EFFECT OF LEAF EXTRACT FROM MANGROVE SPECIESRhizophora mucronataPoir ON HOMOCYSTEINE INDUCED COAGULATION FACTORS
A dissertation submitted to
THE TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY CHENNAI - 600 032
In partial fulfillment of the requirements for the award of the degree of
MASTER OF PHARMACY IN
PHARMACOGNOSY
Submitted by R. HARI REG.No: 261720651
Under the guidance of
DR. R.VADIVU, M.PHARM., Ph.D., ASSISTANT PROFESSOR
DEPARTMENT OF PHARMACOGNOSY
COLLEGE OF PHARMACY MADRAS MEDICAL COLLEGE
CHENNAI-600 003 MAY 2019
DEPARTMENT OF PHARMACOGNOSY COLLEGE OF PHARMACY
MADRAS MEDICAL COLLEGE CHENNAI-600 003
TAMILNADU
DATE:
CERTIFICATE
This is to certify that the dissertation entitled “EFFECT OF LEAF EXTRACT FROM MANGROVE SPECIES Rhizophora mucronata Poir ON HOMOCYSTEINE INDUCED COAGULATION FACTORS” submitted by R.HARI with Register Number: 261720651 to The Tamil Nadu Dr. M.G.R. Medical University examinations is evaluated.
EXAMINERS
1.
2.
COLLEGE OF PHARMACY MADRAS MEDICAL COLLEGE
CHENNAI-600 003 TAMILNADU
DR. A. JERAD SURESH, M.Pharm, Ph.D., M.B.A.
Principal,
College of Pharmacy, Madras Medical College, Chennai-600 003
CERTIFICATE
This is to certify that the dissertation entitled “EFFECT OF LEAF EXTRACT FROM MANGROVE SPECIES Rhizophora mucronata Poir ON HOMOCYSTEINE INDUCED COAGULATION FACTORS” submitted by R.HARI, Reg. No: 261720651 in partial fulfillment of the requirements for the award of the degree of MASTER OF PHARMACY IN PHARMACOGNOSY to The Tamil Nadu Dr. M.G.R Medical University, Chennai, is a bonafide record of work done by him in the Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai-600 003 during the academic year 2018-2019 under the guidance of DR. R.VADIVU, M.Pharm., Ph.D., Assistant Professor, Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai-600 003.
DR. A. JERAD SURESH, M.Pharm., Ph.D., M.B.A., Place: Chennai - 03
Date:
DEPARTMENT OF PHARMACOGNOSY COLLEGE OF PHARMACY
MADRAS MEDICAL COLLEGE CHENNAI-600 003
TAMILNADU
DR. R. VADIVU, M.Pharm., Ph.D., Assistant Professor,
Department of Pharmacognosy, College of Pharmacy,
Madras Medical College, Chennai-600003
CERTIFICATE
This is to certify that the dissertation entitled “EFFECT OF LEAF EXTRACT FROM MANGROVE SPECIES Rhizophora mucronata Poir ON HOMOCYSTEINE INDUCED COAGULATION FACTORS” submitted by R.HARI, Reg. No: 261720651 in partial fulfillment of the requirements for the award of the degree of MASTER OF PHARMACY IN PHARMACOGNOSY to The Tamil Nadu Dr. M.G.R Medical University, Chennai, is a bonafide record of work done by him in the Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai-600003, during the academic year 2018 - 2019 under my guidance and supervision.
DR. R. VADIVU, M.Pharm., Ph.D., Place: Chennai–03
Date:
DEPARTMENT OF PHARMACOGNOSY COLLEGE OF PHARMACY
MADRAS MEDICAL COLLEGE CHENNAI-600 003
TAMILNADU
DR. R. RADHA, M.Pharm., Ph.D., M.B.A., Professor & Head,
Department of Pharmacognosy, College of Pharmacy,
Madras Medical College, Chennai-600003
CERTIFICATE
This is to certify that the dissertation entitled “EFFECT OF LEAF EXTRACT FROM MANGROVE SPECIES Rhizophora mucronata Poir ON HOMOCYSTEINE INDUCED COAGULATION FACTORS’’ submitted by, R.HARI, Reg. No: 261720651 in partial fulfillment of the requirements for the award of the degree of MASTER OF PHARMACY IN PHARMACOGNOSY to The Tamil Nadu Dr. M.G.R Medical University, Chennai, is a bonafide record of work done by him in the Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai-600 003 during the academic year 2018-2019 under the guidance of DR. R.VADIVU, M.Pharm., Ph.D., Assistant Professor, Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai-600 003.
DR. R. RADHA, M.Pharm., Ph.D., M.B.A., Place: Chennai–03
Date:
ACKNOWLEDGEMENT
I express my first and foremost revered regard and obeisance to the ALMIGHTY and my PARENTS with whose blessings I was able to complete my project work. I wish to acknowledge my sincere thanks and express my heartfelt gratitude to the following persons for their help and encouragement.
I whole heartedly express my high esteem and deep sense of gratitude to respectable Dr.Jayanthi, M.D., DEAN, Madras Medical College, Chennai, for providing me all the facilities and support during the period of academic course work.
It is my privilege and great pleasure in expressing my deep sense of gratitude to DR.R.RADHA, M.Pharm.,Ph.D., Professor and Head, Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai -03, DR.R.Vadivu, M.Pharm.,Ph.D., for his gracious guidance, innovative ideas, with constant inspiration, encouragement, suggestions and infinite help throughout my research work.
I greatly thank her valuable support and endless consideration of my project work. I take great pleasure in acknowledging my sincere thanks to all the staff members DR.R.Vijayabharathi M.Pharm.,Ph.D., DR.P.Muthusamy M.Pharm.,Ph.D., B.L., Mrs. Kumudhaveni M.Pharm of the Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai -03 for their valuable suggestions and moral support. I express my sincere thanks to Mr. Thanigainathan, Plant Director, Sai Mirra Innopharm Pvt. Ltd.,Chennai, and also thank the following AR&D Head and Managers, Ms.Rama, Ms. Hema and FR&D Head And Manager Mr.
Murugan ,Mr. Stephen for guidance in carrying out HPTC,GC-MS and IR analysis.
I would like thanks ,Mrs.T.S.Lakshmi, Lab Technicians of Department of Pharmacognosy,Madras Medical College, Chennai for her help during my research work.
I acknowledge my sincere thanks to DR.R. Chelladurai, Botanist, Survey of Medicinal plants for the authentication and to carry out pharmacognostical studies of the plant material.
I wish to thank Mr. Radha Krishna Reddy, Siddha College, Arumbakkam, for the detailed literature review of the plant.
I express my hearty thanks to my all Class mates for their support and especially S.Muthuraj, S.Megala, T.Rajakani, S.Nivedha, T.Sampatkumar, S.Sumitra, A.Yasmin for their encouragement and support during project work.
I am privileged to thank all my dear seniors for their support, especially Prasanna devi, Thangamani and Srinivasan for their suggestions with me during project work.
I express my thanks to my entire dear junior for their support during project work.
