parquetina nigrescens (periplocaceae) on the rabbit

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Konan et al. World Journal of Pharmaceutical Research

IN VITRO EFFECTS OF HYDROETHANOLIC EXTRACT FROM

ROOTS BARK OF PARQUETINA NIGRESCENS (PERIPLOCACEAE)

ON THE RABBIT DUODENUM CONTRACTILE ACTIVITY

André B. Konan1*, Marie L.B. Ahui1, Isidore A. Saraka1, Augustin K. Amonkan1,

Léandre K. Kouakou2, Mathieu N. Bléyéré2, Jacques Y. Datté1

1Laboratory of Nutrition and Pharmacology, UFR-Biosciences, Félix Houphouët-Boigny

University, 22 BP 582 Abidjan 22, Côte d'Ivoire 2Laboratory of Animal Physiology, Phytotherapy and Pharmacology, UFR Sciences de la

Nature, Nangui Abrogoua University, 02 BP 802 Abidjan 02, Côte d’Ivoire

ABSTRACT

Hemorrhoidal disease is one of the most common diseases of the anal

axis. The causes of this disease are numerous. This disease is

manifested by disturbances of the intestinal transit, usually associated

with atony of the handle and which contribute to a reduction in the

force of peristaltic contractions of the large intestine. African

pharmacopoeia in general and in particular the Ivorian pharmacopoeia

contains various plants known anti-hemorrhoidal including Parquetina

nigrescens (AfZ.) Bullock (Periplocaceae). Also, this study was carried

to examine the in vitro effects of hydroethanolic extract from root bark

of P. nigrescens on the contractile activity of rabbit duodenum. The

isometric contractile force of the rabbit duodenum strips (RaDs) was

recorded by using a strain gauge. P. nigrescens hydroethanlolic extract

(EPan, 9.9×10-5 mg/ml – 0.2 mg/ml) showed myostimulant properties.

These myostimulant effects were characterized by the increase of the amplitude and the basal

tone of RaDs in normal Mac Ewen solution and by the development of phasic contractions in

solution without calcium. In this Ca2+-free solution, the addition of EPan (9.9×10-3 mg/ml)

elicited the development of phasic contraction in the presence of EGTA (0.1 mM). This result

suggests that EPan could act on the double calcium flux (intracellular and extracellular). In

conclusion, the hydroethanolic 70 % extract of P. nigrescens (EPan) had myostimulant

World Journal of Pharmaceutical research

Volume 3, Issue 1, 139-150. Research Article ISSN 2277 – 7105

Article Received on 21 October2013 Revised on 23 November 2013, Accepted on 26 December 2013

*Correspondence for

Author:

Dr André B. Konan,

Laboratory of Nutrition and

Pharmacology, UFR-

Biosciences, Félix Houphouët-

Boigny University, 22 BP 582

Abidjan 22, Côte d’Ivoire.

[email protected]

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effects (prostaglandin-like activity) on the rabbit isolated duodenum. The root bark of this

herbal may increase peristalsis of intestinal muscle and thus increasing intestinal transit.

Parquetina nigrescens has mysotimulant property thus supporting its folkloric usage to treat

haemorrhoid.

Keywords: Parquetina nigrescens, Duodenum, Contractile activity, Atropine, EGTA;

INTRODUCTION

Hemorrhoidal disease is one of the most common diseases of the anal axis [1]. This disease is

prevalent worldwide. It is therefore a case of public health, and needs to be studied.

Hemorrhoidal disease represents more than half of the cases of liberal gastroenterological

consultations in France. Less than 5 % of the U.S. population suffers from hemorrhoidal

disease [1].

In Côte d'Ivoire, the prevalence of hemorrhoidal disease is 40 % and some traditional healers

claim that around 60 % of their patients suffer from hemorrhoids [2]. According to a study at

the University Hospital of Cocody, hemorrhoidal disease is recorded in patients aged between

20 and 50 years. Although exceptional in children, hemorrhoids may occur in adolescence

[2]. It affects all social strata and there is no ethnic predisposition.

Hemorrhoidal disease is also not related to sex [3]. Epidemiology recognizes a hereditary

predisposition [4]. The causes of this disease are many whose eating habits, lifestyle, etc.

However, among these various factors, transit disorders, usually associated with atony of the

handle and which contribute to a reduction in the force of peristaltic contractions of the large

intestine is one of the major causes. Hemorrhoidal disease is associated with a disorder of

defecation.

