Folkloric uses of Jatropha gossypifolia in emesis and gut motility disorders: Pharmacological validation

Abstract

Jatropha gossypifolia is used in folkloric system to manage emesis and gastrointestinal motility disorders such as constipation and diarrhea. The present study was undertaken to provide pharmacological evidences for these folkloric uses by in vivo and in vitro experimental setting. Ethanolic extract of J. gossypifolia showed the significant antiemetic potential (p<0.05) against different emetogenic stimuli, when compared with chlorpromazine. The extract (0.01-1.0 mg/mL) on application to isolated rabbit jejunum preparation exerted spasmogenic effect, followed by the spasmolytic effect (3-10 mg/mL). In the presence of atropine, spasmogenic effect was blocked while spasmolytic effect was emerged, suggesting the involvement of muscarinic receptor activation. J. gossypifolia caused relaxation of high K⁺ (80 mM)-induced contraction and shifted the Ca²⁺ concentration-response curve to the right in a manner similar to verapamil, thus conforming its Ca²⁺-channel blocking activity. These findings provide pharmacological validation for the presence of antiemetic and gut modulator (spasmogenic and spasmolytic) activates, validating its folkloric uses.

Introduction

Constipation is a heterogeneous condition associated with infrequent bowel movements and/or difficulty in defecation or both (Talley et al., 2003). Untreated constipation may cause urinary and gastrointestinal disorders (Imanzadeh et al., 2012).

Emesis is protective reflex which expels out the toxic substances from the stomach and intestine, and is associated with indigestion of toxicants, cancer chemotherapy, post-operative procedures and drug adverse effects (Gilman and Goodman, 2001). Commercially available antiemetic may cause excessive sedation, dysphoria, extrapyramidal signs, and hallucination (Griffin et al., 1996).

Jatropha gossypifolia Linn. (Euphorbiaceae) is a bushy gregarious shrub; known as tua-tua (English) and laal bagharenda (Local) and is native to Brazil but cultivated as an ornamental plant in all over world (Khare, 2007).

Phytochemical investigation revealed the presence of histamine, triterpenes, alkaloids, flavonoids, jatropholone A, jatrophatrione, tannins, macrocyclic diterpene, jatrophine, jatrodien, gossypiline (Das et al., 1998; Matsuse et al., 1999), latex contains cyclogossine A and B (Morton, 1980) whereas seeds contain hydroxyl jatrophone and jatropholones A and B (Horsten et al., 1996).

J. gossypifolia has been reported to have hepatoprotective, anti-inflammatory, anticancer, antiseptic, anti-fertility and antipyretic activities (Panda et al., 2009; Falodun et al., 2012). Decoction of the plant has been used as purgative, anti-diarrheal, antiemetic, antidote for snakebite and blood purifier (Kirtikar and Basu, 1996; Khare, 2007). The present study was aimed to pharmacologically evaluate the possible mechanism of action J. gossypifolia in emesis and gastrointestinal motility disorders such as constipation and diarrhea.

Materials and Methods

Collection and extraction of J. gossypifolia

Fully grown aerial parts of J. gossypifolia were collected in the month of November (2013) from the botanical garden of The University of Faisalabad and were identified by the Curator, Botany Department of same University. Adulterants free and electrically grinded coarse powdered material (#40) was subjected to triple maceration (Hussain et al., 2013), with 80% ethanol-aqueous mixtures to produce the ethanolic extract of J. gossypifolia.

Animals used and their housing conditions

Animals (♂/♀) including, Swiss albino mice (32-48 g, 4-5 weeks old), chicks (75-85 g, 10-15 days old) and rabbits (1.2-1.7 kg, 8-10 months old) were used and were provided with standard diet and ad libitum with tap water under controlled environmental condition (25-28°C). Chicks were housed in plastic cages with sawdust as bedding with 12 hours/12 hours dark-light cycle.

Chemicals and drugs used

Research grade and highest purity chemicals, solvents, and drugs were used in the experimental study and were purchased from the Sigma Chemicals (USA), Merck (Germany), Sharlab Labs (Spain) and Highnoon Pharmaceutical (Pakistan).

