PHYTOTHERAPY RESEARCH Phytother. Res. (2012) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.4821
Studies on Prokinetic, Laxative and Spasmodic Activities of Phyllanthus emblica in Experimental Animals Malik Hassan Mehmood,1,2 Abdul Rehman,1 Najeeb-ur-Rehman1 and Anwarul-Hassan Gilani1* 1
Natural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan 2 Department of Pharmacology, Faculty of Pharmacy, University of Karachi-75270, Pakistan
This study was aimed to provide pharmacological basis for the medicinal use of Phyllanthus emblica fruit in indigestion and constipation using the in-vivo and in-vitro assays. The crude extract of the dried fruits of Phyllanthus emblica (Pe.Cr) and its fractions were tested positive for alkaloids, saponins, tannins, terpenes, flavonoids, sterols and coumarins. Pe.Cr at the doses of 100 and 300 mg/kg exhibited the prokinetic and laxative activities in mice, which were found partially sensitive to atropine. In isolated guinea-pig ileum and rabbit jejunum, the crude extract and its aqueous fraction (Pe.Aq) caused concentration-dependent and partially atropine-sensitive stimulatory effects followed by relaxation at higher tested concentrations, being more efficacious in guinea pig, while more potent in rabbit tissues. The petroleum fraction (0.003–0.1 mg/mL) exhibited fully atropine-sensitive contractions in both guinea-pig and rabbit tissues. However, the ethyl acetate and chloroform fractions (0.003–1.0 mg/mL) showed only spasmolytic activity when studied in spontaneously contracting rabbit jejunum. This study showed that the Phyllanthus emblica possesses prokinetic and laxative activities in mice along with spasmodic effect in the isolated tissues of guinea pig and rabbit, mediated partially through activation of muscarinic receptors; thus, this study provides a rationale for the medicinal use of Phyllanthus emblica fruits in indigestion and constipation. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: Phyllanthus emblica; Eurphorbiaceae; prokinetic; laxative; muscarinic receptors.
INTRODUCTION Phyllanthus emblica Linn. Syn. Emblica officinalis Gartn. Belonging to family Eurphorbiaceae, is commonly known as ‘Amla’. The plant is a small or medium-sized deciduous tree found in most areas of Sind and Punjab provinces of Pakistan (Baquar, 1989). In addition to the nutritious value of Phyllanthus emblica and being rich in vitamin C, amino acids and minerals, its different parts are used for multiple health benefits. The dried fruits are equally popular in Ayurvedic, Greco-Arab and Chinese systems of traditional medicine, for the treatment of chronic constipation, dysentery and inflammation (Nadkarni, 1986; Duke et al., 2002; Sabina and Rasool, 2007), while its fresh fruits are made into pickles, which are used to accelerate appetite. This plant is one of the three ingredients of a popular Greco-Arab herbal formulation referred as ‘Trifla’ (Phyllanthus emblic, Terminalia chebula and Terminalia bellerica). It is also frequently used in Ayurvedic medicine to treat constipation, in addition to its usefulness in anemia, jaundice, asthma, fever and chronic ulcers (Singh et al., 2008). The plant contains tannins like glucogallia, corilagin, chebulagic acid, tannins emblicanins A and B (Majeed et al., 2009), and apigeninglucoside (El-Desouky et al., 2008). * Correspondence to: Anwarul-Hassan Gilani, Ph.D, Professor of Pharmacology, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College, Karachi-74800, Pakistan. E-mail:
[email protected]
Copyright © 2012 John Wiley & Sons, Ltd.
