Background
Zanthoxylum armatum DC locally known as timber belongs to family Rutaceae, which comprises about 150 genera and 1,500 species. It grows wildly in hilly areas of Pakistan including district Dir, Hazara division, and Galliyat [
1]. Almost all parts of the plant are aromatic and possess essential oil. Various parts are documented to have ethnomedicinal uses for different ailments. However, the uses of seeds are predominant. The seeds and barks of
Z. armatum are used as aromatic, carminative, tonic in fever, dyspepsia. The fruits and seeds are used for curing cholera, toothache and as leech repellant. The bark, thorns and fruits are used in fish poisoning. The seeds are chewed to cure toothache, added in vegetables for detoxification. The dried seeds can act as effective pesticide against small insects of wheat plants. The aerial parts are extensively used as a carminative, stomachic and anthelmintic, branches are used as toothbrush [
2].
The plant has traditional reputation in the traditional medicine for the management of different ailments including gut and airway.
Z. armatum fruits have stomachic and carminative properties [
1]. In Ayurveda
Z. armatum fruit is considered appetizer, anthelmintic and gives relief from pain, tumors and abdominal troubles
, in diseases of eye and ear [
3]. The berries and the bark are used as aromatic tonic in dealing fevers, heartburn and cholera [
4].
Z. armatum fruits, seeds and stem bark are used traditionally in the treatment of asthma, bronchitis, indigestion [
5]. The various chemical classes of constituents including coumarins, flavonoids, sterols, terpenes, and alkaloids are reported from
Z. armatum.[
4,
6‐
11].
A similar study on
Zanthoxylum armatum has previously been carried out on whole plant extract [
12], there is scarce of information the plant’s leaf, bark and fruit effects separately has on gut, respiratory and cardiovascular smooth muscles. The purpose of this research work was to assess the pharmacological effects of individual extracts from fruit, bark and leaves. Plants-derived flavonoids, alkaloids and terpenes are known having anticholinesterase activities [
13] and such constituents have role in the treatment of different GIT, airway and cardiovascular conditions [
14]. Therefore, the extracts were also evaluated against the butyrylcholine esterase in vitro.
Discussion
The study undertaken describe the pharmacological properties of individual extracts of fruit, bark and leaves of
Zanthoxylum armatum. A similar study of this plant variety has already been conducted previously [
12]. Therefore, purpose of this study was to determine the pharmacological activity and potential mechanism of action by studying the effect of aerial parts of
Z. armatum separately. It is in our best of knowledge that different parts of the plants are used separately for different ailments.
The butyrylcholinesterases are known to induce hydrolysis and lead to reduce acetylcholine level subsequently reducing the motor activity of guts [
21]. Cholinesterases inhibitors elicit a cholinergic action by inhibiting the hydrolysis of endogenous acetylcholine [
22]. More studies will also be useful to discover the medicinal significance of
Z. armatum in Alzheimer’s disease for the reason that both cholinergic and calcium channel blockers are recognized to be beneficial in old age dementia and Alzheimer’s disease [
23,
24].
The present study revealed that the traditional use of Z. armatum to relieve diarrhea and bronchospasm is based on the intestinal spasmolytic and bronchodilatory effects, respectively.
The extracts of
Z. armatum (Zf, Zb, Zl) with dose of 300 and 1000 mg/kg body weight and verapamil inhibited the diarrheal frequency significantly compared to negative control. Verapamil is used as a standard drug and it produce its effect by inhibiting the calcium channels [
25]. Castor oil hydrolyzed to yield recinoleic acid and it in turn induce diarrhea [
26], by altering the water and electrolyte transport and results in hypersecretion and generate oversize contractions of the intestine [
27]. So, the possible way of exhibiting the antidiarrheal mechanism involved the inhibition of gut motility and or inhibition of out flux of electrolyte [
27]. The pattern of antidiarrheal effect of verapamil and extracts was found to be similar and therefore suggests that this effect was due to inhibition of intestinal contractions or on electrolyte out flux. To confirm the mechanism of inhibition of gut contractions, the samples were tested in-vitro.
