Synpsis References

Synopsis of M. Phil (Pharmacology) EFFECT OF ETHANOLIC EXTRACT OF EUGENIA JAMBOLANA ON ACETYLCHOLINE HISTAMINE SEROTONIN AND CALCIUM INDUCED CONTRACTION IN RABBIT ILEUM

By MUHAMMAD NAEEM RIAZ (B. Pham.)

Supervised by

Prof. Dr. Muhammad Azam Zia (MBBS, MCPS, M.PHIL)

Department Of Pharmacology & Therapeutics University Of Health Sciences Lahore

PARTICULARS OF THE RESEARCH SUPERVISOR

Name

Prof. Dr. Muhammad Azam Zia

:

Qualification

:

MBBS, MCPS, M.Phil

Designation

:

Professor and Head of Deptt. Of Pharmacology UHS, Lahore.

Present Place of Posting

:

Department of Pharmacology University of Health Sciences Lahore.

Acceptance of Responsibility _______________________ Signature_____________________da ted____________/2007

Official Stamp

2

PARTICULARS OF THE RESEARCHER

Name

:

MUHAMMAD NAEEM Riaz

Qualification

:

B.PHARMACY

Designation

:

Student M. Phil (Pharmacology)

Present Place of Posting :

Department of Pharmacology University of Health Sciences Lahore.

3

PROJECT SUMMARY Gastrointestinal smooth muscle spasm is the major cause of symptoms of different

gastrointestinal

disorders

such

as

irritable

bowel

syndrome,

inflammatory bowel disease, diverticular disease, motility disorders and colorectal cancer. It has long been recognized that fruits promote good health and have therapeutic properties. Eugenia Jambolana, a fruit of myrtacae family is known to contain flavonoids, phenolic compound and Ellagic acid. Ellagic acid is a phenolic compound that may decrease intestinal spasm. We hypothesize that ethanol extract of fruit pulp of Eugenia jambolana have antispasmodic effects. For testing the hypothesis, we will use isolated rabbit ileum. Acetylcholine, histamine, serotonin and calcium chloride will be used to induce contraction in isolated rabbit ileum. Antispasmodic effects of extract of Eugenia jambolana will be measured on forty rabbits by using kymograph /power lab instrument. It will be a comparative experimental study that will take about one year for completion. Effect of ethanol extract of Eugenia Jambolana fruit pulp on spontaneous activity of rabbit ileum will be found. Dose response curves of acetylcholine, histamine, serotonin and calcium induced contractions will be measured in absence and presence of different doses of extract and possible mode of action of extract will be determined. Results will be entered and processed in MS EXCEL. Muscle contraction will be measured using isometric transducer. Difference in proportions will be examined by using ANOVA. Before and after using the drugs, the changes will be compared and significance of the study will be judged. Value of p<0.05 will be taken as significant. Completion of this study will be a step in search of natural sources of antispasmodic compound in different intestinal disorders.

4

INTRODUCTION Spasm is a sudden, violent, involuntary contraction of muscles, associated with pain and interference with function, producing involuntary movement and distortion.1 Smooth muscle spasm may result from a local disorder in which cellular injury initiates the contractile process and local hormones or other excitatory or irritant substances are released. It may be the result of hyperactivity in efferent excitatory autonomic nerves or electrolyte disturbances that favor increased neuronal and muscle activity.2 Acetylcholine binds to muscarinic receptors on the smooth muscles of ileum and receptor-operated channels to open, thereby allowing sodium influx, which cause a depolarization of cell membrane. This depolarization opens voltage-dependent calcium channels and calcium ions enter the cell to induce the release of calcium from the sarcoplasmic reticulum. The sytosolic calcium then binds to calmodulin and contraction occurs. Histamine, which binds to H1 receptors on gastrointestinal smooth muscles, also initiates the same sequence of events like acetylcholine. Potassium ions directly depolarize the cell membrane, no receptors as such involved. Serotonin is believed to be act directly and indirectly also. Indirectly, acts on neuronal receptors to release Ach, and directly stimulate 5HT receptors in smooth muscles for contraction.3 Intestinal spasm occur in various disorders such as irritable bowel disease, inflammatory bowel disease, diverticular disease, motility disorders and colorectal cancer.4Many different therapeutic approaches have been used to relieve spasm including stool bulking agents, antidiarrheal agents, antiflatulants, dietary modifications and fiber supplements.5 Antimuscarinic agents are used for spastic bowel, calcium for hypocalcemic tetany, calcium channel blockers for various smooth muscle spasms, reflected as antispasmodic agents. Most anticholinergic agents contain

