INTRODUCTION:

Phyllanthus amarus belonging to family Euphorbiaceae and genus Phyllanthus is a tropical annual herb that grows 10-60 cm tall.¹ It is commonly known as carry me seed,stonebreaker.Leaves of the plant is stipulate with numerous small leaflets². Flowers are short pediculled and yellowish in colour. Roots of Phyllanthus amarus are tortuous and woody³.

Phyllanthus seeds: trigonous, pale brown with longitudinal parallel ribs on the back ⁴. Explosive seed capsules are present in the plant, that propel the seeds some distance from the plant. Phyllanthus seeds are 1 mm long and are triangular, light brown in colour.⁵

Phyllanthus amarus is used in traditional medicine for its medicinal properties and is used in metabolic disorders⁶, problems of genitourinary system,liver and kidney. It has diuretic,astringent and antiseptic properties.

The extracts and compounds isolated from Phyllanthus amarus show a wide spectrum of pharmaceutical activities including antibacterial, antifungal, antihepatotoxic antihyperglycemic, antipyretic, antiviral, cytotoxic action.^7,8 Phytochemical composition of Phyllanthus seeds are known to possess antioxidant,anti inflammatory and antimicrobial properties.⁹

The major phytoconstituents present in the seed extract are tannins, alkaloids, lignans, saponins and total phenols.¹⁰Though many analgesic drugs are still in market, novel drug with least side effects are still in search. This study was done to investigate analgesic activity of Phyllanthus amarus seeds in animal models so that this can be beneficial for medicinal use in future.

MATERIALS AND METHODS:

Drugs used for the study:

Normal saline used as control, Drug Morphine as standard drug, Methanolic extract of Phyllanthus amarus seed extract as test drug at three different doses.

Plant material:

Phyllanthus amarus seeds were obtained from Natural remedies company, Bangalore. Batch No: VEX/2012050001 for this study purpose. It was then authenticated in herbarium unit of Botany Department in Davangere University, Karnataka.

Preparation of Extract:

The Phyllanthus seeds were dried in hot air oven to make them suitable for grinding purpose.The seeds were made to coarse powder with mechanical grinder. By using Soxhlet apparatus, powdered seeds were extracted with methanol. The seed material was dried again at 40⁰C and used for next extraction. Methanolic extract of the seed obtained was stored at 4⁰C in suitable container for further analysis.

Source of data:

After the approval from Institutional Animal Ethics Committee of J.J.M. Medical College no: JJMMC/IERB/55/18, this study was done in Department of Pharmacology, J.J.M. Medical College, Davangere, Karnataka, India.

Experimental animals:

Adult albino mice weighing 25-35 gms used for the study. The mice was bred in the central animal house of the Department of Pharmacology, J.J.M. Medical College, Davangere, under suitable conditions of housing, temperature, ventilation and nutrition.

Duration of the study:

6 months in the year 2018 (May 2018-0ct 2018).

Acute toxicity study:

The acute toxicity of the extract was tested using 30 albino mice, divided into 5 groups of 6 mice each, each group receiving graded dose (50-1500 mg/kg body weight) of methanolic extract of Phyllanthus seed extract. After 48 hours , albino mice in each group was observed for toxic effects. The toxicological effects were observed in terms of mortality: expressed as LD50.

Inclusion criteria:

· Adult albino mice (both male and female) weighing between 25-35 gms.

· Age 3-4 months.

· Healthy with normal behaviour and activity.

Exclusion criteria:

· Mice <25gms and >35gms

· Pregnant female mice.

· Animals previously used in other experiments.

Experimental method:

A total of 60 animals were used. They were divided in to 5 groups of 6 animals each in two different models.They were evaluated for analgesic activity using two models - Hot plate method^11,12 and Tail clip method.

Hot Plate Method:

The animals were divided as follows.

Group1: Received 10ml/kg of Normal saline intra peritoneally.

Group 2: Received 10mg/kg of Morphine intraperitoneally.

Group 3: Received 50 mg/kg of methanolic extract of Phyllanthus seeds intraperitoneally.

Group 4: Received 100mg/kg of methanolic extract of seeds of Phyllanthus amarus intraperitoneally.

Group 5: Received 200mg/kg of methanolic extract of Phyllanthus seeds intraperitoneally.

The hot plate method was assessed on 5 groups of mice with 6 animals in each group where response of the mice to the thermal heat stimulus is recorded. The temperature of the metal surface was maintained at 55⁰C. Latency to a discomfort reaction (licking of paws) was determined before and 30, 60, 90 min after drug administration.

Tail Clip Method:

The animals will be divided as follows:

Group 1: Received 10ml/kg of Normal saline intra peritoneally.

Group 2: Received 10mg/kg of Morphine intraperitoneally.

Group 3: Received 50mg/kg of methanolic extract of Phyllanthus seeds intraperitoneally.

Group 4: Received 100mg/kg of methanolic extract of Phyllanthus amarus seeds intraperitoneally.

Group 5: Received 200mg/kg of methanolic extract of Phyllanthus amarus seeds intraperitoneally.

An artery clip is applied to the root of the tail (approximately 1cm from the body) to induce pain. The animals quickly respond to this stimulus by biting the clip or the tail near the location of the clip. The time between stimulus onset and response is noted by a stopwatch. The test was done before the administration of the drug and was repeated 30, 60, 90 min of drug administration and the observations were noted.

STATISTICAL ANALYSIS:

· One way ANOVA followed by Post hoc test used for statistical analysis.