I would like to thank Mr.Ahamed, Shajee Computers for Printing and binding this study.
Finally, I thank all those who helped me directly or indirectly in the successful completion of my thesis. Anyone missed in this acknowledgement are also thanked.
R.HARI
CONTENTS
S.NO TITLE PAGE
NO
1. INTRODUCTION 1
2. LITREATURE REVIEW 15
3. ETHNOBOTANICAL SURVEY 18 4. RATIONALE FOR SELECTION 24
5. AIM AND OBJECTIVE 25
6. PLAN OF WORK 26
7. MATERIALS AND METHODS 28 8. RESULTS AND DISSCUSION 63 9. SUMMARY AND CONCLUSION 102
10. REFERENCES 104
11. ANNEXURES 111
LIST OF TABLES TABLE
NO
TITLE PAGE
NO
1 Risk factors for CVD 2
2 Life-threatening Homocysteine is associated with several conditions as listed in the table
5
3 Diagnostic characters of Rhizophora mucronata Poir 21 4 Geographical coordinates of leaf collection site. 28
5 Grouping of animals 53
6 Kit composition of the Homocysteine bioassay kit 55
7 Biochemical analyzer Setting parameter 63
8 Laminar characters of the mangrove Rhizophora mucronata Poir
66
9 Leaf constants of Rhizophora mucronata Poir 69
10 Physicochemical parameters of Rhizophora mucronata Poir 70 11 Qualitative estimation of inorganic elements of Rhizophora
mucronata Poir
71
12 Quantitative estimation of inorganic elements of Rhizophora mucronata Poir
71
13 Quantitative estimation of Heavy metals 72
14 Percentage yield of successive Rhizophora mucronata Poir 74
15 Qualitative analysis of Phytoconstituents 73
16 Fluorescence characteristic of powdered samples of Rhizophora mucronata Poir at short UV and long UV
74
17 Determination of Total Flavanoid content 75
18 Determination of Total Phenolic Content 76
19 Determination of Total alkaloid Content 77
20 Quantitative estimation of phytoconstituents 79
21 Fluorescence characteristic of extracts of Rhizophora mucronata Poir at short UV and long UV.
79
22 Thin layer chromatography of Various extracts. 80
TABLE NO
TITLE PAGE
NO
23 Key in FT-IR peaks of Ethanolic extract of Rhizophora mucronata Poir
82
24 GC-MS analysis of the ethanolic extract of Rhizophora mucronata Poir.
84
25 Estimated values of antioxidant vitamins in in Rhizophora muucronata Poir leaves
87
26 Parameters of Acute oral toxicity studies 88
27 Weights of the selected group of animals 89
28 Effect of ethanolic extract of Rhizophora mucronata Poir on Homocysteine levels in serum.
92
29 Effect of ethanolic extract ofRhizophora mucronata Poir on Lactate hydrogenase levels in serum.
94
30 Effect of ethanolic extract of Rhizophora mucronata Poir on thromboplastin in blood.
96
31 Effect of ethanolic extract ofRhizophora mucronata Poir onprothrombin in blood.
98
32 Effect of ethanolic extract ofRhizophora mucronata Poir on thromboplastin in blood
100
LIST OF FIGURES FIGURE
NO.
TITLE PAGE
NO
1. Chemical structure of Homocysteine 3
2. Metabolism of homocysteine metabolism. 4 3. Homocysteine effects on blood clotting and
fibrinolysis
8
4. Mechanism of homocysteine and its thiolactones disruption in the endothelial cells.
9
5. Mechanism of homocysteine intrusion in the blood Coagulation pathway
12
6. Ecological Zonation of Mangove 13
7. Rhizophora mucronata Poir morphology 19 8. Distribution of Rhizophora mucronata poir
,Pichavaram Mangrove ,Cuddalore District.
28
9. Shade drying of Leaves 29
10. Soxhlet Extraction and concentration of the crude Rhizophora mucronata poir leaf
41
11. Phytochemical tests of the crude extract 45
12. Estimation of Phytoconstituents 47
13. Macroscopy of leaf 63
14. Micro anatomy of Rhizophora mucronata poir leaves
64
15. Rhizophora mucronata Poir–Stereo microscopy 64 16. TS of Petiole (10X), showing SEM of abaxial side
of Leaf (3000X).
65
17. Transverse Section Of RM 66
18. Powder microscopy of Rhizophora mucronata Poir 68 19. Stomata and Veinislet and Veinlet termination
number
69
FIGURE NO.
TITLE PAGE
NO
20. Total Flavonoid content 76
21. Total Phenolic Content 77
22. Total alkaloid Content 78
23. TLC of various extract of the of Rhizophora mucronata Poir
81
24. IR spectrum of ethanloic extract of Rhizophora mucronata Poir
82
25. GC-MS spectrum of ethanolic extract of Rhizophora mucronata Poir leaves
83
26. High performance liquid chromatography of various extract
85
27. High performance liquid chromatography of various extract
86
28. Standard calibration curve of weight of animals 90 29. Collection of blood sample by retro orbital plexus 90 30. Collected blood in citrated plasma tube and EDTA
palsma tube
91
31. Standard calibration curve of Homocysteine levels in blood
93
32. Standard calibration curve of LDH levels in serum 95 33. Standard calibration curve of APTT result. 97
34. Standard calibration curve of PT 99
35. Standard calibration curve of Prothrombin factors 101
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 1
1. INTRODUCTION
HERBAL MEDICINE
The history of herbal medicine being as old as human civilization and plants have traditionally played an indispensable role in treatment of various pathophysiologic conditions throughout medical history and is being used in several underdeveloped and developing countries in various forms till date.
Human beings are afflicted with more diseases than any other animal species throughout the human civilization and as a result of human beings inquisitive nature they have found the plant sources as a store house of remedies to cure all kinds of ailments of the mankind.
India being home to some of the world's most biodiverse regions and home to four biodiversity hotspots of world :The Himalayas, The Western Ghats, The Indo-Burma region and The Sundaland(Includes Nicobar group of Islands) is enriched with numerous endemic species.
Tribal communities residing in this biodiverse region have traditionally used and relied on herbs for treating various ailments.1
Exponential increase in population and lack of adequate health care facilities to the rural masses necessitates the need to validate the herbal medicines which can be done by doing a meticulous scientific study on medician plants which are under explored and one such work is this research on the
‘EFFECT OF LEAF EXTRACT FROM MANGROVE SPECIES Rhizophora mucronata Poir ON HOMOCYSTEINE INDUCED COAGULATION FACTORS’
CARDIOVASCUAR DISEASES
A key finding from the WHO report on cardiovascular diseases (CVDs) gives us the data that CVDs are the number 1 cause of death globally. An estimated 17.9 million people died from CVDs in 2018, representing 31% of all global deaths. Of these deaths, 85% are due to heart attack and stroke.2
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 2
Out of the 17 million premature deaths (under the age of 70) due to non- communicable diseases in 2015, 82% are in low- and middle-income countries, and 37% are caused by CVDs.
