Antiulcer Screening of Selected Plants of Plumbago Species and the Effect of Plumbagin in Gastric Cell Lines

INDEX

1. Abbreviations

2. Contents

3. List of tables

4. List of figures

5. Introduction

6. Literature review

7. Plant profile

8. Hypothesis, aim & objective

9. Plan of work

10. Materials & methods

11. Results

12. Discussion

13. Conclusion

14. References

15. Appendix

Declaration

I hereby declare that the dissertation work entitled “Antiulcer screening of selected plants of Plumbago species & the effect of Plumbagin in Gastric cell lines” is a original research work carried out in the Post Graduate & Research Laboratory of Dept. of Pharmacology in the year 2011-2012 under the guidance and direct supervision of Dr. SIBI P.I, Assistant Professor & Head, Dept. of Pharmacology, University College of Pharmacy, Cheruvandoor campus of Mahatma Gandhi University, Kottayam. The work embodied in this thesis is original and has not been submitted the basis for the award of any degree/ diploma/associateship/fellowship of any other university/institution.

Ann Shine Paul

University College of Pharmacy, Cheruvandoor campus,

Mahatma Gandhi University

Copyright

Declaration by the candidate

I hereby declare that the University College of Pharmacy, Cheruvandoor campus, Mahatma Gandhi University, Kottayam, Kerala shall have all rights to preserve, use and disseminate this dissertation in print or electronic format for academic/research purpose.

ANN SHINE PAUL

INSTITUTIONAL ANIMAL ETHICAL COMMITTEE University College of Pharmacy

Certificate

This is to certify that the dissertation work entitled “Antiulcer screening of selected plants of Plumbago species & the effect of Plumbagin in Gastric cell lines.” has been approved by the Institutional Animal Ethical Committee, University College of Pharmacy, Cheruvandoor Campus, Mahatma Gandhi University. The IAEC approval No. is [008/MPH/UCP/CVR/12].

IAEC Secretary IAEC Chairman

“For everything God created is good and nothing is to be rejected if it is received with thanksgiving, because it is consecrated by the word of God and prayer”

1 Timothy 4:4-5

ACKNOWLEDGEMENT

When emotions are profound, words may not be sufficient to express thanks and gratitude. The wisdom commitment and the effort of many people source of inspirations to carry out this research work. Many people provided me valuable contributions and gave helpful comments. People world over enjoy growing flowers, but only a few realize that one grow a garden that will tell the time of this day.

Acknowledging a dissertation work represents a silhouette of magnanimity and help rendered by our family and friends. It gives me immense pleasure and contentment to acknowledge and thank all those who in big and small ways have contributed for this effort.

Firstly, I offer my adoration to my internal belief “God Almighty” who created me & always flowers his blessings on me. Grave me the strength and courage to complete my dissertation and gave me the opportunity to thank all those people whom his grace was delivered to me.

I pay my tribute to my beloved parents Mr. Paul K.P and Mrs. Sherly A.L, sisters Ann Linet Paul and Angela Paul whose uncompromising life principles, love, affection, trust, patience, support has been always unshared and showered upon me at all stages of life and giving me more than what I deserved in my life.

With great pleasure and profound sense of gratitude, I express my most cordial and humple thanks to my eminent, respected teacher and guide Dr. Sibi P.I, Asst. Professor & Head,

Department of Pharmacology, University College of Pharmacy, for his valuable guidance, keen interest, inspiration, unflinching encouragement and moral support throughout my dissertation work.

I am extremely thankful to Mr. Krishnakumar K.R, principal for providing adequqte infrastructure and constant encouragement without which I would haven’t comleted my dissertation in time.

It’s my great privilege to express my profound thanks and immense sense of gratefulness to teaching faculty Mr. Pramod C, Mrs. Litty Jeoseph, Mr. A.J Chacko, Mr. Anilkumar, Mr.Sajan Jose, Mrs. Bobby.S. Prasad, for their whole hearted support and encouragement from the beginning to the end. My special thanks to all lab technicians and other faculties for their kind co-operation during my dissertation work.

I extend my deep sense of gratitude to Mr. Joby Paul, Department of environmental Sciences for identifying and authenticating of the plant. I am gratefully indebted to Dr. Renji O. Raphel, Pathologist, Speciality Diagnostic Centre, Irinjalakuda and Dr. Hashim, U-win Life Sciences, Malappuram for their valuable support and suggestions and all the help during my experiments.