Acute toxicity test

Acute toxicity testing was performed by orally administering the 2, 4 and 6 g/kg of ethanolic extract to the mice, as described previously (Hussain et al., 2015a).

In vivo estimation of antiemetic potential

Chick emetic model was adopted to estimate the antiemetic potential of the extract (Eda et al., 2005; Hussain et al., 2015b). Acclimatize and healthy chicks were separated in 8 groups of 4 chicks in each group. Chicks of Group 1 (control) and Group 2 (standard) were given an oral dose of normal saline (0.9%) and chlorpromazine (150 mg/kg body weight) respectively, while the chicks of Group 3 and 4 (experimental) were given an oral dose of 50 and 100 mg/kg body weight of ethanolic extract respectively,dissolved in 10 mL/kg of 0.9% saline containing 5% DMSO and 1% tween 80. After 15 min, 50 mg/kg body weight of copper sulfate was administered orally to chicks of all groups. Same process was repeated by using fresh juice of Brasica compestris (10 mg/kg; oral), instead of copper sulfate while other protocols remain same as before, then number of retches was counted for the next 15 min. The percentage retching inhibition was calculated as:

Retching inhibition (%) = [(A-B)/A] x 100

Where, A and B represents the frequency of retching in control and experimental groups respectively

In vitro assessment of spasmogenic and spasmolytic activities

Possible presence of spasmogenic and spasmolytic activity was assessed by applying ethanolic extract on isolated rabbit jejunum segments (2-3 cm) hanged in tissue baths containing 10 mL of Tyrode’s solution with following composition (mM); KCl (2.68), NaCl (136.9), MgCl₂ (1.05), NaHCO₃ (11.90), NaH₂PO₄ (0.42), CaCl₂ (1.8) and glucose (5.55), aerated with carbogen (37°C) (Gilani et al., 1994; Janbaz et al., 2014). A preload of 1 g was applied and jejunum responses were recorded through isotonic transducers by Power Lab Data Acquisition System (AD Instrument, Sydney, Australia) attached to a computer installed with Lab Chart Reader Software (version 7). The jejunum tissues were allowed to stabilized (spontaneous rhythmic contraction) for at least 30 min. After 30 min, ethanolic extract was added to stabilized jejunum segment in a cumulative fashion, without prior addition of any agonist for the testing of spasmogenic and/or spasmolytic effect (Hussain et al., 2014). Contractile effect of the extract was calculated as the percent of the maximum effect produced by 1.0 μM of acetylcholine.

Assessment and conformation of Ca²⁺ channel blocking activity

For the assessment of calcium channel blockade, jejunum preparations were depolarized by exposing to high K⁺ (80 mM), resulting in sustained contraction (Arshad et al., 2012; Hussain et al., 2015c). Ethanolic extract was applied in a cumulative manner to the sustained contractions to achieve concentration-dependent inhibi-tory response (Farre et al., 1991). The observed relaxant effect of the extract on K⁺ (80 mM)-induced contraction was expressed as percent of the control response mediated by K⁺.

Calcium channel blocking effect of the ethanolic extract was confirmed by the previously reported method (Gilani et al., 2005). The isolated rabbit jejunum preparations were allowed to stabilize in normal Tyrode’s solution, which were subsequently replaced with Ca²⁺-free Tyrode’s solution to which EDTA (0.1 mM) was added for 30 min, in order to remove calcium from the tissues. This bath solution was further replaced with K⁺-rich and Ca²⁺-free Tyrode’s solution, having the following composition (mM): MgCl₂ (1.05), KCl (50), NaHCO₃ (11.90), NaCl (91.04), glucose (5.55), NaH₂PO₄ (0.42), and EDTA (0.1). Subsequent to an incubation period of 30 min, cumulative Ca²⁺ concentra-tions were applied to the tissue bath to obtain control calcium dose-response curves. On achievement of the superimposable control calcium dose-response curves (usually after two cycles), the tissues were then washed and incubated with the plant extract for 60 min. Then concentration response curves of Ca²⁺ were constructed and compared to the control curves. The concentration response curves for Ca²⁺ were developed in the presence of different concentrations of the plant to assess a possible Ca²⁺ channel blocking effect (Bolton, 1979).