Norsesquiterpenoid glycosides (4’-hydroxyphyllaemblicin B, phyllaemblicins E and F, phyllaemblic acid, phyllaemblicin A, B and C) (Liu et al., 2009), quercetin and b-sitosterol (Thakur et al., 1989) have been found in the roots of Phyllanthus emblica. In addition to the presence of bcarotenes, histidine, isolucine, pectin, phyllamblinic acid, selenium and zinc (Duke, 1992), b -caryophyllene, b-bourbonene, 1-octen-3-ol, thymol and methyleugenol from the essential oils of Phyllanthus emblica, most parts of the plant (leaves, bark and fruit) have been found to be enriched in phenolic contents (Chalise et al., 2010). Phyllanthus emblica has been widely studied for its multiple health benefits such as, gastroprotective, antiulcerogenic, hypolipidemic and antidiabetic (Krishnaveni and Mirunalini, 2010), antioxidant (Sharma et al., 2009), hepatoprotective (Srirama et al., 2012), antihypertensive (Bhatia et al., 2011), anti-inflammatory (Nicolis et al., 2008), antidiarrheal and antispasmodic (Mehmood et al., 2011) activities; however, there is no report available in the literature showing its effectiveness in hypomotile gut disorders such as indigestion and constipation. In this study, we showed first time that the plant possesses prokinetic, laxative and spasmodic activities mediated partially through cholinergic pathway, thus providing a rationale to the medicinal use of Phyllanthus emblica in indigestion and constipation. Moreover, the activity-guided fractionation of the crude extract was carried out which showed widely distributed gut stimulant components in the aqueous and petroleum fractions. Received 26 April 2012 Revised 19 July 2012 Accepted 24 July 2012
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MATERIAL AND METHODS Preparation of the crude extract. The fruits of Phyllanthus emblica were purchased from the local market (Jouria Bazaar) of Karachi. Specimen was submitted at the herbarium of Natural Product Research Division, Department of Biological and Biomedical Sciences, the Aga Khan University, Karachi with voucher # Pe-F-0607-100. By following a previously described method (Williamson et al., 1998), the mechanically crushed fruits were soaked in aqueous methanol (30:70) or 3 days and filtered through muslin cloth and Whatman (Maidstone, UK) NO.1 filter paper simultaneously. This procedure was repeated three times, and all the filtrates were pooled and evaporated using rotary evaporator (model RE-111, Buchi, Flawil, Switzerland) under reduced pressure to obtain finally, the crude extract of the fruits of Phyllanthus emblica (Pe.Cr). The yield of thick dark brown pasty like mass was 7–8% w/w. Activity-guided fractionation was carried out by using solvents of increasing polarity. Approximately 20 g of Pe. Cr was dissolved in about 200 mL of distilled water. Petroleum spirit was added into it and shaken vigorously in a separating funnel. The mixture was allowed to separate in two layers. The petroleum spirit layer (upper) was removed. The extraction with petroleum spirit was repeated twice more. All of the petroleum spirit layers were combined and evaporated on rotary evaporator to give the petroleum spirit fraction (Pe.Pet). The other separated layer (lower) was taken in a separating funnel and chloroform, in equal volume, was added into it. Afterwards, it was separated by following the above mentioned procedure. The chloroform layer (lower) was collected thrice and evaporated on rotary evaporator to obtain the chloroform fraction (Pe.CHCl3). The remaining layer (upper) was again taken in a separating funnel and mixed with equal volume of ethyl acetate, separated and evaporated on rotary evaporator to get the ethyl acetate fraction (Pe.EtAc). Last, the lower layer was collected and evaporated to obtain the aqueous fraction (Pe.Aq). Phytochemical screening. Phytochemical analysis of the crude extract of Phyllanthus emblica and its fractions has been performed qualitatively for the presence of alkaloids, flavonoids, tannins, coumarins, sterols, terpenes, saponins and anthraquinones as plant constituents (Evans, 2006). Chemicals. Acetylcholine perchlorate (ACh), atropine sulphate, carbamylcholine (CCh), histamine hydrochloride, pyrilamine maleate, indomethacin and hexamethonium chloride were purchased from Sigma-Aldrich Chemicals Company (St Louis, MO, USA). All chemicals used were of the analytical grade available and solubilized in distilled water. Animals. BALB/c mice (weighing 20–25 g), guinea pigs (weighing 400–600 g) and local breed rabbits (weighing 1–1.5 kg) of either sex were housed at the animal house of the Aga Khan University under a controlled environment (23–25 C). The animals were kept in their respective cages with sawdust (changed at every 48 h) where required, and were fasted for 24 h before starting the experiment. In routine, they were given tap water ad libitum and standard diet. The experiments were performed with the rulings of the Copyright © 2012 John Wiley & Sons, Ltd.
Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (National Research Council, 1996). In-vivo experiments. Charcoal meal GI transit test. By following a method used in earlier study (Mascolo et al., 1994), mice fasted for 12 h were divided into seven different groups (n = 6 animals in each). Two of the groups were treated per oral (p.o.) with increasing doses of Pe.Cr 100 and 300 mg/kg, acting as the test groups. One group was taken as negative control, treated with saline (10 mL/kg, p.o.). The next group was administered CCh (1 mg/kg, p.o.) as the positive control. After 15 min, the animals were given 0.3 mL of charcoal meal of distilled water suspension containing 10% gum acacia, 10% vegetable charcoal and 20% starch. The animals were sacrificed after 30 min, and the abdomen was opened to excise the whole small intestine. The length of the small intestine and the distance between the pylorus region and the front of the charcoal meal was measured to obtain the charcoal transport ratio or percentage. In order to assess the involvement of ACh-like prokinetic effect of the extract and the positive control (CCh), further groups of mice were pretreated with intraperitoneal (i.p.) injection of atropine (10 mg/kg) 15 min prior to the administration of the extract or positive control of CCh. Laxative activity. Mice of either sex fasted for 6 h before the experiment were placed individually in cages lined with clean filter paper. The animals were divided into seven groups (n = 6 animals in each); the first group acting as the negative control and administered saline (10 mL/kg, p.o.), while the next group received CCh (1 mg/kg, p.o.), which served as the positive control. The third and fourth groups received orally, 100 and 300 mg/kg of Pe.Cr, respectively. To determine the mechanism underlying its laxative effect, separate sets of mice (group # 5, 6 and 7) were pretreated with atropine (10 mg/kg, i.p.) 1 h before administration of the extract or CCh. After 18 h, the feces production (total number of feces and total number of wet feces per group) in all animals was counted, and the percentage increase in wet feces relative to that of total fecal output was recorded, which was considered as the laxative effect (Mehmood and Gilani, 2010). In-vitro experiments. Effect on guinea-pig ileum and rabbit jejunum. By following the previously described methods in earlier studies (Ghayur et al., 2005; Mehmood et al., 2010), intestinal preparations (rabbit jejunum and guinea-pig ileum) were obtained subsequent to cervical dislocation of the respective animals; the abdomen was cut opened, required tissues were isolated out. Tissue preparations (ileum or jejunum) of 2–3 cm long were mounted in 10 mL tissue baths containing Tyrode’s solution maintained at 37 C and aerated with a mixture of 5% carbon dioxide and 95% oxygen (carbogen). The composition of Tyrode’s solution (mM) was KCl 2.68, NaCl 136.9, MgCl2 1.05, NaHCO3 11.90, NaH2PO4 0.42, CaCl2 1.8 and glucose 5.55 (pH 7.4). A preload of 1 g was applied to each tissue, and the contractile responses were recorded using isotonic transducer 50–6360 (Harvard Apparatus, Holliston, MA, USA) coupled with PowerLab (ML-845) data acquisition Phytother. Res. (2012)
EFFECTIVENESS OF PHYLLANTHUS EMBLICA IN CONSTIPATION
system (AD Instruments; Sydney, Australia) and a computer using chart software (version 5.3). The tissues were allowed to equilibrate for a period of 30 min, and then stabilized with sub-maximal concentration of ACh (0.3 mM). The tissues were presumed stable only after the reproducibility of the said responses. The crude extract of Phyllanthus emblica and its respective fractions were examined later for its gut stimulatory effects on ileum and jejunum preparations of guinea pig and rabbit, respectively. Statistical analysis. All the data expressed are mean standard error of mean (S.E.M., n = number of experiments) and the median effective concentrations (EC50 values) with 95% confidence intervals. One-way analysis of variance (ANOVA) followed by Dunnett’s test or unpaired t-test was used to assess the laxative activity, while one-way ANOVA followed by Tukey’s test was employed for the effect of plant extract in charcoal meal transit. The concentration–response curves (CRCs) were analysed by non-linear regression, and two-way ANOVA followed by Bonferroni’s posttest correction or unpaired t-test was used for multiple comparisons of CRCs with the respective control. All the graphing, calculations and statistical analysis were performed using GraphPad Prism 4 for windows (GraphPad Software, San Diego, California, USA).