In modern medicine, the treatment of this disease has several components. it goes dietary

hygienic measures to medical methods, with the anti hemorrhoidal chemotherapy that uses

anti-inflammatories, veno-tonic, antibiotics and anesthetic agents and finally instrumental

methods and surgical methods.

African pharmacopoeia in general and in particular the Ivorian pharmacopoeia full of

reputable anti-hemorrhoidal plants those are used in various forms (decocted, macerated or

infused) orally, by enema or sitz bath. Some of these plants have already been the subject of

pharmacological studies, namely, Tamarindus indica, Citrus aurantifolia, Khaya

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senegalensis and Landolphia hirsuta [3, 5]. Among these plants, Parquetina nigrescens

(AfZ.) Bullock (Periplocaceae) caught our attention. The leaves and the powder obtained

from the dried root bark of this plant are used separately in traditional medicine to treat

hemorrhoidal attacks.

In this work, the effects of hydroethanolic extract from dried roots bark of P. nigrescens were

studied on contractile activity of isolated rabbit duodenum.

MATERIALS ET MÉTHODES

Ethics

Experimental procedures and protocols used in this study were approved by Ethical

Committee of Health Sciences, University Félix Houphouet-Boigny. These guidelines were

in accordance with the internationally accepted principles for laboratory animal use and care

[6, 7].

Plant material

The roots barks of P. nigrescens collected locally from the forest of Daoukro in the East of

Côte d’Ivoire in December 2009. Taxonomical identification of the root barks was

established by Professor Aké-Assi Laurent from the National floristic Centre of University of

Felix Houphouet-Boigny, Cocody- Abidjan, Côte d’Ivoire.

Preparation of Hydroethanolic 70 % extract of P. nigrescens

Roots barks of P. nigrescens dried under shade and powdered with a machine (mark

RETSCH, type SM 100, Germany). The extraction process was implemented according to the

method described by some authors [8, 9]. One hundred grams (100 g) of the stem barks

powder were macerated during 24 hours in 1l ethanol-water (70:30 v/v) for 3 times until

complete exhaustion. The mixtures were filtered (Whatman n°1) and concentrated under

reduce pressure using a rotary evaporator (Büchi R110, type MKE 6540/2) at a temperature

of 45 °C. The concentrated extracts (Hydroethanolic 70 % extract of P. nigrescens, EPan)

were stored in dessicators at 45 °C.

Animals

Rabbits Oryctolagus cumuculus (2.2 ± 0.4 kg) were used in our experiments. These animals

were obtained from the Animal House of the Laboratory of Nutrition and Pharmacology of

UFR-Biosciences at Félix Houphouët-Boigny University in Abidjan (Côte d’Ivoire). They

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were housed in a constant temperature rooms with a light/dark cycle of 14/10 hours. All

animals were fed and given water ad libitum.

Recording of the Contractile Activity of Rabbit Isolated Duodenum

This experiment was performed as described by Konan et al. [10-11] and Ahui et al. [12].

After sacrifice of animals, by cervical dislocation, a median laparotomy was practised. The

duodenum was taken and split up in 2 cm length scraps. These Rabbit Duodenum strips

(RaDs, 2 cm) were transferred in a Petri dish containing a normal Mac Ewen solution with

following composition (mM): NaCl, 130; KCL, 2.5; CaCl2, 2.4; NaH2PO4, 1.18; NaHCO3,

11.9; MgCL2, 0.24; glucose, 2.2). The solution (pH of 7.4 and gassed with 95 % O2 + 5 %

CO2) was kept at a temperature of 35 °C [10-14].

A selected fragment (RaDs) was put in an isolated organ bath containing the oxygenated

physiological solution of Mac Ewen, thermostated at a temperature of 37 °C. Using a cotton

yarn, one end of the duodenum strip was vertically attached to a hook and the other was

connected to a stylet inscriptor which transmitted the movements of the duodenum strips on

moving paper (speed 0.1 cm/s). After two hours, the time necessary for stabilization of the

contractile movement, the concentrations to be tested were added directly into the organ bath.

Chemical Used

Atropine (ATR) and EGTA were purchased from Sigma Chemical Company (USA). All

drugs (EPan, ATR and EGTA) were dissolved and/or diluted in distilled water on each day of

our experiments [10-12, 15]. Drugs concentrations quoted in the text refer to final organ-bath

concentration.