The extracts and standard drugs were added in a cumulative fashion and a concentration dependent inhibition was observed in rabbit jejunum (Fig.
2). The experiments revealed spasmolytic activity in smooth muscles. When cytoplasmic free calcium increases, it causes the activation of contractile elements in smooth muscles like jejunum [
28]. The rise in intracellular Ca
++ happens in two ways, one is through influx through voltage-dependent Ca
++ channels and secondly through its release from intracellular stores in the sarcoplasmic reticulum. The spontaneous movements of the intestine are controlled by periodic depolarization and repolarization. When the depolarization is at the peak there is fast influx of Ca
++ results in appearance of action potential [
29]. Thus, the mechanism underlying by which
Z. armatum appeared to produce was calcium channel blocking (CCB) effect involving Ca
++ influx. It has been in our previous observation that spasmolytic activity of plant constituents was mediated through CCB effects [
17,
30,
31]. To observe, whether the spasmolytic property of the
Z. armatum fruit, bark and leaves in this study was also intermediated through a Ca
++ antagonist-like effect, a high concentration of K
+ (80 mM) was used to produce sustained contraction through opening of voltage dependent Ca
++ channels. The crude extracts showed a similar effect as shown by standard drug verapamil and caused the inhibition of K
+ pre-contractions more efficiently than spontaneous contractions (Fig.
1). Therefore, a substance, which cause the inhibition high K
+-induced contractions is possibly considered to be a Ca
++ channels antagonist [
25]. This hypothesis was more supported when pre-treatment of the tissues with plant extract caused a rightward shift in the Ca
++ curves (Fig.
1), similar to verapamil which is in accordance to its known Ca
++ antagonist effect as antidiarrheal [
32]. Thus the extracts effect provides sound pharmacological basis to its antidiarrheal and antispasmodic effects, as the Ca
++ antagonists are considered beneficial in diarrhea and gut spasms [
33].
Histologically, rabbit jejnum, trachea and aorta have smooth muscles that is the only similarity between them. However, the architecture of all the three organs are different that is, in the trachea the smooth muscles are interconnected through skeletal muscles while in the smooth muscles of jejunum and aorta do not. Furthermore, the receptor biology of the three different organs are different. Smooth muscles of the jejunum are regulated predominately through muscarinic receptors, tracheal smooth muscles by the beta-adrenergic receptors while the aortic by alpha-adrenergic receptors [
34].
The fruit, bark and leaves extracts (Zf, Zb and Zl) of
Z. armatum showed inhibition of carbochol (1휇M) and K
+-(80 mM) induced contractions in rabbit isolated trachea. Among the extracts tested, Zf showed more potential of inhibition of tracheal muscles in a concentration dependent manner (Fig.
2). The carbochol caused the activation of muscarinic receptors and is therefore a cholinergic agonist [
35]. The Zf caused the relaxation of tracheal muscles by antagonizing the muscarinic receptors as well as by CCB. The bronchodilator effect may be due to CCB mediation [
36]. It is well-known that muscarinic receptors antagonists are used for the asthma and related airway conditions [
37]. [
38,
39] stated that the parasympathetic division of the ANS regulates the tone of smooth muscles of bronchi. And as the reflex increases in parasympathetic, this can result in bronchoconstriction, since the respiratory tract is abundant in cholinergic innervations through vagal fibers linked to M
1 muscarinic receptors located in the surface of mucosa of the respiratory tract. The submucosal glands in specific, are abundant in parasympathetic innervations generally through M
3 receptors and this also explain for using muscarinic antagonists in chronic COPD as well as asthma.
The extract Zf relaxed completely the phenylephrine induced contractions and partially inhibited the K
+ inducted contractions in aortic strips. The extract Zb proved to be very active and showed very interesting results by causing the relaxation of the aortic strips at very initial dose of 0.1 mg/mL, and when tested on K
+ inducted contractions there was only a partial inhibition at higher doses. The extract Zl found to be least active against the phenylephrine and K
+ induced contractions and relaxation was observed at much higher doses (Fig.
3).
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