Anti Asthmatic Activity Of Phyllanthus Amarus Schum Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Preliminary photochemical screening of MEPA Methanolic extract of Phyllanthus amarus and HFPA Hexane fraction of Phyllanthus amarus was conducted to identify the nature of Phytoconstituents present in it. Bronchodilator activity of MEPA and HFPA was evaluated by using histamine induced bronchospasm in conscious guinea pigs. MEPA Showed most promising result, whose efficacy was evaluated using various animal model i.e. passive paw anaphylaxis in rats model and furthermore to predict the mechanism of MEPA was evaluated in clonidine induced peritoneal mast cell degranulation, milk induced leukocytosis.

Results: Preliminary photochemical screening showed presence of alkaloids, flavanoids, tannins, lignans, steroids and triterpinoids. In histamine induced bronchospasm model, Pre-convulsion time was significantly increased at 400mg/kg of MEPA (61.55 ± 2.82). MEPA showed significant reduction in paw volume at 2nd hour (0.930 ± 0.03) in passive anaphylaxis. Most significant protection against mast cell degranulation was observed at 2 mg/ml of MEPA (45.66 ± 1.03), MEPA significantly reduced total leukocyte count (300 ± 121.10) in milk induced leuckocytosis.

Conclusions: Present study showed MEPA having potential anti-asthmatic activity in acute model of asthma by inhibition of release of inflammatory mediator from mast cell and infiltration of leukocytes during asthmatic condition.

Keywords: MEPA, Bronchospasm, Mast cell, Histamine

INTRODUCTION

Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, in particular masts cells, eosinophils, T lymphocytes, macrophages, neutrophils and epithelial cells. In susceptible individuals, this inflammatory diseases induced recurrent episodes of wheezing, breathlessness, chest tightness and coughing, particularly at night or in the early morning [1,2].

In traditional systems of medicine, many plants have been documented to be useful for the treatment of chronic respiratory disorders including asthma. Current synthetic drugs used in pharmacotherapy of asthma are unable to cure all the stages of asthma and far from satisfactory as they provide only symptomatic relief, produce several adverse effects and may lose effectiveness on continuous use [3,4]. The process of sustain inflammation accomplices chronic disease condition of asthma, which can be ameliorated and prevented by phytomedicine.

Phyllanthus amarus (Euphorbiaceae) is a widely distributed, small tropical herb. It is popular in indigenous system of medicine like ayurveda, siddha, unani and homoeopathy. It possess hepatoprotective, anti-tumour, anti-diabetic, antihypertensive, analgesic, anti-inflammatory and anti-microbial properties [5].

In previous scientific research work, plant is reported to possessed anti-inflammatory activity by inhibiting several inflammatory mediators, till the date there is no scientific research work reported for anti-asthmatic potential by keeping this in view, the present study was aimed to evaluate the anti-asthmatic activity of Phyllanthus amarus Schum and Thonn.

MATERIAL AND METHOD

2.1 Collection of Phyllanthus amarus and preparation of different extract

The fresh, well developed Plants of Phyllanthus amarus were collected from the botanical garden of Saurashtra university,Rajkot,Gujarat.

The plants were authenticated by comparing the morphological and microscopic characters described in the literature[6] and authentification was further conformed with help of botanist from Christ college,Rajkot.

Whole plant samples were dried under shade, reduced powder (60#), and stored in airtight containers.

Preparation of Methanolic Extract

Powdered material was subjected to successive extraction with methanol (95%v/v) in soxhlet apparatus and the extracts were concentrated to ¾ of its original volume by distillation. The concentrated extract were taken in china dish and evaporated on a thermostatic controlled water bath until it forms thick paste. The extract was dried and stored in refrigerator at 4-80C in airtight container throughout study.

Preparation of Hexane Fraction

Methanolic extract was made aqueous by adding distill water (10:1) and extracted with equal volume of hexane in separating funnel and hexane fraction was concentrated to ¾ of its original volume by distillation.

