evaluation of punica grantum stem bark for anti diarrhoeal activity, anti diarrhoeal activity of punica grantum

1. Presented by Under the Guidance of
Mr,Alimudden ,M.Pharm IInd Year Dr. Rajeev Tomar Sir
Uttarakhand Technical University,Dehradun.

2. Content
 Introduction
 Objectives & need
 Methodology
 Result & Discussion
 Summary

3. Introduction
 Diarrhoea is considered a common condition and a relatively minor
one at that. However in reality diarrhoea can be a very serious
affliction and in extreme cases can cause serious internal system
damage.
 Punica granatum Linn. belonging to family Punicaceae is taken for
this antidiorrheal studies.
 The different parts of the plant used traditionally in treatment of
diarrhoea and dysentery.
 The various parts of this plant are reported to be having antioxidant,
antibacterial and antifungal and antidiabetic properties. However,
the anti-diarrhoeal efficiency of the Punica granatum stem bark has
not been scientifically investigated so far. Hence, the present study is
undertaken.

4. Objectives
 To study the extensive review of literature of “Evaluation of Punica
granatum stem bark extract for antidiarrhoeal activity”
 To perform various experiments, which involves:
 Extraction of stem bark of the Punica granatum with 70% hydroalcohol
by Soxhlets extraction.
 Preliminary phytochemical screening of test extract.
 Determination of LD50 of the test extract on female albino mice.
Evaluation of Punica granatum stem bark extract for
antidiarrhoeal activity by employing the following different
experimental animal models of diarrhoea.
 Castor oil induced diarrhoea.
 Magnesium sulphate induced diarrhoea.
 Castor oil induced enteropooling.
 Gastrointestinal motility test (Charcoal meal test).

5. Methodology
Collection of plant material and preparation of extract:
Fresh stem bark collected were cleanedshade dried at room
temperature coarse powdered and then extracted with
70% hydroalcohol by using Soxhlets apparatus. Thereafter,
the extract was concentrated by flash evaporator. The yield
obtained was found to be 23%. The dried crude extract was
stored in refrigerator below 100C for further studies.
The crude EEPGSB was subjected to following studies.
 1.Preliminary phytochemical screening.
 2.Determination of acute toxicity studies.
 3.Antidiarrhoeal activity.

6. Animals used
 Albino rats (Wistar strain) weighing 150-200 g of either
sex and albino mice weighing 20-25g of either sex were
used in the present study.
 The animals were acclimatized for ten days under
standard laboratory condition. They were housed in
polypropylene cages and maintained at 270C± 20C,
relative humidity 65±10% under 12 hr light/dark cycle.
The animals were feed with rodent pellet diet and water
ad libitum.

7. 1.Preliminary phytochemical screening
The preliminary phytochemical investigation
was carried out for the EEPGSB for the
detection of various phytoconstituents. Tests
for the presence of common phytochemicals
were performed by standard methods
described by Kokate C.K. and K.R.
Khandalwal.

8. 2.Determination of acute toxicity (LD50)
The acute toxicity (LD50) of EEPGSB was
determined by fixed dose method (OECD
guide line no. 423) of CPCSEA. The female
albino mice weighing between 20- 25g were
fasted for 24 hr prior to experiment 1/20th,
1/10th and 1/5th LD50 cutoff value of the
extract were selected as screening doses for
the dose dependent study.

9. 3.Antidiarrhoeal activity
(A) Castor oil induced diarrhoea
 Rats of either sex (150-200 g) were fasted for 18 hr and
allocated to five groups of six animals each.
 Group I -Control
 Group II -Standard (loperamide 3 mg/kg p.o.)
 Group III -EEPGSB (10 mg/kg p.o)
 Group IV -EEPGSB (20 mg/kg p.o.)
 Group V -EEPGSB (40 mg/kg p.o.)

10. After 60 min of treatment, the animals of each group
received 1 ml of castor oil by gavage. The onset of
diarrhoea, number of fecal droppings and mean weight
of stool were noted up to 4 hr in the transparent plastic
dishes placed beneath the individual rat cages. Anti-
diarrhoeal activity was determined in terms of
percentage of protection, which was calculated by
following formula:
Percentage of protection (%)=
Mean wt of stool of control animal- Mean wt of stool of drug/extract treated animal_ X 100
Mean wt of stool of control animal

11. (B)Magnesium sulphate induced diarrhoea
 Rats of either sex (150-200 g) were fasted for 18 hr and
segregated into five groups of six animals each.
 The animals were treated as follows
 Group I -Control
 Group II -Standard (loperamide 3 mg/kg p.o.)
GroupIII -EEPGSB (100 mg/kg p.o)
 Group IV -EEPGSB (250 mg/kg p.o.)
 Group V -EEPGSB (500 mg/kg p.o.)