I owe a special thanks to my lovely friends Safeera Hamza, Rosemary jose, Shereen Sunny, Liby Baby, Muhammed Noushad, Sajid K.P, Habbeb Rahman P.K, Ummarul Mukthar A.K, Shenika Mary Simon, Meera Paul, Elizabeth Mathachan, Pilmy Rose Wilson, Veena Vijayan, Jiji K.N and all my batch mates for their good advices and ideas, their helping hands, and notably their friendship.. I express my thanks to juniors especially Anurenj B.A, Krishnadas

M.S and Mithun T. Valiyaveetil for their consistent help. I extend my thanks to my roommate Neelima N. Pillai for being with me during my compiling of thesis work.

From the deepest depth of my heart I articulate my gratitude to my best friend Aneesul Islam N, who constantly inspired me to think upon the problems and to solve them, and for being with moral support in translating this work into reality.

I am expressing humble thanks to all the mentors, well wishers and near & dear ones, who helped me in their own way.

Thanks to one and all.

ANN SHINE PAUL

DEDICATED TO MY BELOVED PARENTS ON

WHOSE DIVINE HANDS, THE GOD ALMIGHTY

HAVE ENTRUSTED ME

ABBREVIATIONS

Abbildung in dieser Leseprobe nicht enthalten

Contents

1. Introduction

2. Literature review
2.1.1. Normal anatomy of the stomach
2.1.2. Histology
2.2.1. Gastric ulcer and peptic ulcer disease
2.2.2. Pathophysiology
2.2.3. The regulation of acid secretion by parietal cells
2.2.4. Alteration of mucosal defense and healing
2.2.5. Abnormal gastric motility
2.3. Etiology-specific pathophysiology
2.3.1. Helicobacter pylori
2.3.2. Nonsteroidal anti-inflammatory drugs
2.3.3. Zollinger-Ellison's syndrome and other hypersecretory 20 conditions
2.3.4. Stress-related erosive syndrome
2.3.5. Gastroesophageal reflux disease
2.3.6. Signs and symptoms
2.4. Treatment
2.5. Gastric cytoprotective effects
2.6. Animal models used in the screening of anti ulcer activity
2.6.1. Aspirin induced ulcers
2.6.2. Ethanol induced ulcers
2.6.3. Pylorus ligation induced ulcers
2.6.4. Water immersion stress induced model
2.6.5. Indomethacin induced ulcers
2.6.6. Histamine induced ulcers
2.6.7. Reserpine induced ulcers
2.6.8. Serotonin induced ulcers
2.6.9. Acetic acid induced ulcers
2.6.10. Hydrochloric acid induced ulcers
2.7. Role of antioxidant studies in antiulcer screening
2.7.1. Free radical and radical reaction
2.7.2. Antioxidant defense
2.7.3. Mode of action antioxidants
2.7.4. Concept of oxidative stress
2.7.5. Molecular damage induced by free radicals
2.7.5.1. Lipid peroxidation
2.7.5.2. Dpph free radical scavenging assay
2.7.6. Oxidative stress in gastric mucosa
2.7.7. Endogenous gastroprotective molecules
2.7.8. Oxidative stress in the stomach and acute ethanol 35 toxicity
2.7.9. Gastric ulcers and erosions caused by ethanol
2.8. Antiulcer properties of specific phytochemicals
2.8.1. Herbal medicines and natural compounds used in treatment of ulcer
2.8.2. Alkaloids
2.8.3. Flavonoids
2.8.4. Saponins
2.8.5. Tannins

3. Plant profile
3. 1. Plumbago species
3.1.1. Classification
3.1.2. India
3.1.3. Western Europe
3.1.4. Traditional South African uses
3.1.5. European medicinal uses
3.2. Plumbago auriculata
3.2.1. Synonyms
3.2.2. Vernacular names
3.2.3. General information
3.2.4. Traditional uses
3.2.5. Pre-clinical data- pharmacology
3.2.6. Toxicities
3.3. Plumbago indica
3.3.1. Synonyms
3.3.2. Vernacular names
3.3.3. General information
3.3.4. Traditional uses
3.3.5. Pre-clinical data- pharmacology
3.3.6. Toxicities
3.4. Plumbago zeylanica
3.4.1. Synonyms
3.4.2. Vernacular names
3.4.3. General information
3.4.4. Traditional uses
3.4.5. Pre-clinical data- pharmacology
3.4.6. Toxicities