The concentrated extract were taken in a china dish and evaporated at 370C by using water bath until it forms thick paste. The extract was dried and stored in refrigerator at 4-80C in airtight container throughout study.

2.2 Preliminary Phytochemical screening.

The phytochemical analysis of both extract was carried out by using qualitative phytochemical test as per method described by Treser and Evans[7,8],to know the nature of phytoconstituents present in it.

2.3 Experimental Animals

Healthy guinea pigs, wistar rats and swiss albino mice of either sex selected for different animal model. All animals were kept at ambient temperature (22±1°C), relative humidity (55±5°%) and 12:12 hrs light/dark cycle in the animal house of S. J. Thakkar Pharmacy college. Animal had free access to standard pellet diet and water ad libitum. The experimental Protocol SJT-046/2012 of pharmacological study was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) and all experimental procedure were conducted according to guideline of CPCSEA.

2.4 Drug and chemicals

Histamine dihydrochloride was obtain from Hi-media Pvt. Ltd (Mumbai, India), Clonidine and egg-albumin was obtain as gift sample from Alembic Pharma Pvt. Ltd. (Baroda, India), Dexamethason and ketotifen was obtain from Cadila Pharma Pvt. Ltd., Ahmadabad. All the chemicals used in the study were of analytical grade.

2.5 Histamine induced bronchospasm in conscious guinea pigs.

Healthy New Zealand guinea pigs of either sex weighing in range of 550-700 g were used and divided into six groups (six animals in each) as following:

• Group I- (Normal Control) Received vehicle (0.5% w/v Sodium CMC,10 ml/kg, p.o.)

• Group II- (Standard) Received Ketotifen (1 mg/kg, p.o)

• Group III- (Test MEPA-Low) Received Methanolic Extract of P. amarus (200 mg/kg, p.o. )

• Group IV - (Test HFPA- Low) Received Hexane fraction of P. amarus (200 mg/kg, p.o. )

• Group V - (Test MEPA- High) Received Methanolic Extract of P. amarus (400 mg/kg, p.o.)

• Group VI - (Test HFPA- High) Received Hexane fraction of P. amarus (400 mg/kg, p.o. )

All test and standard drugs were dispersed in 0.5%w/v sodium CMC.

The animals were kept in a closed chamber (30Ã-30Ã-15cm) and exposed to an aerosol of 0.1%w/v Histamine dihydrochloride and pre-convulsion time (PCT) was noted. Before starting of treatment in each group. Later, all animals of different group treated with respective drug once daily for 7 days. On the 7th days, two hours after the respective drug treatment, animals were exposed to histamine dihydrochloride aerosol and PCT was measured for each animal [4,9].

% Increase in PCT was calculated using following formula.

% Increase in PCT = [1-T1/T2] x 100, where, T1= PCT on day 0 , T2= PCT on 7th day.

2.6 Passive paw anaphylaxis (ppa) in rats.

Preparation of anti-serum from rats

Wistar rats of either sex were injected intraperitoneally with 0.2 ml, 10% egg albumin on day 1, 3 and 5. Ten days after the first immunization, blood was collected from orbital plexus under light ether anesthesia. The collected blood was allowed to coagulate and serum was separated by centrifugation at 1500rpm for 10 min. The separated serum was stored at 2-4°C untill it was used for the experiment.

Procedure

Wistar rats of either sex weighing 150-200 gm were selected and randomly divided into three groups (6 animals in each) and respective treatments were given as follow,

• Group I-(Control) Treated with vehicle (0.5%w/v NaCMC)

• Group II- (Standard) Treated with Dexamethasone (0.27mg/kg, p.o.)

• Group III-(Test) Treated with Methanolic Extract of P. amarus (400 mg/kg, p.o. )

Test and standard drugs were dispersed in 0.5% w/v sodium CMC.

All treatment were given orally once daily for 7 days. 2hr after last dose on 7th day rats were passively sensitized by subplanter administration in to left hind paw with 0.1ml undiluted serum and after 24 hour of sensitization, rats were again challenged with 10 µg of egg-albumin in 0.1ml saline and hind paw volume was measured by plethysmometer for 4hr at interval of 1hr after 30 minutes of sensitization in each animal[10,11].