Statistics

All data was expressed as mean ± SEM of triplicate. EC₅₀ values were given with 95% confidence intervals (95% CI) and the logarithmic dose response curves of different treatments were then plotted using computer software “Graph pad Prism” version 6, (Graph Pad Software, San Diego, CA, USA). Concentration-response curves were analyzed by nonlinear regression sigmoidal response curve (variable slope). Unpaired Student t-test was used for assessment of the observations. p<0.05 and p<0.005 were believed to be statistically significant and most significant values respectively.

Results

Acute toxicity

Acute toxicity testing of J. gossypifolia (2, 4 and 6 g/kg) indicates that the extract is safe up to the maximal tested dose (6 g/kg) because there was no mortality and no toxic effects, such as anorexia, diarrhea and gastrointestinal spasms.

Antiemetic potential

The chicks of control groups, to whom normal saline (10 mL/kg) was administered, followed by copper sulfate and fresh juice of B. compestris, showed the numbers of retches 78 ± 1.10 and 77 ± 1.11 respectively. While in the chicks of the experimental groups (75 mg/kg), number of retches were reduced with %inhibition of emesis of 53.9 and 49.4, whereas at the dose of 150 mg/kg, number of retches were significantly reduced with %inhibition of emesis of 74.4 and 76.6 followed by copper sulfate and fresh juice of B. compestris respectively, which were comparable to standard drug chlorpromazine, indicating the antiemetic potential of J. gossypifolia (Table I).

Spasmogenic and spasmolytic effect

In spontaneously contracting isolated rabbit jejunum preparations, the ethanol extract showed the spasmogenic effect in concentration range of 0.01-1.0 mg/mL, which did not sustained and was subsequently followed by spasmolytic effect at the next higher concentration (3-10 mg/mL) with EC₅₀ value of 1.9 mg/mL (95% CI: 1.40-2.40, n=3) (Figure 1). The observed contractile responses to J. gossypifolia was expressed as percentage of the maximal response to acetylcholine (0.3 µM), i.e. 14.6 ± 1.7, 22.3 ± 0.8, 26.3 ± 1.7, 32.3 ± 1.2, and 10.2 ± 5.2 (mean ± SEM, n=3), at the concentration range of 0.01, 0.03, 0.1, 0.3 and 1.0 mg/mL, respectively (Figure 2). In the presence of atropine (0.1 μM), spasmogenic effect was abolished while the spasmolytic effect was observed with EC₅₀ value of 1.7 mg/mL (95% CI: 0.32-2.95, n = 3).

Figure 1: Concentration–response curves, showing the effect of J. gossypifolia in the absence and presence of atropine (0.1 µM) on spontaneously contracting rabbit jejunum preparation. The spasmogenic responses are expressed as percent of acetylcholine maximum (values are expressed as mean ± SEM, n=3)

Figure 2: Bar diagram showing the effect of J. gossypifolia in comparison to the acetylcholine maximum response in rabbit jejunum preparation

J. gossypifolia was unable to relax the K⁺ (25 mM)-induced spastic contractions, but exhibited complete relaxation of K⁺ (80 mM)-induced contractions in isolated rabbit preparations, with EC₅₀ value of 1.4 mg/mL (95% CI: 0.72-1.95, n=3). Verapamil (standard drug) inhibited the K⁺ (80 mM)-induced contractions, with EC₅₀ value of 0.14 mg/mL (95% CI: 0.07-0.45, n=3) (Figure 3). Moreover, pretreatment of the isolated rabbit jejunum preparations with J. gossypifolia caused a rightward shift of the Ca⁺²-concentration response curves at the dose range of (0.1-1.0 mg/mL, n=3), in a manner similar to that of verapamil (Figure 4)