5

belladonna alkaloids like atropine, which may cause xerostomia, urinary hesitancy, drowsiness, palpitations, blurred vision, dryness of mouth and tachycardia .2 For better health and therapeutic properties, fruits contribute evidence based role. Eugenia Jambolana, a fruit of Myrtacae family, has been reported to contain sufficient amount of sodium, potassium, manganese, copper, cobalt, iron, zinc and phosphorus. The fruits contain glucose, mannose, sucrose, alanine, arginine,

asparagines,

tyrosine,

glutamine

and

cysteine

showed

by

chromatographic analysis.6 The fruit of Eugenia Jambolana also contains citric acid, mallic acid and gallic acid. Polyphenolic phytochemicals (flavanoids) have also been isolated from Eugenia Jambolana which includes Myrcetin, quercetin, kaempherol,delphenidine-3-gentioboside, malvidine-3-laminariboside, oleanolic acid. Flavanoids have been seen to have antispasmodic activity, compounds such as quercetin and isoquercetin are likely to contribute towards effect of Eugenia Jambolana in gastrointestinal tract.8 It has been shown that Eugenia Jambolana has antidiarrheal effect, ripped fruits of Eugenia Jambolana are noted to be the good diet in chronic diarrhea and dysentery and effective in convalescence diarrhea.9 However, its possible mode of action in isolated tissue preparations has not been studied. Therefore, the present study will be undertaken to find out the possible antispasmodic action of Eugenia jambolana to rationalize its use for GIT problems.

6

REVIEW OF LITERATURE Patients frequently seek medical help for functional gastrointestinal disorders. It is believed that many of the symptoms of functional gastrointestinal disorders, including irritable bowel syndrome and gastro-paresis, arise from alterations in smooth muscle function. When normal stimuli produce inappropriate response in the bowel, problems become apparent, continuing irritability or irritation of the gut can produce a spectrum of distressing symptoms including abdominal pain, altered bowel habits, nausea and vomiting as a result of increased motor activity of the intestine. This is the bases of the common “gastro colic” or “gastric spasm” reflex.10 The smooth muscle of the guinea pig ileum when exposed to acetylcholine, it shows a biphasic mechanical response. First phase consists of a rapid increase in tension, which reached a sharp peak, followed by a rapid downfall in tension, which depended on an initial mobilization of intracellular calcium, probably from the sarcoplasmic reticulum or from calcisomes, in an inositol triphosphate (IP3) dependent way. The second phase, which consists of a slower, much sustained increase in tension, usually of a lesser magnitude, called as tonic response. This response is entirely dependent on the extra cellular calcium and is due to an increased calcium influx across the membrane.11 In a study it has been shown that flavonoids obtained from different medicinal plants, such as clausmarin-A12 flavonoids, isoflavones, chalcones13 quercetin and isoquercetin8 have been reported to have spasmolytic activity on isolated tissues like guinea pig trachea and ileum. The flavonoids particularly quercitin is thought to be valuable as antispasmodic agent. It is able to relax intestinal smooth muscles and inhibit bowel contractions. In addition, other flavonoids also show antispasmodic activity.14 These evidences indicated that chemical compounds like flavonoids and coumarin should be investigated for the pharmacological activity.15, 16

7

Dietary antioxidants that compliment the action of enzymatic antioxidants, are now extensively examined including (alpha) tocoferol (vitamin E), ascorbic acid (vitamin C) and B-carotene.17 Eugenia jambolana belongs to family Myrtacae. Its fruit is variable in size up to 2.5cm long, ellipsoid or oblong, black with pink juicy pulp. The fruits are acrid and sweet, cooling, dry and astringent to bowels. Although Eugenia jambolana has been prescribed in various diseases including diabetes, diarrhea and dysentery but scientific proof needs more scientific work. Fruits of Eugenia jambolana have been included with raffinose, glucose, fructose, citric acid, mallic acid, and Gallic acid.7 Composition of water soluble vitamins of Eugenia jambolana fruit (per 100gm of sample) contains thiamine 0.12mg, niacin 0.272mg and ascorbic acid 30.0mg, showing that Eugenia jambolana fruit contain fair amount of minerals, vitamins, free sugars and amino acids and possess good nutritional value.6 The flavonoids isolated from Eugenia jambolana are Myrcetin, Delphenidine, Malvidine, Oleanolic acid, Kaemferol and Quercetin.7 The polyphenolic compounds, Quercetin and Myrcetin have been shown to inhibit the intestinal movement and reduce capillary permeability and this may explain the antidiarrheal and antispasmodic effect.14 The fruits are considered to be the good diet in convalescence diarrhea and dysentery and are effective in chronic diarrhea.9 Ethanol extract of the bark of Eugenia jambolana at dose of 400mg/kg reduced diarrhea by inhibiting gastrointestinal motility.18 It is also suggested that tannins extracted from Eugenia jambolana bark have gastro- protective and anti-ulcerogenic effects.19 However, its exact mode of action is not known so far.