Statistical Analysis

All values were expressed as mean ± standard error on the mean (m ± sem) obtained from n

separate experiments. Statistical analysis of the values and graphical representations of data

were performed respectively by GraphPad Instat software (Microsoft, San Diego, California,

USA) and GraphPad Prism 5 software (Microsoft, San Diego, California, USA). Differences

between the mean statistical validity are assessed through Tukey-Kramer test. The difference

between the averages is considered statistically significant at the 5 % (p < 0.05).

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RESULTS

Dose-Dependent Effects of EPan on Rhythmic Contractions

The effects of EPan were studied on the rhythmic contraction of rabbit duodenum. As shown

in Figure 1, original tracing, EPan (9.9 × 10-5 mg/ml to 0.2 mg/ml) caused a gradual increase

in the contractile activity of isolated rabbit duodenum. Epan-action on the isolated rabbit

duodenum was concentration-dependent. Indeed, the rhythmic contractions of RaDs were not

affected by the low concentration (9.9 × 10-5 mg/ml). As against, the application of higher

concentrations of EPan from 9.9 × 10-4 mg/ml to 0.2 mg/ml induced elevations of contractile

force and basal tone. Thus at a concentration of 9.9 × 10-4 mg/ml, the contractile force was

increased from 24.8 ± 0.86 % (p < 0.001) whereas the basal tone increased and is estimated to

430.4 ± 14.2 mg (p < 0.001). When EPan was administered at 4.9 × 10-3 mg/ml, the increase

in contractile force was 40.8 ± 1.66 % (p < 0.001). At the same concentration, the basal tone

recorded was estimated at 1621 ± 44.28 mg (p < 0.001). Finally, the application of the high

concentration (0.2 mg/ml) caused a sustained contracture and its value was 1844 ± 22.04 mg

(p < 0.001).

Inhibitory effects of atropine on EPan-induced contractile responses in rabbit

duodenum strips

Atropine (ATR), antagonist of the muscarinic receptors, suppressed the stimulating action of

EPan on the isolated duodenum of rabbit (Figure 2). In absence of the ATR, EPan, at 4.9 ×

10-3 mg/ml, increased the force of contractions to 31 ± 1.14 %. In presence of ATR at 2×10-7

mg/ml and 2×10-7 mg/ml, the contractile force induced by EPan increased respectively from

12.6 ± 1.32 % (p < 0.001) and 4.8 ± 1.24 % (p < 0.001). The inhibitory effect of ATR on

EPan-induced contractions on the RaDs was concentration-dependent.

Effects of EPan on rabbit duodenum strips in calcium-free solution

Removing extracellular calcium affected the contractile activities of muscles. It abolished

totally or strongly spontaneous contractions, whereas the basal tone was maintained (Figure

3). When EPan was introduced into the isolated organ bath, it caused the appearance of

phasic contractions and a slight increase in basal tone. It was observed an elevation of basal

tone of RaDS and the measured amplitude (force) of contraction was 907 ± 86,33 mg (p

<0.001) compared to its value before EPan-application (Figure 3a). The pre-treatment of

RaDs with EGTA (0.1 mM) did not abolish the EPan-induced contractile responses. Indeed,

in the presence of this specific Ca2+ chelator, EPan (4.9 × l0 3 mg/ml) induced rhythmic and

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regular contractions of RaDs. These phasic contractions reached a value of 627 ± 67.73 mg (p

< 0.001) (Figure 3b).

Figure 1: Original tracing showing in vitro effect of hydroethanolic extract from roots back of P. nigrescens (EPan) on the rabbit duodenum contractile activity. The arrows indicate administration of EPan. EPan increased the contractile activity of this intestinal smooth muscle concentration-dependent manner. Horizontal scale: 30 s; vertical scale: 0.5 g.

0 2x10 -7 2x10 -5

0

10

20

30

40 EPan, 4.9 x 10-3 mg/ml

ATR [mg/ml]

Incr

ease

of C

F (%

)

Figure 2: Influence of atropine on the contractile activity of the isolated rabbit duodenum induced by hydroethanolic extract from roots back of P. nigrescens (EPan). Atropine (ATR) inhibited in concentration-dependent manner the effects of EPan used at 4.9 10-3 mg/ml. Data shown are mean ± S.E.M. (n = 6; *** p < 0.001)

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Figure 3: Original tracing showing the effect of EPan on the contractile activity of the isolated rabbit duodenum placed in the calcium-free solution (0 Ca2+). The arrows indicate administration of the studied substances. Used alone, the plant extract caused the onset of phasic contractions of isolated rabbit duodenum (a). The addition of EGTA (0.1 mM) in this solution did not suppress the contractile activities induced by EPan (b). Horizontal scale: 30 s; vertical scale: 0.5 g. DISCUSSION

The hydroaethnolic extract of P. nigrescens (EPan) increased the contractile activity of the

rabbit duodenum strips (RaDs), an intestinal smooth muscle. EPan could have a

myostimulant action. This myostimulant effect of ESera was characterized by the increase of

the basal tone and the force of the contractions of the RaDs in normal solution and by the

development of a contracture in calcium-free solutions.