Most cardiovascular diseases can be prevented by addressing behavioural risk factors such as tobacco use, unhealthy diet and obesity, physical inactivity and harmful use of alcohol using population-wide strategies.3
People with cardiovascular disease or who are at high cardiovascular risk (due to the presence of one or more risk factors such as hypertension,hyperhomocystenemia, diabetes, hyperlipidemia or already established disease) need early detection and management using counselling and medicines, as appropriate.4
The WHO report envisages the immediate need for an immediate action to be taken by the health care professionals in weeding out the causes of the risk factors for cardiovascular diseases of which homocysteine is a novel but potent risk factor.
The risk factors causing the cardiovascular diease can be calssified as modifiable, non-modifiabe and novel risk factor as listed below.
Table .1 RISK FACTORS FOR CVD
Modifiable Non-modifiable Novel Risk Factors
• Advancing age
• Heredity or family history
• Gender
• Ethnicity or race
• High blood
pressure.
• Abnormal blood lipids
• Tobacco use.
• Physical inactivity
• Obesity
• Diabetes mellitus
• Unhealthy diets
Hyperhomocysteinemia
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 3
HOMOCYSTEINE
Homocysteine (Hcy) is a sulphur amino acid derived from methionine during transmethylation.
It is a by-product of methionine metabolism, first reported in 1932.For a number of years, some researchers demonstrated that vascular disease of various forms is associated with abnormal methionine metabolism, leading to elevated plasma levels of Hcy.5
Due to its association with various pathological conditions, Hcy gained widespread attention, leading to clarification of the methionine metabolism pathway.
Fig.1 Chemical structure of Homocysteine
METHIONINE METABOLISM PATHWAY.
6-9Methionine is converted to S-adenosylmethionine (SAM) via the enzyme methionine adenosyltransferase, which is the only methyl-donating pathway in humans.
This pathway is essential in the provision of methyl groups to activate biomolecules such as DNA, creatine, phospholipids etc. SAM is demethylated to S- adenosylhomocysteine (SAH), as a product of these methyl-transferase reactions.
SAH is hydrolyzed to Hcy in a reversible reaction, in which SAH formation is favored. Once Hcy is formed, it is metabolized through two metabolic pathways:
remethylation and transsulfuration.
Remethylation is the vitamin-dependent pathway, which converts Hcy back to methionine via the enzyme 5-MethylTetrahydrofolate Reductase (MTHFR) andtheenzymemethioninesynthase(MS).
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 3
HOMOCYSTEINE
Homocysteine (Hcy) is a sulphur amino acid derived from methionine during transmethylation.
It is a by-product of methionine metabolism, first reported in 1932.For a number of years, some researchers demonstrated that vascular disease of various forms is associated with abnormal methionine metabolism, leading to elevated plasma levels of Hcy.5
Due to its association with various pathological conditions, Hcy gained widespread attention, leading to clarification of the methionine metabolism pathway.
Fig.1 Chemical structure of Homocysteine
METHIONINE METABOLISM PATHWAY.
6-9Methionine is converted to S-adenosylmethionine (SAM) via the enzyme methionine adenosyltransferase, which is the only methyl-donating pathway in humans.
This pathway is essential in the provision of methyl groups to activate biomolecules such as DNA, creatine, phospholipids etc. SAM is demethylated to S- adenosylhomocysteine (SAH), as a product of these methyl-transferase reactions.
SAH is hydrolyzed to Hcy in a reversible reaction, in which SAH formation is favored. Once Hcy is formed, it is metabolized through two metabolic pathways:
remethylation and transsulfuration.
Remethylation is the vitamin-dependent pathway, which converts Hcy back to methionine via the enzyme 5-MethylTetrahydrofolate Reductase (MTHFR) andtheenzymemethioninesynthase(MS).
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 3
HOMOCYSTEINE
Homocysteine (Hcy) is a sulphur amino acid derived from methionine during transmethylation.
It is a by-product of methionine metabolism, first reported in 1932.For a number of years, some researchers demonstrated that vascular disease of various forms is associated with abnormal methionine metabolism, leading to elevated plasma levels of Hcy.5
Due to its association with various pathological conditions, Hcy gained widespread attention, leading to clarification of the methionine metabolism pathway.
Fig.1 Chemical structure of Homocysteine
METHIONINE METABOLISM PATHWAY.
6-9Methionine is converted to S-adenosylmethionine (SAM) via the enzyme methionine adenosyltransferase, which is the only methyl-donating pathway in humans.
This pathway is essential in the provision of methyl groups to activate biomolecules such as DNA, creatine, phospholipids etc. SAM is demethylated to S- adenosylhomocysteine (SAH), as a product of these methyl-transferase reactions.
SAH is hydrolyzed to Hcy in a reversible reaction, in which SAH formation is favored. Once Hcy is formed, it is metabolized through two metabolic pathways:
remethylation and transsulfuration.
Remethylation is the vitamin-dependent pathway, which converts Hcy back to methionine via the enzyme 5-MethylTetrahydrofolate Reductase (MTHFR) andtheenzymemethioninesynthase(MS).
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 4 Remethylation appears to be the primary modulator of fasting and elevated plasma Hcy concentrations. Transsulfuration requires vitamin B12 to convert Hcy to cysteine via a two- step process involving the vitamin B6-dependent enzyme cystathionine β-synthase (CBS) and cystathionase. Ulti- mately cysteine is converted to sulfate and excreted into the urine (Figure.2)
Figure 2. Metabolism of homocysteine formation. MS: Methionine synthetase; CBS:
Cystathionine β-synthase; CSE: Cystathionine γ- lyase; MTHFR:
Methylenetetrahydrafolate reductase; BHMT: Betaine-homocysteine methyltransferase; MG: Dimethylglycine; 5, 10-CH3-THF: 5, 10-Methylene- Tetrahydrofolate; 5-CH3-THF: 5-Methyl-Tetrahydrofolate; THF: Tetrahydrofolate;
SH: Serine hydroxymethyl transferase; Pi: Orthophosphate; PPi: Pyrophos- phate In the human body, total Hcy (tHcy) reflects the combined pool of free, bound, reduced and oxidized forms of Hcy in the blood.
Normal tHcy levels ranges between 5 and 15 μmol/L (12 μmol/L being the upper reference limit for populations on a folic-acid-fortified diet, as in North America) with elevations of 16 to 30 μmol/L, 31 to 100 μmol/L, and >100 μmol/L classified as mild, moderate and severe hyperhomocysteinemia (HHcy), respectively.
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 5 Table. 2 Life-threatening Homocysteine is associated with several conditions as listed in the table10
GENETIC FACTORS
Cystathionine 3-synthase deficiency Methionine synthase deficiency Cobalamin C disease.
