Anti-emetic activity of some members of the family, Euphorbiaceae of Lahore region

The present investigation aimed to evaluate the anti-emetic activity of some members of the family Euphorbiaceae of Lahore region. The selected plants were Euphorbia prostrata, Euphorbia splendens, Euphorbia hirta, Euphorbia helioscopia, Ricinus communis, Jatropha integerrima, Putranjiva roxburghii and Phyllanthus emblica. Anti-emetic activity was carried out by the preparation of aqueous extract of these plants. Anti-emetic investigations showed that aqueous extracts of E. prostrata, P. roxburghii and P. emblica have high potential to reduce the frequency of retching in CuSO4 induced emesis in four days old chicks. Frequency of retching after treatment of aqueous extracts of E. prostrata, P.a roxburghii and P. emblica were 24.8 ± 0.89, 17.4 ± 0.89, 15.2 ± 0.58, respectively, which statistically proved significant at p<0.01 from the control (87.2 ± 1.46, 82.6 ± 1.46, 82.6 ± 1.45, respectively) values.


INTRODUCTION
The use of plants by traditional people has started long time ago, which has laid basis for the discovery of modern medicines.Some years earlier, mankind was well aware of the medicinal characteristics of few plants growing around him (Sofowora, 1982;Hill, 1989).Drug discovery programs have started from the earliest times of mankind's history (Cotton, 1996).
A chemical substance that induces motion sickness, nausea and vomiting is called emetic.Emesis occurs in such cases when a person ingests certain chemical substances such as salt water, mustard water and copper sulphate, zinc sulphate, yellow mercuric subsulphate, alumen and apomorphine.When a toxic substance has been ingested immediately, an emetic is used for their removal from the body (Decker, 1971;Moder and Hurley, 1991).
An anti-emetic is a drug that is widely used to control motion sickness, severe nausea and vomiting.Various types of anti-emetic drugs are used to control nausea and vomiting.Some of these works on the brain by preventing the activation of the medullar chemoreceptor trigger zone or vomiting centre and other drugs work on the gut by speeding up the rate at which the stomach empties and so help to move food through the intestines more quickly (Zachary et al., 2004).
The family Euphorbiaceae has been selected because many of the members of family Euphorbiaceae has been used as folk medicine in the treatment of different diseases like Croton tiglium used as a pain reliever and dry cough (Koche et al., 2010) and Eclipta alba (Saxene et al., 1993), Euphorbia fusiformis used for Hepatitis (Anusuya et al., 2010), etc.

Selection of plants
Eight plants were selected (Euphorbia prostrata, Euphorbia *Corresponding author.E-mail: drtahiramughal@gmail.com or ssass85@yahoo.com.Tel: 042-99203801-8.Ext: 207.Fax: 042-99203810. splendens, Euphorbia hirta, Euphorbia helioscopia, Ricinus communis, Jatropha integerrima, Putranjiva roxburghii and Phyllanthus emblica) of the family Euphorbiaceae for the study of anti-emetic activity.These plants were selected on the basis of easy approach, easy growth and easy availability and mainly on the basis of ethnomedicinal importance.

Collection of plants
These plants were collected from different areas of Lahore (Lahore College for Women University, Lahore, Lawrence Garden Lahore and Harbanspura, Lahore).These plants were authenticated by Dr. Tahira Aziz Mughal Assistant Professor in Botany Department of Lahore College for Women University, Lahore.Plant specimens were deposited as voucher specimen in Prem Madam Herbarium of Lahore College for Women University, Lahore, for the record and further reference.

Drying and grinding of plants
The plant material was washed with water and dried in shade, then cut into small pieces and ground in an electrical grinder to be obtained in a powder form.

Aqueous extract preparation
The crude aqueous extract of the powdered plant material was prepared by using the standard methods (Onyeyili et al., 2001).Twenty grams of the powdered plant material was mixed with 100 ml of distilled water in a 1 L beaker and boiled for 1.5 h.After boiling, it was allowed to cool at room temperature and then filtered by using Whatman 1 filter paper.

Evaporation of solvent
The solvent was removed by rotary evaporator to obtain crude extracts of plant materials.These extracts were stored at 4°C.