In the modified and calcium-free solutions, the addition of EPan provoked the development

of phasic contractions in presence of EGTA, a specific chelator of Ca2+. According to

previous studies [10-11,13-14,16-17], this result permits to suggest that EPan would act on

the double entering and intracellular calcium flux. Therefore EPan could be a spasmogenic

substance. It would mobilize the calcium that seems to be the determining and inescapable

element of the intracellular mechanisms that underlies the contractile activity of the smooth

muscles [18-20].

Indeed, the contractile activity requires the presence of Ca2+ from two sources that are

calcium influx through calcium channels (responsible for the phasic contraction) and the

mobilization of internal calcium from intracellular stores responsible for the tonic contraction

[21]. The increase in basal tone may be caused by the presence of cytosolic free calcium that

activates the contractile elements [22]. The effects of P. nigrescens (EPan) on the RaDs were

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similar to those of various medicinal plants, among others Citrus aurantifolia (Rutaceae),

Khaya senegalensis (Meliaceae), Bridelia ferruginea (Euphorbiaceae) and Sesamum

radiatum (Pedaliaceae) on the isolated Taenia caeci of guinea-pig [10,16-17,23], Mareya

micrantha (Euphorbiaceae) on the isolated intestine of guinea-pig [24], Euphorbia hirta

(Euphorbiaceae) on the isolated intestine of rat [25] and Sesamum radiatum (Pedaliaceae) on

the uterine smooth muscle isolated from pregnant rat [11].

The probable presence of cholinomimetic coumpounds in the plant extract could explain the

usefulness of P. nigrescens as anti-haemoroidal plant as indicated by the ethnobotanical

information. This cholinomimetic action of EPan corroborates the results obtained by Datté et

al. [26] with the P. nigrescens leaves. These authors showed the cholinomimetic effect of this

herbal.

The intestinal transit is controlled by both neural and myogenic mechanisms [27]. An

increase of the contractile activity of the smooth cellular layers in general is responsible for

the acceleration of intestinal propulsion. Several mediators and neurotransmitters govern

these motor patterns. Acetylcholine is the main excitatory neurotransmitter in the enteric

nervous system [28]. Acetylcholine (ACh) causes through its M3 muscarinic receptors in the

intestine an increase of the basal tone with sometimes an increase of the peristaltic

contractions.

CONCLUSION

EPan is a spasmogenic substance. It induced an increase of the contractile activity of the

rabbit isolated duodenum smooth muscle. It could probably act by the mobilization of the

double calcium flux. This spasmodic action could be assigned partly to the presence of

cholinomimetic substances in the plant extract. To summarize, our study could justify the

folkloric use of Parquetina nigrescens roots back to treat haemorrhoid, disease causing a

disorder of intestinal transit. Considering its myostimulant action, Parquetina nigrescens

could use in people suffering from this disease by increasing intestinal transit. Parquetina

nigrescens has mysotimulant property thus supporting its folkloric usage to treat

haemorrhoid.

ACKNOWLEDGEMENT

The authors are grateful to Professor Ake-Assi Laurent (National floristic Centre of of Felix

Houphouet-Boigny University, Cocody- Abidjan, Côte d’Ivoire) for botanical identification

of Parquetina nigrescens (AfZ.) Bullock (Periplocaceae).

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ABBREVIATIONS

EPan: Hydroethanolic extract of P. nigrescens; RaDs, Rabbit duodenum strips, ATR:

Atropine; EGTA: ethyleneglycol-bis(aminoethyl ether) N,N,N’,N’-tetraacetic acid.

FUNDING

This research did not receive any specific grant from any funding agency in the public,

commercial, or not-for-profit sector.

AUTHORS’ CONTRIBUTIONS

All authors contributed equally in the study. They made substantial contributions to the

design of the study, the collection of the data as well as the preparation and analysis of the

data. They also drafted the manuscript and gave final approval for its submission to the

journal for consideration of publication.

DECLARATION OF INTEREST

The authors declare that there is no conflict of interest that could be perceived as prejudicing

the impartiality of the research reported.

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