Methylene tetrahydrofolate reductase Deficiency (congenital deficiency) Methylene tetrahydrofolate reductase Variant (decreased enzyme activity) DEMOGRAPHIC FACTORS Ethnic origin
Increased age Male
Postmenopause
ACQUIRED FACTORS
Deficiencies in folate, vitamin B6, vitamin B12
Health conditions End-stage renal disease Impaired renal function Post organ transplantation Hypothyroidism
Malignant diseases Hypertension Therapeutic drugs
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 6 LIFESTYLE FACTORS
Chronic alcohol consumption Chronic illicit substance use Excessive coffee consumption Lack of exercise
Smoking
PREANALYTICAL FACTORS Delay in separating plasma from cells Nonfasting sample
Despite considerable advances in our understanding of the etiology of CVD, about 30% of CVD cannot be explained by conventional risk factors.
It has been suggested that HHcy accounts for the higher prevalence of CVD that is not explained by traditional risk factors.
HHcy is known to cause multi-disease manifestations such as premature occlusive vascular disease, smooth muscle proliferation, progressive arterial stenosis, haemostatic changes, nephritic syndrome, placental vasculopathy, birth defects, impaired cognitive function, dementia and type-2 diabetes. HHcy is also a risk factor for osteoporotic fractures. In 1969, observations in patients with homocystinuria led McCully to suggest that Hcy may be involved in the pathogenesis of arteriosclerosis In general, clinical and epidemiologic studies show an independent and graded association between Hcy levels and CVD, as well as peripheral artery disease, myocardial infarction and venous thromboembolism.
HAEMOSTASIS
11-13Haemostasis is a integrated process that regulates in vivo flowing properties of the blood.
Conventionally primary and secondary haemostasis only comprised the sequential formation of the white and red thrombus, but there is increasing awareness that haemostasis is intact to fibrinolytic processes, inflammatory reactions as well as to initiation of angiogenesis and wound healing.
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 7 Radically, three components need to interact to assure effective primary and secondary haemostasis together with the normal sequelae, fibrinolysis and tissue repair:
1. The vascular wall (smooth muscle, matrix connective tissue and endothelial cells).
2. Formed elements of the blood (blood platelets, granulocytes, monocytes,lymphocytes),
3. The plasmatic clotting and fibrinolytic systems .
Haemostatic abnormalities can lead to excessive bleeding, thrombosis or other cardiovascular diseases.
Recent haematological studies have proven that homocysteine (Hcy), which is an intermediate formed during the catabolism of the essential dietary amino acid methionine induces changes in haemostasis, including blood clotting and fibrinolysis.
Elevated level of Hcy may disrupt functions of the vascular endothelium, changing the character of its surface from anticoagulant to procoagulant .
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 8 In vitro studies results suggest that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of S- homocysteinylated factor V(Labile factor) by activated protein C (Fig. 3).
Fig3. Homocysteine effects on blood clotting and fibrinolysis
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 8 In vitro studies results suggest that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of S- homocysteinylated factor V(Labile factor) by activated protein C (Fig. 3).
Fig3. Homocysteine effects on blood clotting and fibrinolysis
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 8 In vitro studies results suggest that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of S- homocysteinylated factor V(Labile factor) by activated protein C (Fig. 3).
Fig3. Homocysteine effects on blood clotting and fibrinolysis
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 9 Physiological modifications associated with Hyperhomocysteine conditions:
Homocysteine inhibits cell-surface thrombomodulin expression and irreversibly inactivates thrombomodulin and protein C in a sulphydryl-dependent process. By inhibiting both thrombomodulin surface expression and protein C activation, homocysteine may contribute to the development of thrombosis
Endothelial anticoagulant pathway is based onheparin-like glycosaminoglycan- antithrombin III interaction. Homocysteine, cysteine and 2-mercaptoethanol reduce antithrombin binding activity of endothelial cells. This effect might be causedby generation of hydrogen peroxide. Endothelial cells modulate fibrinolysis by synthesis and secretion of tissue plasminogen activator and its inhibitor, plasminogen activator inhibitor-1.
Fig 4 Mechanism of homocysteine and its thiolactones disruption in the endothelial cells.
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 10 Homocysteine blocks the tissue plasminogen activator binding domain of annexin II, by binding to Cys-9 of annexin .
Clots from plasma or fibrinogen of homocysteinemic patients are composed of thin, tightly packed fibers that are more resistant to lysis by plasmin than control clots. The formation of such clots could directly contribute to the increased risk of thrombosis in hyperhomocysteinemia.
In humans, plasma homocysteine affects clot permeability and resistance to lysis, most likely by a mechanism involving fibrinogen modification by HTL.
Homocysteine inhibits the function of thrombomodulin, an anticoagulant glycoprotein on the endothelial surface that serves as a cofactor for the activation of protein C by thrombin. By inhibiting both thrombomodulin surface expression and protein C activation, homocysteine may contribute to the development of thrombosis in patients with cystathionine beta-synthase deficiency.
Homocysteine, coagulation and fibrinolysis
14-17Fibrinogen is the main substrate for coagulation cascade and form a polymerized fibrin clot.
Fibrinogen is a 340 kDa glycoprotein, consisting of three pairs of nonidentical polypeptide chains, Aa, Bb and g, interconnected by disulfide bonds. In the course of blood coagulation, fibrinogen, after the thrombin-induced cleavage of short fibrinopeptides A and B from the amino termini of a and b chains, is converted to fibrin monomers.
The monomers thereafter interact spontaneously and form half staggered protofibrils flowed by the laterally associated fibers. The initial clot is stabilized by the formation of covalent cross-links in a reaction catalyzed by activated coagulation factor XIII.
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 11 Fibrinogen and other plasma proteins can be covalently modified by Hcy or its thiolactone. Lysine homocysteinylation is a plausible mechanism for protein modification in vivo and is likely an important pathogenic mechanism.
Mass spectrometric analysis of fibrinogen treated with Hcy have revealed that twelve lysines that were homocysteinylated. Several of these are close to tissue plasminogen activator (tPA) and plasminogen binding sites. homocysteinylation of lysine residues in fibrinogen may be linked to three important functional consequences.
1. Modification in the lysine residue could alter the lateral association of fibers and thereby alter clot structure.
2. The alteration of the protein conformation may interfere with calcium binding, which could contribute to alterations in fibrin clot structure.
3. Modification of lysine sites that are directly involved in fibrinolitic enzyme binding and activity could lead to increased resistance to fibrinolysis – the process by which a fibrin clot is dissolved.
In mildly hyperhomocysteinemic human subjects dysfibirinogemia (which is characterized by formation of clots composed of abnormally thin, tightly packed fibers with an increased resistance to fibrinolysis) could play a role in the increased risk of atherothrombotic disease. On the other hand,the increased risk of cardiovascular diseases in hyperhomocysteinemia is mainly related to elevated coagulation factor VIII levels.
Moreover, functional activities of coagulation factors XII, X and II in hyperhomocysteinemia is reduced-induced by folate deficiency in rat, whereas the functional factor VII activity was unchanged and levels of coagulation factor VIIa and Hcy correlate in plasma of acute coronary syndrome patients. This changes may enhanced risk of thrombotic events in hyperhomocysteinemic patients.
Recent reports showed that activity of antithrombin, which
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 12 is one of the most important inhibitors of blood coagulation, is inhibited by Homocsteine thiolactone. Proposed targets of Hcy and HTL action on coagulation and fibrinolysis process are presented in Figure 5.