RESULTS Phytochemical analysis Preliminary phytochemical analysis of the fruit extract of Phyllanthus emblica, its aqueous, chloroform, ethyl acetate and petroleum fractions revealed the presence of alkaloids, flavonoids, tannins, coumarins, sterols, terpenes and saponins. On the account of froth formation, the saponins were observed relatively in higher proportion in the aqueous fractions than the crude extract and petroleum fraction, respectively. In-vivo findings. Effect of Pe.Cr on charcoal meal GI transit The crude extract of Phyllanthus emblica (Pe.Cr) dose dependently (100–300 mg/kg) propelled charcoal meal through the small intestine of mice (Fig. 1). The distance travelled by the saline-treated group was 56.1 3.4% (mean S.E.M, n = 6) of total length of small intestine, while the positive control group treated with CCh (1 mg/kg) significantly enhanced the movement of charcoal meal to 94.7 2.8% (p < 0.001 versus saline). The plant extract at the doses of 100 and 300 mg/kg moved the charcoal meal to the respective levels of 77.1 1.6% (p < 0.001) and 90.3 4.5% (p < 0.001), when compared with the saline-treated group. When the plant extract (100 and 300 mg/kg) and the positive control groups were re-determined in mice pretreated with atropine, all the excitatory effects were markedly decreased as seen in Fig. 1. Copyright © 2012 John Wiley & Sons, Ltd.
Figure 1. Bar diagram showing the dose-dependent effect of the crude extract of Phyllanthus emblica (Pe.Cr) in mice on propulsion of charcoal meal through small intestine of mice, in the absence and presence of atropine.*p < 0.05, **p < 0.01 and ***p < 0.001, one-way ANOVA followed by Tukey’s test. Each bar shown represents mean S.E.M of the results obtained from six different animals per group.
Laxative effect of Pe.Cr Administration of Pe.Cr to mice produced 35.7% and 44.1% (n = 6) wet feces at 100 and 300 mg/kg, respectively. The positive control, CCh (1 mg/kg) produced 48.2% wet feces, while the saline-treated group formed only 10.5% wet feces. When Pe.Cr (100 and 300 mg/ kg) was studied in mice pretreated with atropine, the production of wet feces declined to 25.8% and 34.4%, respectively, and detail results are shown in Table 1. In-vitro findings. Effect of Pe.Cr and its fractions on guinea-pig ileum Pe.Cr (1–5 mg/mL) caused stimulatory effect to the magnitude of 71.6 8.4% (mean S.E.M, n = 5) expressed in terms of contractions induced by ACh (0.3 mM). The stimulant effect was followed by relaxation at next tested concentration (10 mg/mL). The stimulatory effect was significantly (p < 0.001) attenuated in tissues pretreated with atropine with maximum effect declined to 28 4.5%, while, it remained unaltered (p > 0.05) in the presence of hexamethonium (0.3 mM), pyrilamine (1 mM) or indomethacin (1 mM) as shown in Fig. 2A. The aqueous fraction (Pe.Aq) exerted contractile effect at 0.3–10 mg/mL attaining the highest response of 85.7 2.8% of ACh. When studied in the presence of atropine, the control effect of Pe.Aq was markedly (p < 0.001) decreased to 43.3 3.2%; however, it remained unchanged (p > 0.05) in the presence of hexamethonium, pyrilamine or indomethacin pretreatment (Fig. 2B). The petroleum fraction (Pe.Pet) exhibited contractile effect at 0.03 and 0.01 mg/mL attaining maximum of 27.3 4.3% of ACh, at 0.01 mg/mL, p < 0.001 versus baseline status of the tissue (Fig. 2C), followed by relaxation (data not shown). Amongst fractions, Pe.Pet was found the most potent; its stimulatory effect was apparent at distinctly lower concentrations (0.03 and 0.1 mg/ml), but less efficacious, as its maximum contractile effect was 27.3 4.3% Phytother. Res. (2012)
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Table 1. Effect of atropine on the laxative activity of the crude extract of Phyllanthus emblica (Pe.Cr) in mice.
