Analysis of the central and peripheral mechanisms underlying the analgesic effects of the extracts of Phyllanthus amarus and Phyllanthus fraternus

Atul R. Chopade* and F. J. Sayyad

Dept. of Pharmacology and Pharmacognosy, Government College of Pharmacy, Karad, Dist. Satara. 415124.

*Corresponding Author E-mail: chopadearv@gmail.com

ABSTRACT:

Ethnopharmacological relevance:

A great number of preclinical and clinical studies have not only confirmed but have also extended the medicinal uses of species of the genus Phyllanthus mentioned in traditional medicine.

Aim of the study:

To evaluate the central and peripheral mechanisms underlying the analgesic effects of the extracts of Phyllanthus amarus and Phyllanthus fraternus in mice using the acetic acid-induced writhing and hot plate tests.

Materials and methods:

a) Writhing was induced by intraperitoneal injection of 0.6% acetic acid (v/v) (80 mg/kg body wt.) and the number of muscular contractions were counted for 30 min following acetic acid injection with animals treated with the extracts of Phyllanthus amarus and Phyllanthus fraternus.

b) The assay was performed according to the classical hot-plate technique of Eddy and Leimbach. The parameter evaluated was the latency time for paw licking and jumping responses after exposure to the hot plate surface.

Results:

Results showed that the extracts of Phyllanthus amarus and Phyllanthus fraternus given intraperitoneally can significantly attenuate acetic acid-induced writhing in mice in a dose-dependent manner. In the hot plate latency test, Phyllanthus extracts showed common activity in prolonging duration time and caused marked inhibition of acetic acid induced pain.

Conclusions: These findings of the current study imply the involvement of both peripheral and central antinociceptive mechanisms.

KEYWORDS: Phyllanthus amarus, Phyllanthus fraternus, analgesic activity, writhing test and hot plate test.

INTRODUCTION:

The plants belonging to the genus Phyllanthus (Euphorbiaceae) are widely distributed throughout tropical and subtropical countries.[1-3] These plants are used in folk medicine for treatment of several diseases, such as disturbances of kidney and bladder calculi, intestinal infections, diabetes and hepatitis B virus. A great number of preclinical and clinical studies have not only confirmed but have also extended the medicinal uses of species of the genus Phyllantus mentioned in traditional medicine. [1-3]

Previous studies showed that the extracts of genus Phyllanthus revealed potent, dose related and long-lasting antinociceptive properties when tested in several models of pain and nociception. [4-9] However, the mechanisms that underlying their analgesic effect still remains unclear. In the present study we therefore attempt to evaluate the central and peripheral mechanisms underlying the analgesic effects of the extracts of Phyllanthus amarus and Phyllanthus fraternus in mice using the acetic acid-induced writhing and hot plate tests.

MATERIALS AND METHODS:

Plant material

Phyllanthus amarus Schum and Thonn and Phyllanthus fraternus Webster family- Euphorbiaceae were obtained from different places in Karad western Maharashtra. The plant species were identified and authenticated by Botanical survey of India, Pune [Reference No:BSI/WC/ Tech./2012/644].

Preparation of the P. fraternus extract

The dried leaves, stems and roots of P. fraternus was minced and extracted with 70% ethanol-water in the proportion of 70:30, being stirred and macerated at room temperature (22-28°C) for 15 days. The ethanol was evaporated and the extract (yield 5-7%) was concentrated to the desired level and stored in a refrigerator. The extract was dissolved in DMSO [dimethyl sulphoxide] to the desired concentration just before use.

Extracts of Phyllanthus amarus

The standardized extract of Phyllanthus amarus whole plant (water extract) Reference No: SR/KN/CL/1/2012-L12030241, was procured as a gift sample from Chemiloids Ltd., Vijaywada.