2.7 Milk induced leucocytosis in mice

Procedure

Swiss albino mice of either sex weighing 25-30gm were divided into three groups.(n=6)

• Group I -(Control) Treated with vehicle (0.5% w/v Na CMC)

• Group II- (Diseases control) Treated with vehical (0.5% w/v Na CMC)

• Group III-(Test) Treated with Methanolic Extract of P. amarus (400 mg/kg, p.o.)

Blood samples were collected from retro-orbital plexus. Total leukocyte was performed in each group and then milk (boiled, 4 mg/kg) was injected subcutaneously in each group except control group and leukocyte count was measured 24 hour after milk injection in all group. Difference in Total leukocyte count before and 24 hours after milk administration was compared for animal of each group[11,12].

2.8 Clonidine induced mast cell degranulation

Normal saline containing 5 units/ml of heparin was injected in the peritoneal cavity of male rats lightly anaesthetized with ether. After gentle abdominal massage, the peritoneal fluid were collected in centrifuge tubes. Peritoneal fluid of 4 - 5 rats was collected and centrifuged at 2,000 rpm for 5 min. Supernatant solution was discarded and the cells were washed twice with saline and resuspended in 1 ml of saline. 0.1 ml of the peritoneal cell suspension was transferred to 5 test tubes and all standard and test drug were added in following way[4].

• Test tube no. 1 - Positive control

• Test tube no. 2 - 0.1 ml of 10 µg/ml Ketotifen in Saline

• Test tube no. 3 - 0.1 ml of 1000 µg/ml methanolic of P.amarus in Saline

• Test tube no. 4 - 0.1ml of 1500 µg/ml methanolic extract of P.amarus in Saline

• Test tube no. 5 - 0.1ml of 2000 µg/ml methanolic extract of P.amarus in Saline

Each test tube was incubated for 15 min at 37°C and then Clonidine (0.1 ml, 80 μg/ml) was added to each test tube. After again incubation for 10 minutes at 37°C, the mast cells were stained with 0.1% toluidine blue solution in distilled water in each test tube and resultant mixture was kept on slide and examined under the high power of light microscope [13].

Protection of the mast cells in the control group and the treated groups was calculated by counting the number of degranulated mast cells from total 100 mast cells counted from different region of slide.

2.9 Statistical analysis

All value were expressed as mean ± SEM (standard error mean). The statistical analysis was done by analysis of variance (ANOVA) followed by Tukey's test when compared to Disease control or control respectively. Value of p < 0.5 was considered as significant. The statistical software Graph pad Prism (version 5.0) was used to perform all statistical analysis.

RESULTS

3.1 Extraction yield

The yields of methanolic extract and hexane fraction of whole herb of Phyllanthus amarus were found to be 16.3% w/w and 4.6 % w/w respectively.

3.2 Preliminary Phytochemical screening

MEPA and HFPA were quantitatively tested for presence of phytochemical, result of tests were mention in Table 1.

Table 1: Results of different chemical constituents:

Constituents

Results

MEPA

HFPA

Alkaloids

+

+

Lignans

+

+

Tannins

+

-

Steroids

+

+

Triterpinoids

+

-

Flavanoids

+

+

MEPA= Methanolic extract of Phyllanthus amarus, HFPA= Hexane Fraction of Phyllanthus amarus

3.3 Histamine induced bronchospasm in conscious guinea pigs

When guinea pigs were exposed to 0.1% histamine aerosol, there were a bronchospasm seen in the form of pre-convulsion dyspnoea. Treatment with MEPA (200mg/kg, 400mg/kg) and HFPA (200mg/kg, 400mg/kg), delayed onset of pre-convulsion time (Table 2) but most significant % increases in pre-convulsion time was seen with MEPA (400mg/kg) as compare to normal control group and that was comparable to standard Ketotifen (1mg/kg) (p<0.01) (Table 2).