12. After 60 min of treatment, the animals of each group
received magnesium sulphate (2 g/kg) by gavage. The
onset of diarrhoea, number of fecal droppings and
mean weight of stool were noted up to 4 hr in the
transparent plastic dishes placed beneath the individual
rat cages.

13. (C)Castor oil induced enteropooling
Rats of either sex (150-200 g) were fasted for 18 hr and
divided into five groups of six animals each.
 Group I -Control
 Group II -Standard (Atropine sulphate 5 mg/kg i.m.)
Group III -EEPGSB (100 mg/kg p.o
 Group IV -EEPGSB (250 mg/kg p.o.)
 Group V -EEPGSB (500 mg/kg p.o.)

14. The above shown treatment was made 1 hr
prior to castor-oil administration to all the
rats. Thirty minutes later, the rats were
sacrificed, exsanguininated after ligating at
both the pyloric and the ileocaecal junctions.
The intestinal contents were expelled into a
graduated measuring cylinder to record the
volume of intestinal fluid.

15. (D)Gastro intestinal motility test in rats
(Charcoal meal test)
Wistar rats of either sex (180-200 g) were fasted for 18
hr but allowed free access to water. The animals were
randomly allotted to five groups of six animals each.
 Group I -Control
 Group II -Standard (atropine sulphate 5 mg/kg, i.p.)
 Group III -EEPGSB (10 mg/kg p.o)
 Group IV -EEPGSB (20 mg/kg p.o.)
 Group IV -EEPGSB (40 mg/kg p.o.)

16. After 30 min of the above treatment, each rat was
administered orally 1 ml charcoal meal (3% charcoal in
5% aqueous tragacanth). The animals of all the groups
were killed 30 min later by cervical dislocation. The
small intestine from pylorus to ceacum rapidly
dissected out and placed on a clean surface. The
distance traversed by the charcoal meal from the
pylorus to caecum was measured. The percentage
movement of charcoal meal and percentage of
inhibition was calculated by following formulas.
Percentage of travelled=Distance travelled by charchoal meal X 100
Total length of small intestine

17. Results
Results of preliminary phytochemical screening of
EEPGSB
Phytoconstituents Inference
Alkaloids +
Carbohydrates ++
Flavonoids ++
Glycosides +
Tannins +++
Steroids -

18. Result
Effect of EEPGSB on castor oil induced diarrhoea in rats
Grps Treatment
Dose mg/kg Onset of
diarrhoea
(min) ± SEM
Mean No of
fecal drops ±
SEM
Mean wt of
feaces (g) ±
SEM after 4hr
%
protection
1 Control --
31.33 ±
03.48
8.33 ±
0.80
3.78 ±
0.37 --
2
Standard
(Loperamide) 03
63.50 ±
03.10**
2.83 ±
0.47***
1.15 ±
0.14*** 69.57
3 EEPGSB 10
38.66 ±
02.26ns
6.16 ±
0.83ns
3.30 ±
0.48ns
12.69
4 EEPGSB 20
56.66 ±
02.23*
5.01 ±
0.25**
2.55 ±
0.29ns 32.53
5 EEPGSB 40
53.16 ±
10.67ns
3.66 ±
0.42***
2.22 ±
0.31*
41.26

19. Result
Grps Treatment
Dose mg/kg
Onset of
diarrhoea
(min) ±
SEM
Mean No of
fecal drops ±
SEM
Mean wt of
feaces (g) ±
SEM after 4hr
%
protection
1
Control --
39.50
±1.96
7.16 ±0.40 2.53 ±0.27 --
2 Standard
(Loperamide)
03
86.01
±2.65***
3.16
±0.30***
1.50 ±0.12** 40.71
3
EEPGSB 10 43.50
±6.2ns
5.33 ±0.66* 2.44 ±0.17ns 03.82
4 EEPGSB 20
62.16
±3.16**
3.66±0.21*** 1.86 ±0.14ns 26.48
5 EEPGSB 40
73.33
±2.61***
4.01
±0.44***
1.68 ±0.18* 33.59
Effect of EEPGSB on MgSo4 induced diarrhoea in rats