While the standardized methanolic extract of Phyllanthus amarus leaf (Methanol extract contains >2.5% of Phyllanthin and Hypophyllanthin) Report No: FP1112042- PA/11LOT05 and the standardized hydro methanolic extract of Phyllanthus amarus leaf (60% Methanol Hydroalcoholic extract contains >5% of Corilagen) - Report No: FP1102034 -PA/11LOT/02 were procured as a gift sample from Natural Remedies Pvt. Ltd., Bangalore.

Experimental Animals

Swiss albino mice (25-30 g) were used for the study. The groups of animals (n = 4) animals were maintained under standard environmental conditions and were fed with standard diet and water ad libitum. Twenty-four hours before the experiments, they had access only to water ad libitum. A prior approval [Approval number- GCOPK/2011-12/ CPCSEA/616] was obtained from the Animal Ethics Committee of Govt. College of Pharmacy, Karad. (GCOPK) for the study [Protocol Reference No: CPCSEA-IAEC/2011-NOV/01 and CPCSEA-IAEC/2012-MAR/01]. GCOPK is registered under Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA), Govt. of India, CPCSEA Registration no- 209/GO/a/2000/ CPCSEA.

Acetic acid-induced writhing test [10-11]

The assay was performed according to the classical technique of Koster et al. with slight modifications. Writhing was induced by intraperitoneal injection of 0.6% acetic acid (v/v) (80 mg/kg body wt.) into a group of 4 mice. Animals were treated with standardized aqueous extract of P. amarus whole plant (PAAE), standardized methanolic extract of P. amarus leaf (PAME) and the standardized hydro methanolic extract of P. amarus leaf (PAHME) at dose of 50-100 mg/kg, i.p. and the standardized hydro ethanolic extract P. fraternus (PFHEE) (50-100 mg/kg, i.p.) 10 -15 min before the injection of 0.6% acetic acid. Control animals received a similar volume of vehicle (DMSO). Aceclofenac was used as positive control and administered intraperitoneally at a dose of 30 mg/kg. The number of muscular contractions was counted for 30 min following acetic acid injection. Data represent the average of the total number of writhes observed for 30 minutes are expressed as mean ± SEM (standard error of mean).

Hot plate test [12-13]

The assay was performed according to the classical hot-plate technique of Eddy and Leimbach the method described previously with little modification. The parameter evaluated was the latency time for paw licking and jumping responses after exposure to the hot plate surface. The hot plate temperature was maintained at 55 ± 1 ºC. The animal was kept on the hot plate until it lifted one of its hind paws. The response was determined over 150 min after the injection of the phyllanthus extracts and the data represent the mean reaction time for the animals. Latency time was recorded and the results are expressed as the hot plate analgesic index.

The phyllanthus extracts were administered intraperitoneally at a dose of 100 mg/kg body wt. The standard (Pentazocine lactate, 10 mg/kg body wt.) was administered to by intraperitoneal route. The latency time for paw licking was recorded as pain threshold when mice were exposed to the hot plate surface which is kept at 55 ± 1°C. Basic pain threshold was measured, then all treatments were given 10-15 min before the thermal stimulus and the response was determined at 30, 60, 120 and 180 min. Pain threshold inhibition (%) = (Pt − P0) × 100/P0, P0 and Pt separately presents basic pain threshold and pain threshold at time interval.

Statistical analysis

All the Statistical calculation were performed using Graphpad Prism software version 6.01, © 1992-2012. Results are presented as means + SEM (standard error of mean). Data was analyzed statistically using Dunnett’s Multiple Comparison test, with the level of significance set at p < 0.05.