Figure 3: Concentration-dependent inhibitory effect of J. gossypifolia and verapamil against K⁺(80 mM)-induced contractions in isolated rabbit jejunum preparations (values are expressed as mean ± SEM, n=3)

Figure 4: Concentration-response curves of Ca²⁺ in the absence and presence of different concentrations of (A) J. gossypifolia and (B) verapamil in isolated rabbit jejunum preparations (values are expressed as mean ± SEM, n=3)

Discussion

Ethanolic extract of J. gossypifolia was tested for acute toxicity on healthy mice at oral dose ranges of 2, 4 and 6 g/kg and it was found that J. gossypifolia is safe because it did not produced the lethality, behavioral changes and toxic effect, even at the dose range of 6 g/kg, which is more greater than normal therapeutic dose range. J. gossypifolia has folkloric repute for use in the management of emesis in traditional system of medicine; hence, in vivo pharmacological study was carried out by adopting chick emetic model to evaluate its possible antiemetic effect. Emesis is either caused by the activation of the vomiting centre located in medulla oblongata or by the activation of motor pathway. Additionally, signals from following four principal regions, i.e., chemoreceptor trigger zone, GIT, cortex, thalamus, cerebral and vestibular) region may also cause emesis (Hussain et al., 2015a). The chemoreceptor trigger zone is in the proximity to medulla and it is not protected by the blood brain barrier (Becker, 2010). Results clearly indicate that J. gossypifolia (150 mg/kg) showed more significant antiemetic response against copper sulfate and B. compestris induced emesis which was comparable to chlorpromazine. So, it can be hypothesized that the antiemetic effect of J. gossypifolia can be likely mediated through inhibition of chemoreceptor trigger zone while other mechanism cannot be ruled out.

For the inspection of conventional uses of J. gossypifolia in gastrointestinal motility disorders, pharmacological study of J. gossypifolia was carried out on spontaneously contracting isolated rabbit jejunum preparations, to evaluate its possible effect and mechanism of action. Initially it produced the spasmogenic effect (jejunum contractile effect), which is generally mediated through the action of acetylcholine on cholinergic system (Janbaz et al., 2012; Hussain et al., 2014). For the conformation of this mechanism, spontaneously contracting rabbit jejunum preparations were pre-exposed to 0.1 µM of atropine (Muscarinic receptor blocker) (Ghayur and Gilani , 2005), which resulted in blocking the stimulatory effect of extract, indicating that the J. gossypifolia exerted the gut stimulation via cholinergic pathway (Hussain et al., 2015b). Acetylcholine (neurotransmitter) acts on the M₃ muscarinic receptor and regulate the peristaltic movement of GIT while atropine antagonize the muscarinic receptors (Brown and Taylor, 1996). The observed spasmogenic effect of J. gossypifolia validates its traditional use as anti-constipative agent in the hypomotility disorder of the gut. The spasmogenic effect was followed by the spasmolytic effect at next higher doses of the extract, indicating the co-existence of spasmogenic, and spasmolytic constituent (s), which is probably meant by the nature not to allow the spasmogenic effect going beyond the constipation, particularly at higher doses (Janbaz et al., 2012).

Addition of J. gossypifolia to the tissue bath completely relaxed the K⁺(80 mM)-induced contractions, suggesting that relaxant activity might be due to blockade of Ca²⁺ channels (Gilani et al., 2005). The contractile elements in rabbit jejunum are activated through increase in cytoplasmic free Ca²⁺ concentration via opening of the voltage dependent Ca²⁺ L-type channels (Karaki et al., 1997) or release of Ca²⁺ from sarcoplasmic stores (Godfraind et al., 1986). The extract exhibited relaxant effect through Ca²⁺ channels blockade and further inhibition of sequences of events including decrease of cytosolic Ca²⁺ concentration, decrease in Ca²⁺ binding to calmodulin, decrease in Ca²⁺ calmodulin complex formation, decrease in activation of myosin light chain kinase, decrease in phosphorylation of myosin light chains, decrease in interaction between actin and myosin and inhibition of contractile process. These speculations were confirmed as pretreatment of rabbit jejunum preparation with extract caused rightward shift of Ca²⁺-concentration response curves, like that caused by verapamil (Fleckenstein, 1977).