20. Result
Groups Treatment Dose mg/kg Mean volume of
intestinal fluid
(ml) ±
SEM
Mean weight of
intestinal fluid
(ml) ±
SEM
%
Inhibit ion
1 Control --- 02.10 ±
0.19
6.05 ±
0.31
---
2 Standard
(Atropine
sulphate)
05 0.75 ±
1.18***
4.06 ±
0.09***
42.97
3 EEPGSB 10 1.60 ±
0.50*
4.94 ±
0.36*
21.15
4 EEPGSB 20 1.26 ±
0.83***
4.77 ±
0.25**
26.61
5 EEPGSB 40 1.16 ±
0.93***
4.60 ±
0.49***
32.89
Effect of EEPGSB on castor oil induced enteropooling in rats

21. Result
Groups Treatment Dose mg/kg
Mean distance
traveled by
charcoal meal
(cm) ±
SEM
Mean
%movement of
charcoal ± SEM
%
Inhibition
1 Control --- 66.58 ± 1.77 72.60 ± 4.15 ---
2 Standard
(Atropine
sulphate)
05 49.33 ± 2.72 52.43 ±
2.70***
47.61
3 EEPGSB 10 65.33 ± 0.62 70.00 ±
0.19ns
29.86
4 EEPGSB 20 56.08 ± 2.35 60.72 ±
3.33*
39.53
5 EEPGSB 40 51.41 ± 2.44 55.46 ±
2.69***
44.57
Effect of EEPGSB on gastro-intestinal motility (Charcoal meal test) in rats

22. DISCUSSION
 In this context, the investigation of the antidiarrhoeal effect
of Punica granatum for its effect on intestinal transit and
fluid accumulation was carried out.
 Literature study reveals that, the castor oil will induce
diarrhoea through different mechanisms. These
mechanisms includes due to its hypersecretory functions
mediated by ricinoleic acid, the most active component of
castor oil, through reduction of normal fluid absorption by
inhibiting intestinal Na+, K+ -ATPase, stimulation of
prostaglandin formation, platelet activating factor and
though nitric oxide.
 Though these several mechanisms have been proposed,
mechanism of action of castor oil induced diarrhoea is still
not clear.

23. DISCUSSION
 In the present study, the ethanolic extract of Punica
granatum significantly inhibited castor oil induced
diarrhoea.
 This antidiarrhoeal activity of the extract may be because of
its antisecretory mechanism and it was also evident from
the significant delay in the onset of diarrhoea and decrease
in purging frequency (reduction of number of wet stool,
weight of wet stool and severity of dirrhoea).
 Further it was also supported by antidiarrhoeal index
(ADI).
 The higher ADI value the greater the effectiveness in the
treatment of diarrhoea.

24. DISCUSSION
 In magnesium sulphate induced model, the diarrhoea is
induced by increasing the volume of intestinal content
through prevention of reabsorption of water.
 It has also been proposed that the magnesium sulphate
promotes the cholecystokinin released from the duodenal
mucosa which in turn stimulates the secretion and
increased the motality of small intestine and their by
prevents the reabsorption of sodium chloride and water.
 The plant extract was exhibited a significant anti-diarrheoal
property which may be mediated through absorption of
water and electrolyte from the GIT, since the extract delayed
the GIT transist time in mice.

25. DISCUSSION
 In the present investigation also the observed
significant antidiarrhoeal property of stem bark extract
of title plant may be attributed to flavonoids and
tannins content in the test extract which was evident by
preliminary phytochemical investigation.

26. CONCLUSION
 In conclusion, the result of this investigation revealed
that 70% hydroalcoholic stem bark extract of Punica
granatum contains pharmacologically active
substance(s) with antidiarrhoeal efficacy.
 Scope for the further study
Further research has to be carried out to fractionate
and purify the test extract, in order to find out the
molecule responsible for observed antidiarrhoeal
activity.

27. SUMMARY
 In the present investigation Petroleum ether, 70% ethanolic
extract of Punica granatum stem bark have been investigated for
the preliminary phytochemical analysis, acute toxicity, and anti-
diarrhoeal activity.
 The results are summarized as follows:
 In preliminary phytochemical screening ethanolic extract showed
presence of carbohydrates, flavonoid and tannins.
 Acute toxicity study was carried out on 70% extracts of Punica
granatum stem bark and the doses selected for the evaluation of
anti-diarrhoeal activity were based on LD50 cutoff value.
 Ethanolic extract (40 mg/kg doses) of Punica granatum showed
more potent anti- diarrhoral activity against all tested models.
 The anti-diarrhoeal efficacy of the test extract could be due to the
presence of flavonoids and tannins.

28. Thank You