4. Hypothesis, aim & objective

5. Plan of work

6. Materials & methods
6.1. Materials
6.2. Extraction
6.2.1. Herbal plant collection
6.2.2. Plumbgin extraction
6.2.3. Preparation of plumbagin free alcoholic extract
6.2.4.Peliminary phytochemical analysis
6.2.5.Estimation of the amount of plumbagin in the extracts
6.3. In-vitro methods:-
6.3.1. Micro culture tetrazolium(mtt) assay
6.3.2. DPPH assay
6.3.3. Lipid peroxidase assay
6.3.4. Acid nuetralising capacity
6.3.5. Ealuation of ethanol induced antiulcer activity
6.3.6. Statistical analysis
6.4. In-vivo methods
6.4.1.Aspirin induced model
6.4.2. Ethanol induced model
6.4.3. Histological studies
6.4.5. Statistical analysis

7. Results
7.1. Extraction
7.1.1. Herbal plant collection
7.1.2. Plumbagin extraction
7.1.3. Preparation of Plumbagin free alcoholic extract
7.1.4.Preliminary phytochemical analysis
7.1.5.Estimation of the amount of Plumbagin in the extracts
7.2. In-vitro methods:-
7.2.1. Micro culture tetrazolium(mtt) assay
7.2.2. DPPH assay
7.2.3. Lipid peroxidase assay
7.2.4. Acid nuetralising capacity
7.2.5. Evaluation of ethanol induced antiulcer activity
7.2.6.Statistical analysis
7.3. In-vivo methods
7.3.1.aspirin induced model
7.3.2. Ethanol induced model
7.3.3. Histological studies
7.3.4. Statistical analysis