Group No. 1 2 3 4 5 6 7
Treatment Saline (p.o., ml/kg) Carbachol (p.o.) Pe.Cr (p.o.) Carbachol (p.o.) + Atropine (i.p.) Pe.Cr (p.o.) + Atropine (i.p.)
Dose (mg/kg)
Mean defecation/group
Mean number of wet feces/group
Mean % of wet feces
10 1 100 300 1 + 10 100 + 10 300 + 10
1.9 0.17 8.7 2.20*** 3.2 0.65* 6.8 0.32*** 1.5 0.56*** 2.7 0.33* 2.4 0.85***
0.2 0.01 4.0 0.35*** 1.5 0.57* 3.7 0.42*** 0.5 0.26*** 0.8 0.21* 1.0 0.74**
10.5 48.2 35.7 44.1 33.3 25.8 34.4
Values shown are mean S.E.M of the results obtained from 6 different animals per group;*p < 0.05,**p < 0.01 and***p < 0.001 show a comparison of group # 2,3 and 4 versus group # 1 (One-way ANOVA followed by Dunnett’s test), group # 5 versus group # 2, group # 6 versus group # 3 and group # 7 versus group # 4 (unpaired t- test). The term p.o. represents per oral, while i.p. is for intraperitoneal injection.
at 0.01 mg/mL versus Pe.Cr, 71.6 8.4% and Pe.Aq, 85.7 2.8% at 10 mg/mL. The stimulatory effect of Pe.Pet was abolished in the presence of atropine (0.1 mM), as shown in Fig. 2C. The ethyl acetate and chloroform fractions did not show any contractile effects in guineapig ileum (data not shown).
Effect of Pe.Cr and its fractions on rabbit jejunum In spontaneously contracting isolated rabbit jejunum, Pe.Cr caused stimulatory effect at low concentrations (0.1–1 mg/mL) reaching its maximum 56.2 5.2% of ACh followed by relaxation at next higher doses (3–10 mg/mL). The spasmodic effect was markedly (p < 0.001) reduced when studied in the presence of atropine (0.1 mM) with resultant maximum of 12.5 7.5%, while remained unchanged in the presence of hexamethonium, pyrilamine or indomethacin (Fig. 3A). Amongst resultant fractions of parent extract, the aqueous fraction exhibited stimulatory effect at 0.1 and 0.3 mg/mL attaining maximum of 66.6 8.1% of ACh contraction at 0.3 mg/mL followed by relaxation at 1–10 mg/mL, similar to the effect of parent extract. The contractile effect was greatly (p < 0.001) reduced in the presence of atropine with declining response to 29 5.6%. This stimulatory effect was devoid of any change when studied in tissue pretreated with hexamethonium, pyrilamine or indomethacin (Fig. 3B). The petroleum fraction exhibited contractile effect at 0.01–0.1 mg/mL with maximum 28.8 6.5% of ACh contraction followed by relaxation, while in atropinized tissue, the contractile effect was abolished (Fig. 3C). The chloroform (Pe.CHCl3) and ethyl acetate (Pe.EtAc) fractions showed only spasmolytic effect with respective EC50 values of 0.14 mg/mL (0.07–0.28, n = 4) and 0.30 mg/mL (0.21–0.42, n = 3) and as shown in Fig. 3D. The relaxant effects of Pe.EtAc and Pe.CHCl3 were devoid of any change in the tissue pretreated with atropine, pyrilamine or indomethacin (data not shown). DISCUSSION The crude extract of Phyllanthus emblica propelled charcoal meal through the small intestine and increased the production of wet feces in mice, showing prokinetic and laxative activities, similar to the effect of carbachol, a standard cholinergic agonist and accelerator of Copyright © 2012 John Wiley & Sons, Ltd.