Fig.5 Mechanism of homocysteine intrusion in the blood Coagulationpathway
MANGROVES
18-25Mangroves are highly dynamic ecological entities which supply energy to aquatic as well as terrestrial habitats through their production and decomposition of plant debris.
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 13 A mangrove is a shrub or small tree that grows in coastal saline or brackish water.
The term is also used for tropical coastal vegetation consisting of such species.
Mangroves occur worldwide in the tropics and subtropics, mainly between latitudes 25° Nand 25° S. The total mangrove forest area of the world in 2000 was 137,800 square kilometres (53,200 sq.m), spanning 118 countries and territories.
Mangroves are salt-tolerant trees, also called halophytes and are adapted to life in harsh coastal conditions. They contain a complex salt filtration system and complex root system to cope with salt water immersion and wave action. They are adapted to the low oxygen conditions of waterlogged mud.
Fig. 6 Ecological Zonation of Mangove
MSL-Mean Sea Leval ; HWSL-High Sea Level
INTRODUCTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 14 Local communities residing in thecoastal areas(mangrove forest)have traditionally used and relied on herbs for treating various ailments and as a health drink.
Rhizophora mucronata Poir26-31
Rhizophora mucronata Poir(Rhizoporaceae) is a mangrove commonly found in the coastal intertidal zones of Indo-pacific and East Africa. It is popularly known as Asiatic mangrove and regionally (Tamil Nadu) as ‘Peykkanthal’.The natural habitat of Rhizophora mucronata Poir is estuaries, tidal creeks and flat coastal areas subject to daily tidal flooding. Mangroves that need no introduction in today’s world with a variety of bioactive metabolites have been the interest of marine researchers all over the world. Apart from the resources those flourishes in the dense tangle of roots, mud and tidal water, mangroves are known for its medicinal wealth that have been successfully employed for treating a variety of diseases over hundreds of years.
The specific medicinal properties of Rhizophora mucronataPoir depend on the part employed and finds its use to treat human ailments like angina, dysentery, hematuria and many more. Rhizophoras species are gradually disappearing due to increasing salinity and biotic pressures. It is placed In the Red list of IUCN placed Rhizophora mucronata Poir in least concern category but in Indus delta due number of reason Rhizophora mucronata Poir facing extinction.
Taking into account the ethnomedicinal uses owing to its potential antioxidant and nutritional value this study is a meticulous research to overcome the destructive and life threatening effects of homocysteine induced coagulation factors by employing this loop root mangrove.
LITERATURE REVIEW
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 15
2. LITERATURE REVIEW PHARMACOGNOSTICAL REVIEW
Batool et al.,( 2014) studied an overview of the Asiatic mangrove (Rhizophora mucronata Poir)32
Nurul Auni Zainal Abidin et,al.,(2013) studied the Basic Chemical Constituents of the Rhizophora Mangrove species.33
Kathiresan K. (2010) studied about the Globally threatened mangrove species in India.34
PHYTOCHEMICAL ANAYSIS
P.Thirunavukkarasu et al., (2017) have done the phytochemical analysis of mangrove derived crude plant extract -Rhizophora mucronata Poir35
Silpa Sreedhar et al., (2015) has done the Phytochemical analysis of Rhizophora mucronataPoir36
PHARMACOLOGICAL REVIEW
Mariappan Premananthan et al.,(2016) studied the Invitro anti human immune deficiency virus activity of polysaccharide from Rhizophora mucronata.37
Rohini R M et al., (2015) has done A comparative evaluation of analgesic and anti-inflammatory activities of Rhizophora mucronata bark Extracts.38
Yunita E. et al., (2015) studies the The Potency of Rhizophora mucronata Leaf Extract as Antidiarrheal agent.39
S.Kusuma, (2014) has studied the Potent antimicrobial activity of Rhizophora mucronata.40
Kaori Taniguchi et al. (2013) have isolated two new coumarins and a new xanthone from the leaves of Rhizophora mucronata.41
Mahmiah, Giman et al.,(2012) has studied the Potential of methanolic extract from the stem bark of mangrove Rhizophora mucronata against bacteria Escherichia coli and Aeromonas Hydrophylla .42
LITERATURE REVIEW
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 16
Ammanamanchi S. R. (2012) has studied Rhizophorin A,A Novel Secolabdane Diterpenoid from the Indian Mangrove Plant Rhizophora mucronata.43
A .Moumita Ray, et al.,(2011) has done Pharmacognostic and anti- hyperglycemic evaluation of the leaves of sunderban mangrove, Rhizophora mucronata l.44
Andy Ramu (2012) .has studied the Antidiabetic activity of 7- methoxycoumarin from the bark of Marine plant Rhizophora mucronata.45
Md. Atiqur Rahman et al.,(2011) has studied the antinociceptive , antidiarrhoeal and cytotoxic activities of Rhizophora mucronata lamk .46
S.Priscilla Helen Christy et al.,(2010) has done Cellulase enzyme production from Rhizophora mucronata.47
Hardoko, Suprayitno et al., (2009) has studied of Ripe Rhizophora mucronata fruit flour as functional food for antidiabetic.48
Suganthy N and Devi KP.(2009) has reported In vitro antioxidant and anti- cholinesterase activities of Rhizophora mucronata.49
Y. Narasimha Rao et al.(2008) has reportedIn-vitro anthelmintic activity of Rhizophora mucronata leaf extract.50
Cortez, J.R, Dalabajan, A.K. and Montinola, T.R. mangrove ( Rhizophora mucronata) leaf extracts as larvicidal agents for mosquito population.51
Haque et al.(2007) has reported that The extract of Rhizophora mucronata have anti-diabetic activity in Long Evans rats (500 mg/kg bw).52
Vigneswaran T, et al.,(2005) has determined the in-vitro antioxidant activity of leaf extracts of Rhizophora mucronata Lam. (Rhizophoraceae).53
Adhikari A et al., (2003) has studied the ( Antidiabetic and antioxidant activity of Rhizophora mucronata leaves (Indian sundarban mangrove): An in vitro and in vivo study.54
LITERATURE REVIEW
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 17
ANTIHYPERHOMOCYSTEINE ACTIVITY
Shaker E et al., (2013) studied the Hypo- Homocysteinemia Effect of Some Ethanolic Plant Extracts.55
M. Nazam Ansari et al.,(2008)studied the Hypo- Homocysteinemia Effect of Some Ethanolic Plant Extracts of embelica ribes.56
Richard N. Podell, M.D.(2000) has proved that Antioxidant Vitamins Blocks the acute toxic effects of homocysteiene .57
ANTIOXIDANT ACTIVITY
Nappo F et,al.(2013) studeid the Impairment of endothelial functions by acute hyperhomocysteinemia and reversal by antioxidantvitamins.58
T Vigneswaran et al(2012) has determined the in-vitro antioxidant activity of leaf extracts of Rhizophora mucronata Poir(Rhizophoraceae).59