8. Discussion

9. Conclusion

10. References

LIST OF TABLES

2.1 Different stages in tissue damage

3.1 Classifications of plants used in the study

3.2 Vernacular names of P.auriculata

3.3 Vernacular names of P.indica

3.4 Vernacular names of P.zeylanica

6.1 Materials required in the study

6.2 Ulcer scoring in the in-vivo models

7.1 Percentage yield of 3 Plumbaginales

7.2 Phytochemical analysis of different extracts of 3 73 Plumbaginales

7.3 Optical density obtained for plotting calibration curve

7.4 Estimation of Plumbagin in the extracts

7.5 Optical density of the extracts in MTT assay

7.6 Percentage viability of the extracts in MTT assay

7.7 Percentage Cytotoxicity of the extracts in MTT assay

7.8 Mean Percentage Cytotoxicity ± SD shown in MTT assay

7.9 Summary of Tukey’s Multiple comparison test in MTT assay

7.10 Probit values of each samples

7.11 IC50 of the extracts

7.12 Percentage inhibition of Chloroform extract in DPPH 81 assay

7.13 Percentage inhibition of P. indica in DPPH assay

7.14 Percentage inhibition of P. zeylanica in DPPH assay

7.15 Percentage inhibition of P.auriculata in DPPH assay

7.16 Summary of Tukey’s Multiple comparison test in DPPH 82 assay

7.17 IC50 values of the extracts using DPPH assay

7.18 Percentage inhibition of chloroform extract in Lipid 84 peroxidase assay

7.19 Percentage inhibition of P. indica in Lipid peroxidase assay

7.20 Percentage Inhibition of P. zeylanica in Lipid peroxidase 85 assay

7.21 Percentage Inhibition of P.auriculata in Lipid peroxidase assay

7.22 Summary of Tukey’s Multiple comparison test in Lipid 86 peroxidase assay

7.23 IC50 values of the extracts using DPPH assay

7.24 Acid nuetralising capacity of the extracts

7.25 Number of ulcer spots in ethanol induced in-vitro models

7.26 ANOVA summary of ethanol induced in-vitro models

7.27 Antiulcer effect in Aspirin induced ulcer models in rats

7.28 Antiulcer effect in Ethanol induced ulcer models in rats

7.29 Histopathological scoring in aspirin induced models

7.30 Histopathological scoring in ethanol induced models

LIST OF FIGURES

2.I Normal anatomy of stomach

2.2 Normal gastric histology

2.3 Histology showing parietal cells

2.4 Histology showing chief cells

2.5 Stages of cell errosional damage

2.6 H.pylori in the stomach

2.7 Changes in gastric endoscopy after H.pylori infection

2.8 Mechanism of gastric protection

2.9 Gastric protection before & after treatment

2.10 Oxidative stress & physiological consequences of acute ethanol intoxication in gastric mucosa

3.1 Plumbago auriculata & roots

3.2 Plumbago indica & roots

3.3 Plumbago zeylanica & roots

7.1 Authentification of P.auriculata

7.2 Authentification of P.zeylanica

7.3 Authentification of P.indica

7.4 Calibration curve of Plumbagin

7.5 Estimation of Plumbagin present in the extracts

7.6 Percentage Cytotoxicity of the extracts

7.7 IC50 calculation of the extracts

7.8 Graph showing Percentage inhibition of four extracts in DPPH assay

7.9 IC50 calculation of the extracts using DPPH assay

7.10 Graph showing Percentage inhibition of extracts in Lipid Peroxidase Assay

7.11 IC50 calculation of the extracts using Lipid peroxidase assay

7.12 Acid nuetralising capacity shown by different extracts.

7.13 In-vitro evaluation of Ethanol induced ulcer models

7.14 Ulcer shown in goat ileum a) Plumbagin b) P.indica C) P.zeylanica d) P. Auriculata e) Normal saline

7.15 Graph of ulcer index of aspirin induced model

7.16 Gastric ulcers shown by a) Positive control B) Ranitidine c) P. Auriculata extract (300mg/ml)

7.17 Graph of ulcer index of Ethanol induced model

7.18 Gastric ulcers shown by a) Positive control B) Ranitidine c) P. Auriculata extract (300mg/ml)

7.19 Comparison of effect of Aspirin in histology

7.20 Histopathological changes shown by a) Positive control b) Ranitidine c) P. Auriculata extract (300mg/ml)

7.21 Comparison of effect of Ethanol in histology

7.22 Histopathological changes shown by a) Positive control b) Ranitidine c) P. Auriculata extract (300mg/ml)

ABSTRACT

ANTIULCER SCREENING OF SELECTED PLANTS OF PLUMBAGO SPECIES & THE EFFECT OF PLUMBAGIN IN GASTRIC CELL LINES

Objectives: The objective of the study is to evaluate the antiulcer effect of the Plumbago species and to investigate the most effective plant among the selected Plumbaginales, P.auriculata, P.indica & P.zeylanica after the nvitro evaluation.

Materials and Methods: MTT assay was used to perform the cytotoxic effect of the Plumbagin and to scrutinize the least cytotoxic among the Plumbaginales for gastroprotective effect. The DPPH scavenging assay, Lipid peroxidase inhibition assay, Acid neutralising capacity test and Ethanol induced ulcer model were used for assessing the antiulcer effective plant for invivo studies. Ranitidine (20mg/kg, p.o) was used as reference drug in the study.

Results and Discussion: MTT results revealed that the Plumbagin showed cytotoxic activity at

40.18µ g/ml, P.auriculata is having least cytotoxic effect . Even though significant antiulcer activity were exhibited by all the Plumbaginales and Plumbagin showed significant antioxidant and acid neutralising assays, P.auriculata had decreased the number of ulcers and increased in the percentage gastric protection.

Conclusion: P.auriculata showed antiulcer study and the results were compared by Plumbagin, P.indica and P.zeylanica. A future prospect on mechanism based studies and isolation of the constituents is needed for the individual plants.

1. INTRODUCTION

Peptic ulcer disease (PUD) is one of the most common, chronic gastrointestinal disorder in modern era. It has become a common global health problem affecting a large number of people worldwide and also still a major cause of morbidity and mortality. An estimated 15,000 deaths occur each year as a consequence of PUD. A report of the Indian Council of Medical Research on the epidemiology of peptic ulcer in India showed that the overall incidence of the disease ranged from 1 to 6.5 per thousand in the age group of 15 years and above in the selected urban population.

Ulcer is an open sore that develops on the inside lining of the stomach (a gastric ulcer) or the small intestine (a duodenal ulcer). Both types of ulcers are also referred to as PUD and can be characterized by inflamed lesions or excavations of the mucosa and tissue that protect the gastrointestinal tract. The most common symptom of a peptic ulcer is a burning or gnawing pain in the center of the abdomen (stomach). It was a belief that lifestyle factors, such as diet, smoking, alcohol and stress were the main causes of peptic ulcers. While these factors may play a limited role, it is known that the leading cause of peptic ulcers is a type of bacteria called Helicobacter pylori (H. pylori) can infect the stomach and small intestine; and in some people, the bacteria can irritate the inner layer of the stomach and small intestine, leading to the formation of an ulcer. Painkillers known as nonsteroidal anti-inflammatory drugs (NSAIDs), which include aspirin and ibuprofen, are the second most common cause of peptic ulcers that can irritate the lining of the stomach and small intestine in some people, particularly if they are taken on a long-term basis.

Antacids of different formulas may produce mild laxative effect or result in constipation. Side effects of anticholinergics include glaucoma, urinary retention, rapid heartbeat and mouth dryness. H receptor antagonists have fewer side effects and the major ones are the loss of directional sense and allergic reactions. Such side effects will subside if medication stops.