J. gossypifolia exhibited the anticholinergic and Ca²⁺ channels blocking activities, which can be attributed due to presence of alkaloids and flavonoids, as these phytoconstituents have been reported to possess the anticholinergic (Broadley, 1996) and Ca²⁺ channel blocking (Revuelta et al., 1997) activities, respectively, while other mechanisms and phytoconstituents cannot be neglected.

Conclusion

This study provides the sound pharmacological back ground for the traditional use of J. gossypifolia as aperients (mild laxative) and anti-diarrheal drug, mediated possibly through the activation of M₃-muscarinic receptors and blockade of voltage-dependent Ca²⁺ channels respectively. The observed results also validate its folkloric use in emesis.

References

Arshad U, Janbaz KH, Bashir S, Rehman NU, Mehmood MH, Gilani AH. Ethnopharmacological studies on Chrozophora prostrata in perspective of its folkloric reputation as purgative. Bangladesh J Pharmacol. 2012; 7: 243-48.

Becker DE. Nausea, vomiting, and hiccups: A review of mechanisms and treatment. Anesth Prog. 2010; 57: 150-57.

Bolton TB. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Rev. 1979; 59: 606-718.

Broadley KJ. Autonomic Pharmacology. London, Taylor and Francis, 1996.

Brown JH, Taylor P. Muscarinic receptor agonists and antagonists. In: Gilman AG, Hardman JG, Limbird LE, Molinoff PB, Ruddon RW. (Eds.)The Pharmacological Basis of Therapeutics. New York: McGraw-Hill, 1996, pp 141-59.

Das R, Das B and Kashinatham A. Gossypiline, a new lignin from Jatropha gossypifolia. Nat Prod Sci. 1998; 4: 238-40.

Eda M, Hayashi Y, Kinoshita K, Koyama K, Takahashi K, Akutu K. Anti-emetic principles of water extract of Brazilian Propolis. Pharm Biol. 2005; 43: 184-88.

Falodun A, Sheng-Xiang Q, Parkinson G, Gibbons S. Isolation and characterization of a new anticancer diterpenoid from Jatropha gossypifolia. Pharm Chem J. 2012; 45: 636-39.

Farre AJ, Colombo M, Fort M, Gutierrez B. Differential effects of various Ca²⁺ antagonists. Gen Pharmacol. 1991; 2: 177-81.

Fleckenstein A. Specific pharmacology of Ca⁺⁺ in myocardium, cardiac pacemakers and vascular smooth muscles. Rev Pharm Toxic. 1977; 17: 149-66.

Ghayur MN, Gilani AH. Pharmacological basis for the medicinal use of ginger in gastrointestinal disorders. Dig Dis Sci. 2005; 50: 1889-97.

Gilani AH, Bashir S, Janbaz KH, Shah AJ. Presence of cholinergic and calcium channel blocking activities explains the traditional use of Hibiscus rosasinensis in constipation and diarrhea. J Ethnopharmacol. 2005; 102: 289-94.

Gilani AH, Janbaz KH, Zaman M, Lateef A, Suri A, Ahmed HR. Possible presence of calcium channel blocker(s) in Rubia cordifolia: An indigenous medicinal plant. J Pakistan Med Assoc. 1994; 44: 82-85

Gilman AG, Goodman LS. The pharmacological basis of therapeutics. 10th ed. New York, The McGraw-Hill Companies, 2001, p 1029.

Godfraind T, Miller R, Wibo M. Calcium antagonism and calcium entry blockade. Pharmacol Rev. 1986; 38: 321-26.

Griffin AM, Butow PN, Coates AS, Childs AM, Ellis PM, Dunn SM, Tattersall MHN. On the receiving end V: Patient perceptions of the side effects of cancer chemotherapy in 1993. Ann Oncol. 1996; 7: 189-95.