Chitra Jairaman1 et al., have performed chromatographic analysis of Rhizophora mucronata Poirbark- based on antioxidant and free radical scavenging potential.60
Wulan tri wahyuni et al.,(2007) studied the Potency of Rhizophora mucronata Poir species Extracts as antioxidant and inhibitor of acethylcholinesterase.61
HEAMOSTASIS
Chwatko G et al.,(2005) has studied The determination of Homocysteine- Thiolactone in human plasma.62
Cafolla A et al .(2002)has studied the Effect of folic acid and vitamin C supplementation on folate status and homocysteine level: a randomised controlled trial in Italain smoker-blood donors.63
ETHNOBOTANICAL SURVEY
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 18
3. ETHNOBOTANICAL SURVEY PLANT PROFILE
TAXONIMIC POSITION OF RHIZOPHORA SPECIES
64Plantae : Plants
Subkingdom : Tracheobionta–Vascular plants Superdivision : Spermatophyta–Seed plants Division : Magnoliophyta–Flowering plants Class : Magnoliopsida–Dicotyledons Subclass : Rosidae
Order : Rhizophorales
Family : Rhizophoraceae–Red Mangrove family Genus : Rhizophora P.–mangrove
Species : Rhizophora mucronata Poir.–Asiatic mangrove
MORPHOLOGICAL DESCRIPTION OF Rhizophora mucronata Poir
65Rhizophora mucronata Poirbelongs to family Rhizophoraceae and commonly known as the looproot mangrove, red mangrove and Asiatic mangrove An evergreen, medium to a tall tree that may reach 25 to 30 m height. As in the case of Rhizophora apiculata numerous, much-branched, hoop or pile-like stilt roots loop from branches and stems and provide support to trees. These stilt roots also function as above-ground breathing roots. The bark is brown or reddish, smooth and sometimes scaly. Leaves are single, opposite, leathery, broadly elliptic to oblong-elliptic in shape with very distinct black dots on the under surface and tipped with a fine spine (mucor). Leaf stalk is 3 to 5 cm long. The inflorescence is dichotomously branched cyme, four to eight flowered and axillary in position; peduncle is slender, yellow and 2 to 3 cm long.
ETHNOBOTANICAL SURVEY
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 19 Fig.7 Rhizophora mucronata Poir morphology (A) Habit (B) Stem base with stilt roots (C) Bark (D) Branches (E) Leafy branch end with flowers (F)Leaf apex with mucro (G) Inflorescence (H) Minute bract at dichotomous inflorescence branch (I) Mature bud with minute bracteole below calyx (J) Cross section of bud (K) Mature propagules (L) Thick leathery petal (M) Stamens (N) Flower (O) Pistil showing four-
ETHNOBOTANICAL SURVEY
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 20 sided ovary (P) Flower with one petal removed (Q) Pear-shaped fruit (R) Stamens with pollen.
PHYSIOLOGICAL DISTINCTION
66The photosynthetic characteristics of mangroves are consistent with those of C3 plants photosynthetic biochemistry. Photosynthesis of mangroves is typically saturated at moderate light intensities with photosynthetic rates maximal at leaf temperatures less than 35 °C. Carbon dioxide fixation is done by the use of intermediates containing 3 carbon atoms which is converted into glucose (C6H12O6) using light that at most 10% of the salinity of seawater were found in the sap of the tree. Due to the presence of salt glands on the leaves, the excess of salt can be secreted .Water use efficiency is one outstanding feature of the gas exchange characteristics in mangroves and when compared with other C3 plants grown under similar conditions it is unusually high.
ECOLOGY
67Grows up in an environment similar to R.apiculata, but is more tolerant to sandy and harder substrate. Generally grows in clusters near or on the banks of creeks and tidal estuaries, rarely far from the tides. Optimal growth occurs in deep submerged areas, on the hard ground and rich in humus. This species is one of the most important and widespread mangrove. Flowering occurs throughout the year. Seedlings are often eaten by crab, preventing new growth. Seeds dried in the shade for a few days before planting are less favourable for crab. This process is likely to cause the accumulation of protective tannins. The presence is very abundant
ETHNOBOTANICAL SURVEY
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 21
DIAGNOSTIC FEATURES
68-69Table.3. Diagnostic characters of Rhizophora mucronata Poir
S.No. Parts Description
1. Leaf shape ovate, broader at
base
2. Leaf apex broadly acute
3. Leaf base Rounded
4. Leaf mucro Spike
5. Leaf L/W 1.6 cm
6. Bark Brown to bark grey rough friable
horizontally fissured
7. Petiole L 2.61 cm
8. Petiole W 0.31 cm
9. Inflorescence branch no. 3 cm
10. No. of flowers per
inflorescences 2 to 8 cm
11. Bract condition Smooth
12. Bud L 1.47 cm
13. Bud W 0.8 cm
14. Bud L/W 1.81 cm
15. Mature bud X section Rounded
16. Bud shape Ellipsoidal
17. Peduncle L 3.15 cm
18. Peduncle W 0.3 cm
19. Petal L 0.8 cm
20. Petal W 0.3 cm
21. Petal X section Folded
22. Petal margin Hairy
23. Stamen no. 8
ETHNOBOTANICAL SURVEY
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 22
S.No. Parts Description
24. Style L 0.1 cm
25. Fruit L 4.5 cm
26. Fruit W 3 cm
27. Fruit L/W 1.5 cm
28. Hypocotyl L 51 cm
29. Hypocotyl W 1.7 cm
30. Collar L 1.5 cm
L-Length; W-Width
DISTRIBUTON
70Rhizophora species has a wide geographic range of distribution and are primarily divided into two genetically isolated global regions by two currently effective dispersal barriers, namely the Atlantic East Pacific and Indo West Pacific. In the AEP, two chief Rhizophora taxa dominate, namely R. mangle and R. racemosa there are also two groupings of taxa in the IWP, including R. apiculata and a sub-group combining Rhizophora mucronata Poir and R. stylosa. Taxa in the genus Rhizophora are primarily divided into IWP and AEP groups based on morphological characters, notably the presence/absence of leaf mucro. The total number of species is around five, but there are also comparable wide-ranging hybrids in each region, as well as wide variation in diagnostic morphological characters for the known entities. This combination of factors has created considerable confusion for those wanting to identify the genetic composition of particular mangrove stands. Rhizophora species comprise four distinct groupings of taxa: “mangle” and “racemosa” in the AEP and
“Rhizophora mucronata Poir-stylosa” and “apiculata” in the IWP. There are also at least four reported hybrids in the world and there are likely to be more. In the IWP, there are four reported species [R.apiculata. Rhizophora mucronata Poir, R. Stylosa ].
PHYTOCONSTITUENTS
Leaves:
Carbohydrates, Proteins, Antioxidant vitamins (A, C, E), Alkaloids, Flavanoids, Carotenoids, Chlorophyll A, B, A and B.
ETHNOBOTANICAL SURVEY
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 23 Bark:
Condensed tannins, Saponins, Anthocyanins, Inositol, 7-Methoxycoumarin.