intestinal contents (Brown and Taylor, 2006). These gut stimulatory actions of the extract were found partially sensitive to atropine, a muscarinic receptor blocker (Gilani et al., 1997), indicating the presence of some ACh-like component(s), in addition to other gut stimulant constituent(s). ACh is a neurotransmitter of the parasympathetic nervous system and is known to cause gastrointestinal stimulation through the activation of muscarinic receptors (Brown and Taylor, 2006), hence, the presence of ACh-like constituents explains its medicinal use in constipation. In addition, ACh is known to correct indigestion partly through stimulation of salivary secretion and gastric acid release, thus the presence of ACh-like activity in the plant may support its medicinal use as digestive add (Hoogerwerf and Pasricha, 2006). To further explore the possible mode of the observed prokinetic and laxative properties of the extract, we used isolated rabbit jejunum and guinea-pig ileum preparations. The rabbit jejunum showing spontaneous rhythmic contractions is considered a suitable preparation to assess the presence of gut stimulant and relaxant constituents in test materials without the use of any antagonist, while guinea-pig ileum is a quiescent preparation and is widely used for characterization of the gut stimulant effects (Mehmood et al., 2010). In line with the in-vivo findings, the spasmodic effect of the plant extract both in guinea-pig ileum (1–10 mg/mL) and rabbit jejunum (0.1–1 mg/mL) was found mediated through a common pathway involving cholinergic receptor activation, which was evident by its partial sensitivity when studied in atropinized tissues. The muscarinic receptors arbitrate human circular and longitudinal gut smooth muscle contractions; thus, these are important in controlling the gastrointestinal smooth muscle tone (Kerr et al., 1995). When the stimulant effect of the plant extract was studied in the presence hexamethonium, a ganglion blocker (Wien et al., 1952), pyrilamine, a histaminic type-1 (H1) receptor blocker (Sharif et al., 1994) or indomethacin, a prostaglandin synthesis inhibitor (Ruff and Secrist, 1984), it remained unaltered, suggesting the presence of ACh-like constituents along with some additional elements independent of histamine, nicotine receptors activation or prostaglandin synthesis inhibition. The activity guided fractionation revealed distribution of combination of gut stimulant and relaxant components in the aqueous and petroleum fractions similar to that of parent extract, while petroleum Phytother. Res. (2012)
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Figure 2. (A) The stimulatory effects of (A) the crude extract of Phyllanthus emblica (Pe.Cr), (B) its aqueous (Pe.Aq) in the absence and presence of atropine, hexamethonium, pyrilamine and indomethacin and in (C) the effect of petroleum fraction (Pe.Pet) without and with atropine only, in isolated guinea-pig ileum preparations. Values shown represent mean s.e.m. of 5–6. *p < 0.05, **p < 0.01 and ***p < 0.001 (Two-way ANOVA, followed by bonferoni post-test correction or unpaired t-test).
fraction was the most potent in its gut stimulatory effects than the aqueous fraction and crude extract, respectively. The aqueous fraction was found the most efficacious in producing maximum contractile responses when compared with that of the crude extract and petroleum fraction, indicating that the stimulant constituents accumulated in the aqueous fraction. The stimulant effect was blunted at high concentrations, most probably because of the accompanied relaxant action, and this was also observed exclusively in its ethyl acetate and chloroform fraction, which is probably meant Copyright © 2012 John Wiley & Sons, Ltd.