8

HYPOTHESIS Ethanolic extract of Eugenia jambolana fruit pulp has antispasmodic effect in acetylcholine, histamine, serotonin and calcium chloride- induced contractions in isolated rabbit’s ileum.

9

AIMS AND OBJECTIVES -To study the effect of ethanolic extract of Eugenia jambolana fruit pulp on acetylcholine, histamine, serotonin and calcium chloride induced contractions in isolated rabbit’s ileum. - To compare the dose response curves of different groups.

10

MATERIAL AND METHODS Study Design: It is a comparative interventional experimental study.

Setting: Study will be carried out in the Department of Pharmacology and Therapeutics, University of Health Sciences, Lahore.

Duration of Study: Study will be carried out in about one year. First six months will be utilized in data collection, next six months for data analysis and thesis writing.

Sample Size: Study will be carried out on forty rabbits, purchased and kept in animal house of University of Health Sciences, Lahore.

Preparation of Ethanol Extract of Eugenia Jambolana Fruit: Two kilograms fruit of Eugenia jambolana (jamun) will be procured from fruit shop and identified with the help of botanist and University of Punjab. It will be washed well and pulp will be separated from the seeds. One kilogram of Fruit pulp will be stirred in 95% ethanol in an electrical stirrer for 15 hrs at room temperature. It will be then filtered with the help of a filter funnel and the filtrate will be evaporated to paste like mass using rotary evaporator at 45oC under reduced pressure20. The extract will be standardized from PCSIR.

11

PREPARATION OF EXPERIMENTAL ANIMAL Local rabbits of 1-1.5 kg will be used in this study. Animals will be kept in the experimental research laboratory of UHS under standard environment (23-25°C). All the animals will be fed and water ad libitum.

Animals will be kept fasting overnight. In the morning animal will be sacrificed by cervical dislocation, and dissection will be carried out to obtain the desired part of intestine. Ileum from rabbits will be used for the study of spasmolytic activity. Intestinal contents will be washed with Tyrode’s solution if present after overnight fasting. The respective intestinal portions of the animals will be removed and the segments of about 2cm long will be hanged in a 10 ml tissue bath containing Tyrode’s solution, bubbled with a mixture of 95% oxygen and 5% carbon dioxide (carbogen gas) and maintained at 37ºC. The tissue will be allowed to equilibrate for 30 minutes. One end of the segment will be attached to the metal tissue hook and the other will be attached by a cotton thread to an isometric transducer and connected to a Power Lab data recording system. A preload of 1 g will be applied to each tissue. The time cycle of 6 minutes duration will be operated and a particular step will be performed at particular time.3 Responses will be recorded and labeled using Power Lab data recording system coupled with Chart Software.21 Tissues will be stabilized with maximal dose of acetylcholine, histamine, serotonin and Kcl.3 The plant material will be applied in different concentrations ranging from 0.5-4mg/ml to see whether it has spasmolytic activity. The possible mode of action(s) and comparison of spasmolytic activity of extract will be carried out in this experiment. Shift of different dose-response curves of agonists will be recorded and analyzed in presence and absence of different doses of Eugenia jambolana extract.

12

The following sequence explains the operation of a 6 minutes cycle. STEPS IN 6 MINUTES CYCLE

At 0 min

start Chart software

At 0-30 min

Stabilized tissue to get baseline recordings

30 sec-60 sec 1 min 2 min

add drug and mark Wash the preparation Wash again if the contractions do not

3 min- 4 min 5 min

return to baseline. Repeat the dose of drug / extract wash preparation with Tyrode,s

6 min

solution Start Chart software

GROUPING OF ANIMALS Animals will be divided into four groups.

GROUP A: EFFECT OF ACETYLCHOLINE 1. Tissue will be stabilized using sub maximal dose of Ach (0.1 μmol )after every 3-5 minutes with washing after 30 sec.the tissue will be considered stabilized when 2-3 doses give equal response. 2. Start will be with lowest concentration of Ach, wait for 30 sec,and washing will occur. Same cycle will be repeated for each dose, till to find no further increase in response on increasing the dose (produced ceiling effect). 3. Minimum dose of extract of Eugenia jambolana fruit extract will be added to the tissue bath and wait for 10 min.Repeat step (2) in the presence of extract. Extract will be added immediately after washing of each dose of Ach (45 sec). Same method will be performed for each increasing dose of extract. The graded dose response curve will be presented in absolute values or as a percentage of the maximal response. The concentration-response curve will be plotted in Arithmetic or logarithmic scale. 13