Preparation of stock solution
Stock solution was prepared from crude extracts of plants and stock solution was further diluted into different dilutions according to the requirement of the activity either in milligram or microgram.

Anti-emetic activity
Anti-emetic activity of the plant extracts was examined by the methodology of Yang et al. (1999).

Selection of animals
Four days old chicks (male) weighing 35 to 45 g were selected for anti-emetic activity.They are purchased from Tollinton Market, Lahore.

Preparation of animal house for chicks
Chicks were kept in a wire cage.The wire cage was 17 inches in length and 15 inches in width.The chick housing facility was maintained at standard conditions.Water pot was kept in chick house.Chicks need at least two inches of bedding material beneath them, so straw or dry leaves were spread beneath them.

Feeding routine
Chicks were fed at regular intervals with poultry feeding purchased from Tollinton Market Lahore.The feed was left with them all the time because they will stop eating when they have had enough.

Record of chicks
The information about each chick's group and its age, weight (grams) was recorded at the start and at end of the experiment.

Chemical used to induce the emesis
CuSO4 was used to induce the emesis.For this purpose 1% CuSO4 was prepared by dissolving 1 g CuSO4 in 100 ml distilled water.
Chemical used as diluter 0.9 % saline water drip (Medisol Pvt Ltd, Pakistan) of 1 liter was used.

Preparation of dilutions
Four different concentrations (25, 50, 75 and 100 mg/kg) from crude aqueous plant extract were prepared in saline water.

Induction of emesis
Emesis was induced by 1% CuSO4 in the chicks orally by 1 ml/20 g body weight.

Experimental plan
The antiemetic activity was determined by calculating the mean decrease in number of retching in contrast with those of the control.All the young male chicks were divided into 12 groups (A, B, C, D, E, F, G, H, I, J, K and L) and groups B, C, D, E, F, G, H, and I were further divided into subgroups.Each group or subgroups had 3 chicks.

Group A
The group A treated as control, was not given any treatment.

Groups B, C, D, E, F, G, H and I
These groups of chicks were administrated with 1% CuSO4 to induce emesis and after 10 min the number of retching reflexes or emetic action was recorded, and then plant extracts were given in the form of dilution of 25, 50, 75, and 100 mg/kg with the help of a dropper.

Group B
Chicks of group B were administered aqueous extract of E. prostrata.This group was was further divided into four subgroups.

Group H
Aqueous extract of P. roxburghii was given to chicks of group H.This group was further divided into four subgroups.H1: Aqueous extract of P. roxburghii at the dose of 25 mg/kg was given to chicks.H2: Aqueous extract of P. roxburghii at the dose of 50 mg/kg was given to chicks.H3: Aqueous extract of P. roxburghii at the dose of 75 mg/kg was given to chicks.H4: Aqueous extract of P. roxburghii at the dose of 100 mg/kg was given to chicks.

Group I
Chicks of group I were treated with methanolic extract of P. emblica.This group was further divided into four subgroups.

Group J
Chicks of group J were administrated 50 mg/kg Motilium or Domperidone (Janssen and Janssen Pakistan (Pvt) Ltd.) which served as positive control.

Group K
125 mg/4 ml Gravinate or Dimenhydrinate syrup (Searle pharmaceutical Pakistan (Pvt) Ltd.) was given to chicks of group K which served as positive control.

Group L
Chicks of group L weretreated with 50 mg/kg Metodine or Diiodohydroxyquinoline which served as positive control.

Dosage administration
After dosage administration, the results were recorded by counting number of retching reflexes for the next 10 min.

Calculation of antiemetic activity
The percent inhibition was calculated by the following formula: Where A is frequency of retching before treatment and B is frequency of retching after plant treatment.

Statistical application
The standard error means (SEM) and statistical significance difference was determined by unpaired student t-test with the help of statistical software SPSS 19.