by nature to offset the excessive stimulant effect which could have been otherwise harmful in terms of producing abdominal cramps, common side-effects of chemical drugs when used to treat constipation (Pasricha, 2006). The data in ileum and jejunum preparations of guinea pig and rabbit showed similar nature of the gut stimulatory activities of the crude extract and its fractions; however, the efficacy was found higher in guinea-pig ileum, while greater potency was observed in rabbit jejunum. Similarly, the chloroform fraction was found slightly more potent than the ethyl acetate fraction in displaying its relaxant action on spontaneously contracting rabbit jejunum. Such differences in the gut stimulatory effects of this plant extract might be due to multiple reasons: (i) the presence of unidentified species or tissue-selective gut stimulant component(s), (ii) the dominant behavior of species or tissue-selective gut stimulant component(s) and (iii) the heterogeneity of muscarinic receptors along the gastrointestinal tract (Raj and Goyal, 1988) or amongst the gut tissues from different animals (Tobin et al., 2009), though further studies are needed to conclude with confidence. The tissue and/or species-specific gut stimulant effects of Phyllanthus emblica are also in agreement with various earlier similar studies (McLeod et al., 1994; Ghayur et al., 2005; Mehmood et al., 2010; Rehman et al., 2011; 2012). The presence of saponins amongst the chemical constituents, which are known for their spasmogenic effect (Akah et al., 1997), may explain the gut stimulant actions of Phyllanthus emblica. Keeping in view of the popularity of the plant for its medicinal use, alone or in formulation (Trifla, Jawaresh-e-tamar Hindi), in the Unani (Greco-Arab) and Ayurveda systems of medicine, this study validates its medicinal use in constipation (Krishnaveni and Mirunalini, 2010), in addition to its application as digestive aid, which is in line with WHO guidelines recommending medicinal use of herbal products based on scientific validation (Norman et al., 1985; World Health Organization, WHO, 2000). This study may be a step forward towards the evidence-based use of phytomedicine. The plant has been widely studied for its safety including an earlier study conducted in our lab (Mehmood et al., 2011), as well as multiple other reports (Itthipanichpong et al., 1987; Xia et al., 1997; Jaijoy et al., 2010; Huabprasert et al., 2012) showing the safety of Phyllanthus emblica, while testing it in acute and chronic models, up to a dose of as high as 10–15 times than the normal doses (300–1200 mg/kg/day) used for therapeutic purposes (Mehmood et al., 2011; Huabprasert et al., 2012). Moreover, in a clinical trial, Phyllanthus emblica fruit at a dose of 100 g/day up to four months of study period showed no sign of toxicity (Chen et al., 2009). CONCLUSION This study shows that the prokinetic and laxative activities of Phyllanthus emblica in mice are mediated in part through activation of muscarinic receptors, and the in-vitro findings have also shown similar mechanism responsible for its spasmodic activities both in rabbit and guinea-pig tissues. Thus, this study provides sound mechanistic basis for the medicinal use of Phyllanthus emblica in gut disorders, such as indigestion and constipation. Phytother. Res. (2012)
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Figure 3. The stimulant and relaxant effects of (A) the crude extract of Phyllanthus emblica (Pe.Cr), (B) its aqueous (Pe.Aq) in the absence and presence of atropine, hexamethonium, pyrilamine and indomethacin, (C) shows the effect of petroleum fraction (Pe.Pet) in the absence and presence of atropine only, while (D) represents the effects of ethyl acetate (Pe.EtAc) and chloroform (Pe.CHCl3) fractions on spontaneously contracting isolated rabbit jejunum preparations. Values shown represent mean s.e.m. of 6–7 determinations. **p < 0.01 and***p < 0.001 (Two-way ANOVA followed by Bonferroni’s post-test correction).
Acknowledgement
Conflict of Interest
The study was carried out with the financial support from the Higher Education Commission, Government of Pakistan, under the scheme of Distinguished National Professor research allowance.
The authors have declared that there is no conflict of interest.
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