Moreover, development of drug tolerance and incidence of recurrences make the efficacy of allopathic drugs arguable. Therefore, there is urgent need to find alternatives that have antiulcerogenic properties. This necessitates the need of new herbal anti-ulcer agents, which is required for the development of new drugs. However, the clinical evaluation of these drugs showed development of tolerance, incidence of relapses and side effects that make their efficacy arguable. Furthermore, most of these drugs produce several serious adverse reactions including toxicities, arrhythmias, impotence, gynaecomastia, arthralgia, hypergastrinemia, haemopoeitic changes and even may alter biochemical mechanisms of the body upon chronic usage. This has been the rationale for the development of alternative approach in recent days for the research of new antiulcer drugs medicaments from traditional medicinal system, with lesser toxicities.

India has an ancient heritage of traditional medicine. The material medica of India provides a great deal of the information of folklore practices and traditional aspects of therapeutically important natural products. Indian traditional medicine is based on various systems including Ayurveda, Sidha and Unani, which are unique but there is a common thread in their fundamental principles and practices with the emerging worldwide interest in adopting and studying traditional systems and exploiting their potential based on different health care systems, the evaluation of rich heritage of traditional medicine is essential. So we have to exploit all the possibilities for the perfect evaluation of these systems in order to effectively adopt the therapeutic approaches available in original systems of medicines as well as to help in generating the data to put these systems as seeking attention.

Several medical products of natural origin were conceived in traditional systems of knowledge and practice that has been transmitted over centuries and which continuously change. Traditional indigenous medicine is discovering a reliable ‘living tradition’ rather than relying upon second hand account of their value and use. In many parts of the world, the indigenous systems of medicine have almost completely broken down and disappeared. This system is fragmented with the use of indigenous materials being limited to small tribal and geographical areas. Traditional drugs have an important role in bioprospecting of new medicines. Globally there is an increasing interest in traditional system of medicines. Traditional use of herbals is the very basis and integral part of various cultures, which was developed within ethnic groups before the development and spread of modern medicines. In view of the importance of finding new plant compounds for the management of gastric ulcers in the context of current health, this research aims to show plant species and crud drug preparations with antiulcer activity identified within the ethnopharmacological approach.

It has been systematically showed that aleatory screening of plants used traditionally by pharmaceutical industries in the search for new leads or drugs is vastly expensive and requires much time. On the other hand, ethnodirected approach to traditional knowledge has been extremely useful in screening and identification of plants with bioactive compounds with potential application in drug development. This approach consists in selecting species according to the indication of specific population groups in certain contexts of use. The ethnodirected approach has significantly increased the chances of discovery of new biomolecules with potential therapeutic application while reduce the cost and time involved in this process. Beyond this approach provide a shortcut to the discovery of active compounds that could serve as a basis for rational drug development, it also provides a mechanism for pre-screening on the therapeutic properties of the species collected. Most of these compounds are part of routinely used traditional medicines and hence their tolerance and safety are relatively better known than any other chemical entities that are new for human use. Thus, traditional medicine based on ethnodirected bioprospecting offers an unmatched structural variety as promising new leads.

In the context of ethnodirected studies, the ethnopharmacological research has shown a great contribution in selecting plants and discovery of compounds with pharmacological potential. Ethnopharmacology is a strategy used in the investigation of plants with medicinal properties, combining information acquired from users of medicinal plants (traditional communities and experts), with chemical and pharmacological studies. While in the past the typical industrial drug discovery process made the use of aleatory selection and systematic bioassays to find promising compounds for a particular target, Ethnopharmacology goes the opposite way, tries to understand the pharmacological basis of culturally important medicinal plants, testing their efficacy in the laboratory. Currently, research centers and pharmaceutical industries have driven the search for new drugs of plant origin with effective activity to fight several diseases that today present a limited treatment, including gastrointestinal ailments. In the case of gastric ulcers, several plants extracts described in the specific cultural context are being investigated in the search for sources of effective biomolecules in reducing the damage to gastric mucosa.

For many years, herbal medicines were generally indicated only as co-adjutant gastrointestinal therapy to conventional drugs and when these drugs presented adverse effects and are used during a long-term. Due to several plants encountered in many countries have been reported to posses marked antiulcer activity, the role of natural medicine in management of gastrointestinal diseases has been rethought. Thus, the investigation of traditional knowledge, popular medicine and the development of new medicaments based in natural products for the treatment of diseases like peptic ulcer have been indicated as a absolute requirement of our time. Medicinal plants and their derivatives have been an invaluable source of therapeutic agents to treat various disorders including PUD.