RESULTS:

Writhing test

In the writhing test, which is more sensitive for non-steroidal analgesics, the effect of phyllanthus extracts was signiﬁcant at studied doses as compared to the control and standard. The intraperitoneal injection of phyllanthus extracts reduced, the number of stretching episodes induced by the intraperitoneal injection of acetic acid (0.6%) Fig. 1 shows the effect of the phyllanthus extracts on acetic acid-induced writhing in mice. The administration of phyllanthus extracts significantly inhibited writhing response in a dose dependent manner. PAAE, PAME, PAHME and PFHEE produced 80.00 %, 89.143 %, 83.429 % and 86.857 % inhibition of writhing at dose of 50 mg/kg respectively. Whereas, PAAE, PAME, PAHME and PFHEE produced 85.714 %, 96.00 %, 89.143 and 94.857% inhibition of writhing at dose of 100 mg/kg respectively. Whereas the standard drug acelofenac produced 96.00% inhibition of writhing.

Hot plate test

The intraperitoneal injection of phyllanthus extracts increased the reaction time of the mouse to the hot plate painful stimulus (Fig. 2). Phyllanthus extracts showed maximum analgesic activity at 30, 60, 120 and 150 min for 100 mg/kg dose. The mean reaction time in normal control group at 30, 60, 120 and 150 min were found to be 2.995 ± 0.4517, 2.332 ± 0.332, 1.8325 ± 0.1675, 1.747 ± 0.1606 and 2.165 ± 0.0952 Seconds respectively. The reaction time (paw licking / jumping response) in rats pretreated with phyllanthus (100mg/kg) and Pentazocine (10 mg/kg) were found to be highly elevated, when compared to the control group rats. The duration of analgesic effect was good compared to the control and reference drug pentazocine which significantly increased the reaction time at each time point of testing. Effect of phyllanthus extracts expressed as the % inhibition threshold of pain response to heat stimuli induced by hot plate is summarised in figure 3.

DISCUSSION:

The previously reported results show that the active principles present in some plants belonging to the genus Phyllanthus, exhibit potent and long-lasting systemic analgesic effect when analyzed in various models of inflammation and pain. By both hot plate and writhing tests, we showed that the extracts of Phyllanthus amarus and Phyllanthus fraternus has marked analgesic properties. Deraedt et al. have described the quantification of prostaglandins in the peritoneal exudates of rats by radioimmunoassay, obtained after intraperitoneal injection of acetic acid.[14] They found high levels of prostaglandins PGE2α e PGF2α during the first 30 min after acetic acid injection. Nevertheless, it was found that intraperitoneal administration of acetic acid induces not only the liberation of prostaglandins, but also the liberation of sympathetic nervous system mediators [15-16]. Therefore, we may state that anti-inflammatory substances may also be involved in peripheral analgesic activity. The phyllanthus extracts inhibited acetic acid induced writhing in mice. It can therefore be suggested that the analgesic effect of the extract is peripherally mediated. Phyllanthus extracts produced a reduction in the number of writhes at two doses used, but the most significant effect was obtained at the dose of 100 mg/kg body wt. (Fig. 1). The other algesimetric test used was hot plate test that reveals the central analgesic response. The extracts showed a significant increase in the pain threshold, corresponding to a significant increase in the percentage of protection, as compared to the control group and the pentazocine standard (Fig. 2). Therefore, the analgesic effect of the extracts was significant by both measures, a fact that can be explained by the presence of a mixture of substances in the extract, bearing both peripheral and central analgesic properties. Different classes of organic compounds of medicinal importance have been isolated in phyllanthus extracts including alkaloids, flavonoids, hydrolysable, tannins (Ellagitannins), major lignans, polyphenols, triterpenes, sterols and volatile oil.[1-8] The lignins flavanoids and tannins may be the active principles responsible for the observed analgesic effect. [1-8]The literature contains numerous reports on the analgesic and anti-inflammatory properties of these classes of compounds [1-8]. Overall, it can be concluded that the phyllanthus extract possesses central and peripheral analgesic activity, probably mediated by the inhibition of prostaglandin synthesis, as well as by central inhibitory mechanisms.

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Received on 20.01.2013 Accepted on 12.02.2013

Asian J. Pharm. Res. 3(1): Jan.-Mar. 2013; Page 10-14