Flower:
Gallic acid, and Polyphenols, Triterpenes, Carbohydrates and Quercetin Stem:
Hydrolysable tannins, Minerals, Polysaccharide, and Steroids.
ETHANOMEDICAL USES
71Fruits:
The fruit juiceis used in the treatment of diaarhoea
Fruits are used in diabetes.
Leaves
Leaves are used to treat microbial infections.
Leaves are used in case of haematuria.
The leaves decoctions are used as health drinks.
Leaves are used in curing ulcer, inflammation and as have cytoprotective effect.
Leaves are used to hyperglyceamic conditions.
Bark
Used to extract tannins.
And used as antioxidant, laxatives, antifouling agent
Reduce cholesterol level.
Root:
The driedpowder is uses as astringent, and in treatment of malaria and as laxative
RATIONALE FOR SELECTION
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 24
4. RATIONALE FOR SELECTION
This plant Rhizophora mucronata Poir belonging to the family Rhizoporaceae was chosen for its abundance in antioxidant vitamins.
The tradionally claimed properties associated with the plant includes antiviral, anti- bacterial, anti-diarrheal, antifungal, antioxidant properties. anti-oxidant, anti- microbial, antinociceptive, activities and also used to treat diabetes, asthma, cancer, ulcer and wounds.
Pharmacological study relating to homocysteine was not done earlier.
Toxicity studies of this mangrove palnt have shown to be safe for animal experimental studies.
Medicinally active moieties were underexplored in this mangrove which necessitates its exploration.
Hence, this plant was chosen for our study on homocysteine induced cogulation factors.
AIM AND OBJECTIVE OF THE STUDY
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 25
5. AIM AND OBJECTIVE OF THE STUDY
The present investigation is to find a natural alternative to the current allopathic treatment coupled with its nutritional benefits as Antioxidant vitamins are known to increase the immunity and reduce ageing process.
THE CORE OBJECTIVE OF THE PRESENT STUDY ARE AS FOLLOWS
• To abate the prothrombotic property of Homocysteine and its free radicals on the blood by lowering its levels in the blood through the administration of antioxidant vitamins.
• To mitigate the risk of cardiovascular diseases caused due to increased levels of Homocysteine in the blood by reducing the levels of Homocysteine and its pro-coagulant property through the administration of antioxidant vitamins.
On proving the efficiency of this plant extract on lowering the levels of homocysteine this will provide an alternative natural treatment for hyperhomocysteine and diseases caused by pro-coagualant property of blood.
Once the activity is proved the leaves can be used as a source for health drink preparations as it is rich in antioxidants.19
Adding the leaf extracts in daily diet will keep the destructive effect of the free radicals in check.
PLAN OF WORK
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 26
6. PLAN OF WORK
COLLECTION OF PLANT MATERIAL
AUTHENTICATION
PHARMACOGNOSTICAL STUDIES
Macroscopy
Microscopy
Powder Microscopy
Quantitative Microscopy- Stomatal Index, Veinislet and veinlet termination number
Determination of Physico-chemical constants.
Ash values
Total ash value
Acid insoluble ash value
Water soluble ash value
Sulphated ash value Extractive values
Alcohol soluble extractive value
Water soluble extractive value Determination of Loss on Drying Determination of Swelling index Determination of Foaming index Determination of Volatile oil content
Qualitative and Quantitative Estimation of Heavy metals and Inorganic element
PLAN OF WORK
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 27
PHYTOCHEMICAL STUDIES
Preparation of Extracts
Preliminary phytochemical screening of powder and extracts
Behaviour of drug powder with different chemical reagents
Quantitative Estimation of phytoconstituents
Fluorescence analysis of Plant powder and extracts
Chromatographic Studies
Thin Layer Chromatography
GC-MS
High Performance liquid column Chromatography
Spectroscopic Studies
Infra Red Spectroscopy
PHARMACOLOGICAL STUDIES
In vivo Methionine induced Hyperhomocysteinemia
Serum Analysis
Homocysteine.
Lactate dehydrogenase (LDH).
Blood Analysis
Activated partial thromboplastin time (APTT)
Thromboplastin time (PT)
Thrombin time (TT)
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 28
7. MATERIALS AND METHODS COLLECTION OF PLANT
The leaves of Rhizophora mucronata Poirwere collected from Pichavarammangrove, Cuddalore district ,Tamilnadu and was placed in a air tight container to prevent loss of moisture during transportation to the laboratory and the geographical coordinates are given below
Table.4. Geographical coordinates of leaf collection site.
District Location Coordinates
CUDDALORE PICHAVARAM
MANGROVE
11.4319°N, 79.7810° E
Fig 8. Distribution of Rhizophora mucronata poir ,Pichavaram Mangrove ,Cuddalore District.
IDENTIFICATION AND AUTHENTICATION OF PLANT
Taxonomic identification of the plant was carried out by Professor Dr.R.
Chelladurai ,botanist ,survey of Medicinal plants and authenticated as leaves of Rhizophora mucronata Poir Fresh leaves were air dried, powdered and stored in an air tight container at room temperature which wasthen subjected to pharmacognostical and phytochemical studies.
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 29
DRYING AND POWDERING
72The purpose of drying is to reduce the moisture content andto prevent the plant from being affected by any microbial growth . Almostall plants contain 60-8-% of moisture when harvested and must be dried to keep the moisture below 5% limit.
The leaves were then air dried and pulverised coarsely.
Fig 9. Shade drying of Leaves
7.1 PHARMACOGNOSTICAL STUDIES
Pharmacognosy is the systemic study of natural source, it is preliminary step in the standardization of crude drugs, which consist of identification, authentication and standardization of herbal medicinal plants through organoleptic character, histological character, powder microscopy, quantitative microscopy, histochemical analysis and physicochemical observations as prescribed by an authoritative sources.
Evaluation of crude drugs involves determination of authenticity, quality, purity, potency, safety, efficacy, reliabity and reproducibility of the results for varying batch of crude drug. It is also used in the detection of nature of adulteration. Systemic identification of crude drugs and their quality assurance gives an integral part of drug description.
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 30
7.1.1 MACROSCOPICAL EVALUATION
73Macroscopical characters includes organoleptic characters and various morphological features of the plant were studied. This observation is primary importance before any further testing can be carried out.
Organoleptic characters like as color, odour, taste and nature were studied.
Morphological characters such as size, shape, surface, fracture and thicknes were observed for the determination of safety, efficacy and purity of crude drugs.
7.1.2 MICROSCOPICAL EVALUATION
POWDER MICROSCOPY
The shade dried, powdered plant material was used for powder microscopic analysis. The organoleptic charcters were observed and to identify the different characteristic features various staining reagent were used. Powder was stained with 1% phloroglucinol in 90% ethanol, concentrated hydrochloric acid and observed through microscope. All the lignified cells stained with pink color. Calcium oxalate crystals were observed under polarized light microscope.
7.1.3 QUANTITATIVE MICROSCOPY
73-75Determination of leaf constants and linear Measurements of crude drugs helps in the identification, characterization and standardization of leaves.