The use of plant extracts used in popular medicine and their phyto-constituents as drug therapy to treat major ailments has proved to be clinically effective for the treatment of PUD. Furthermore, the use of plants and their phytoconstituents in the treatment of gastrointestinal diseases is promising due to the broad spectrum of action on various defensive mechanisms like antioxidant, anti inflammatory, immunomodulatory, cytoprotective and anti secretory. Although several plants have showed beneficial gastroprotective effects, earlier publications, and researchers from around the world, have pointed out that relatively little of the world’s plant biodiversity has been extensively screened for bioactivity, this scenario extends to most plants that have traditional indication for the management of gastric ulcers.

The research oriented thoughts should be farsighted on the status of plant based products globally and underline the thinking and thus trying to reflect to our scenario. The roots of P. auriculata and P. zeylanica are used for edema and piles by the tribals of Tirunelveli.¹ The tribals of Maharashtra are having the habit of consuming the root juice or extract of P. auriculata for gastric acidity before each meal for a weak.²

P . zeylanica is used for centuries in Ayurvedic medicine to increase longitivity and vitality. It is the most important medicinal plant extensively used in herbal formulations. It is used against diarrhoea, dyspepsia, piles; gastro-intestinal complaints and against parasitic diseases, scabies and ulcers in India and Northwest-Ethiopia, in South-Western Nigerian folk medicine respectively.³ The root and root bark of P.zeylanica used as are bitter, stomachic carminative, astringent to bowels, anthelmintic, piles in West Bengal, Bihar and peninsular India.⁴ A review on medicinal plants used by local community of Jodhpur district of Thar desert, it was reported that the plant powder is used for gastrointestinal disturbances.⁵ The root is bitter, carminative and astringent to the bowels. It is also used as laxative, to cure piles. Traditionally the infusion of the roots and leaves are used to cure stomachache and gastric trouble as tea.⁶

P. zeylanica has been evaluated for their wound healing effects of in rats.⁷,⁸ Ayurvedicaly prepared tablets or pills of the same plant (2 tablets or pills) are taken orally thrice in a day after meal preparably with luke warm water or butter milk for period of two weeks relieves dyspepsia, indigestion, peptic ulcers, dysentery and diarrhoea. The hot infusion (15-30 ml) is taken orally twice a day for a period of 7-10 days, relieves dyspepsia, peptic ulcers and indigestion. The decoction prepared out of the root bark churnam is taken orally (30-60 ml) twice in day for about 1-2 weeks gives relief from dysentery, abdominal disorders, peptic ulcers, piles and improves the appetite.⁹

In a review of the drugs derived from herbal plant which are more commonly used in the world for treatment of peptic ulcer and H. pylori, which can say as anti-ulcer activity and having gastroprotective effects; it is reported that the P.indica is having antibacterial activity and Mullu Kuruma tribe of Wayanad district in Kerala uses etnomedicines like P.indica as antibacterial herb.¹⁰,¹¹ P. zeylanica is believed to kill intestinal parasites¹² and it is also used as antibacterial agent in panchakarma ayurvedic therapy¹³ and also in Taiwanese folk medicine for anti- Helicobacter activity.¹⁴ Antibacterial reports point outs the possibilities of the plants against H. pylori infection. In a review of Ethnomedicinal plants to fight neoplastic diseases by P.zeylanica is used for treating diarrhea, dysentery, piles and peptic ulcers, which can later develops to neoplasm. These reviews suggests that these selected plants of plumbago species may protect the gastric mucosa may cure gastric ulcers.¹⁵

A retrospection of healing power of plants, a return to natural remedies is absolute need of our time. Medicine of plant origin is based upon the premise that herbals can pr omote health and alleviate illness. In order to establish the above assertion about their validity, these medicinal plants must be subjected to extensive study in different research works. This study tries to forward the kind attention on the antiulcer drug of natural origin with their suggested medicinal part, screening methodology and type of extract used for evaluation and investigation to prove its use.

An enzymatic screening made with the root extracts of P.indica, P.zeylanica, P.auriculata has shown effect as gastro-intestinal flora normalizer. The investigations were directed into the possibilities of the presence of some powerful enzymes in the root of Plumbago species. The P.zeylanica flowers showed greater effect on digestive stimulus activity than the other Plumbago species.¹⁶

A variety of botanical products have been reported to possess antiulcer activity but the documented literature has centered primarily on pharmacological action in experimental animals. Despite this, there are several botanical products with potential therapeutic applications because of their high efficacy and low toxicity. Finally, it should be noted that substances such as flavonoids, possess antiulcer activity are of particular therapeutic importance as most of the antiinflammatory drugs used in modern medicine are ulcerogenic; flavonoids, tannins and terpenoids are active principles of antiulcer activity.¹⁷ Preliminary photochemical screening of these medicinal plants identified the presence of important secondary metabolites like flavonoids and tannins. Although the Plumbagin is having antioxidant property, its actions are dose dependent; has a dual nature that it is having cytotoxic effects at high doses and no effect on low doses; and it also produces blemishes and other toxicities¹⁸, the current study is evaluating the antiulcer activity by removing Plumbagin by treatment with excess of limewater. Hence Further studies are required to assess the significance of these observations to give a scientific explanation to the antiulcer activity also by performing antioxidant and cytoprotective effects of the plant.