Table 2: MEPA and HFPA on Histamine induced bronchospasm in conscious guinea pigs

Sr.no.

Groups

% Increase in PCT

1

Normal Control

2.891 ± 0.370

(0.5% w/v NaCMC)

2

MEPA-Low

57.653± 0.817**

(200mg/kg)

3

HFPA-Low

39.731 ± 1.625**

(200mg/Kg)

4

MEPA-High

61.553 ± 2.821***

(400mg/kg)

5

HFPA-High

40.633 ± 2.258**

(400mg/Kg)

6

Ketotifen

73.476 ± 1.780***

(1mg/kg)

All values represent as mean + SEM (n=6)

**p < 0.01 & ***p < 0.001, All treated group were compared to normal control group.

MEPA= Methanolic extract of Phyllanthus amarus, HFPA= Hexane Fraction of Phyllanthus amarus

3.4 Passive paw anaphylaxis (ppa) in rats

There were significant reduction in paw volume when treated with most effective dose of MEPA (400mg/kg) (p<0.01) as compared to control group animals at 2nd hour interval, while standard drug Dexamethasone (0.27mg/kg) was also showed significant reduction in paw volume at interval of 2nd ,3rd and 4th hour (p<0.5, p<0.01) respectively (Table 3).

Table 3: Effect of MEPA (400mg/kg) on passive paw anaphylaxis in rats

Sr.

no.

Groups

Paw edema volume (ml)

1st hr

2nd hr

3rd hr

4th hr

1

Normal Control

(0.5%w/v NaCMC)

0.970 ± 0.040

1.031 ± 0.027

0.983 ± 0.033

0.922 ± 0.035

2

MEPA

(400mg/kg)

0.960 ± 0.034

0.930 ± 0.038**

0.930 ± 0.036

0.920 ± 0.031

3

Dexamethasone

(0.27mg/kg)

0.820 ± 0.028*

0.771 ± 0.025**

0.801 ± 0.036**

0.771± 0.044**

All values represent as mean + SEM (n=6)

*p<0.05 & **p < 0.01, All treated group were compared with control group.

MEPA= Methanolic extract of Phyllanthus amarus

3.5 Milk induced leukocytosis in mice

There were significant increases in total leukocyte count in disease control (2850.00 ± 645.10) (p<0.001), as compared to control (416.66 ± 84.32) group and when MEPA (400mg/kg) (300±121.10) (p<0.001) treated group showed significant reduction in total leukocytes counts as compared to disease control(Table 4).

Table 4: Effect of MEPA (400mg/kg) on Total leukocytes counts in mice

Sr.no.

Groups

Total leukocytes per cu.mm.

Before

After

Difference

1

Control

(0.5% w/v NaCMC)

3250.00 ± 416.73

3666.00 ± 391.50

416.66 ± 84.32

2

Diseases control

3425.00± 149.30

6208.33 ± 504.54

2850.00 ± 645.10# # #

3

MEPA

(400mg/kg)

3566.01 ± 275.58

3850.00 ± 310.64

300 ± 121.10***

All values represent as mean + SEM (n=6)

# # #p<0.001 Disease control group compared with control group

***p < 0.001, Treated group was compared with Diseases control group.

MEPA= Methanolic extract of Phyllanthus amarus

3.6 Clonidine induced rat peritoneal mast cell degranulation

Clonidine (80µg/ml) produced disruption of the peritoneal mast cell which was significantly inhibited by pretreatment with MEPA (1.5-2mg/) (p<0.01) as compared to Positive control,This protection was comparable to reference standard Ketotifen (10µg/ml) (p<0.001) (Table 5).

Table 5: Effect of MEPA (1-2mg/ml) on Clonidine induced rat peritoneal mast cell degranulation

Sr. no.