GROUP B: EFFECT OF HISTAMINE 1. Tissue will be stabilized using sub maximal dose of histamine (0.1 μmol) after every 3-5 minutes with washing after 30 sec.the tissue will be considered stabilized when 2-3 doses give equal response. 2. Start will be with lowest concentration of histamine, wait for 30 sec,and washing will occur. Same cycle will be repeated for each dose, till to find no further increase in response on increasing the dose(produced ceiling effect). 3. Minimum dose of extract of Eugenia jambolana fruit extract will be added to the tissue bath and wait for 10 min.Repeat step (2) in the presence of extract. Extract will be added immediately after washing of each dose of histamine (45 sec). Same method will be performed for each increasing dose of extract. The graded dose response curve will be presented in absolute values or as a percentage of the maximal response. The concentration-response curve will be plotted in Arithmetic or logarithmic scale.

GROUP C: EFFECT OF SEROTONIN 1. Tissue will be stabilized using sub maximal dose of serotonin (0.1 μmol) after every 3-5 minutes with washing after 30 sec.the tissue will be considered stabilized when 2-3 doses give equal response. 2. Start will be with lowest concentration of serotonin, wait for 30 sec,and washing will occur. Same cycle will be repeated for each dose, till to find no further increase in response on increasing the dose(produced ceiling effect). 3. Minimum dose of extract of Eugenia jambolana fruit extract will be added to the tissue bath and wait for 10 min.Repeat step (2) in the presence of extract. Extract will be added immediately after washing of each dose of serotonin (45 sec). Same method will be performed for each increasing dose of extract. The graded dose response curve will be presented in absolute values or as a percentage of the maximal response. The concentration-response curve will be plotted in Arithmetic or logarithmic scale. 14

.

GROUP D: EFFECT OF CALCIUM & POTASSIUM Effect of extract will be seen on CaCl2 induced contractions in this group. 1. Tissue will be pretreated with K+ (50mM) and lower dose of plant extract will be added and changes will be changed. Dose of extract will be increased in cumulative fashion to obtain inhibitory responses. 2. The normal tyrode’s solution will replaced with Ca 2 free tyrode’s solution containing EDTA (0.1 mM) for 30 minutes, in order to remove calcium from the tissues. 3. This solution will be further replaced with K+ -rich and Ca2 free tyrode’s solution which will be in following composition in mM; Kcl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.87, NaH2PO4 0.41, glucose 5.55 & EDTA 0.1. 4. Following the incubation period of 30 min,control dose response curve of Ca+ will be obtained, till to get no further increase in response on increasing the dose (ceiling effect). 5. The tissue will be pretreated with plant extract and wait for 20 minutes. The dose response curve of Ca2 will be obtained in presence of different doses of extract.

Drugs 1. Ethanol extract of Eugenia jambolana fruit pulp 2. Acetylcholine chloride 3. Histamine dihydrochloride

15

4. Serotonin creatine sulfate 5. CaCl2 6. KCl 7. EDTA

Doses 1)

Dose of fruit extract: 3

0.5-4mg/ml.

2)

Dose of Acetylcholine: 3

1ml of 10 -9 M to 10-4

3)

Dose of Histamine 3

1ml of 10 -8 M to 10-4

4)

Dose of Serotonin 3

1ml of 10-8 to 10-5 M

5)

Dose of CaCl2 3

6)

1ml of 10-3 to 3.8 x 10-1

Dose of KCl 3

7)

50 mM

Dose of EDTA3

0.1 Mm

POWER LAB DATA SYSTEM SETTING: Number of channels:

1

Range:

2-10 mv or as appropriate

Sampling speed:

20-40 samples/sec

Filters:

50 Hz, Low pass 10Hz

Preload:

1 gram.

Composition of Tyrode’s Solution The Tyrode,s solution will be in the following Com;mM: NaCl 136.8; KCl 2.7; CaCl2 1.3; NaHCO 3 12.0; MgCl 2 0.5; Na 2 PO 4 0.14 and Glucose 5.5.22

STATISTICAL ANALYSIS

16

The result will be expressed as mean ± SEM. All statistical comparison will be made by means of students’t test or ANOVA when appropriate and a P value smaller than 0.05 regarded as significant.

OUTCOME & UTILIZATION This study would be a step forward in search of natural sources as spasmolytic agent and other gastrointestinal disorders.

17

REQUIRMENTS Sr. No.

Requirements

18

Amount(Rs.)

1

40 Rabbits

2

Diet for Rabbits

3

Power lab instrument and transducer Graph paper Kymograph

4

Drugs Eugenia Jambolana fruit Acetylcholine Histamine Serotonin EDTA KCl & CaCl2 Tyrode’s solution chemicals

5

6

Miscellaneous (Syringes, Gloves, Chloroform, Ethanol, jars and pipettes) Computer papers and accessories

Total

19

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