· p<0.05 was considered as statistically significant.

RESULTS:

In our study, Mean ± SEM of time values between stimulus onset and response of animals (albino mice) among various groups in hot plate method were compared and shown in Table 1.

Table 1: Showing Mean± SEM of time values of stimulus onset and response time of mice in Hot plate method

Groups

Baseline

30 min after inj.

60 min after inj.

90 min after inj.

Control

2.33±

0.47

2.37±

0.48

2.40±

0.46

2.35±

0.46

Standard

2.35±

0.46

11.18±

2.15^#

8.23±

1.36^#

7.98±

2.66^#

Test1 50mg/kg

2.98±

0.23

3.92±

0.95

4.85±

0.60

5.08±

0.91

Test 2 100mg/kg

2.18±

0.27

4.93±

0.88

7.00±

1.00^#

5.55±

1.65

Test 3 200mg/kg

2.72±

0.37

5.82±

0.73

7.32±

1.21^#

4.73±

0.52

SEM-standard error of mean

One way ANOVA followed by post hoc Tukey test. #p<0.05 statistically significant when compared to control.

Reaction time of animals among various groups in hot plate model in mice is depicted in Fig. 1

Fig. 1: Showing reaction time of mice (in secs) among different groups in hot plate method

Extract of P.amarus increased the reaction time of mice at 30 min and 60 min and 90 min after administration of the extract in hot plate method and showed a dose dependent analgesic effect at 100mg/kg and 200 mg/kg dose when compared to control group (p<0.05).

The acute toxicity study done according to the guidelines of Organization for Economic Co-operation and Development (OECD). The animals were fasted overnight with free access to water, weighed and then test drug (methanolic extract of Phyllanthus seeds) was administered. Animals were observed individually during first 30 min and then periodically during 48 hours with more attention given during first 4 hours (short-term toxicity). The mice were found safe after observation except in the group that was administered with 1000 mg/kg where one animal was found dead. The median acute toxicity (LD50) of the compound was determined to be 1000 mg/Kg as per the observations done using software of probit analysis.¹³

In tail clip method, Mean ± SEM of reaction time of animals among control, standard and three test groups are depicted in Table 2. Increase in reaction time of animals occurs in 3 doses of test group (methanolic extract of Phyllanthus seeds) when compared to control,though statistically significant differences occurs at 30 min and 60 min of test drug administration (P<0.05) in the dose range of 100mg/kg and 200 mg/kg.

Table 2: Showing Mean± SEM of time values between stimulus onset and response time of mice in Tail clip method:

Groups	Baseline	30 min after inj.	60 min after inj.	90 min after inj.
Control	0.72±0.15	0.55± 0.15	0.53± 0.10	0.47± 0.16
Standard	0.63±0.20	3.87± 0.23^#	5.05± 0.61^#	4.13± 0.21^#
Test1 50mg/kg	0.63±0.17	1.22± 0.26	2.22± 0.38	1.18± 0.31
Test 2100mg/kg	0.67±0.23	2.95± 0.32^#	4.13± 0.39^#	1.10± 2.35
Test 3200mg/kg	0.95±030	3.2± 0.32^#	4.65± 0.52^#	1.20± 0.41

SEM-Standard error of mean

One way ANOVA followed by post hoc Tukey test. #p<0.05 statistically significant when compared to control

DISCUSSION:

In this study, methanolic extract of seeds (Phyllanthus amarus) were evaluated for analgesic activity in albino mice using hot plate method and tail clip model. These methods are used to evaluate central analgesic activity in animal models.

In this study, Phyllanthus seed extract showed analgesic effect with onset of action at 30 min after the injection; Peak action seen at 60 min and antinociceptive effect started to fade away by 90 min of test drug administration in both tail clip and hot plate method as shown in Table 1 and 2 respectively. Dose dependant increase in reaction time of animals occurs in three different doses of test group when compared to control group,though not comparable to standard Morphine group. In a study done by Sijuade A ¹⁴ methanolic extract of Phyllanthus showed dose dependant analgesic activity when compared to control group. Similarly in a study by Chopade AR, Phyllanthus amarus extract showed increase in reaction time of mice in hot plate method.¹⁵

Phytochemicals like alkaloids, saponins, triterpenoids, carbohydrates, flavonoids, tannins, phenolic compounds were seen in Phyllanthus seed extract. Quercetin, kaempferol, astragalin, quercetin-3-O-glucoside, quercitrin are the bioactive constituents present in flavonoids. Phyllanthus exhibits long lasting analgesic effect in neurogenic pain model in a study done by Santos et al¹⁶where flavanoids of plant extract are known to target prostaglandins. PGs are involved in pain perception and in the late phase of acute inflammation. Tannins and saponins also play a role in anti nociceptive activity.^17,18

In our study, methanolic extract of Phyllanthus amarus seeds has shown significant analgesic action in both hot plate and tail clip methods. Phyllanthus amarus extract showed central analgesic activity in albino mice when compared to control. It may be useful for newer development of analgesic drug with least side effects, but there is definitely a need for further studies on other experimental animals and human beings to establish its usefulness, exact mode of action.

ACKNOWLEDGEMENTS:

I thank my fellow colleagues who helped in completing this study and in preparing the manuscript.

CONFLICT OF INTEREST:

None declared.

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Received on 04.11.2020 Modified on 18.03.2021

Research J. Pharm.and Tech 2022; 15(2):713-716.

DOI: 10.52711/0974-360X.2022.00118