.Indeed, an increase in oxidative stress is observed in hypertensive patients (32).The polyphenols content in this extract might play a key role in inhibiting the action of activated oxygen species.Moreover, the antidiabetic activity of H. floribunda (22) might also have positive preventive effects, by improving the insulin sensitivity.

Conclusion
This study showed the presence of various active phytochemical groups in the bark of the trunk and the leaves of H. floribunda specimen of the Zone IV of Togo.So, the phytochemical screening revealed alkaloids, tannins (catechics, gallic), flavonoids, leucoanthocyanins, saponosides, free quinones, anthrax Cand O-heterosides), reducing compounds, mucilages, cardiotonic heterosides, sterols, and terpenoids in the two parts of H. floribunda.The hydro-ethanolic extract of the trunk bark of H. floribunda exhibited in vitro antioxidant activity and in vivo antihypertensive effect.The two observations might prove the use of H. floribunda extract in traditional medicine against cardiocardiovascular diseases.However, further study might be envisaged in order to elucidate the mechanism of action of the observed effects.This could provide strong pharmacological arguments that converge in favor of the use of H. floribunda as an alternative treatment against high blood pressure (hypertension).In the same way, it might be very interesting to carry out a more thorough phytochemical study in the hope of isolating some active principles responsible for this hypotensive activity.
Density of the control; OD: Optical Density of the sample (extract or quercetin).The anti-radical power was defined as the concentration of extract needed to induce the reduction of 50% of the free radical DPPH (IC 50 ).The IC 50 value was calculated by the linear regression method shown in Figure2: %I = f (concentration) = ax + b, with a and b constants and x a concentration; %I = 50% = f (IC 50 ).Standard deviations were calculated from three sets of experiments.The IC 50 value depends on the nature of the sample and the final residual concentration of the DPPH.

Figure 2 :Figure 3 .
Figure 2: a) Linear regression line of quercetin, b) Linear regression line of Holarrhena floribunda extract with DPPH

Figure 5 .
Figure 5.Effect of dose-response of Holarrhena floribunda extract on MAP.

Figure 6 .
Figure 6.a) MAP of normal guinea pigs development under NaCl 9‰ and doses of Holarrhena floribunda extract 15 minutes after injection; b) Impact of NaCl 9‰ and extract of Holarrina floribunda on fall in MAP of normal guinea pigs.Each point represents an average ± SD (standard deviation).With n = 5, *P < 0,05; **P < 0,01 and ***P < 0,001 significantly different compared to the initial value of the MAP.

Table 1 .
Chemical groups detected in infuse and decocted powders of the leaves and the bark of the trunk

Table 2 .
Phyto-constituents from different macerated phases of leaves and trunk bark +: Detected; -: Not detected.