Groups

Mast cell Degranulation

% Mast Cell Degranulation

% Inhibition of Degranulation

1

Positive control

71.00 ± 0.57

2

MEPA

(1mg/ml)

67.66 ± 1.02

33.33%

3

MEPA

(1.5mg/ml)

57.64 ± 1.08**

43.36%

4

MEPA

(2mg/ml)

45.66 ± 1.03**

55.33%

5

Ketotifen

(10µg/ml)

26.66 ± 0.88***

73.33%

All values represent as mean + SEM (n=6)

**p <0.01 & ***p < 0.001, All treated group were compared with Positive control group.

MEPA= Methanolic extract of Phyllanthus amarus

DISCUSSION

Bronchial asthma is characterized by increased airway reactivity to spasmogens. An initial event in asthma appears to be the release of inflammatory mediators (e.g. Histamine, Tryptase, Leukotrienes and prostaglandins). Some of these mediators directly cause acute bronchoconstriction, airway hyper responsiveness and bronchial airway inflammation.

In present study, for primary evaluation of bronchodilator activity, % protection in pre-convulsion time was measure in histamine induced bronchospasm model in guinea pigs. Many previous study showed histamine induced bronchospasm model is one of the standard evaluation technique for bronchodilatory activity. There was significant increase in pre-convulsion time in dose dependent manner was observed after treatment with MEPA and HFPA for 7 days.This result revealed that MEPA and HFPA produced powerful bronchodilator activity as like standard antiasthmatic drug ketotifen which posses mast-cell stabilizing and anti-histaminic activity by inhibiting stimulation and release of inflammatory cells like mast cell, eosinophils, neutrophils, macrophage.[4]. Most promising protection was observed in MEPA which was selected for further evaluation.

Anti-asthmatic agents are use-full in the treatment of asthma by inhibiting antigen-antibody reaction (AG-AB) and there by inhibiting the release of inflammatory mediator releasing in asthma[10,11]. Effectiveness of drug against allergic asthma is generally evaluated using passive paw anaphylaxis in rats. So in this study, MEPA was checked to find efficacy against Passive paw induced anaphylaxis model.

MEPA treatment shown significantly reduction in paw volume in passively sensitized rat, which indicates potential of MEPA in allergic condition of asthma. Hence this data proved the fact that Phyllanthus amarus methanolic extract might produced this effect in antibody mediated hypersensitivity reaction by inhibition of release of major inflammatory mediators in antigen-antibody mediated hypersensitivity reaction.

Mast cell degranulation is important event that initiation of immediate responses after exposure to allergens. Degranulated mastcells release many mediators of inflammation such as histamine, leukotrienes, platelet activating factors and chemotactic factor from mast cells. They play a significant role in airway inflammatory response such immediate hypersensitivity reaction like bronchial contraction, airway hyperresponsiveness as well as airway eiosinophilia[4,13].MEPA showed significant protection of rat peritoneal mast cells in dose dependent manner against clonidine in our study. This results indicate MEPA produced significant mast cell stabilizing potential in asthmatic animals and that's why also reduce release of important inflammatory mediators and ameliorate the symptoms of asthma.

Excessive stress or nerve debility may aggravate the symptoms of asthma, normalization effect of adaptogen can be observed in milk induced leukocytosis after administration of milk[11,12]. In milk induced leukocytosis, MEPA (400mg/kg) showed significant reduction in total leukocyte count indicating its effectiveness in stressful condition.

Phyllanthus amarus reported to possessed significantly anti-inflammatory activity against carrageenan, bradykinin, serotonin and prostaglandin E1-induced paw oedema[14]. In support to that, results of our study showed that Phyllanthus amarus was found to be effective in various experimental model allergic asthma. Further more, MEPA was potent Anti-allergic and Mast cell stabilizing activity, Bronchodilator and anti-eosinophilic activity appear to be involved in its mode of action.

Results of present study provide evidence that methanolic extract of whole plant of Phyllanthus amarus Schum and Thonn produced powerful bronchodilator activity, which might be due to suppression of many inflammatory mediators release from mast cell.

Probably above mention effect of methanolic extract Phyllanthus amarus Schum and Thonn might be due to inhibition of mast cell degranulation and infiltration of important inflammatory cells.

However, further investigation may be carried out to indentify active constituents; which are responsible for anti-asthmatic activity.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.