DISCUSSION
Phytochemical screening showed the presence of alkaloids, phytosterol, phenols, flavonoid, tannins and phlobatannins in the extracts of E. prostrata, E. splendens, E. hirta, E. helioscopia, R. communis, J. integerrima, P. roxburghii and P.s emblica (Tahira et al., 2010).Four days old chick is a useful animal model selected for anti-emetic effects because this animal possesses acute emesis that is observed in man.This animal also serves as a useful animal for evaluating the involvement of the brain in the observed anti-emetic effects of the extracts (Tijani et al., 2008).
Emesis (nausea and vomiting) is caused by interaction of gastrointestinal system, the vestibular system and the various areas of the brain like chemoreceptor trigger zone (CTZ) in the brain (Gan et al., 2003).Emesis occurred due to copper sulfate, because copper sulphate causes the excitation of visceral afferent nerve fibers of gastrointestinal tract.It has also been observed that the peripheral 5HT 4 plays a major role in copper sulphate induced emesis (Andrews et al., 1990).An aqueous extract of E. prostrata exhibited significant inhibition (71.5%) of emesis at the dose of 100 mg/kg (Table 1).
Lower concentrations of aqueous extract of E. splendens showed insignificant results but at the dose of 100 mg/kg it showed significant inhibition (61.9%) of emesis (Table 2).59.9% inhibition of emesis was observed by the therapeutic treatment of aqueous extract of E. hirta at the dose 100 mg/kg (Table 3).An aqueous extract of E. helioscopia at the dose of 25, 50, 75 and 100 mg/kg exhibit 23.5, 35.1, 49.8 and 55.2% inhibition of emesis respectively (Table 4).The frequency of retching in young chicks reduced (61%) significantly by the administration of aqueous extract of R. communis only at higher concentration (100 mg/kg) (Table 5).An aqueous extract of J. integerrima therapeutically reduced the copper sulfate induced emesis in young chicks by increasing the concentration of plant extract (Table 6).Significant inhibition of emesis observed by the application of aqueous extract of P. roxburghii even at lower concentration like 25 mg/kg showed 61.7% inhibition of emesis in young chicks (Table 7).An aqueous extract of P. emblica at the dose of 25, 50, 75 and 100 mg/kg showed 59.9, 63.7, 70.8 and 81.6 % inhibition of emesis, respectively (Table 8).
The most important findings of the present study are that, all the selected plants significantly reduced the frequency of copper sulphate induced retching in four days old chicks as compared to commercially available medicines: Gravinate, Metodine and Motilium (Table 9).These medicines showed anti-emetic activity by acceleration of gastrointestinal tract movement (Akita et al., 1998).
Therefore, it is concluded that these plants E. prostrata, E. splendens, E. hirta, E. helioscopia, R. communis, J. integerrima, P. roxburghii and P. emblica showed significant anti-emetic activity and thus provides the basis for their use in traditional or folk medicines for the treatment of emesis.

Table 1 .
Anti-emetic effect of aqueous extract of E. prostrata on copper sulfate induced emesis in young chicks.
*Significantly different from the control value p<0.01.

Table 2 .
Anti-emetic effect of aqueous extract of E. splendens on copper sulfate induced emesis in young chicks.
*Significantly different from the control value p< 0.01.

Table 3 .
Anti-emetic effect of aqueous extract of E. hirta on copper sulfate induced emesis in young chicks.
*Significantly different from the control value p< 0.01.

Table 4 .
Anti-emetic effect of aqueous extract of E. helioscopia on copper sulfate induced emesis in young chicks.

Table 5 .
Anti-emetic effect of aqueous extract of R. communis on copper sulfate induced emesis in young chicks.

Table 6 .
Anti-emetic effect of aqueous extract of J. integerrima on copper sulfate induced emesis in young chicks.

Table 7 .
Anti-emetic effect of aqueous extract of P. roxburghii on copper sulfate induced emesis in young chicks.

of plant extract (mg/kg) Average weight (g) *Mean ± S.E.M. of no. of retching/10 min Inhibition (%) Remark Before plant treatment After plant treatment
*Significantly different from the control value p< 0.01.

Table 8 .
Anti-emetic effect of aqueous extract of P. emblica on copper sulfate induced emesis in young chicks.
*Significantly different from the control value p< 0.01.

Table 9 .
Anti-emetic effect of commercial medicines on copper sulfate induced emesis in young chicks.Significantly different from the control value p< 0.01. *