Determination of leaf constants
The important histological features on the epidermal surfaces of leaves are the stomata, trichomes and palisade cells. The measurements like Stomatal number, Stomatal index, Palisade ratio, Veinislet number and Veinlet termination number are considered to be important parameters in the microscopical evaluation.
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 31 STOMATAL NUMBER
The stomatal number is the number stomata per sq.mm of epidermis of the leaf.
Procedure:
The leaf (middle part) was cleaned by boiling with potassium hydroxide solution. Upper and lower epidermis was separated by means of forceps and mounted on slide with glycerine water. Camera lucida and drawing board wasarrangedfor making the drawing. Square of 1 mm was drawn by means of stage micrometer. The cleared leaf (epidermis) was placed on the stage.Epidermis cells and Stomata were traced and the number of stomata present in the area of 1 sq.mm was counted. The results for each of the ten fields were recorded and the average number of stomata per sq.mm was calculated.
STOMATAL INDEX
The Stomatal Index is the percentage which the numbers of stomata from tothe total number of epidermal cells; each stomata being counted as one cell.
It is calculated by using the following equation S
S.I = --- X 100 E+S
Where,
S.I = Stomatal Index
S = Number of stomata per unit area
E = Number of epidermal cells in the same unit area.
PROCEDURE:
The leaf (middle part) was cleaned by boiling with potassium hydroxide solution. Upper and lower epidermis was separated by means of forceps and mounted on slide with glycerine water. Camera lucida and drawing board wasarrangedfor making the drawing . Square of 1 mm was drawn by means of stage micrometer. The
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 32 cleared leaf (epidermis) was placed on the stage. Epidermis cells and Stomata were traced and the number of stomata present in the area of 1 sq.mm was counted . The stomatal index was calculated using the above formula. The values for upper and lower surface(epidermis) was determined separately.
VEIN ISLET AND VEINLET TERMINATION NUMBER76-77 VEIN ISLET NUMBER:
The Vein islet number is defined as the number of vein-islets per sq.mm of the leaf surface midway between the midrib and the margin.
PROCEDURE:
The leaf (middle part) was cleaned by boiling with potassium hydroxide solution for about 30 mins. Camera lucida and drawing board was arranged for making the drawing. Stage micrometer was placed on the microscope using 16 mm objectives, and a line equivalent to 1mm was drawn as seen through the microscope. A square on this line was constructed .The paper is moved so that the square is seen in the eye piece, in the centre of the field.The veins were traced off which were within the square. The number of vein islets in the square millimetre were counted and found the average number of vein islets form the four adjoining squares, to get the values for one sq.mm.
VEINLET TERMINATION NUMBER
The Veinlet number is defined as the number of veinlets terminations per sq.mm of the leaf surface midway between the midrib and the margin.
PROCEDURE:
The leaf (middle part) was cleaned by boiling with potassium hydroxide solution for about 30 mins. Camera lucida and drawing board was arranged for making the drawing. Stage micrometer was placed on the microscope using 16 mm objectives, and a line equivalent to 1mm was drawn as seen through the microscope. A square on this line was
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 33 constructed . The paper is moved so that the square is seen in the eye piece, in the centre of the field. The veins were traced off which were within the square. The number of veinlet terminations present within the square were countedand found the average number of veinlet termination number from the four adjoining squares, to get the values for one sq.mm
7.1.4 PHYSICO-CHEMICAL CONSTANTS
78Shade dried powdered plant materials of the (leaves of Rhizophora mucronata Poir) was used for the determination of the physicochemical constants in accordance with the WHO guidelines.
DETERMINATION OF ASH VALUE
Ash values are helpful in determining the quality and purity of a crude drug in the powdered form. The residue remaining after incineration is the ash content of the drug, which simply represents inorganic salts, naturally occurring in drug or adhering to it or deliberately added to it as a form of adulteration.
Ash value of a crude drug is defined as the inorganic residue remaining after incineration, which complies of inorganic salts, naturally occurring in drug or adhering to it or deliberately added to it as a form of adulteration.
Hence is used for the determination of the quality and purity of the crude drug in the powdered form.
TOTAL ASH
Total ash method is designed to measure the total amount of material remaining after ignition. They include both physiological ash which is derived from plant tissue itself and non-physiological ash which is the residue of extraneous matter adhering to plant surface.
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 34 Procedure
Silica crucible was heated to red hot for 30 minutes and cooled in the desiccators incinerate about 2 to 3g accurately weighed, of the ground drug in a tarred silica dish at a temperature not exceeding 450˚C until the sample is free from carbon, cooled in desiccators and weighed. The ash obtained and weighed. The percentage of total ash was calculated.
The percentage of ash value = (weight of total ash/ weight of drug taken×
100)
WATER SOLUBLE ASH
The difference in weight between the total ash and the residue after treatment of the total ash in water.
Procedure
Total ash obtained was boiled for 5 minutes with 25ml of water, insoluble matter were collected in ash less filter paper, washed with hot water and ignite for 15 min at a temperature not exceeding 450˚C. Subtract the weight of the residue in mg from the weight of total ash. Calculate the content of water-soluble ash in mg per gram of air-dried material.
Percentage of water soluble ash = (Weight of residue obtained/ Weight of sample taken) × 100.
ACID INSOLUBLE ASH
The residue obtained after boiling the total ash with dilute hydrochloric acid, the remaining insoluble matters are ignited and measured. This measures the amount of silica present, especially as sand and siliceous earth.
Procedure
To the crucible containing total ash of the sample, 25ml of dilute hydrochloric acid was added. The insoluble matter was collected on ash less filter paper (Whatmann 41) and washed with hot water until the filtrate is neutral. Filter paper containing the insoluble matter to the original crucible
MATERIALS AND METHODS
Department of Pharmacognosy, COP, MMC, Chennai-03. Page 35 dry on hot plate and ignite to constant weight. Allow the residue to cool in suitable desiccators for 30 min and weighed without delay. Content of acid- insoluble ash with reference to the air dried drug is calculated.
Percentage of acid insoluble ash = (Weight of the residue obtained/
Weight of the sample taken) ×100.
SULPHATED ASH
Sulphated ash test is used to measure the amount of residual substance not volatilized from a sample. These tests are usually to determine the content of inorganic substance.
Procedure
Silica crucible dish were heated to redness for 10 minutes, allowed to cool in a desiccators and weigh. 2g of sample were accurately weighed, ignited gently until the white fumes are no longer evolved and ignite at 800 ± 25˚C until all black particles have disappeared. Crucible is allowed to cool, and few drops of sulphuric acid and heat. Ignite as before, allow to cool and weighed. This process was repeated until two successive weighing differ by more than 0.5mg.
Percentage of sulphated ash = (Weight of the residue obtained/ Weight of the sample taken) × 100.
DETERMINATION OF EXTRACTIVE VALUE
Extractive values are useful for the evaluation of phytoconstituents especially when the constituents of a drug cannot be really estimated by and other means. Further these values indicate the nature of the active constituents present in a crude drug.