Horsten SFAJ, Van AJJ, Kettenes-van JJ, Leeflang BR, Labadie RP. Cyclogossine A: A novel cyclic heptapeptide isolated from the latex of Jatropha gossypifolia. Planta Med. 1996; 62: 46-50

Hussain M, Bakhsh H, Aziz A, Majeed A, Khan IA, Mujeeb A, Farooq U. Comparative in vitro study of antimicrobial activities of flower and whole plant of Jasminum officinale against some human pathogenic microbes. J Pharm Alternative Med.2013; 2: 33-43.

Hussain M, Raza S.M, Khan MRU, Majeed A. Assessment of antiemetic potential of crude extract of Vigna trilobata (Linn.) against different emetogenic stimuli: An in vivo study. Indo Am J Pharmaceut Res. 2015b; 5: 1588-93.

Hussain M, Raza SM, Janbaz KH. Pharmacological basis for the folkloric uses of Buxus wallichiana Baill. (Buxaceae) in gastrointestinal, respiratory and vascular disorders. Bangladesh J Pharmacol. 2015c; 10: 260-66.

Hussain M, Raza SM, Janbaz KH. Pharmacological evaluation and validation for the folkloric use of Oligochaeta ramose (Roxb.) in constipation and diarrhea. Bangladesh J Pharmacol. 2014; 9: 617-23.

Hussain M, Raza SM, Janbaz KH. Pharmacologically mecha-nistic basis for the traditional uses of Rumex acetosa (Linn) in gut motility disorders and emesis. Bangladesh J Pharmacol. (2015a) (accepted).

Imanzadeh F, Sayyari AA, Sharifian M, Javaherizadeh H, Aghasi P. Study of factors affecting resolution of urinary tract infection following treatment of constipation in Iranian children who visited a tertiary referral hospital. Prz Gastroenterol. 2012; 7: 78-80.

Janbaz KH, Haider S, Imran I, Zia-Ul-Haq M, De-Martino L, De-Feo V. Pharmacological evaluation of Prosopis cineraria (L.) Druce in gastrointestinal, respiratory, and vascular disorders. Evid Based Complement Alternat Med. 2012; 12: 1-7.

Janbaz KH, Shabbir A, Mehmood MH, Gilani AH. Pharma-cological basis for the medicinal use of Rhus coriaria in hyperactive gut disorders. Bangladesh J Pharmacol. 2014; 9: 636-44.

Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano K, Harada K, Miyamoto S, Nakazawa H, Won KJ, Sato K. Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev. 1997; 49: 157-230.

Khare CP. Indian medicinal plants: An illustrated dictionary. New Delhi, Springer, Berlin/ Heidelberg, 2007, p 346

Kirtikar KR, Basu BD. Indian Medicinal Plants, vol. 2. India, International Book Distributors, 1996, p 2247.

Matsuse IT, Lim YA, Hattori M, Correa M, Gupta MP. A search for anti-viral properties in Panamanian medicinal plants, the effects on HIV and its essential enzymes. J Ethnopharmacol. 1999; 64: 15-22.

Morton JF. Caribbean and Latin American folk medicine and its influence in the United States. Q J Crude Drug Res. 1980; 18: 57-75.

Panda BB, Gaur KA, Nema RK, Sharma CS, Jain AK, Jain CP. Hepatoprotective activity of Jatropha gossypifolia against carbon tetrachloride-induced hepatic injury in rats. Asian J Pharm Clin Res. 2009; 2: 50-54.

Revuelta MP, Cantabrana B, Hidalgo A. Depolarization-dependent effect of flavonoids in rat uterine smooth muscle contraction elicited by CaCl₂. Gen Pharmacol Vasc Syst. 1997; 29: 847-57.

Talley NJ, Jones M, Nuyts G, Dubois D. Risk factors for chronic constipation based on a general practice sample. Am J Gastroenterol. 2003; 98: 1107-11.