2.LITERATURE REVIEW

2.1.1. Normal anatomy of the stomach

The stomach is located in the left superior abdominal quadrant. It is a muscular sac that lies between the esophagus and the duodenum of the small intestine. The stomach begins at the lower esophageal sphincter and ends at the pyloric sphincter. It has two edges: the lesser curvature, connected to the liver by the lesser omentum, and the greater curvature, closer to the spleen. There are five regions: the cardia, fundus, body, antrum, and pylorus. The stomach receives a rich blood supply largely from the celiac trunk, specifically the left gastric artery. This branches into the hepatic and splenic arteries, that supply the stomach through the right gastric artery, right and left gastroepiploic arteries and short gastric arteries respectively.¹⁹

Abbildung in dieser Leseprobe nicht enthalten

Figure 2.I: Normal anatomy of stomach19

2.1.2. Histology

The mucosal surface of the stomach is lined with simple columnar epithelium. There are numerous funnel-shaped indentations called gastric pits. The base of each gastric pit opens to several tubular gastric glands. The lamina propria is very thin, containing reticular fibers. The muscularis mucosae sits under the base of the gastric glands. The submucosa consists of loose collagenous tissue and arterioles. It has many folds, rugae, that flatten when the stomach is distended. The highly developed muscularis propria has 2 inner layers, inner oblique and middle circular, and an outer longitudinal layer. The serosal layer is thin and covers the peritoneal surface of the stomach

Abbildung in dieser Leseprobe nicht enthalten

Figure 2.2: Normal gastric histology²⁰

There are three types of glands.

The cardia region has cardiac glands, which contain surface mucous cells. The cells have small microvilli and secrete mucus to serve as a protective lining for both the lumen and gastric pits. They also secrete bicarbonate to help maintain a higher pH than gastric acid near the stomach surface in order to protect it from acidic and proteolytic damage. The fundus and body of the stomach have gastric or oxyntic glands, containing several cell types.

In addition to the surface mucous cells, there are neck mucous cells. These are small columnar or triangular shape cells with a basal nuclues, larger granules that stain light purple, and more ribosomes than the surface mucous cells.

University College of Pharmacy, M.G. University, Kottayam Stem cells are also present but cannot be histologically identified. These have a large nucleolus and abundant ribosomes. Stomach stem cells are located in the neck of the gastric glands, which is different than stem cells of the intestine that are located at the base of the crypts. If damaged, these cells can show prominent mitotic figures. The daughter cells differentiate to continuously renew the gastric mucosa. For example, the turnover of surface mucous cells is 3-6 days.

Parietal cells have a broad pyramidal base with central nuclei, clear cytoplasm and abundant mitochondria.. The cells have a fried egg appearance and are concentrated in the upper and middle portions of the gastric glands. Parietal cells have an apical membrane invagination, called a secretory or intracellular canaliculus, where HCl is released through a unique H+/K+ ATPase. Parietal cells also secrete intrinsic factor, a glycoprotein that aids in Vitamin B12 absorption. Bicarbonate is released at the basal surface into the capillary blood flow to neutralize hydrogen ions.

Abbildung in dieser Leseprobe nicht enthalten

Figure 2.3: Histology showing parietal cells19

Chief cells are large, cubodial cells with basophilic cytoplasm and a basally located nucleus. The cells are concentrated in the middle to lower portions of the gastric glands. They have abundant RER, well developped Golgi and numerous apical secretory granules which release pepsinogen I and II. These precursors are activated by the stomach's low luminal pH into the proteolytic enzyme pepsin.

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Figure 2.4: Histology showing chief cells19

Endocrine cells are triangular shaped with basally concentrated eosinophilic granules. They release gastrin, a potent stimulator of acid secretion and other regulatory horomones. The pylorus region has pyloric glands, which are branched and coiled with deeper irregularly shaped pits. There are mucous cells, endocrine cells. The pyloric sphincter, which separates the stomach from the duodenum of the small intestine, is formed from the middle circular layer of the musculars propria/externa of the pyloric region.19

2.2.1. Gastric ulcer and peptic ulcer disease

There are millions of people across the globe suffering from stomach ulcers. It is widely accepted that the pathogenesis of the peptic ulcers is still not fully understood. Increased acid secretion and pepsin activity, reduced mucus, bicarbonate secretion, enhanced contractility of the gastric wall and reduced gastric mucosa blood flow represent some of the established pathogenic factors of gastric ulceration. There are several different kinds of ulcers, and their names usually describe their locations in the digestive tract. A duodenal ulcer or peptic ulcer can be found in the small intestines, just past the stomach while a gastric ulcer is located inside the stomach itself. The peptic and duodenal ulcers are usually benign, but gastric stomach ulcers can be cancerous. Stomach ulcer can sometimes become so raw that it will start to bleed.

Peptic ulcer disease (PUD) refers to a group of ulcerative disorders of the upper gastrointestinal tract that require acid and pepsin for their formation. Ulcers differ from gastritis and erosions in that they extend deeper into the muscularis mucosa. The three common forms of peptic ulcers include Helicobacter pylori associated ulcers, nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers, and stress-related mucosal damage (also called stress ulcers).²¹

There are several mechanisms within our body to protect GIT from the aggressive factors. These defensive forces include the following: (l) the secretion of surface mucus layer by epithelial cells. The nature and adherent characteristics of the mucous, a glycoprotein get secreted by surface epithelium over the gastric wall, is responsible in protecting the underlying cell from the damage caused by the mechanical forces of digestion. Likewise, it lubricates the mucosa, assisting the movement of food over the surface and retains water with in its gelatinous matrix; (2) secretion of bicarbonate in to the mucus layer, thus producing a pH gradient from the highly acidic gastric lumen to the almost neutral mucosal surface; (3) protection against diffusion of hydrogen ions (H+) into the mucosa by the specialized apical surface of the gastric mucosal cells; (4) remarkable regenerative capacity of mucosal epithelial cells for rapid repair of injuries; and (5) mucosal elaboration of prostaglandins having cytoprotective activity, possibly by maintaining adequate mucosal blood flow and by stimulating the secretion of mucus and bicarbonate. Possible causes include increased parietal cell mass and impaired inhibition of stimulatory mechanisms like gastrin discharge.

Table 2.1: Different stages in tissue damage

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2.2.2. Pathophysiology

a) The pathogenesis of duodenal ulcers (DU) and gastric ulcers (GU) is multifactorial and most likely reflects a combination of pathophysiologic abnormalities and environmental and genetic factors.

b) Most peptic ulcers occur in the presence of acid and pepsin when H. pylori, NSAIDs, or other factors disrupt normal mucosal defense and healing mechanisms. Acid is an independent factor that contributes to disruption of mucosal integrity. Increased acid secretion has been observed in patients with DU and may result from H. pylori infection. Patients with GU usually have normal or reduced rates of acid secretion.

c) Alterations in mucosal defense induced by H. pylori or NSAIDs are the most important cofactors in peptic ulcer formation. Mucosal defense mechanisms include mucus and bicarbonate secretion, intrinsic epithelial cell defense, and mucosal blood flow. Maintenance of mucosal integrity and repair is mediated by endogenous prostaglandin production.

d) H. pylori infection causes gastritis in all infected individuals and is causally linked to PUD. However, only about 20% of infected persons develop symptomatic PUD. Most non- NSAID ulcers are infected with H. pylori, and its eradication markedly decreases ulcer recurrence, may cause ulcers by direct mucosal damage, impairing mucosal defense via elaboration of toxins and enzymes, altering the immune/inflammatory response, and by increasing antral gastrin release, which leads to increased acid secretion.

e) Nonselective NSAIDs (including aspirin) cause gastric mucosal damage by two mechanisms: (1) a direct or topical irritation of the gastric epithelium, and (2) systemic inhibition of the cyclooxygenase-1 (COX-1) enzyme, which results in decreased synthesis of protective prostaglandins.

f) Use of corticosteroids alone does not increase the risk of ulcer or complications, but ulcer risk is doubled in corticosteroid users taking NSAIDs concurrently.

g) Epidemiologic evidence links cigarette smoking to PUD, impaired ulcer healing, and ulcer-related GI complications. The risk is proportional to the amount smoked per day. Smoking does not increase ulcer recurrence after H. pylori eradication.

h) Although clinical observation suggests that ulcer patients are adversely affected by stressful life events, controlled studies have failed to document a cause-and-effect relationship.

i) Coffee, tea, cola beverages, beer, milk, and spices may cause dyspepsia but do not increase PUD risk. Ethanol ingestion in high concentrations is associated with acute gastric mucosal damage and upper GI bleeding but is not clearly the cause of ulcers21.

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Figure 2.5: Stages of cell errosional damage21

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